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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Vet. Sci.</journal-id>
<journal-title>Frontiers in Veterinary Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Vet. Sci.</abbrev-journal-title>
<issn pub-type="epub">2297-1769</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fvets.2022.935307</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Veterinary Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Cryptorchidism: The dog as a study model</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name><surname>Hern&#x000E1;ndez-Jard&#x000F3;n</surname> <given-names>Norma</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/1872227/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name><surname>Rojas-Casta&#x000F1;eda</surname> <given-names>Julio C&#x000E9;sar</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="corresp" rid="c002"><sup>&#x0002A;</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/848501/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Landero-Huerta</surname> <given-names>Daniel</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/658001/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Reyes-Cruz</surname> <given-names>Estefan&#x000ED;a</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/1982145/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Reynoso-Robles</surname> <given-names>Rafael</given-names></name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/278809/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Ju&#x000E1;rez-Mosqueda</surname> <given-names>Mar&#x000ED;a del Lourdes</given-names></name>
<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
</contrib>
<contrib contrib-type="author">
<name><surname>Medrano</surname> <given-names>Alfredo</given-names></name>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/84919/overview"/>
</contrib>
<contrib contrib-type="author">
<name><surname>Reyes-Delgado</surname> <given-names>Fausto</given-names></name>
<xref ref-type="aff" rid="aff6"><sup>6</sup></xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name><surname>Vigueras-Villase&#x000F1;or</surname> <given-names>Rosa Mar&#x000ED;a</given-names></name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="corresp" rid="c001"><sup>&#x0002A;</sup></xref>
<uri xlink:href="http://loop.frontiersin.org/people/809278/overview"/>
</contrib>
</contrib-group>
<aff id="aff1"><sup>1</sup><institution>Programa Doctorado en Ciencias de la Producci&#x000F3;n y de la Salud Animal, Universidad Nacional Aut&#x000F3;noma de M&#x000E9;xico</institution>, <addr-line>Mexico City</addr-line>, <country>Mexico</country></aff>
<aff id="aff2"><sup>2</sup><institution>Laboratorio de Biolog&#x000ED;a de la Reproducci&#x000F3;n, Instituto Nacional de Pediatr&#x000ED;a, SS</institution>, <addr-line>Mexico City</addr-line>, <country>Mexico</country></aff>
<aff id="aff3"><sup>3</sup><institution>Laboratorio de Morfolog&#x000ED;a Celular y Tisular, Instituto Nacional de Pediatr&#x000ED;a, SS</institution>, <addr-line>Mexico City</addr-line>, <country>Mexico</country></aff>
<aff id="aff4"><sup>4</sup><institution>Departamento de Morfolog&#x000ED;a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut&#x000F3;noma de M&#x000E9;xico</institution>, <addr-line>Mexico City</addr-line>, <country>Mexico</country></aff>
<aff id="aff5"><sup>5</sup><institution>Laboratorio de Reproducci&#x000F3;n Animal, Facultad de Estudios Superiores Cuautitl&#x000E1;n, Universidad Nacional Aut&#x000F3;noma de M&#x000E9;xico</institution>, <addr-line>Cuautitl&#x000E1;n Izcalli</addr-line>, <country>Mexico</country></aff>
<aff id="aff6"><sup>6</sup><institution>Banfield Pet Hospital-Universidad Nacional Aut&#x000F3;noma de M&#x000E9;xico</institution>, <addr-line>Mexico City</addr-line>, <country>Mexico</country></aff>
<author-notes>
<fn fn-type="edited-by"><p>Edited by: Peter DOVC, University of Ljubljana, Slovenia</p></fn>
<fn fn-type="edited-by"><p>Reviewed by: Rita Payan Carreira, University of Evora, Portugal; Alessandra Pelagalli, University of Naples Federico II, Italy; Caterina Squillacioti, University of Naples Federico II, Italy</p></fn>
<corresp id="c001">&#x0002A;Correspondence: Rosa Mar&#x000ED;a Vigueras-Villase&#x000F1;or <email>rmvigueras&#x00040;yahoo.com.mx</email></corresp>
<corresp id="c002">Julio C&#x000E9;sar Rojas-Casta&#x000F1;eda <email>rocajc1&#x00040;yahoo.com.mx</email></corresp>
<fn fn-type="other" id="fn001"><p>This article was submitted to Comparative and Clinical Medicine, a section of the journal Frontiers in Veterinary Science</p></fn></author-notes>
<pub-date pub-type="epub">
<day>13</day>
<month>09</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2022</year>
</pub-date>
<volume>9</volume>
<elocation-id>935307</elocation-id>
<history>
<date date-type="received">
<day>03</day>
<month>05</month>
<year>2022</year>
</date>
<date date-type="accepted">
<day>22</day>
<month>08</month>
<year>2022</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x000A9; 2022 Hern&#x000E1;ndez-Jard&#x000F3;n, Rojas-Casta&#x000F1;eda, Landero-Huerta, Reyes-Cruz, Reynoso-Robles, Ju&#x000E1;rez-Mosqueda, Medrano, Reyes-Delgado and Vigueras-Villase&#x000F1;or.</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Hern&#x000E1;ndez-Jard&#x000F3;n, Rojas-Casta&#x000F1;eda, Landero-Huerta, Reyes-Cruz, Reynoso-Robles, Ju&#x000E1;rez-Mosqueda, Medrano, Reyes-Delgado and Vigueras-Villase&#x000F1;or</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p></license> </permissions>
<abstract>
<p>Cryptorchidism (CO) or undescended testicle is an abnormality of male gonadal development that can generate long-term repercussions in men, such as infertility and germ cell neoplasia <italic>in situ</italic> (GCNIS). The origin of these alterations in humans is not completely clear, due to the absence of an animal model with similar testicular development as in humans with CO. This work intends to describe the testicular histological development of dogs with congenital CO, and determine whether the species could adequately serve as a study model for this pathology in humans. The study was carried out with 36 dogs, equally distributed in two groups: healthy control (CTRL) and CO groups. The contralateral testis to the undescended one in CO group of the animals was considered and analyzed. Each group was subdivided in three stages of development: (1) peripubertal stage (6&#x02013;8 months), (2) young adult (9&#x02013;48 months) and (3) senile (49&#x02013;130 months). Histological development, the presence of cells with gonocyte morphology, cell proliferation, testicular lipoperoxidation and hormonal concentrations of testosterone, estradiol, FSH and LH were evaluated and described. In the cryptorchid testes, the first histological alterations appeared from the first stage of development and were maintained until the senile stage. A pronounced testicular lipoperoxidation occurred only in the second stage of development. The histological alterations due to CO were markedly evident in the young adult stage. Testosterone concentrations witnessed a decrease starting from in the second stage and kept on until the last stage. The contralateral testes of the CO animals showed alterations that positioned them between the control and CO testes. Testicular development of dogs with CO is similar to that of humans. The results of the study suggest that this species could serve as a suitable model for the study of CO in humans.</p></abstract>
<kwd-group>
<kwd>cryptorchidism</kwd>
<kwd>testis</kwd>
<kwd>dog</kwd>
<kwd>human</kwd>
<kwd>seminiferous epithelium</kwd>
<kwd>Leydig cell</kwd>
<kwd>vimentin</kwd>
</kwd-group>
<contract-num rid="cn001">INP 830420-3F7</contract-num>
<contract-num rid="cn002">student:297349</contract-num>
<contract-sponsor id="cn001">Instituto Nacional de Pediatria<named-content content-type="fundref-id">10.13039/100016928</named-content></contract-sponsor>
<contract-sponsor id="cn002">Consejo Nacional de Ciencia y Tecnolog&#x00ED;a<named-content content-type="fundref-id">10.13039/501100003141</named-content></contract-sponsor>
<counts>
<fig-count count="5"/>
<table-count count="2"/>
<equation-count count="0"/>
<ref-count count="84"/>
<page-count count="13"/>
<word-count count="9091"/>
</counts>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="s1">
<title>Introduction</title>
<p>Cryptorchidism (CO) or undescended testis is the most common abnormality of male reproductive development (<xref ref-type="bibr" rid="B1">1</xref>). In premature children, the incidence is 1.1&#x02013;45.3% (<xref ref-type="bibr" rid="B2">2</xref>) while in term newborn and 1-year aged males, it is 3&#x02013;5 and 1.0&#x02013;1.5% respectively (<xref ref-type="bibr" rid="B3">3</xref>&#x02013;<xref ref-type="bibr" rid="B5">5</xref>). The etiology is associated with environmental, anatomical and genetic factors (<xref ref-type="bibr" rid="B6">6</xref>). Its importance resides on the long-term effects, since it predisposes patients to the development of infertility and germ cell neoplasia <italic>in situ</italic> (GCNIS) (<xref ref-type="bibr" rid="B7">7</xref>, <xref ref-type="bibr" rid="B8">8</xref>). GCNIS is the histological lesion that precedes the development of testicular germ cell tumor (TGCT) (<xref ref-type="bibr" rid="B9">9</xref>).</p>
<p>Reported fertility in men who underwent CO and orchidopexy is 89.7% for unilateral CO and 65.3% for bilateral CO (<xref ref-type="bibr" rid="B2">2</xref>). Moreover, oligospermia is documented in 43 and 73% of patients with unilateral and bilateral CO respectively (<xref ref-type="bibr" rid="B10">10</xref>). It is mentioned that the oxidative stress generated by the high temperature in the suprascrotal area can damage the Sertoli cells function (<xref ref-type="bibr" rid="B11">11</xref>). This condition deprives the germ cells of the factors necessary for their differentiation, or their binding proteins (<xref ref-type="bibr" rid="B12">12</xref>). Although, it is possible that free radicals directly affect germ cells by inhibiting spermatogenesis (<xref ref-type="bibr" rid="B13">13</xref>).</p>
<p>The prevalence of GCNIS is estimated to be 2.9% in men with a history of CO (<xref ref-type="bibr" rid="B14">14</xref>). Undifferentiated gonocytes with pluripotent capacity are suggested to be the target of malignancy, and thus the origin of this neoplasm (<xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B15">15</xref>). A gonocyte is a cell of embryonic origin that differentiates into a spermatogonia <italic>stem cell</italic> in the last third of gestation period and continues through the first 10 months of postnatal period to give rise to the germ line that will initiate spermatogenesis in the future (<xref ref-type="bibr" rid="B16">16</xref>&#x02013;<xref ref-type="bibr" rid="B19">19</xref>).</p>
<p>Knowledge about the etiopathogenesis of GCNIS and infertility in patients with CO is still limited (<xref ref-type="bibr" rid="B20">20</xref>). This may be attributed to the current lack of an animal model that show similar testicular development as in human. Inevitably, this situation may have affected the efforts to find alternative ways to reduce the risk of developing testicular cancer and fertility problems in these patients.</p>
<p>The most used animal models for the study of CO in man have been the rat, mouse and rabbit. In these species, CO is induced by the administration of drugs or surgical manipulation, which can generate stress, surgical iatrogenesis and tissue adhesions, thus compromising the results (<xref ref-type="bibr" rid="B21">21</xref>). The rat and the mouse have the disadvantages that the period of gonocyte differentiation to spermatogonia (4&#x02013;10 days of postnatal life) and the so-called &#x0201C;minipuberty&#x0201D; (the first postnatal hours) are short and without quiescence period (<xref ref-type="bibr" rid="B22">22</xref>). Therefore, its experimental management implies challenges.</p>
<p>The domestic dog (<italic>Canis familiaris</italic>) presents the advantage of spontaneous CO development, with a frequency of 6.8% (<xref ref-type="bibr" rid="B23">23</xref>). In dogs, the CO presentation can be unilateral or bilateral, the retained testis positioned in inguinal or abdominal the right unilateral inguinal region being the most frequent as in humans (<xref ref-type="bibr" rid="B23">23</xref>, <xref ref-type="bibr" rid="B24">24</xref>). In addition, CO in the dog increases the risk of presenting testicular neoplasia from 3.6 to 13.6 times (<xref ref-type="bibr" rid="B25">25</xref>). Coupled with this, is the likelihood of developing tumors of the germ cells; such as Sertoli-Leydig cells tumor (<xref ref-type="bibr" rid="B26">26</xref>), and classic (<xref ref-type="bibr" rid="B26">26</xref>) and spermatocytic seminomas (<xref ref-type="bibr" rid="B27">27</xref>). Also, the differentiation period of the gonocytes in dogs, as a target of malignancy, extends up to the first 4 months of age (<xref ref-type="bibr" rid="B28">28</xref>).</p>
<p>Based on the above exposed points, the objective of this study was to describe the histological development of the canine testis with spontaneous CO, at different stages of development, to determine if this species can be best suited as a study model for this pathology in humans.</p>
</sec>
<sec sec-type="materials and methods" id="s2">
<title>Materials and methods</title>
<sec>
<title>Animals</title>
<p>Thirty-six small-breed dogs (&#x02264; 10 kg) were included in the study. The animals were from several private veterinary clinics, UNAM-Banfield Veterinary Hospital and the Office of the Mobile Sterilization Unit for Dogs and Cats in Alvaro Obregon borough, Mexico City, Mexico. Animals that presented infections, chronic degenerative diseases, malnutrition, testicular tumors and problems in the reproductive system unrelated to CO were excluded from the study.</p>
<p>For the purposes of the study, the dogs were divided into two equal groups of 18 animals each, consisting of healthy control (CTRL) and unilateral inguinal CO animals, clinically diagnosed by palpation and ultrasound imaging study. Each group was subdivided with an equal number of animals into three stages of development as follows: (1) peripuberal (6&#x02013;8 months), representing the age of the spermatogenic onset and also the limit for testicular descent into the scrotum (<xref ref-type="bibr" rid="B28">28</xref>). The average age of the CTRL group was 6.42 &#x000B1; 0.48 months and that of the CO group was 5.6 &#x000B1; 0.41 months. (2) The age range of young adult animals was (9&#x02013;48 months), which is the age of sexual maturity (<xref ref-type="bibr" rid="B26">26</xref>). The average age of the CTRL group was 41.14 &#x000B1; 5.14 months and that of the CO group was 29.85 &#x000B1; 5.35 months; and (3) while the age of senile animals ranged 49&#x02013;130 months (<xref ref-type="bibr" rid="B26">26</xref>, <xref ref-type="bibr" rid="B28">28</xref>), which encompasses the age of possible development of neoplasia (<xref ref-type="bibr" rid="B29">29</xref>, <xref ref-type="bibr" rid="B30">30</xref>). The average age of the CTRL group was 102.85 &#x000B1; 6.85 months and that of the CO group was 122.57 &#x000B1; 11.04 months. In addition, in the CO group, the contralateral testicle (CONTRA) to the cryptorchid one, which was always in scrotal position, was analyzed. The testicular samples were obtained at orquiectomy; from each gonad, three samples of testicular tissue, &#x0007E;0.25 cm<sup>3</sup> each, from the mid-portion, opposite to the testicular mediastinum were obtained for morphological analysis, two samples of &#x0007E;0.75 cm<sup>3</sup> for immunohistochemistry and two of &#x0007E;0.75 cm<sup>3</sup> for the determination of lipoperoxidation. In addition, peripheral blood samples were obtained and used to hormonal determination.</p>
</sec>
<sec>
<title>Morphological analysis</title>
<p>The histological alterations induced by canine spontaneous CO condition were used to compare for resemblance with the histological pattern of human CO development.</p>
<p>The evaluation was performed by fixing testicular tissue samples in Karnovsky solution for 24 h, postfixed in 1% OsO<sub>4</sub>, dehydrated in gradual ethanol solutions and embedded in EPON 812 (Ted Pella, INC. CA, USA). Semi-thin sections of 1 &#x003BC;m thickness were cut using an Ultracut UCT ultramicrotome (Leica, Vienna, Austria) and stained with 0.5% toluidine blue. Histological analysis of the seminiferous tubules was performed using a light microscope (BX 51, Olympus, Tokyo, Japan). From each tissue sample, a slide with 5 tissue sections was obtained. Therefore, 5 cross-sectioned tubules were evaluated, resulting in a total of 15 tubules per animal. The area of the seminiferous epithelium was determined by subtracting the internal area from the external area. The cellular area of at least 15 Leydig cells per animal was also determined using an image analysis system (Image-Pro Plus 6.2. Media Cybernetics, INC. MD, USA). Leydig cells were evaluated using the 100x objective lens and identified by their central location in the interstitial space, its polygonal shape of round nucleus, with an evident nucleolus. The maturation index (MI) or Johnsen index (<xref ref-type="bibr" rid="B31">31</xref>) and the histopathological index (HI) were determined (<xref ref-type="bibr" rid="B32">32</xref>).</p>
<p>To confirm the presence of gonocytes and cellular alterations, the testicular tissue was sectioned at 60&#x02013;70 nm thickness. Sections were counterstained with uranyl acetate and lead citrate and analyzed with an electron microscope (JEM-1011, JEOL, Osaka, Japan). The presence of gonocytes was determined through their ultramorphological characteristics, among which are large round cells with the presence of cytoplasmic extensions in some cases, prominent nucleus and nucleolus, and mitochondria peripheral to the nucleus.</p>
</sec>
<sec>
<title>Determination of cell proliferation and the functional status of Sertoli cells</title>
<p>Immunoreactivity to proliferating cell nuclear antigen (PCNA) and to vimentin antigen were evaluated using immunohistochemical techniques to determine if the spontaneous CO in the dogs presents cell hypoplasia due to low cell proliferation and alterations in the functional status of Sertoli cells.</p>
<p>The determination was carried out by fixing the two testicular samples per gonad in 4% paraformaldehyde for 2 h. Later, these were dehydrated, clarified and embedded in paraffin. Sections of 4 &#x003BC;m thickness were cut and mounted on slides with poly-L- lysine (Sigma-Aldrich, St. Louis, MO, USA). Tissue sections were deparaffinized with xylene and hydrated with graded ethanol solutions. Subsequently, tissue sections were exposed to sodium citrate (pH 7.6) for 5 min in a microwave oven at 800 W. Afterwards, sections were outlined with PAP pen for immunostaining (Dako, Carpinteria, CA, USA). The tissue sections were then treated with 3% hydrogen peroxide for 10 min. Subsequently, they were placed in 0.1% Tween 20 (Sigma-Aldrich) in phosphate buffered saline (PBS, pH 7.4) for 10 min and blocked with 1% bovine serum albumin in PBS for 2 h. Thereafter, they were incubated overnight at room temperature with mouse monoclonal primary antibody against PCNA (sc-56 B1717, Santa Cruz, Biotechnology, CA, USA) and the primary monoclonal antibody against vimentin (sc-6260, Santa Cruz Biotechnology) both at a dilution of 1:500. This was followed by another incubation with biotinylated anti-mouse IgG (Invitrogen, Carlsbad, CA,USA) at a dilution of 1:100 for 1 h. Finally, the samples were exposed to the streptavidin-horseradish peroxidase conjugate for 1 h (Vector Laboratories, Peterborough, UK). To visualize the immunoreaction, tissue sections were incubated in a peroxidase substrate solution containing 1.6 ml distilled H<sub>2</sub>O, 20 &#x003BC;l 10x substrate buffer and 40 &#x003BC;l 50x diaminobenzidine (chromogen) (Santa Cruz Biotechnology) and 1% H<sub>2</sub>O<sub>2</sub> (Merck, Darmstadt, Germany) in methanol for 30 min. Subsequently, sections were counterstained with hematoxylin, dehydrated through a graded ethanol series and washed with xylene. All dilutions and washes between steps were done with PBS. The number of immunoreactive cells per transversely cut seminiferous tube was determined. 5 seminiferous tubes per section were evaluated, making a total of 10 tubes evaluated per animal. The distribution pattern of immunoreactivity to vimentin in the cytoplasm of Sertoli cells was also determined. In addition, the Masson trichrome technique was performed on one slide of each animal to show the collagen fibers.</p>
</sec>
<sec>
<title>Determination of lipoperoxidation</title>
<p>Malondialdehyde, the final product of testicular lipoperoxidation, was determined using TBARS technique with the objective to know if the spontaneous CO in the dogs presents an increase in lipoperoxidation as an indirect way of oxidative damage.</p>
<p>For this purpose, 1 ml aliquot containing homogenized testicular tissue was added to 2 ml of thiobarbituric acid reagent (TBA; MP, Illkirch, France) (0.375 g of TBA &#x0002B; 15 g of trichloroacetic acid &#x0002B; 2.5 ml of HCl diluted in 100 ml distilled water). The resulting solution (3 ml total volume) was heated in a boiling water bath for 30 min. Samples were cooled on ice and centrifuged at 3,000 g for 15 min. The absorbance was measured in the supernatants by spectrophotometry at 532 nm. The concentrations of substances reactive with thiobarbituric acid were calculated by interpolation on a standard curve of periodic oxidation of malondialdehyde. The final result was expressed as nmol of substances reactive to thiobarbituric acid per mg of protein. Protein content in testicular tissue samples was measured with Folin&#x00027;s phenol reagent (<xref ref-type="bibr" rid="B33">33</xref>). Lipid peroxidation results were corrected for protein content in each sample and expressed as pg MDA/mg protein.</p>
</sec>
<sec>
<title>Hormonal determination</title>
<p>Peripheral blood (5 ml) was performed at 9:00 AM to avoid circadian fluctuations and collected in sterile BD vacutainer tubes for serum extraction and centrifuged at 3500 rpm for 10 min. The serum obtained was stored at &#x02212;70&#x000B0;C until analysis. The determination of testosterone, estradiol, LH, and FSH was performed for each individual without replicates and by automated chemiluminescent microparticle immunoassay, using the Architect R 2nd generation (Abbott Diagnostics, Abbott Park, IL, USA), with strict adherence to the manufacturers&#x00027; instructions. The detection limits and the intra- and inter-assay coefficients of variation for each hormone, respectively, were as follows: testosterone 0.01 ng/ml, 3.10 and 3.60%; estradiol 10.00 pg/ml, 2.98 and 3.60%; LH 0.50 mIU/ml, 2.07 and 3.74%; FSH 0.05 mIU/mL, 2.99 and 3.80%.</p>
</sec>
<sec>
<title>Statistical analysis</title>
<p>The data were expressed as the mean &#x000B1; SEM. A parametric ANOVA test, followed by a multiple comparison using the Tukey test (seminiferous epithelial area, MI, HI, proliferation index, and hormone concentrations) and non-parametric Kruskal&#x02013;Wallis (lipoperoxidation) test were used for statistical analysis with <italic>P</italic> values &#x0003C; 0.05 considered statistically significant.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<sec>
<title>Stage 1: Peripuberal</title>
<p>The CTRL group animals presented different degrees of development of spermatogenesis. The 16.6% of the animals (1/6) presented few gonocytes (verified with electron microscopy), spermatogonia and spermatocytes. In other specimens, it was possible to observe round and elongated spermatids. Slight histological alterations such as scaling were observed (<xref ref-type="fig" rid="F1">Figure 1A</xref>). The ultrastructure showed completely healthy Sertoli and germ cells (<xref ref-type="fig" rid="F2">Figure 2A</xref>).</p>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption><p>Seminiferous tubules of dogs from the control (CTRL) <bold>(A&#x02013;C)</bold>, cryptorchid (CO) <bold>(D&#x02013;F)</bold> and contralateral (CONTRA) <bold>(G&#x02013;I)</bold> groups at peripubertal (first column); young adult (second column) and senile (third column) stages. In CTRL dogs, complete spermatogenesis is observed, although gonocytes were still observed in the peripubertal stage <bold>(A)</bold>. In the senile stage <bold>(C)</bold>, some specimens showed histological alterations such as thickening and folding in the basement membrane (arrow) and even complete disorganization of the seminiferous epithelium (<bold>C</bold> insert). In animals with CO in the peripubertal stage, different degrees of maturation of the epithelium are observed, from the presence of gonocytes to spermatocytes (<bold>D</bold> insert). Histological alterations become evident from the young adult stage <bold>(E)</bold>, which continued until senile <bold>(F)</bold> with vacuolization (V), desquamation (D), folding of the basement membrane (arrow), presence of atypical cells <bold>(F)</bold>, cellular degeneration and arrest of spermatogenesis, as well as Sertoli-cell-only syndrome (<bold>E</bold> insert). In the contralateral testes, the degree of histological damage was not as severe as in the cryptorchid ones, presenting alterations such as hypoplasia and cellular degeneration, thickening and folding of the basement membrane (arrow) with cellular desquamation (D). The CONTRA group images inserts show a panoramic view of the tubes. Gonocyte (G), Spermatogonia (Es) spermatocyte (Ec), Round spermatid (Er), elongated spermatid (Ed), atypical cells (a), Sertoli cell nuclei (S). Toluidine Blue. Bar scale: 20&#x003BC;m.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-935307-g0001.tif"/>
</fig>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption><p>Electron microscopy (EM) of canine testes from the control groups (CTRL) <bold>(A&#x02013;C)</bold> and from cryptorchidism (CO) <bold>(D&#x02013;F)</bold>. It is observed in the peripubertal stage, the presence of gonocytes in migration phase <bold>(A)</bold> and with small pseudopodia touching the basement membrane <bold>(D)</bold>. In the young adult stage of the second row, a clear spermatogenesis is observed in the CTRL testis <bold>(B)</bold>, unlike the CO testis, which shows the presence of a gonocyte-like cell with thickening of the basement membrane <bold>(E)</bold>. In the senile stage, the CTRL testes still preserve spermatogenesis <bold>(C)</bold> with disorganization of the epithelium, and cells morphologically similar to gonocytes <bold>(F)</bold> were observed in the CO. Gonocyte (G), round spermatid (Er), elongated spermatid (Ed), basement membrane (arrow), vacuoles (V), glycogen (Gl). Electron microscopy. Bar scale: 2&#x003BC;m.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-935307-g0002.tif"/>
</fig>
<p>Fifty percent (3/6) of the animals of CO group, presented cells with gonocyte morphology (<xref ref-type="fig" rid="F1">Figure 1D</xref>). Spermatogenesis did not develop beyond spermatocytes (<xref ref-type="fig" rid="F1">Figure 1D</xref>) and slight histological alterations, such as tubular hypoplasia and cellular degeneration, mainly in spermatocytes, were observed in their seminiferous tubules. The animals of the CO group presented a significant reduction in the maturation index (MI) of 2.57 times as well as in cell proliferation index 4.1 times when compared with the CTRL group (<xref ref-type="table" rid="T1">Table 1</xref>; <xref ref-type="fig" rid="F1">Figures 1D</xref>, <xref ref-type="fig" rid="F3">3A</xref>,<xref ref-type="fig" rid="F3">D</xref>). However, histopathological index (HI) and seminiferous epithelial area did not show differences between both groups (<italic>p</italic> &#x0003E; 0.05). The result of TBARS technique did not show significant difference (<italic>p</italic> &#x0003E; 0.05) between both groups, although there was a tendency for greater lipoperoxidation in the CO group (2.07 times more. The analysis of the hormones show that T concentrations experienced a downward trend in the CO group when compared with the CTRL group, nevertheless, this was not significant (<xref ref-type="table" rid="T2">Table 2</xref>). The same trend was observed with the rest of the hormones (<xref ref-type="table" rid="T2">Table 2</xref>).</p>
<table-wrap position="float" id="T1">
<label>Table 1</label>
<caption><p>Parameters evaluated in the studied groups at different stages of development of the dogs.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Development period</bold></th>
<th valign="top" align="center"><bold>Group</bold></th>
<th valign="top" align="center"><bold>Seminiferous epithelial area</bold><break/> <bold>(&#x003BC;m<sup>2</sup> X10<sup>2</sup>)</bold></th>
<th valign="top" align="center"><bold>Histopathologic index (HI)</bold></th>
<th valign="top" align="center"><bold>Maturation index (MI)</bold></th>
<th valign="top" align="center"><bold>Proliferation Index (PI)</bold></th>
<th valign="top" align="center"><bold>Lipoperoxidation</bold><break/> <bold>(pg MDA/mg of protein</bold></th>
<th valign="top" align="center"><bold>Leydig cells area</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Peripuberal</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">177.46 &#x000B1; 39.42</td>
<td valign="top" align="center">4.76 &#x000B1; 0.62</td>
<td valign="top" align="center">8.11 &#x000B1; 0.80</td>
<td valign="top" align="center">96.57 &#x000B1; 10.97</td>
<td valign="top" align="center">0.14 &#x000B1; 0.01</td>
<td valign="top" align="center">165.51 &#x000B1; 13.05</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO</td>
<td valign="top" align="center">84.78 &#x000B1; 19.17</td>
<td valign="top" align="center">10.20 &#x000B1; 1.45</td>
<td valign="top" align="center">3.15 &#x000B1; 0.15<sup>a</sup></td>
<td valign="top" align="center">24.05 &#x000B1; 3.85<sup>a</sup></td>
<td valign="top" align="center">0.29 &#x000B1; 0.11</td>
<td valign="top" align="center">127.78 &#x000B1; 21.59</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CONTRA</td>
<td valign="top" align="center">137.55 &#x000B1; 42.82</td>
<td valign="top" align="center">10.85 &#x000B1; 0.94</td>
<td valign="top" align="center">4.00 &#x000B1; 0.40</td>
<td valign="top" align="center">29.55 &#x000B1; 7.88</td>
<td valign="top" align="center">0.16 &#x000B1; 0.02</td>
<td valign="top" align="center">128.98 &#x000B1; 16.95</td>
</tr>
<tr>
<td/>
<td valign="top" align="center"><italic>p</italic>-value</td>
<td valign="top" align="center">&#x0003E;0.05</td>
<td valign="top" align="center">&#x0003E;0.05</td>
<td valign="top" align="center">0.013</td>
<td valign="top" align="center">0.030</td>
<td valign="top" align="center">&#x0003E;0.05</td>
<td valign="top" align="center">&#x0003E;0.05</td>
</tr>
<tr>
<td valign="top" align="left">Young adult</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">369.27 &#x000B1; 26.99</td>
<td valign="top" align="center">3.07 &#x000B1; 0.33</td>
<td valign="top" align="center">9.35 &#x000B1; 0.29</td>
<td valign="top" align="center">115.83 &#x000B1; 11.53</td>
<td valign="top" align="center">0.13 &#x000B1; 0.00</td>
<td valign="top" align="center">205.29 &#x000B1; 14.87</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO</td>
<td valign="top" align="center">100.56 &#x000B1; 17.73<sup>a</sup></td>
<td valign="top" align="center">13.45 &#x000B1; 1.12<sup>a</sup></td>
<td valign="top" align="center">3.46 &#x000B1; 0.77<sup>a</sup></td>
<td valign="top" align="center">25.56 &#x000B1; 7.36<sup>a</sup></td>
<td valign="top" align="center">0.40 &#x000B1; 0.14<sup>a</sup></td>
<td valign="top" align="center">122.94 &#x000B1; 14.27<sup>a</sup></td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CONTRA</td>
<td valign="top" align="center">172.96 &#x000B1; 43.67</td>
<td valign="top" align="center">6.56 &#x000B1; 2.90</td>
<td valign="top" align="center">6.16 &#x000B1; 1.70</td>
<td valign="top" align="center">102.20 &#x000B1; 32.49</td>
<td valign="top" align="center">0.39 &#x000B1; 0.18</td>
<td valign="top" align="center">198.15 &#x000B1; 15.46<sup>c</sup></td>
</tr>
<tr>
<td/>
<td valign="top" align="center"><italic>p</italic>-value</td>
<td valign="top" align="center">0.030</td>
<td valign="top" align="center">0.008</td>
<td valign="top" align="center">0.0001</td>
<td valign="top" align="center">0.002</td>
<td valign="top" align="center">0.0001</td>
<td valign="top" align="center">&#x0003C;0.037</td>
</tr>
<tr>
<td valign="top" align="left">Senile</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">494.19 &#x000B1; 128.40</td>
<td valign="top" align="center">3.42 &#x000B1; 0.45</td>
<td valign="top" align="center">9.19 &#x000B1; 0.22</td>
<td valign="top" align="center">122.00 &#x000B1; 13.78</td>
<td valign="top" align="center">0.17 &#x000B1; 0.01</td>
<td valign="top" align="center">197.44 &#x000B1; 10.84</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO</td>
<td valign="top" align="center">114.72 &#x000B1; 13.41<sup>a</sup></td>
<td valign="top" align="center">16.72 &#x000B1; 2.22<sup>a</sup></td>
<td valign="top" align="center">2.93 &#x000B1; 0.70<sup>a</sup></td>
<td valign="top" align="center">43.28 &#x000B1; 10.13<sup>a</sup></td>
<td valign="top" align="center">0.22 &#x000B1; 0.03</td>
<td valign="top" align="center">123.10 &#x000B1; 16.32<sup>a</sup></td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CONTRA</td>
<td valign="top" align="center">156.09 &#x000B1; 25.74<sup>b</sup></td>
<td valign="top" align="center">13.35 &#x000B1; 6.20<sup>b</sup></td>
<td valign="top" align="center">7.20 &#x000B1; 1.73<sup>c</sup></td>
<td valign="top" align="center">57.68 &#x000B1; 22.30</td>
<td valign="top" align="center">0.21 &#x000B1; 0.02</td>
<td valign="top" align="center">180.70 &#x000B1; 13.08</td>
</tr>
<tr>
<td/>
<td valign="top" align="center"><italic>p</italic>-value</td>
<td valign="top" align="center">&#x0003C;0.017</td>
<td valign="top" align="center">&#x0003C;0.040</td>
<td valign="top" align="center">&#x0003C;0.03</td>
<td valign="top" align="center">0.002</td>
<td valign="top" align="center">&#x0003E;0.05</td>
<td valign="top" align="center">0.036</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>The results are expressed as mean &#x000B1; standard error. Control (CTRL), with cryptorchidism (CO) and contralateral (CONTRA) to the undescended testicle.</p>
<p><sup>a</sup> p &#x0003C; 0.05 CTRL vs. CO of the same period of development.</p>
<p><sup>b</sup> p &#x0003C; 0.05 CTRL vs. CONTRA of the same period of development.</p>
<p><sup>c</sup> p &#x0003C; 0.05 CO vs. CONTRA the same development period.</p>
</table-wrap-foot>
</table-wrap>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption><p>Immunohistochemistry for PCNA in seminiferous tubes of dogs from the control group (CTRL) <bold>(A&#x02013;C)</bold>; from cryptorchidism (CO) <bold>(D&#x02013;F)</bold> and contralateral (CONTRA) <bold>(G&#x02013;I)</bold> in the peripubertal stages (first column); young adult (second column) and senile (third column). A marked immunoreactivity (arrow) is observed in the testes of the CTRL animals unlike the CO, mainly in the young adult stages. The testes of the CONTRA group show an immunoreactivity that is neither as marked as in the CTRL nor as weak as in the CO. Bar scale: 50 &#x003BC;m.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-935307-g0003.tif"/>
</fig>
<table-wrap position="float" id="T2">
<label>Table 2</label>
<caption><p>Hormone concentrations in the control and cryptorchid groups at different developmental stages in the dog.</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th valign="top" align="left"><bold>Development period</bold></th>
<th valign="top" align="center"><bold>Group</bold></th>
<th valign="top" align="center"><bold>Testosterone (ng/ml)</bold></th>
<th valign="top" align="center"><bold>Estradiol (pg/mL)</bold></th>
<th valign="top" align="center"><bold>FSH (mUI/mL)</bold></th>
<th valign="top" align="center"><bold>LH</bold><break/> <bold>(mUI/mL)</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Peripuberal</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">3.85 &#x000B1; 1.39</td>
<td valign="top" align="center">10.86 &#x000B1; 0.70</td>
<td valign="top" align="center">0.05 &#x000B1; 0.00</td>
<td valign="top" align="center">0.50 &#x000B1; 0.01</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO<break/> <italic>p</italic>-value</td>
<td valign="top" align="center">0.87 &#x000B1; 0.36<break/> &#x0003E;0.05</td>
<td valign="top" align="center">10.50 &#x000B1; 0.28<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.06 &#x000B1; 0.02<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.50 &#x000B1; 0.03<break/> &#x0003E;0.05</td>
</tr>
<tr>
<td valign="top" align="left">Young adult</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">5.97 &#x000B1; 1.01</td>
<td valign="top" align="center">13.75 &#x000B1; 1.40</td>
<td valign="top" align="center">0.05 &#x000B1; 0.00</td>
<td valign="top" align="center">0.50 &#x000B1; 0.05</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO<break/> <italic>p</italic>-value</td>
<td valign="top" align="center">1.22 &#x000B1; 0.67<sup>a</sup><break/> 0.044</td>
<td valign="top" align="center">11.00 &#x000B1; 0.40<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.05 &#x000B1; 0.00<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.50 &#x000B1; 0.06<break/> &#x0003E;0.05</td>
</tr>
<tr>
<td valign="top" align="left">Senile</td>
<td valign="top" align="center">CTRL</td>
<td valign="top" align="center">9.19 &#x000B1; 1.34</td>
<td valign="top" align="center">13.83 &#x000B1; 0.47</td>
<td valign="top" align="center">0.05 &#x000B1; 0.00</td>
<td valign="top" align="center">0.50 &#x000B1; 0.01</td>
</tr>
<tr>
<td/>
<td valign="top" align="center">CO<break/> <italic>p</italic>-value</td>
<td valign="top" align="center">1.41 &#x000B1; 0.47<sup>a</sup><break/> 0.0001</td>
<td valign="top" align="center">12.00 &#x000B1; 1.18<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.06 &#x000B1; 0.008<break/> &#x0003E;0.05</td>
<td valign="top" align="center">0.50 &#x000B1; 0.01<break/> &#x0003E;0.05</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>The results are expressed as mean &#x000B1; standard error. Control (CTRL) and with cryptorchidism (CO).</p>
<p><sup>a</sup>p &#x0003C; 0.05 CTRL vs. CO of the same period of development.</p>
</table-wrap-foot>
</table-wrap>
<p>In the CO group, the contralateral testes showed seminiferous tubes with different stages of development, which go from gonocytes to round spermatids (<xref ref-type="fig" rid="F1">Figure 1G</xref>). There were no differences in the variables studied when compared to the CTRL and CO groups (<italic>p</italic> &#x0003E; 0.05) (<xref ref-type="table" rid="T1">Table 1</xref>; <xref ref-type="fig" rid="F3">Figure 3G</xref>).</p>
<p>In the CTRL, CO groups and the contralateral testes, Leydig cells were observed occupying the central spaces of the tubular interstitium. They were characterized by their polygonal shape of central nuclei that may present lipid droplets (<xref ref-type="fig" rid="F4">Figures 4A</xref>,<xref ref-type="fig" rid="F4">D</xref>,<xref ref-type="fig" rid="F4">G</xref>).</p>
<fig id="F4" position="float">
<label>Figure 4</label>
<caption><p>Seminiferous tubules of dogs from the control (CTRL) <bold>(A&#x02013;C)</bold>, cryptorchid (CO) <bold>(D&#x02013;F)</bold> and contralateral (CONTRA) <bold>(G&#x02013;I)</bold> groups at peripubertal (first column); young adult (second column) and senile (third column) stages. The interstitial spaces with their characteristic Leydig cells (arrow) are observed. The young adult and senile stage CO group showed small Leydig cell hyperplasia, when compared with the other groups. In some cases in the CO group in the senile stage, completely degenerated seminiferous tubes can be seen with some degenerating or absent Leydig cells (<bold>F</bold> insert). Toluidine Blue, except <bold>(B,C)</bold> Masson trichrome technique. Bar scale: 50 &#x003BC;m.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-935307-g0004.tif"/>
</fig>
</sec>
<sec>
<title>Stage 2: Young adult</title>
<p>The CTRL group complete spermatogenesis with slight histological alterations, such as vacuolization and desquamation, in the seminiferous epithelium were observed. The seminiferous tubules showed marked proliferation (<xref ref-type="fig" rid="F3">Figure 3B</xref>), however, there was absence of gonocytes in all of them (<xref ref-type="fig" rid="F1">Figures 1B</xref>, <xref ref-type="fig" rid="F2">2B</xref>).</p>
<p>In the CO group, a marked hypoplasia of germ cells was observed in the seminiferous epithelium, vacuolization, desquamation, folding and thickening of the basement membrane with cell degeneration and cytoplasmic extensions of Sertoli cells directed toward the center of the tubules (<xref ref-type="fig" rid="F1">Figure 1E</xref>). Spermatogenesis did not continue beyond the spermatocytes, and in some specimens, no germ cells were observed. Sertoli-cell-only syndrome was identified in 50% of the animals (3/6) (<xref ref-type="fig" rid="F1">Figure 1E</xref> insert). In addition, they depicted the presence of cells with gonocyte morphology in 16.6% of the specimens (1/6) (<xref ref-type="fig" rid="F1">Figures 1E</xref>, <xref ref-type="fig" rid="F2">2E</xref>). The animals of the CO group presented a significant reduction of 3.67 times in the area of the seminiferous epithelium, de 2.70 times in MI and de 4.53 times in PI (<xref ref-type="fig" rid="F3">Figure 3E</xref>) with a significant increase in HI of 4.38 times when compared with the CTRL group (<xref ref-type="table" rid="T1">Table 1</xref>).</p>
<p>At this age, there was a reduction of 4.89 times in testosterone concentrations in the CO group, without significant changes in estradiol, LH and FSH concentrations (<italic>p</italic> &#x0003E; 0.05, <xref ref-type="table" rid="T2">Table 2</xref>).</p>
<p>The TBARS technique showed a significant increase (<italic>p</italic> &#x0003C; 0.05) of 3.07 times in lipid peroxidation in CO testes when compared with the CTRL group (<xref ref-type="table" rid="T1">Table 1</xref>).</p>
<p>The contralateral testes did not show a significant difference in any of the variables studied, when compared to the CTRL and CO groups (<xref ref-type="fig" rid="F1">Figures 1H</xref>, <xref ref-type="fig" rid="F3">3H</xref>).</p>
<p>In the CTRL groups and contralateral testes to CO, Leydig cells located in the central spaces of the tubular interstices were observed. In the CO group, a clear hyperplasia of this cell type was observed, interspersed between collagen fibers (<xref ref-type="fig" rid="F4">Figures 4B</xref>,<xref ref-type="fig" rid="F4">E</xref>). Leydig cells area were smaller compared to the other groups (<xref ref-type="fig" rid="F4">Figures 4B</xref>,<xref ref-type="fig" rid="F4">E</xref> insert, <xref ref-type="fig" rid="F4">H</xref>).</p>
</sec>
<sec>
<title>Stage 3: Senile</title>
<p>In senile CTRL animals the seminiferous tubules presented complete spermatogenesis, slight thickening of the basement membrane, and evident cell proliferation (<xref ref-type="fig" rid="F3">Figure 3C</xref>). However, 33.3% of the specimens (2/6) showed damaged histological features characterized by arrest of spermatogenesis, degenerating cells, thickening and folding of the basement membrane and abundant cell desquamation (<xref ref-type="fig" rid="F1">Figures 1C</xref> insert, <xref ref-type="fig" rid="F2">2C</xref>) without the presence of gonocytes.</p>
<p>In the senile CO group, the alterations were similar to those observed in young adult CO animals, with arrest of spermatogenesis in spermatocyte stage and the presence of atypical cells (<xref ref-type="fig" rid="F1">Figure 1F</xref>). In 16.6% of the animals (1/6), electron microscopy confirmed the presence of cells with persistent gonocyte morphology (<xref ref-type="fig" rid="F2">Figure 2F</xref>) and in 16.6% (1/6), Sertoli-cell-only syndrome was appreciated. When compared with the CTRL group, a significant reduction in the seminiferous epithelium area of 4.30 times, MI of 3.13 times and proliferation of 2.81 times (<xref ref-type="fig" rid="F3">Figure 3F</xref>) was determined. Contrarily, the HI registered a significant increase of 4.88 times (<xref ref-type="table" rid="T1">Table 1</xref>) while lipid peroxidation did not show significant difference (<italic>p</italic> &#x0003E; 0.05) (<xref ref-type="table" rid="T1">Table 1</xref>). Testosterone concentrations showed a significant reduction of 6.51 times with respect to the CTRL group. The estradiol, LH, and FSH did not present a significant difference (<italic>p</italic> &#x0003E; 0.05) (<xref ref-type="table" rid="T2">Table 2</xref>).</p>
<p>In terms of the contralateral testes to the retained one in the CO group (<xref ref-type="fig" rid="F1">Figure 1I</xref>), there was a significant reduction in the area of the seminiferous epithelium of 3.16 times with respect to the CTRL group. On the other hand, the HI increased by 3.90 times with respect to the CTRL group. Histological changes consisted of germ cell hypoplasia, folding and thickening of the basement membrane, some tubes with arrested spermatogenesis in spermatocytes, and other sections of tubules with complete spermatogenesis. Moreover, a significant increase in MI of 2.45 times was appreciated when compared to the CO testes. The rest of the variables were found in a position between the CTRL and CO groups with no significant difference (<italic>p</italic> &#x0003E; 0.05, <xref ref-type="fig" rid="F3">Figure 3I</xref>).</p>
<p>In all groups, the Leydig cells remained in the same location (<xref ref-type="fig" rid="F4">Figure 4</xref>). However, in the CO group, the cells were observed with hyperplasia and smaller. In the cases where the seminiferous tubules showed clear atrophy, their Leydig cells were observed to be degenerated or absent (<xref ref-type="fig" rid="F4">Figures 4C</xref>,<xref ref-type="fig" rid="F4">F</xref> insert,<xref ref-type="fig" rid="F4">I</xref>), interspersed between a large number of collagen fibers (<xref ref-type="fig" rid="F4">Figures 4B</xref>,<xref ref-type="fig" rid="F4">E</xref> insert).</p>
<p>In all stages of development, vimentin immunoreactivity was present in the cytoplasm of Sertoli cells, mainly perinuclear and in the basal compartment, forming linear columns toward the tubular lumen in the control group. In the CO group, the vimentin immunoreactivity was more evident, due to the absence of germ cells, showing the cytoplasmic extensions of the free Sertoli cells all along the tubular lumen. In the senile stage, organization of Sertoli cells was compromised. In the contralateral testicles, its distribution was similar to the control group (<xref ref-type="fig" rid="F5">Figure 5</xref>).</p>
<fig id="F5" position="float">
<label>Figure 5</label>
<caption><p>Immunohistochemistry for vimentin in seminiferous tubes of dogs from the control group (CTRL) <bold>(A&#x02013;C)</bold>; from cryptorchidism (CO) <bold>(D&#x02013;F)</bold> and contralateral (CONTRA) <bold>(G&#x02013;I)</bold> in the peripubertal stages (first column); young adult (second column) and senile (third column). A marked immunoreactivity is observed in all stages of development with variation in its distribution, mainly in animals with CO. Bar scale: 50 &#x003BC;m.</p></caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fvets-09-935307-g0005.tif"/>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>Cryptorchidism occurs in dogs with high frequency (6.8%) (<xref ref-type="bibr" rid="B23">23</xref>) and shows anatomical and physiological similarities, as well as risk of infertility and development of neoplasia as in humans (<xref ref-type="bibr" rid="B26">26</xref>, <xref ref-type="bibr" rid="B34">34</xref>). Therefore, this species can be considered as a suitable study model of CO.</p>
<p>In this study, the first seminiferous epithelium alterations in the dogs with CO appeared in the peripubertal stage. These alterations were decreased MI and delay in gonocyte differentiation to spermatogonia, evidenced by the presence of gonocytes in 50% of the specimens (3/6). These alterations generated a decrease in the number of germ cells. It has been reported in children with CO that the first histological alterations of the seminiferous epithelium begin, in subtle form, at 18 months of age with cell desquamation, folding of the basement membrane, vacuolization, lack of gonocyte differentiation to spermatogonia, with germ cell hypoplasia from the third year (<xref ref-type="bibr" rid="B35">35</xref>&#x02013;<xref ref-type="bibr" rid="B37">37</xref>). Thus, at peripubertal stage, these alterations become evident. However, in this work, we did not include animals younger than 6 months of age. Hence, we were unable to determine the precise moment in which the first histological lesions appear in the pre-peripubertal stage.</p>
<p>In the young adult stage, alterations to the seminiferous epithelium generated by CO were evident, which was in accordance with what was reported for dogs (<xref ref-type="bibr" rid="B38">38</xref>, <xref ref-type="bibr" rid="B39">39</xref>). These findings also coincide with what have been reported for seminiferous epithelium in humans where the thickening of the basement membrane was found (<xref ref-type="bibr" rid="B40">40</xref>, <xref ref-type="bibr" rid="B41">41</xref>). In addition, various alterations such as germinal hypoplasia, vacuolization, desquamation, cell degeneration, presence of atypical cells, arrest in germ cell maturation (<xref ref-type="bibr" rid="B40">40</xref>, <xref ref-type="bibr" rid="B42">42</xref>), reduction in cell proliferation (<xref ref-type="bibr" rid="B43">43</xref>), Sertoli-cell-only syndrome (<xref ref-type="bibr" rid="B40">40</xref>, <xref ref-type="bibr" rid="B41">41</xref>) leading to the cessation of spermatogenesis with reduction in the MI and epithelial area have been found in humans with CO.</p>
<p>In our study, the presence of CO testes with Sertoli-cell-only syndrome in 22.2% of the dogs (4/18) support that this syndrome is particularly characteristic for men with a history of cryptorchidism (<xref ref-type="bibr" rid="B44">44</xref>). The presence of Sertoli-cell-only syndrome has also been reported in 7/10 dogs with CO in young adult animals (<xref ref-type="bibr" rid="B39">39</xref>), which is consistent with our results with 3/6 animals with CO in young adult status with this syndrome.</p>
<p>The arrest of spermatogenesis with a reduction in the area of the seminiferous epithelium, as well as in MI and cell proliferation with marked histological and ultrastructural alterations can be related to heat stress, which is known to affect the functioning of Sertoli cells in different species. The heat stress leads to downregulation in the expression of proteins of tight junctions (<xref ref-type="bibr" rid="B11">11</xref>, <xref ref-type="bibr" rid="B45">45</xref>); thus, causing failures in spermatogenesis and cell desquamation (<xref ref-type="bibr" rid="B11">11</xref>, <xref ref-type="bibr" rid="B45">45</xref>) as was observed in our study. Apoptotic pathways in germ cells can also be activated through redistribution of Bax protein from the cytoplasm to perinuclear of germ cells (<xref ref-type="bibr" rid="B46">46</xref>) and expression of the p53 mRNA (<xref ref-type="bibr" rid="B47">47</xref>). In this work, we observed in the CO group that spermatogenesis is arrested in spermatocytes. This is consistent with what was reported by Hirano et al. (<xref ref-type="bibr" rid="B48">48</xref>) who showed that pachytene spermatocytes are the most sensitive cells to temperature increases, generating DNA double-strand breaks and asynapsis, which leads to discontinuation of meiosis. Consequently, the cells are eliminated by apoptosis.</p>
<p>The effect of heat stress is likely due to oxidative stress generated by the production of superoxide anion, hydroxyl radical, nitric oxide and hydrogen peroxide (<xref ref-type="bibr" rid="B49">49</xref>&#x02013;<xref ref-type="bibr" rid="B52">52</xref>). This phenomenon can be determined by increased malondialdehyde, the final product of lipid peroxidation (<xref ref-type="bibr" rid="B53">53</xref>) and which leads to the degeneration of germ cells (<xref ref-type="bibr" rid="B13">13</xref>). The increase in this compound observed in our animals with CO has also been seen in children with CO (<xref ref-type="bibr" rid="B54">54</xref>, <xref ref-type="bibr" rid="B55">55</xref>). Oxidative stress in the CO patient has been shown to participate in germ cell damage (<xref ref-type="bibr" rid="B54">54</xref>, <xref ref-type="bibr" rid="B55">55</xref>). Moreover, oxidative stress can be generated by the inactivation of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase, demonstrated in animal models (<xref ref-type="bibr" rid="B56">56</xref>, <xref ref-type="bibr" rid="B57">57</xref>). Low SOD enzyme activity has also been found in dogs with CO (<xref ref-type="bibr" rid="B58">58</xref>).</p>
<p>These alterations are possibly owing to the fact that spermatocytes and spermatids are the germ cells most vulnerable to damage caused by reactive oxygen species because, even though they are capable of converting super oxide anion to hydrogen peroxide, they have difficulty metabolizing peroxide, which saturates its protective system against peroxide that becomes a highly toxic hydroxyl radical (<xref ref-type="bibr" rid="B59">59</xref>) when compared to spermatogonia or Sertoli cells. This coincides with what was observed in this work.</p>
<p>From young adult to senile development, CO determined histological alterations remained constant, well above the CTRL group, despite the fact that 33.3% of the CTRL animals (2/6) in this study also were experiencing alterations. None of the control animals showed testicular tumor development. There is controversy about the development of testicular tumor in senile dogs. Some authors mentioned a correlation with tumor development (<xref ref-type="bibr" rid="B60">60</xref>) while others assumed a contrary position (<xref ref-type="bibr" rid="B61">61</xref>). Seminiferous tubes without germ cells are reported in humans with unilateral and bilateral undescended testicle until old age (<xref ref-type="bibr" rid="B62">62</xref>) who, in some cases, did not develop testicular tumor (<xref ref-type="bibr" rid="B62">62</xref>).</p>
<p>The presence of cells with morphological and ultrastructural characteristics of gonocytes in 16.6% of the animals (2/12) in the CO dogs in the young and senile stages is uncommon. Gonocytes are not expected to be present in these stages of development; although the presence of gonocytes has been reported in 2/10 young dogs with CO (<xref ref-type="bibr" rid="B39">39</xref>). Hence, this event may suggest that gonocytes predisposes the development of testicular tumor (<xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B15">15</xref>). It has been reported that in dogs with CO, the main types of testicular tumors that can develop are seminoma and mixed germ cell-sex cord stromal cell tumor, both with germ cell components, and to a lesser extent Sertoli cell tumor and interstitial cell tumor (<xref ref-type="bibr" rid="B26">26</xref>).</p>
<p>This opinion is concurrent with the proposition that the failure in the differentiation of the gonocyte to spermatogonia may be the cause of the precursor lesion of GCNIS (<xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B15">15</xref>, <xref ref-type="bibr" rid="B19">19</xref>, <xref ref-type="bibr" rid="B63">63</xref>) reported that 5.4% of patients human with CO have germ cells with immunohistochemical characteristics of gonocytes.</p>
<p>Regarding the endocrine behavior of the patient with CO, some authors mentioned a decrease in testosterone concentrations (<xref ref-type="bibr" rid="B2">2</xref>, <xref ref-type="bibr" rid="B64">64</xref>), while others found it unchanged (<xref ref-type="bibr" rid="B65">65</xref>, <xref ref-type="bibr" rid="B66">66</xref>).</p>
<p>This reduction in testosterone concentrations in boys with cryptorchidism suggests a malfunction in the Leydig cells or the hypothalamic-pituitary-gonadal axis (<xref ref-type="bibr" rid="B67">67</xref>&#x02013;<xref ref-type="bibr" rid="B69">69</xref>). Such event may alter the period of &#x0201C;minipuberty,&#x0201D; in which some authors indicate that gonocyte differentiation takes place (<xref ref-type="bibr" rid="B19">19</xref>, <xref ref-type="bibr" rid="B70">70</xref>). However, the role of gonadotropins and testicular hormones in this process remains to be clarified (<xref ref-type="bibr" rid="B70">70</xref>). In CO dogs, low concentrations of testosterone have been reported from prepubertal to senile stages (<xref ref-type="bibr" rid="B71">71</xref>&#x02013;<xref ref-type="bibr" rid="B73">73</xref>), as it was observed in this work, which leads to altered spermatogenesis. Leydig cell hyperplasia in the animals of CO group has been previously described in dogs with CO (<xref ref-type="bibr" rid="B30">30</xref>) and coincides with what has been reported for humans with this condition (<xref ref-type="bibr" rid="B74">74</xref>). Controverselly, in the current study we observed that the Leydig cells presented a smaller size and signs of degeneration despite being present in large numbers. A reduction in Leydig cell function has been shown in CO testes in rats (<xref ref-type="bibr" rid="B75">75</xref>) and humans (<xref ref-type="bibr" rid="B76">76</xref>), probably due to depletion of the smooth endoplasmic reticulum as it was described in humans (<xref ref-type="bibr" rid="B74">74</xref>).</p>
<p>In humans, 6.33% of patients with unilateral CO present contralateral damage, which was attributed to the impact of this pathology on both gonads (<xref ref-type="bibr" rid="B38">38</xref>, <xref ref-type="bibr" rid="B77">77</xref>). In the contralateral testes, the reduction in the area of the seminiferous epithelium when compared with CTRL group and the increase in MI when compared to the CO group suggest that the contralateral position conditions testicular damage. The level of damage places the contralateral testes between the CTRL and CO groups as reported by Kawakami et al. (<xref ref-type="bibr" rid="B78">78</xref>) in dogs. However, some authors mentioned that this may be caused by deficient cooling system in the venous and arterial circulation between both gonads, thus, constant heat stress is maintained (<xref ref-type="bibr" rid="B75">75</xref>). In any case, in this study, no difference in lipid peroxidation was observed when compared to the CTRL and CO groups. Hence, we suggest that the mechanism of damage may be through another pathway such as Sertoli cell dysfunction determined by low concentrations of transferrin, which has been associated with low concentrations of testosterone, and which has been reported in the contralateral testis of dogs with surgically induced CO (<xref ref-type="bibr" rid="B78">78</xref>).</p>
<p>Greater intensity and disorganization of vimentin immunoreactivity have been reported in rats and humans with CO (<xref ref-type="bibr" rid="B79">79</xref>&#x02013;<xref ref-type="bibr" rid="B81">81</xref>) and in animals exposed to oxidative stress (<xref ref-type="bibr" rid="B81">81</xref>, <xref ref-type="bibr" rid="B82">82</xref>). Although Pecile et al. (<xref ref-type="bibr" rid="B83">83</xref>) do not report changes in vimentin immunoreactivity in the testes of dogs with CO, their images show a loss in the linear organization of immunoreactivity to this intermediate filament (<xref ref-type="bibr" rid="B83">83</xref>). Damage and restoration of spermatogenesis are related to the disintegration or recovery of these filaments (<xref ref-type="bibr" rid="B84">84</xref>).</p>
<p>The results of this work demonstrate that the testicular development of the dog with CO is similar to that of the human with this pathology, provoking alterations of the seminiferous epithelium that lead to problems in fertility and possible risk of developing neoplasia. Therefore, we suggest that this species could be used as a suitable model for the study of CO. This may allow research for compounds that would reduce fertility problems and the risk of developing testicular tumor, as well as provide early risk markers for the development of GCNIS in patients with CO.</p>
</sec>
<sec sec-type="data-availability" id="s5">
<title>Data availability statement</title>
<p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p>
</sec>
<sec id="s6">
<title>Ethics statement</title>
<p>The animal study was reviewed and approved by INP: 54/2020. Written informed consent was obtained from the owners for the participation of their animals in this study.</p>
</sec>
<sec id="s7">
<title>Author contributions</title>
<p>Experiments were conceived and designed and the paper was written by RMV-V, JCR-C, and NH-J. Experiments were performed by NH-J, ER-C, and FR-D. Data were analyzed by RMV-V, NH-J, JCR-C, DL-H, MJ-M, RR-R, and AM. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec sec-type="funding-information" id="s8">
<title>Funding</title>
<p>This work was supported by Instituto Nacional de Pediatria (Grant Number INP: 54/2020).</p>
</sec>
<sec sec-type="COI-statement" id="conf1">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s9">
<title>Publisher&#x00027;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
</body>
<back>
<ack><p>We express our gratitude to the Mobile Dog and Cat Sterilization Unit of Alvaro Obregon Borough Office, and to UNAM-Banfield Veterinary Hospital, CDMX. In addition, our thanks go to Pedro Medina Granados and Edna Garc&#x000ED;a Cruz for their technical support in processing the samples. In the same, our gratitude to CONACyT for granting the doctoral scholarship to NH-J (Student: 297349) to carry out the research and publication of this article, which is required for her doctorate degree.</p>
</ack>
<ref-list>
<title>References</title>
<ref id="B1">
<label>1.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Radcliffe</surname> <given-names>J</given-names></name></person-group>. <article-title>Hospital Cryptorchidism Study Group. Cryptorchidism: a prospective study of 7500 consecutive male births, 1984-8. John Radcliffe Hospital Cryptorchidism Study Group</article-title>. <source>Arch Dis Child.</source> (<year>1992</year>) <volume>67</volume>:<fpage>892</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1136/adc.67.7.892</pub-id><pub-id pub-id-type="pmid">1355643</pub-id></citation></ref>
<ref id="B2">
<label>2.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname> <given-names>PA</given-names></name> <name><surname>Coughlin</surname> <given-names>MT</given-names></name></person-group>. <article-title>Fertility after bilateral cryptorchidism. evaluation by paternity, hormone, and semen data</article-title>. <source>Horm Res.</source> (<year>2001</year>) <volume>55</volume>:<fpage>28</fpage>&#x02013;<lpage>32</lpage>. <pub-id pub-id-type="doi">10.1159/000049960</pub-id><pub-id pub-id-type="pmid">11423739</pub-id></citation></ref>
<ref id="B3">
<label>3.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ghirri</surname> <given-names>P</given-names></name> <name><surname>Ciulli</surname> <given-names>C</given-names></name> <name><surname>Vuerich</surname> <given-names>M</given-names></name> <name><surname>Cuttano</surname> <given-names>A</given-names></name> <name><surname>Faraoni</surname> <given-names>M</given-names></name> <name><surname>Guerrini</surname> <given-names>L</given-names></name> <etal/></person-group>. <article-title>Incidence at birth and natural history of cryptorchidism: a study of 10,730 consecutive male infants</article-title>. <source>J Endocrinol Invest.</source> (<year>2002</year>) <volume>25</volume>:<fpage>709</fpage>&#x02013;<lpage>15</lpage>. <pub-id pub-id-type="doi">10.1007/BF03345105</pub-id><pub-id pub-id-type="pmid">12240903</pub-id></citation></ref>
<ref id="B4">
<label>4.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Sijstermans</surname> <given-names>K</given-names></name> <name><surname>Hack</surname> <given-names>WW</given-names></name> <name><surname>Meijer</surname> <given-names>RW</given-names></name> <name><surname>van der Voort-Doedens</surname> <given-names>LM</given-names></name></person-group>. <article-title>The frequency of undescended testis from birth to adulthood: a review</article-title>. <source>Int J Androl.</source> (<year>2008</year>) <volume>31</volume>:<fpage>1</fpage>&#x02013;<lpage>11</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-2605.2007.00770.x</pub-id><pub-id pub-id-type="pmid">18380786</pub-id></citation></ref>
<ref id="B5">
<label>5.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Virtanen</surname> <given-names>HE</given-names></name> <name><surname>Toppari</surname> <given-names>J</given-names></name></person-group>. <article-title>Epidemiology and pathogenesis of cryptorchidism</article-title>. <source>Hum Reprod Update.</source> (<year>2008</year>) <volume>14</volume>:<fpage>49</fpage>&#x02013;<lpage>58</lpage>. <pub-id pub-id-type="doi">10.1093/humupd/dmm027</pub-id><pub-id pub-id-type="pmid">18032558</pub-id></citation></ref>
<ref id="B6">
<label>6.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mitsui</surname> <given-names>T</given-names></name></person-group>. <article-title>Effects of the prenatal environment on cryptorchidism: a narrative review</article-title>. <source>Int J Urol.</source> (<year>2021</year>) <volume>28</volume>:<fpage>882</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1111/iju.14600</pub-id><pub-id pub-id-type="pmid">34075642</pub-id></citation></ref>
<ref id="B7">
<label>7.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Giwercman</surname> <given-names>A</given-names></name> <name><surname>Grindsted</surname> <given-names>J</given-names></name> <name><surname>Hansen</surname> <given-names>B</given-names></name> <name><surname>Jensen</surname> <given-names>OM</given-names></name> <name><surname>Skakkebaek</surname> <given-names>NE</given-names></name></person-group>. <article-title>Testicular cancer risk in boys with maldescended testis: a cohort study</article-title>. <source>J Urol.</source> (<year>1987</year>) <volume>138</volume>:<fpage>1214</fpage>&#x02013;<lpage>6</lpage>. <pub-id pub-id-type="doi">10.1016/s0022-5347[17]43553-1</pub-id><pub-id pub-id-type="pmid">2889833</pub-id></citation></ref>
<ref id="B8">
<label>8.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Piltoft</surname> <given-names>JS</given-names></name> <name><surname>Larsen</surname> <given-names>SB</given-names></name> <name><surname>Dalton</surname> <given-names>SO</given-names></name> <name><surname>Johansen</surname> <given-names>C</given-names></name> <name><surname>Baker</surname> <given-names>JL</given-names></name> <name><surname>Cederkvist</surname> <given-names>L</given-names></name> <etal/></person-group>. <article-title>Early life risk factors for testicular cancer: a case-cohort study based on the Copenhagen School Health Records Register</article-title>. <source>Acta Oncol.</source> (<year>2017</year>) <volume>56</volume>:<fpage>220</fpage>&#x02013;<lpage>4</lpage>. <pub-id pub-id-type="doi">10.1080/0284186X.2016.1266085</pub-id><pub-id pub-id-type="pmid">28080187</pub-id></citation></ref>
<ref id="B9">
<label>9.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Skakkebaek</surname> <given-names>NE</given-names></name> <name><surname>Berthelsen</surname> <given-names>JG</given-names></name> <name><surname>Giwercman</surname> <given-names>A</given-names></name> <name><surname>M&#x000FC;ller</surname> <given-names>J</given-names></name></person-group>. <article-title>Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma</article-title>. <source>Int J Androl.</source> (<year>1987</year>) <volume>10</volume>:<fpage>19</fpage>&#x02013;<lpage>28</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-2605.1987.tb00161.x</pub-id><pub-id pub-id-type="pmid">3034791</pub-id></citation></ref>
<ref id="B10">
<label>10.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hadziselimovic</surname> <given-names>F</given-names></name> <name><surname>Hoecht</surname> <given-names>B</given-names></name></person-group>. <article-title>Testicular histology related to fertility outcome and postpubertal hormone status in cryptorchidism</article-title>. <source>Klin Padiatr.</source> (<year>2008</year>) <volume>220</volume>:<fpage>302</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.1055/s-2007-993194</pub-id><pub-id pub-id-type="pmid">18401814</pub-id></citation></ref>
<ref id="B11">
<label>11.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname> <given-names>WR</given-names></name> <name><surname>Li</surname> <given-names>BB</given-names></name> <name><surname>Hu</surname> <given-names>Y</given-names></name> <name><surname>Zhang</surname> <given-names>L</given-names></name> <name><surname>Wang</surname> <given-names>XZ</given-names></name></person-group>. <article-title>Oxidative stress mediates heat-induced changes of tight junction proteins in porcine sertoli cells via inhibiting CaMKK&#x003B2;-AMPK pathway</article-title>. <source>Theriogenology</source>. (<year>2020</year>) <volume>142</volume>:<fpage>104</fpage>. <pub-id pub-id-type="doi">10.1016/j.theriogenology.2019.09.031</pub-id><pub-id pub-id-type="pmid">31586867</pub-id></citation></ref>
<ref id="B12">
<label>12.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>He</surname> <given-names>C</given-names></name> <name><surname>Sun</surname> <given-names>J</given-names></name> <name><surname>Yang</surname> <given-names>D</given-names></name> <name><surname>He</surname> <given-names>W</given-names></name> <name><surname>Wang</surname> <given-names>J</given-names></name> <name><surname>Qin</surname> <given-names>D</given-names></name> <etal/></person-group>. <article-title>Nrf2 activation mediates the protection of mouse Sertoli Cells damage under acute heat stress conditions</article-title>. <source>Theriogenology.</source> (<year>2022</year>) <volume>177</volume>:<fpage>183</fpage>&#x02013;<lpage>94</lpage>. <pub-id pub-id-type="doi">10.1016/j.theriogenology.2021.10.009</pub-id><pub-id pub-id-type="pmid">34715543</pub-id></citation></ref>
<ref id="B13">
<label>13.</label>
<citation citation-type="journal"><person-group person-group-type="author"><collab>Vigueras-Villase&#x000F1;or RM Ojeda I Gutierrez-P&#x000E9;rez O Chavez-Salda&#x000F1;a M Cuevas O Maria DS </collab></person-group>. <article-title>Protective effect of &#x003B1;-tocopherol on damage to rat testes by experimental cryptorchidism</article-title>. <source>Int J Exp Pathol.</source> (<year>2011</year>) <volume>92</volume>:<fpage>131</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-2613.2010.00757.x</pub-id><pub-id pub-id-type="pmid">21314742</pub-id></citation></ref>
<ref id="B14">
<label>14.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>R&#x000F8;rth</surname> <given-names>M</given-names></name> <name><surname>Rajpert-De Meyts</surname> <given-names>E</given-names></name> <name><surname>Andersson</surname> <given-names>L</given-names></name> <name><surname>Dieckmann</surname> <given-names>KP</given-names></name> <name><surname>Foss&#x000E5;</surname> <given-names>SD</given-names></name> <name><surname>Grigor</surname> <given-names>KM</given-names></name> <etal/></person-group>. <article-title>Carcinoma in situ in the testis</article-title>. <source>Scand J Urol Nephrol Suppl.</source> (<year>2000</year>) <volume>205</volume>:<fpage>166</fpage>&#x02013;<lpage>86</lpage>. <pub-id pub-id-type="doi">10.1080/00365590050509896</pub-id><pub-id pub-id-type="pmid">11144894</pub-id></citation></ref>
<ref id="B15">
<label>15.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hutson JM Li</surname> <given-names>R</given-names></name> <name><surname>Southwell</surname> <given-names>BR</given-names></name> <name><surname>Petersen</surname> <given-names>BL</given-names></name> <name><surname>Thorup</surname> <given-names>J</given-names></name> <name><surname>Cortes</surname> <given-names>D</given-names></name></person-group>. <article-title>Germ cell development in the postnatal testis: the key to prevent malignancy in cryptorchidism?</article-title> <source>Front Endocrinol (Lausanne).</source> (<year>2013</year>) <volume>3</volume>:<fpage>176</fpage>. <pub-id pub-id-type="doi">10.3389/fendo.2012.00176</pub-id><pub-id pub-id-type="pmid">23316184</pub-id></citation></ref>
<ref id="B16">
<label>16.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hamilton-Dutoit</surname> <given-names>SJ</given-names></name> <name><surname>Lou</surname> <given-names>H</given-names></name> <name><surname>Pallesen</surname> <given-names>G</given-names></name></person-group>. <article-title>The expression of placental alkaline phosphatase (PLAP) and PLAP-like enzymes in normal and neoplastic human tissues. an immunohistological survey using monoclonal antibodies</article-title>. <source>APMIS.</source> (<year>1990</year>) <volume>98</volume>:<fpage>797</fpage>&#x02013;<lpage>811</lpage>. <pub-id pub-id-type="doi">10.1111/j.1699-0463.1990.tb05000.x</pub-id><pub-id pub-id-type="pmid">2171580</pub-id></citation></ref>
<ref id="B17">
<label>17.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Huff</surname> <given-names>DS</given-names></name> <name><surname>Hadziselimovic</surname> <given-names>F</given-names></name> <name><surname>Snyder</surname> <given-names>HM</given-names></name> <name><surname>Blythe</surname> <given-names>B</given-names></name> <name><surname>Ducket</surname> <given-names>JW</given-names></name></person-group>. <article-title>Histologic maldevelopment of unilaterally cryptorchid testes and their descended partners</article-title>. <source>Eur J Pediatr</source>. (<year>1993</year>) 152:S11-4.<pub-id pub-id-type="pmid">8101802</pub-id></citation></ref>
<ref id="B18">
<label>18.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Culty</surname> <given-names>M</given-names></name></person-group>. <article-title>Gonocytes, the forgotten cells of the germ cell lineage</article-title>. <source>Birth Defects Res C Embryo Today.</source> (<year>2009</year>) <volume>87</volume>:<fpage>1</fpage>&#x02013;<lpage>26</lpage>. <pub-id pub-id-type="doi">10.1002/bdrc.20142</pub-id><pub-id pub-id-type="pmid">19306346</pub-id></citation></ref>
<ref id="B19">
<label>19.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Manku</surname> <given-names>G</given-names></name> <name><surname>Culty</surname> <given-names>M</given-names></name></person-group>. <article-title>Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges</article-title>. <source>Reproduction.</source> (<year>2015</year>) <volume>149</volume>:<fpage>R139</fpage>&#x02013;<lpage>57</lpage>. <pub-id pub-id-type="doi">10.1530/REP-14-0431</pub-id><pub-id pub-id-type="pmid">25670871</pub-id></citation></ref>
<ref id="B20">
<label>20.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Batool</surname> <given-names>A</given-names></name> <name><surname>Karimi</surname> <given-names>N</given-names></name> <name><surname>Wu</surname> <given-names>XN</given-names></name> <name><surname>Chen</surname> <given-names>SR</given-names></name> <name><surname>Liu</surname> <given-names>YX</given-names></name></person-group>. <article-title>Testicular germ cell tumor: a comprehensive review</article-title>. <source>Cell Mol Life Sci.</source> (<year>2019</year>) <volume>76</volume>:<fpage>1713</fpage>&#x02013;<lpage>27</lpage>. <pub-id pub-id-type="doi">10.1007/s00018-019-03022-7</pub-id><pub-id pub-id-type="pmid">30671589</pub-id></citation></ref>
<ref id="B21">
<label>21.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname> <given-names>RD</given-names></name> <name><surname>Wen</surname> <given-names>XH</given-names></name> <name><surname>Kong</surname> <given-names>LS</given-names></name> <name><surname>Deng</surname> <given-names>XZ</given-names></name> <name><surname>Peng</surname> <given-names>B</given-names></name> <name><surname>Huang</surname> <given-names>AP</given-names></name> <etal/></person-group>. <article-title>A quantitative (stereological) study of the effects of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis</article-title>. <source>Reproduction.</source> (<year>2002</year>) <volume>124</volume>:<fpage>95</fpage>&#x02013;<lpage>105</lpage>. <pub-id pub-id-type="doi">10.1530/rep.0.1240095</pub-id><pub-id pub-id-type="pmid">12090923</pub-id></citation></ref>
<ref id="B22">
<label>22.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Picut</surname> <given-names>CA</given-names></name> <name><surname>Ziejewski</surname> <given-names>MK</given-names></name> <name><surname>Stanislaus</surname> <given-names>D</given-names></name></person-group>. <article-title>Comparative aspects of pre- and postnatal development of the male reproductive system</article-title>. <source>Birth Defects Res.</source> (<year>2018</year>) <volume>110</volume>:<fpage>190</fpage>&#x02013;<lpage>227</lpage>. <pub-id pub-id-type="doi">10.1002/bdr2.1133</pub-id><pub-id pub-id-type="pmid">29063715</pub-id></citation></ref>
<ref id="B23">
<label>23.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yates</surname> <given-names>D</given-names></name> <name><surname>Hayes</surname> <given-names>G</given-names></name> <name><surname>Heffernan</surname> <given-names>M</given-names></name> <name><surname>Beynon</surname> <given-names>R</given-names></name></person-group>. <article-title>Incidence of cryptorchidism in dogs and cats</article-title>. <source>Vet Rec.</source> (<year>2003</year>) <volume>152</volume>:<fpage>502</fpage>&#x02013;<lpage>4</lpage>. <pub-id pub-id-type="doi">10.1136/vr.152.16.502</pub-id><pub-id pub-id-type="pmid">12733559</pub-id></citation></ref>
<ref id="B24">
<label>24.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Tannouz</surname> <given-names>VGS</given-names></name> <name><surname>Mamprim</surname> <given-names>MJ</given-names></name> <name><surname>Lopes</surname> <given-names>MD</given-names></name> <name><surname>Santos-Sousa</surname> <given-names>CA</given-names></name> <name><surname>Souza Junior</surname> <given-names>P</given-names></name> <name><surname>Babinski</surname> <given-names>MA</given-names></name> <etal/></person-group>. <article-title>Is the right testis more affected by cryptorchidism than the left testis? an ultrasonographic approach in dogs of different sizes and breeds</article-title>. <source>Folia Morphol (Warsz).</source> (<year>2019</year>) <volume>78</volume>:<fpage>847</fpage>&#x02013;<lpage>52</lpage>. <pub-id pub-id-type="doi">10.5603/FM.a2019.0022</pub-id><pub-id pub-id-type="pmid">30835343</pub-id></citation></ref>
<ref id="B25">
<label>25.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hayes HM</surname> <given-names>Jr</given-names></name> <name><surname>Pendergrass</surname> <given-names>TW</given-names></name></person-group>. <article-title>Canine testicular tumors: epidemiologic features of 410 dogs</article-title>. <source>Int J Cancer.</source> (<year>1976</year>) <volume>18</volume>:<fpage>482</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.1002/ijc.2910180413</pub-id><pub-id pub-id-type="pmid">977190</pub-id></citation></ref>
<ref id="B26">
<label>26.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Liao</surname> <given-names>AT</given-names></name> <name><surname>Chu</surname> <given-names>PY</given-names></name> <name><surname>Yeh</surname> <given-names>LS</given-names></name> <name><surname>Lin</surname> <given-names>CT</given-names></name> <name><surname>Liu</surname> <given-names>CH</given-names></name></person-group>. <article-title>A 12-year retrospective study of canine testicular tumors</article-title>. <source>J Vet Med Sci.</source> (<year>2009</year>) <volume>71</volume>:<fpage>919</fpage>&#x02013;<lpage>23</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.71.919</pub-id><pub-id pub-id-type="pmid">19652479</pub-id></citation></ref>
<ref id="B27">
<label>27.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Grieco</surname> <given-names>V</given-names></name> <name><surname>Riccardi</surname> <given-names>E</given-names></name> <name><surname>Greppi</surname> <given-names>GF</given-names></name> <name><surname>Teruzzi</surname> <given-names>F</given-names></name> <name><surname>Ierman&#x000F2;</surname> <given-names>V</given-names></name> <name><surname>Finazzi</surname> <given-names>M</given-names></name></person-group>. <article-title>Canine testicular tumours: a study on 232 dogs</article-title>. <source>J Comp Pathol.</source> (<year>2008</year>) <volume>138</volume>:<fpage>86</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1016/j.jcpa.2007.11.002</pub-id><pub-id pub-id-type="pmid">18295787</pub-id></citation></ref>
<ref id="B28">
<label>28.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kawakami</surname> <given-names>E</given-names></name> <name><surname>Tsutsui</surname> <given-names>T</given-names></name> <name><surname>Ogasa</surname> <given-names>A</given-names></name></person-group>. <article-title>Histological observations of the reproductive organs of the male dog from birth to sexual maturity</article-title>. <source>J Vet Med Sci.</source> (<year>1991</year>) <volume>53</volume>:<fpage>241</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.53.241</pub-id><pub-id pub-id-type="pmid">1830795</pub-id></citation></ref>
<ref id="B29">
<label>29.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Taha</surname> <given-names>MB</given-names></name> <name><surname>Noakes</surname> <given-names>DE</given-names></name></person-group>. <article-title>The effect of age and season of the year on testicular function in the dog, as determined by histological examination of the seminiferous tubules and the estimation of peripheral plasma testosterone concentrations</article-title>. <source>J Small Anim. Pract</source>. (<year>1982</year>) <volume>23</volume>:<fpage>351</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.1111/j.1748-5827.1982.tb01677.x</pub-id></citation>
</ref>
<ref id="B30">
<label>30.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Merz</surname> <given-names>SE</given-names></name> <name><surname>Klopfleisch</surname> <given-names>R</given-names></name> <name><surname>Breithaupt</surname> <given-names>A</given-names></name> <name><surname>Gruber</surname> <given-names>AD</given-names></name></person-group>. <article-title>Aging and senescence in canine testes</article-title>. <source>Vet Pathol.</source> (<year>2019</year>) <volume>56</volume>:<fpage>715</fpage>&#x02013;<lpage>24</lpage>. <pub-id pub-id-type="doi">10.1177/0300985819843683</pub-id><pub-id pub-id-type="pmid">31060479</pub-id></citation></ref>
<ref id="B31">
<label>31.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Johnsen</surname> <given-names>SG</given-names></name></person-group>. <article-title>Testicular biopsy score count-a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males</article-title>. <source>Hormones.</source> (<year>1970</year>) <volume>1</volume>:<fpage>2</fpage>&#x02013;<lpage>25</lpage>. <pub-id pub-id-type="doi">10.1159/000178170</pub-id><pub-id pub-id-type="pmid">5527187</pub-id></citation></ref>
<ref id="B32">
<label>32.</label>
<citation citation-type="journal"><person-group person-group-type="author"><collab>Vigueras-Villase&#x000F1;or RM Molina-Ortiz D Reyes-Torres G del Angel DS Moreno-Mendoza NA Cruz ME </collab></person-group>. <article-title>Effect of allopurinol on damage caused by free radicals to cryptorchid testes</article-title>. <source>Acta Histochem.</source> (<year>2009</year>) <volume>111</volume>:<fpage>127</fpage>&#x02013;<lpage>37</lpage>. <pub-id pub-id-type="doi">10.1016/j.acthis.2008.05.004</pub-id><pub-id pub-id-type="pmid">18621413</pub-id></citation></ref>
<ref id="B33">
<label>33.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Lowry</surname> <given-names>OH</given-names></name> <name><surname>Rosebrough</surname> <given-names>NJ</given-names></name> <name><surname>Farr</surname> <given-names>AL</given-names></name> <name><surname>Randall</surname> <given-names>RJ</given-names></name></person-group>. <article-title>Protein measurement with the Folin phenol reagent</article-title>. <source>J Biol Chem</source>. (<year>1951</year>) <volume>93</volume>:<fpage>265</fpage>&#x02013;<lpage>75</lpage>.<pub-id pub-id-type="pmid">14907713</pub-id></citation></ref>
<ref id="B34">
<label>34.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ortega-Pacheco</surname> <given-names>A</given-names></name> <name><surname>Rodr&#x000ED;guez-Buenfil</surname> <given-names>JC</given-names></name> <name><surname>Segura-Correa</surname> <given-names>JC</given-names></name> <name><surname>Bolio-Gonzalez</surname> <given-names>ME</given-names></name> <name><surname>Jim&#x000E9;nez-Coello</surname> <given-names>M</given-names></name> <name><surname>Linde Forsberg</surname> <given-names>C</given-names></name></person-group>. <article-title>Pathological conditions of the reproductive organs of male stray dogs in the tropics: prevalence, risk factors, morphological findings and testosterone concentrations</article-title>. <source>Reprod Domest Anim.</source> (<year>2006</year>) <volume>41</volume>:<fpage>429</fpage>&#x02013;<lpage>37</lpage>. <pub-id pub-id-type="doi">10.1111/j.1439-0531.2006.00688.x</pub-id><pub-id pub-id-type="pmid">16984349</pub-id></citation></ref>
<ref id="B35">
<label>35.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mengel</surname> <given-names>W</given-names></name> <name><surname>Wronecki</surname> <given-names>K</given-names></name> <name><surname>Schroeder</surname> <given-names>J</given-names></name> <name><surname>Zimmermann</surname> <given-names>FA</given-names></name></person-group>. <article-title>Histopathology of the cryptorchid testis</article-title>. <source>Urol Clin North Am</source>. (<year>1982</year>) 9:331-8.</citation>
</ref>
<ref id="B36">
<label>36.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Huff</surname> <given-names>DS</given-names></name> <name><surname>Hadziselimovic</surname> <given-names>F</given-names></name> <name><surname>Snyder</surname> <given-names>HM</given-names> <suffix>3rd</suffix></name> <name><surname>Duckett</surname> <given-names>JW</given-names></name> <name><surname>Keating</surname> <given-names>MA</given-names></name></person-group>. <article-title>Postnatal testicular maldevelopment in unilateral cryptorchidism</article-title>. <source>J Urol.</source> (<year>1989</year>) <volume>142</volume>:<fpage>546</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1016/s0022-5347(17)38811-0</pub-id><pub-id pub-id-type="pmid">2568502</pub-id></citation></ref>
<ref id="B37">
<label>37.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Verkauskas</surname> <given-names>G</given-names></name> <name><surname>Malcius</surname> <given-names>D</given-names></name> <name><surname>Dasevicius</surname> <given-names>D</given-names></name> <name><surname>Hadziselimovic</surname> <given-names>F</given-names></name></person-group>. <article-title>Histopathology of Unilateral Cryptorchidism</article-title>. <source>Pediatr Dev Pathol.</source> (<year>2019</year>) <volume>22</volume>:<fpage>53</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1177/1093526618789300</pub-id><pub-id pub-id-type="pmid">30012073</pub-id></citation></ref>
<ref id="B38">
<label>38.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mattos</surname> <given-names>FMR</given-names></name> <name><surname>Simoes-Mattos</surname> <given-names>L</given-names></name> <name><surname>Domingues</surname> <given-names>SSF</given-names></name></person-group>. <article-title>Cryptorchidism in dog</article-title>. <source>Ciencia Animal.</source> (<year>2000</year>) <volume>10</volume>:<fpage>61</fpage>&#x02013;<lpage>70</lpage>.</citation>
</ref>
<ref id="B39">
<label>39.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Veronesi</surname> <given-names>MC</given-names></name> <name><surname>Riccardi</surname> <given-names>E</given-names></name> <name><surname>Rota</surname> <given-names>A</given-names></name> <name><surname>Grieco</surname> <given-names>V</given-names></name></person-group>. <article-title>Characteristics of cryptic/ectopic and contralateral scrotal testes in dogs between 1 and 2 years of age</article-title>. <source>Theriogenology.</source> (<year>2009</year>) <volume>72</volume>:<fpage>969</fpage>&#x02013;<lpage>77</lpage>. <pub-id pub-id-type="doi">10.1016/j.theriogenology.2009.06.016</pub-id><pub-id pub-id-type="pmid">19700187</pub-id></citation></ref>
<ref id="B40">
<label>40.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Amat</surname> <given-names>P</given-names></name> <name><surname>Paniagua</surname> <given-names>R</given-names></name> <name><surname>Montero</surname> <given-names>J</given-names></name></person-group>. <article-title>Seminiferous tubule degeneration in human cryptorchid testes</article-title>. <source>J Androl.</source> (<year>1985</year>) <volume>6</volume>:<fpage>1</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1002/j.1939-4640.1985.tb00810.x</pub-id><pub-id pub-id-type="pmid">2857707</pub-id></citation></ref>
<ref id="B41">
<label>41.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Govender</surname> <given-names>D</given-names></name> <name><surname>Sing</surname> <given-names>Y</given-names></name> <name><surname>Chetty</surname> <given-names>R</given-names></name></person-group>. <article-title>Sertoli cell nodules in the undescended testis: a histochemical, immunohistochemical, and ultrastructural study of hyaline deposits</article-title>. <source>J Clin Pathol.</source> (<year>2004</year>) <volume>57</volume>:<fpage>802</fpage>&#x02013;<lpage>6</lpage>. <pub-id pub-id-type="doi">10.1136/jcp.2004.015982</pub-id><pub-id pub-id-type="pmid">15280399</pub-id></citation></ref>
<ref id="B42">
<label>42.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>M&#x000FC;ller</surname> <given-names>J</given-names></name> <name><surname>Skakkebaek</surname> <given-names>NE</given-names></name> <name><surname>Nielsen</surname> <given-names>OH</given-names></name> <name><surname>Graem</surname> <given-names>N</given-names></name></person-group>. <article-title>Cryptorchidism and testis cancer. Atypical infantile germ cells followed by carcinoma in situ and invasive carcinoma in adulthood</article-title>. <source>Cancer.</source> (<year>1984</year>) <volume>54</volume>:<fpage>629</fpage>&#x02013;<lpage>34</lpage>. <pub-id pub-id-type="doi">10.1002/1097-0142(</pub-id><year>1984</year>)54:4 &#x0003C;629::aid-cncr2820540407&#x0003E;3.0.co;2-e<pub-id pub-id-type="pmid">6146398</pub-id></citation></ref>
<ref id="B43">
<label>43.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ofordeme</surname> <given-names>KG</given-names></name> <name><surname>Aslan</surname> <given-names>AR</given-names></name> <name><surname>Nazir</surname> <given-names>TM</given-names></name> <name><surname>Hayner-Buchan</surname> <given-names>A</given-names></name> <name><surname>Kogan</surname> <given-names>BA</given-names></name></person-group>. <article-title>Apoptosis and proliferation in human undescended testes</article-title>. <source>BJU Int.</source> (<year>2005</year>) <volume>96</volume>:<fpage>634</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1111/j.1464-410X.2005.05698.x</pub-id><pub-id pub-id-type="pmid">16104924</pub-id></citation></ref>
<ref id="B44">
<label>44.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Fedder</surname> <given-names>J</given-names></name></person-group>. <article-title>History of cryptorchidism and ejaculate volume as simple predictors for the presence of testicular sperm</article-title>. <source>Syst Biol Reprod Med.</source> (<year>2011</year>) <volume>57</volume>:<fpage>154</fpage>&#x02013;<lpage>61</lpage>. <pub-id pub-id-type="doi">10.3109/19396368.2010.550796</pub-id><pub-id pub-id-type="pmid">21284574</pub-id></citation></ref>
<ref id="B45">
<label>45.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Cai</surname> <given-names>H</given-names></name> <name><surname>Ren</surname> <given-names>Y</given-names></name> <name><surname>Li</surname> <given-names>XX</given-names></name> <name><surname>Yang</surname> <given-names>JL</given-names></name> <name><surname>Zhang</surname> <given-names>CP</given-names></name> <name><surname>Chen</surname> <given-names>M</given-names></name> <etal/></person-group>. <article-title>Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF-&#x003B2; expression</article-title>. <source>Int J Androl.</source> (<year>2011</year>) <volume>34</volume>:<fpage>352</fpage>&#x02013;<lpage>62</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-2605.2010.01089.x</pub-id><pub-id pub-id-type="pmid">20633196</pub-id></citation></ref>
<ref id="B46">
<label>46.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Yamamoto</surname> <given-names>CM</given-names></name> <name><surname>Sinha Hikim</surname> <given-names>AP</given-names></name> <name><surname>Huynh</surname> <given-names>PN</given-names></name> <name><surname>Shapiro</surname> <given-names>B</given-names></name> <name><surname>Lue</surname> <given-names>Y</given-names></name> <name><surname>Salameh</surname> <given-names>WA</given-names></name> <etal/></person-group>. <article-title>Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia</article-title>. <source>Biol Reprod.</source> (<year>2000</year>) <volume>63</volume>:<fpage>1683</fpage>&#x02013;<lpage>90</lpage>. <pub-id pub-id-type="doi">10.1095/biolreprod63.6.1683</pub-id><pub-id pub-id-type="pmid">11090436</pub-id></citation></ref>
<ref id="B47">
<label>47.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Absalan</surname> <given-names>F</given-names></name> <name><surname>Movahedin</surname> <given-names>M</given-names></name> <name><surname>Mowla</surname> <given-names>SJ</given-names></name></person-group>. <article-title>Germ cell apoptosis induced by experimental cryptorchidism is mediated by molecular pathways in mouse testis</article-title>. <source>Andrologia.</source> (<year>2010</year>) <volume>42</volume>:<fpage>5</fpage>&#x02013;<lpage>12</lpage>. <pub-id pub-id-type="doi">10.1111/j.1439-0272.2009.00947.x</pub-id><pub-id pub-id-type="pmid">20078510</pub-id></citation></ref>
<ref id="B48">
<label>48.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hirano</surname> <given-names>K</given-names></name> <name><surname>Nonami</surname> <given-names>Y</given-names></name> <name><surname>Nakamura</surname> <given-names>Y</given-names></name> <name><surname>Sato</surname> <given-names>T</given-names></name> <name><surname>Sato</surname> <given-names>T</given-names></name> <name><surname>Ishiguro</surname> <given-names>KI</given-names></name> <etal/></person-group>. <article-title>Temperature sensitivity of DNA double-strand break repair underpins heat-induced meiotic failure in mouse spermatogenesis</article-title>. <source>Commun Biol.</source> (<year>2022</year>) <volume>5</volume>:<fpage>504</fpage>. <pub-id pub-id-type="doi">10.1038/s42003-022-03449-y</pub-id><pub-id pub-id-type="pmid">35618762</pub-id></citation></ref>
<ref id="B49">
<label>49.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Zini</surname> <given-names>A</given-names></name> <name><surname>Schlegel</surname> <given-names>PN</given-names></name></person-group>. <article-title>Cu/Zn superoxide dismutase, catalase and glutathione peroxidase mRNA expression in the rat testis after surgical cryptorchidism and efferent duct ligation</article-title>. <source>J Urol.</source> (<year>1997</year>) <volume>158</volume>:<fpage>659</fpage>&#x02013;<lpage>63</lpage>. <pub-id pub-id-type="doi">10.1016/S0022-5347(01)64578-6</pub-id><pub-id pub-id-type="pmid">9224387</pub-id></citation></ref>
<ref id="B50">
<label>50.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ikeda</surname> <given-names>M</given-names></name> <name><surname>Kodama</surname> <given-names>H</given-names></name> <name><surname>Fukuda</surname> <given-names>J</given-names></name> <name><surname>Shimizu</surname> <given-names>Y</given-names></name> <name><surname>Murata</surname> <given-names>M</given-names></name> <name><surname>Kumagai</surname> <given-names>J</given-names></name> <etal/></person-group>. <article-title>Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress</article-title>. <source>Biol Reprod.</source> (<year>1999</year>) <volume>61</volume>:<fpage>393</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1095/biolreprod61.2.393</pub-id><pub-id pub-id-type="pmid">10411517</pub-id></citation></ref>
<ref id="B51">
<label>51.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kumagai</surname> <given-names>A</given-names></name> <name><surname>Kodama</surname> <given-names>H</given-names></name> <name><surname>Kumagai</surname> <given-names>J</given-names></name> <name><surname>Fukuda</surname> <given-names>J</given-names></name> <name><surname>Kawamura</surname> <given-names>K</given-names></name> <name><surname>Tanikawa</surname> <given-names>H</given-names></name> <etal/></person-group>. <article-title>Xanthine oxidase inhibitors suppress testicular germ cell apoptosis induced by experimental cryptorchidism</article-title>. <source>Mol Hum Reprod.</source> (<year>2002</year>) <volume>8</volume>:<fpage>118</fpage>&#x02013;<lpage>23</lpage>. <pub-id pub-id-type="doi">10.1093/molehr/8.2.118</pub-id><pub-id pub-id-type="pmid">11818514</pub-id></citation></ref>
<ref id="B52">
<label>52.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ishii</surname> <given-names>T</given-names></name> <name><surname>Matsuki</surname> <given-names>S</given-names></name> <name><surname>Iuchi</surname> <given-names>Y</given-names></name> <name><surname>Okada</surname> <given-names>F</given-names></name> <name><surname>Toyosaki</surname> <given-names>S</given-names></name> <name><surname>Tomita</surname> <given-names>Y</given-names></name> <etal/></person-group>. <article-title>Accelerated impairment of spermatogenic cells in SOD1-knockout mice under heat stress</article-title>. <source>Free Radic Res.</source> (<year>2005</year>) <volume>39</volume>:<fpage>697</fpage>&#x02013;<lpage>705</lpage>. <pub-id pub-id-type="doi">10.1080/10715760500130517</pub-id><pub-id pub-id-type="pmid">16036348</pub-id></citation></ref>
<ref id="B53">
<label>53.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Gasparovic</surname> <given-names>AC</given-names></name> <name><surname>Jaganjac</surname> <given-names>M</given-names></name> <name><surname>Mihaljevic</surname> <given-names>B</given-names></name> <name><surname>Sunjic</surname> <given-names>SB</given-names></name> <name><surname>Zarkovic</surname> <given-names>N</given-names></name></person-group>. <article-title>Assays for the measurement of lipid peroxidation</article-title>. <source>Methods Mol Biol.</source> (<year>2013</year>) <volume>965</volume>:<fpage>283</fpage>&#x02013;<lpage>96</lpage>. <pub-id pub-id-type="doi">10.1007/978-1-62703-239-1_19</pub-id><pub-id pub-id-type="pmid">23296666</pub-id></citation></ref>
<ref id="B54">
<label>54.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Durairajanayagam</surname> <given-names>D</given-names></name> <name><surname>Agarwal</surname> <given-names>A</given-names></name> <name><surname>Ong</surname> <given-names>C</given-names></name></person-group>. <article-title>Causes, effects and molecular mechanisms of testicular heat stress</article-title>. <source>Reprod Biomed Online.</source> (<year>2015</year>) <volume>30</volume>:<fpage>14</fpage>&#x02013;<lpage>27</lpage>. <pub-id pub-id-type="doi">10.1016/j.rbmo.2014.09.018</pub-id><pub-id pub-id-type="pmid">25456164</pub-id></citation></ref>
<ref id="B55">
<label>55.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Imamoglu</surname> <given-names>M</given-names></name> <name><surname>B&#x000FC;lb&#x000FC;l</surname> <given-names>SS</given-names></name> <name><surname>Kaklikkaya</surname> <given-names>N</given-names></name> <name><surname>Sarihan</surname> <given-names>H</given-names></name></person-group>. <article-title>Oxidative, inflammatory and immunologic status in children with undescended testes</article-title>. <source>Pediatr Int.</source> (<year>2012</year>) <volume>54</volume>:<fpage>816</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1111/j.1442-200X.2012.03695.x</pub-id><pub-id pub-id-type="pmid">22783848</pub-id></citation></ref>
<ref id="B56">
<label>56.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Ahotupa</surname> <given-names>M</given-names></name> <name><surname>Huhtaniemi</surname> <given-names>I</given-names></name></person-group>. <article-title>Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally cryptorchid rat testis</article-title>. <source>Biol Reprod.</source> (<year>1992</year>) <volume>46</volume>:<fpage>1114</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1095/biolreprod46.6.1114</pub-id><pub-id pub-id-type="pmid">1356492</pub-id></citation></ref>
<ref id="B57">
<label>57.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Peltola</surname> <given-names>V</given-names></name> <name><surname>Huhtaniemi</surname> <given-names>I</given-names></name> <name><surname>Ahotupa</surname> <given-names>M</given-names></name></person-group>. <article-title>Abdominal position of the rat testis is associated with high level of lipid peroxidation</article-title>. <source>Biol Reprod.</source> (<year>1995</year>) <volume>53</volume>:<fpage>1146</fpage>&#x02013;<lpage>50</lpage>. <pub-id pub-id-type="doi">10.1095/biolreprod53.5.1146</pub-id><pub-id pub-id-type="pmid">8527519</pub-id></citation></ref>
<ref id="B58">
<label>58.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kawakami</surname> <given-names>E</given-names></name> <name><surname>Hirano</surname> <given-names>T</given-names></name> <name><surname>Hori</surname> <given-names>T</given-names></name> <name><surname>Tsutsui</surname> <given-names>T</given-names></name></person-group>. <article-title>Testicular superoxide dismutase activity, heat shock protein 70 concentration and blood plasma inhibin-alpha concentration of dogs with a Sertoli cell tumor in a unilateral cryptorchid testis</article-title>. <source>J Vet Med Sci.</source> (<year>2007</year>) <volume>69</volume>:<fpage>1259</fpage>&#x02013;<lpage>62</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.69.1259</pub-id><pub-id pub-id-type="pmid">18176022</pub-id></citation></ref>
<ref id="B59">
<label>59.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bauch&#x000E9;</surname> <given-names>F</given-names></name> <name><surname>Fouchard</surname> <given-names>MH</given-names></name> <name><surname>J&#x000E9;gou</surname> <given-names>B</given-names></name></person-group>. <article-title>Antioxidant system in rat testicular cells</article-title>. <source>FEBS Lett.</source> (<year>1994</year>) <volume>349</volume>:<fpage>392</fpage>&#x02013;<lpage>6</lpage>. <pub-id pub-id-type="doi">10.1016/0014-5793(94)00709-8</pub-id><pub-id pub-id-type="pmid">8050602</pub-id></citation></ref>
<ref id="B60">
<label>60.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mosier</surname> <given-names>JE</given-names></name></person-group>. <article-title>Effect of aging on body systems of the dog</article-title>. <source>Vet Clin North Am Small Anim Pract.</source> (<year>1989</year>) <volume>19</volume>:<fpage>1</fpage>&#x02013;<lpage>12</lpage>. <pub-id pub-id-type="doi">10.1016/s0195-5616(89)50001-9</pub-id><pub-id pub-id-type="pmid">2646811</pub-id></citation></ref>
<ref id="B61">
<label>61.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Peters</surname> <given-names>MA</given-names></name> <name><surname>de Rooij</surname> <given-names>DG</given-names></name> <name><surname>Teerds</surname> <given-names>KJ</given-names></name> <name><surname>van Der Gaag</surname> <given-names>I</given-names></name> <name><surname>van Sluijs</surname> <given-names>FJ</given-names></name></person-group>. <article-title>Spermatogenesis and testicular tumours in ageing dogs</article-title>. <source>J Reprod Fertil.</source> (<year>2000</year>) <volume>120</volume>:<fpage>443</fpage>&#x02013;<lpage>52</lpage>.<pub-id pub-id-type="pmid">11787185</pub-id></citation></ref>
<ref id="B62">
<label>62.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kassir</surname> <given-names>R</given-names></name> <name><surname>Dubois</surname> <given-names>J</given-names></name> <name><surname>Berremila</surname> <given-names>SA</given-names></name> <name><surname>Baccot</surname> <given-names>S</given-names></name> <name><surname>Boueil-Bourlier</surname> <given-names>A</given-names></name> <name><surname>Tiffet</surname> <given-names>O</given-names></name> <etal/></person-group>. <article-title>rare variant of inguinal hernia: Cryptorchid testis at the age of 50 years. etiopathogenicity, prognosis and management</article-title>. <source>Int J Surg Case Rep.</source> (<year>2014</year>) <volume>5</volume>:<fpage>416</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1016/j.ijscr.2014.03.015</pub-id><pub-id pub-id-type="pmid">24892247</pub-id></citation></ref>
<ref id="B63">
<label>63.</label>
<citation citation-type="journal"><person-group person-group-type="author"><collab>Vigueras-Villase&#x000F1;or RM Cort&#x000E9;s-Trujillo L Ch&#x000E1;vez-Salda&#x000F1;a M V&#x000E1;zquez FG Carrasco-Daza D Cuevas-Alpuche O </collab></person-group>. <article-title>Analysis of POU5F1, c-Kit, PLAP, AP2&#x003B3; and SALL4 in gonocytes of patients with cryptorchidism</article-title>. <source>Acta Histochem.</source> (<year>2015</year>) <volume>117</volume>:<fpage>752</fpage>&#x02013;<lpage>61</lpage>. <pub-id pub-id-type="doi">10.1016/j.acthis.2015.08.004</pub-id><pub-id pub-id-type="pmid">26315991</pub-id></citation></ref>
<ref id="B64">
<label>64.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Baker</surname> <given-names>BA</given-names></name> <name><surname>Morley</surname> <given-names>R</given-names></name> <name><surname>Lucas</surname> <given-names>A</given-names></name></person-group>. <article-title>Plasma testosterone in preterm infants with cryptorchidism</article-title>. <source>Arch Dis Child.</source> (<year>1988</year>) <volume>63</volume>:<fpage>1198</fpage>&#x02013;<lpage>200</lpage>. <pub-id pub-id-type="doi">10.1136/adc.63.10.1198</pub-id><pub-id pub-id-type="pmid">2904244</pub-id></citation></ref>
<ref id="B65">
<label>65.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Barthold</surname> <given-names>JS</given-names></name> <name><surname>Manson</surname> <given-names>J</given-names></name> <name><surname>Regan</surname> <given-names>V</given-names></name> <name><surname>Si</surname> <given-names>X</given-names></name> <name><surname>Hassink</surname> <given-names>SG</given-names></name> <name><surname>Coughlin</surname> <given-names>MT</given-names></name> <etal/></person-group>. <article-title>Reproductive hormone levels in infants with cryptorchidism during postnatal activation of the pituitary-testicular axis</article-title>. J Urol. (<year>2004</year>) <volume>172</volume>:<fpage>1736</fpage>&#x02013;<lpage>41</lpage>. <pub-id pub-id-type="doi">10.1097/01.ju.0000138523.24337.be</pub-id><pub-id pub-id-type="pmid">15371802</pub-id></citation></ref>
<ref id="B66">
<label>66.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Iwatsuki</surname> <given-names>S</given-names></name> <name><surname>Kojima</surname> <given-names>Y</given-names></name> <name><surname>Mizuno</surname> <given-names>K</given-names></name> <name><surname>Kamisawa</surname> <given-names>H</given-names></name> <name><surname>Umemoto</surname> <given-names>Y</given-names></name> <name><surname>Sasaki</surname> <given-names>S</given-names></name> <etal/></person-group>. <article-title>Endocrine assessment of prepubertal boys with a history of cryptorchidism and/or hypospadias: a pilot study</article-title>. <source>J Urol.</source> (<year>2011</year>) <volume>185</volume>:<fpage>2444</fpage>&#x02013;<lpage>50</lpage>. <pub-id pub-id-type="doi">10.1016/j.juro.2011.01.018</pub-id><pub-id pub-id-type="pmid">21555006</pub-id></citation></ref>
<ref id="B67">
<label>67.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Suomi</surname> <given-names>AM</given-names></name> <name><surname>Main</surname> <given-names>KM</given-names></name> <name><surname>Kaleva</surname> <given-names>M</given-names></name> <name><surname>Schmidt</surname> <given-names>IM</given-names></name> <name><surname>Chellakooty</surname> <given-names>M</given-names></name> <name><surname>Virtanen</surname> <given-names>HE</given-names></name> <etal/></person-group>. <article-title>Hormonal changes in 3-month-old cryptorchid boys</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2006</year>) <volume>91</volume>:<fpage>953</fpage>&#x02013;<lpage>8</lpage>. <pub-id pub-id-type="doi">10.1210/jc.2004-2318</pub-id><pub-id pub-id-type="pmid">16394094</pub-id></citation></ref>
<ref id="B68">
<label>68.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Bay</surname> <given-names>K</given-names></name> <name><surname>Virtanen</surname> <given-names>HE</given-names></name> <name><surname>Hartung</surname> <given-names>S</given-names></name> <name><surname>Ivell</surname> <given-names>R</given-names></name> <name><surname>Main</surname> <given-names>KM</given-names></name> <name><surname>Skakkebaek</surname> <given-names>NE</given-names></name> <etal/></person-group>. <article-title>Nordic Cryptorchidism Study Group, Toppari J. Insulin-like factor 3 levels in cord blood and serum from children: effects of age, postnatal hypothalamic-pituitary-gonadal axis activation, and cryptorchidism</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2007</year>) <volume>92</volume>:<fpage>4020</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.1210/jc.2007-0974</pub-id><pub-id pub-id-type="pmid">17666478</pub-id></citation></ref>
<ref id="B69">
<label>69.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hamdi</surname> <given-names>SM</given-names></name> <name><surname>Almont</surname> <given-names>T</given-names></name> <name><surname>Galinier</surname> <given-names>P</given-names></name> <name><surname>Mieusset</surname> <given-names>R</given-names></name> <name><surname>Thonneau</surname> <given-names>P</given-names></name></person-group>. <article-title>Altered secretion of Sertoli cells hormones in 2-year-old prepubertal cryptorchid boys: a cross-sectional study</article-title>. <source>Andrology.</source> (<year>2017</year>) <volume>5</volume>:<fpage>783</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1111/andr.12373</pub-id><pub-id pub-id-type="pmid">28544660</pub-id></citation></ref>
<ref id="B70">
<label>70.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Becker</surname> <given-names>M</given-names></name> <name><surname>Hesse</surname> <given-names>V</given-names></name></person-group>. <article-title>Minipuberty: why does it happen?</article-title> <source>Horm Res Paediatr.</source> (<year>2020</year>) <volume>93</volume>:<fpage>76</fpage>&#x02013;<lpage>84</lpage>. <pub-id pub-id-type="doi">10.1159/000508329</pub-id><pub-id pub-id-type="pmid">32599600</pub-id></citation></ref>
<ref id="B71">
<label>71.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pathirana</surname> <given-names>IN</given-names></name> <name><surname>Yamasaki</surname> <given-names>H</given-names></name> <name><surname>Kawate</surname> <given-names>N</given-names></name> <name><surname>Tsuji</surname> <given-names>M</given-names></name> <name><surname>B&#x000FC;llesbach</surname> <given-names>EE</given-names></name> <name><surname>Takahashi</surname> <given-names>M</given-names></name> <etal/></person-group>. <article-title>Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: changes with age and effects of cryptorchidism</article-title>. <source>Theriogenology.</source> (<year>2012</year>) <volume>77</volume>:<fpage>550</fpage>&#x02013;<lpage>7</lpage>. <pub-id pub-id-type="doi">10.1016/j.theriogenology.2011.08.030</pub-id><pub-id pub-id-type="pmid">22015156</pub-id></citation></ref>
<ref id="B72">
<label>72.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Hornakova</surname> <given-names>L</given-names></name> <name><surname>Vrbovska</surname> <given-names>T</given-names></name></person-group>. <article-title>Pavl&#x00027;ak M, Valencakova-Agyagosova A, Halo M, Hajurka J. The evaluation of blood concentrations of testosterone, 17&#x003B2;-oestradiol and anti-Mu&#x000A8;llerian hormone in dogs with cryptorchidism and testicular tumours</article-title>. <source>Pol J Vet Sci.</source> (<year>2017</year>) <volume>20</volume>:<fpage>677</fpage>&#x02013;<lpage>85</lpage>. <pub-id pub-id-type="doi">10.1515/pjvs-2017-0085</pub-id><pub-id pub-id-type="pmid">29611653</pub-id></citation></ref>
<ref id="B73">
<label>73.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kawakami</surname> <given-names>E</given-names></name> <name><surname>Tsutsui</surname> <given-names>T</given-names></name> <name><surname>Saito</surname> <given-names>S</given-names></name> <name><surname>Kakimoto</surname> <given-names>T</given-names></name> <name><surname>Ogasa</surname> <given-names>A</given-names></name></person-group>. <article-title>Changes in peripheral plasma luteinizing hormone and testosterone concentrations and semen quality in normal and cryptorchid dogs during sexual maturation</article-title>. <source>Lab Anim Sci.</source> (<year>1995</year>) <volume>45</volume>:<fpage>258</fpage>&#x02013;<lpage>63</lpage>.<pub-id pub-id-type="pmid">7650895</pub-id></citation></ref>
<ref id="B74">
<label>74.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Gotoh</surname> <given-names>M</given-names></name> <name><surname>Miyake</surname> <given-names>K</given-names></name> <name><surname>Mitsuya</surname> <given-names>H</given-names></name></person-group>. <article-title>A study of cryptorchidism. I. Light and electron microscopic study of Leydig&#x00027;s cells in the testes of cryptorchid patients</article-title>. <source>Hinyokika Kiyo.</source> (<year>1984</year>)<volume>3</volume>:<fpage>327</fpage>&#x02013;<lpage>47</lpage>.<pub-id pub-id-type="pmid">6147073</pub-id></citation></ref>
<ref id="B75">
<label>75.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Aldahhan</surname> <given-names>RA</given-names></name> <name><surname>Stanton</surname> <given-names>PG</given-names></name></person-group>. <article-title>Heat stress response of somatic cells in the testis</article-title>. <source>Mol Cell Endocrinol.</source> (<year>2021</year>) <volume>527</volume>:<fpage>111216</fpage>. <pub-id pub-id-type="doi">10.1016/j.mce.2021.111216</pub-id><pub-id pub-id-type="pmid">33639219</pub-id></citation></ref>
<ref id="B76">
<label>76.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Regadera</surname> <given-names>J</given-names></name> <name><surname>Mart&#x000ED;nez-Garc&#x000ED;a</surname> <given-names>F</given-names></name> <name><surname>Gonz&#x000E1;lez-Peramato</surname> <given-names>P</given-names></name> <name><surname>Serrano</surname> <given-names>A</given-names></name> <name><surname>Nistal</surname> <given-names>M</given-names></name> <name><surname>Su&#x000E1;rez-Quian</surname> <given-names>C</given-names></name></person-group>. <article-title>Androgen receptor expression in sertoli cells as a function of seminiferous tubule maturation in the human cryptorchid testis</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2001</year>) <volume>86</volume>:<fpage>41321</fpage>. <pub-id pub-id-type="doi">10.1210/jcem.86.1.7109</pub-id><pub-id pub-id-type="pmid">11232033</pub-id></citation></ref>
<ref id="B77">
<label>77.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Akre</surname> <given-names>O</given-names></name> <name><surname>Pettersson</surname> <given-names>A</given-names></name> <name><surname>Richiardi</surname> <given-names>L</given-names></name></person-group>. <article-title>Risk of contralateral testicular cancer among men with unilaterally undescended testis: a mta analysis</article-title>. <source>Int J Cancer.</source> (<year>2009</year>) <volume>124</volume>:<fpage>687</fpage>&#x02013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1002/ijc.23936</pub-id><pub-id pub-id-type="pmid">18973229</pub-id></citation></ref>
<ref id="B78">
<label>78.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Kawakami</surname> <given-names>E</given-names></name> <name><surname>Hori</surname> <given-names>T</given-names></name> <name><surname>Tsutsui</surname> <given-names>T</given-names></name></person-group>. <article-title>Function of contralateral testis after artificial unilateral cryptorchidism in dogs</article-title>. <source>J Vet Med Sci.</source> (<year>1999</year>) <volume>61</volume>:<fpage>1107</fpage>&#x02013;<lpage>11</lpage>. <pub-id pub-id-type="doi">10.1292/jvms.61.1107</pub-id><pub-id pub-id-type="pmid">10563287</pub-id></citation></ref>
<ref id="B79">
<label>79.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Abd-El-Hafez</surname> <given-names>MA</given-names></name> <name><surname>El-Shafee</surname> <given-names>MD</given-names></name> <name><surname>Omar</surname> <given-names>SH</given-names></name> <name><surname>Aburahma</surname> <given-names>AA</given-names></name> <name><surname>Kamar</surname> <given-names>SS</given-names></name></person-group>. <article-title>The ameliorative effect of curcumin on cryptorchid and non-cryptorchid testes in induced unilateral cryptorchidism in albino rat: histological evaluation</article-title>. <source>Folia Morphol (Warsz).</source> (<year>2021</year>) <volume>80</volume>:<fpage>596</fpage>&#x02013;<lpage>604</lpage>. <pub-id pub-id-type="doi">10.5603/FM.a2020.0084</pub-id><pub-id pub-id-type="pmid">32748946</pub-id></citation></ref>
<ref id="B80">
<label>80.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Rogatsch</surname> <given-names>H</given-names></name> <name><surname>Jezek</surname> <given-names>D</given-names></name> <name><surname>Hittmair</surname> <given-names>A</given-names></name> <name><surname>Mikuz</surname> <given-names>G</given-names></name> <name><surname>Feichtinger</surname> <given-names>H</given-names></name></person-group>. <article-title>Expression of vimentin, cytokeratin, and desmin in Sertoli cells of human fetal, cryptorchid, and tumour-adjacent testicular tissue</article-title>. <source>Virchows Arch.</source> (<year>1996</year>) <volume>427</volume>:<fpage>497</fpage>&#x02013;<lpage>502</lpage>. <pub-id pub-id-type="doi">10.1007/BF00199510</pub-id><pub-id pub-id-type="pmid">8624579</pub-id></citation></ref>
<ref id="B81">
<label>81.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Mohammed</surname> <given-names>M</given-names></name> <name><surname>Elfadeel</surname> <given-names>KA</given-names></name> <name><surname>Abdel-Aziz Fawzy</surname> <given-names>A</given-names></name> <name><surname>Samy</surname> <given-names>W.</given-names></name></person-group> <article-title>The effect of chronic stress on the testis of adult albino Rats and the possible protective effect of astaxanthin supplementation (Histological, immunohistochemical and biochemical studies)</article-title>. <source>J Med Histol.</source> (<year>2019</year>) <volume>2</volume>:<fpage>115</fpage>&#x02013;<lpage>30</lpage>. <pub-id pub-id-type="doi">10.21608/jmh.2019.7416.1048</pub-id></citation>
</ref>
<ref id="B82">
<label>82.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname> <given-names>F</given-names></name> <name><surname>Li</surname> <given-names>XL</given-names></name> <name><surname>Lin</surname> <given-names>T</given-names></name> <name><surname>He</surname> <given-names>DW</given-names></name> <name><surname>Wei</surname> <given-names>GH</given-names></name> <name><surname>Liu</surname> <given-names>JH</given-names></name> <etal/></person-group>. <article-title>The cyclophosphamide metabolite, acrolein, induces cytoskeletal changes and oxidative stress in Sertoli cells</article-title>. <source>Mol Biol Reprod.</source> (<year>2012</year>) <volume>39</volume>:<fpage>493</fpage>&#x02013;<lpage>500</lpage>. <pub-id pub-id-type="doi">10.1007/s11033-011-0763-9</pub-id><pub-id pub-id-type="pmid">21553225</pub-id></citation></ref>
<ref id="B83">
<label>83.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>Pecile</surname> <given-names>A</given-names></name> <name><surname>Groppetti</surname> <given-names>D</given-names></name> <name><surname>Pizzi</surname> <given-names>G</given-names></name> <name><surname>Banco</surname> <given-names>B</given-names></name> <name><surname>Bronzo</surname> <given-names>V</given-names></name> <name><surname>Giudice</surname> <given-names>C</given-names></name> <etal/></person-group>. <article-title>Immunohistochemical insights into a hidden pathology: canine cryptorchidism</article-title>. <source>Theriogenology.</source> (<year>2021</year>) <volume>176</volume>:<fpage>43</fpage>&#x02013;<lpage>53</lpage>. <pub-id pub-id-type="doi">10.1016/j.theriogenology.2021.09.011</pub-id><pub-id pub-id-type="pmid">34571397</pub-id></citation></ref>
<ref id="B84">
<label>84.</label>
<citation citation-type="journal"><person-group person-group-type="author"><name><surname>ElGhamrawy</surname> <given-names>TA</given-names></name> <name><surname>Helmy</surname> <given-names>D</given-names></name> <name><surname>Elall</surname> <given-names>HF</given-names></name></person-group>. <article-title>Cadherin and vimentin immunoexpression in the testis of normal and induced infertility models of albino rats</article-title>. <source>Folia Morphol (Warsz).</source> (<year>2014</year>) <volume>73</volume>:<fpage>339</fpage>&#x02013;<lpage>46</lpage>. <pub-id pub-id-type="doi">10.5603/FM.2014.0050</pub-id><pub-id pub-id-type="pmid">25343410</pub-id></citation></ref>
</ref-list> 
</back>
</article> 