Edited by: Ishwar Parhar, Monash University Malaysia, Malaysia
Reviewed by: Toni R. Pak, Loyola University Chicago, United States; Gustavo M. Somoza, Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico de Chascomus (IIB-INTECH), Argentina
Specialty section: This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology
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Gonadotropin-releasing hormone (GnRH) is a key regulatory molecule of the hypothalamus–pituitary (PIT)–gonadal (HPG) axis that ultimately leads to the downstream release of estradiol (E2) and progesterone (P). These gonadal steroids feed back to the hypothalamus and PIT to regulate reproductive function and behavior. While GnRH is thought to be the master regulator of reproduction, its metabolic product GnRH-(1–5) is also biologically active. Thimet oligopeptidase 1 (also known as EP24.15) cleaves GnRH to form GnRH-(1–5). GnRH-(1–5) is involved in regulation of the HPG axis, exerting its actions through a pair of orphan G protein-coupled receptors, GPR101 and GPR173. The physiological importance of GnRH-(1–5) signaling has been studied in several contexts, but its potential role during reproductive senescence is poorly understood. We used an ovariectomized (OVX) rat model of reproductive senescence to assess whether and how GnRH-(1–5) signaling genes in hypothalamic subnuclei change in response to aging and/or different estradiol replacement regimens designed to model clinical hormone replacement in women. We found that
Gonadotropin-releasing hormone (GnRH) is a key regulatory molecule of the hypothalamus–pituitary (PIT)–gonadal (HPG) axis. Neurons in the hypothalamus release GnRH which acts downstream on the PIT to stimulate transcription and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (
While GnRH is thought to be the master regulator of reproduction, its metabolic product GnRH-(1–5) is also shown to be biologically active. GnRH-(1–5) is produced after thimet oligopeptidase 1 (also known as EP24.15) cleaves the covalent bond linking the fifth and sixth amino acids of GnRH (
It is thought that during aging, changes in the hypothalamic GnRH system, as well as PIT and ovarian processes, are key components that contribute to reproductive senescence (
Utilizing a reproductive aging female rat model (
Tissue samples assayed in this study were previously generated and utilized for separate publications (
Female Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) were purchased at 3–4 months [reproductively mature (MAT); virgin] and 10–11 months old [reproductively aging (AG); retired breeder]. On arrival, rats were pair housed at random with same-age partners in a controlled room temperature (22°C) and light cycle (12-h light, 12-h dark, lights on at 7 a.m.). Food and water were available
The animal procedures were previously described (
Experimental design used to study the effects of age, timing, and duration of hormone treatment. Ovariectomy (OVX) surgery, followed by vehicle (VEH) or estradiol (E2) capsule implantation, was performed at age 4–5 [mature (MAT)] and 11–12 months [aging (AG)]. Animals in groups 1–6 were MAT or AG rats that were given VEH or E2 for 3 or 6 months. Animals in groups 7 and 8 were AG rats that were given VEH or E2 post-OVX and then switched after 3 months to the opposite treatment for an additional 3 months. Comparisons between groups 1–4, 3–6, and 5–8 were made as described in Section “
At the time of surgery, Silastic capsules containing either 100% cholesterol (VEH) or 5% 17β-estradiol/95% cholesterol (E2) were implanted subcutaneously between the shoulder blades. Delivery of E2
After 3 or 6 months of hormone treatment, animals were euthanized by rapid decapitation between 1 and 3 p.m. (4–6 h before lights off at 7 p.m.). Brains were quickly extracted and briefly cooled on ice. Coronal brain sections (eight total) were taken at 1 mm intervals throughout the entire hypothalamus using an ice-cold brain matrix (Ted Pella, Inc., Redding, CA, USA). Sections were quickly immersed in 1.5 mL of RNAlater (cat. no. AM7021M, Invitrogen, Waltham, MA, USA) and stored overnight at 4°C. After overnight storage, each section was mounted on plain glass slides and stored at −20°C before micropunching. Additionally, at time of euthanasia, the PIT was removed, immersed in RNAlater overnight at 4°C, and stored at −20°C prior to RNA extraction.
Brains treated with RNAlater were thawed once for micropunching. Hypothalamic regions containing the mPOA (bregma −0.26 to −1.80) and ARC (bregma −2.12 to −4.52) (
The mRNA expression of
Primer sequences used for real-time PCR.
Gene | Accession number | Primer sequence | Amplicon size (bp) |
---|---|---|---|
NM_017008.4 | (F) 5′-GTGCCAGCCTCGTCTCATAG-3′ | 122 | |
(R) 5′-CGTTGATGGCAACAATGTCCA-3′ | |||
NM_012767.2 | (F) 5′-GGCTTTCACATCCAAACAGAATG-3′ | 181 | |
(R) 5′-TGATCCTCCTCCTTGCCCAT-3′ | |||
NM_031038.3 | (F) 5′-TCAGGACCCACGCAAACTAC-3′ | 182 | |
(R) 5′-CTGGCTCTGACACCCTGTTT-3′ | |||
NM_001108258.1 | (F) 5′-ATAGCCATCCTGAGCTTCGC-3′ | 167 | |
(R) 5′-CGGTGCGCTGAATAGAAAGC-3′ | |||
NM_022255.1 | (F) 5′-CGAGTATCGTCACCGCAAGA-3′ | 119 | |
(R) 5′-CAAAGCCAGCGATCCAGTTG-3′ | |||
NM_172075.2 | (F) 5′-GTGTACCAGAGGGTCGTGTG-3′ | 142 | |
(R) 5′-TGATCTTCTCCTGTGTGTCCTG-3′ |
Statistical analyses were conducted using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Differential tissue expression of
The relative abundance of
Relative mRNA expression of
To determine the mechanisms by which GnRH-(1–5) signaling was altered after aging, ovarian hormone loss, and E2 treatment, we quantified mRNA expression of its receptors,
Effects of age and estradiol on mRNA expression of
Effects of age and estradiol on mRNA expression of
Unlike
Effects of age and estradiol on mRNA expression of
In order to better understand the changes in key GnRH-(1–5) signaling genes, it was important to also assess whether there were changes in the expression of
Effects of age and estradiol on mRNA expression of
This study analyzed the effects of aging and the timing and duration of E2 treatment on the expression of key GnRH-(1–5) signaling genes in the OVX rat. Gene expression of receptors that GnRH-(1–5) binds to,
The present study showed tissue-specific expression of
Similar to
Previous studies demonstrated that GnRH-(1–5) is biologically active and has important roles in the facilitation of lordosis (
Unlike
It is within the ARC that our study identified the most significant changes in
Serum hormone levels in the rats used in the present study were measured previously. Serum LH levels were lowered by E2 treatment, independent of age, consistent with expected estrogen negative feedback effects (
This study assessed the effects of age, hormone, treatment duration, and the timing of treatment on the expression of key GnRH-(1–5) signaling genes. The serum E2 levels for the rats studied (see Figure
The results reported here are the first to systematically assess the effects of E2 replacement regimens on the mRNA expression of
The importance of the colocalization of EP24.15 and GPR173 with GnRH and Kiss1 neurons is slowly emerging as recent research suggests a more complex regulation of reproduction and GnRH release by a multitude of factors. Aside from cleaving GnRH to GnRH-(1–5), EP24.15 is responsible for the cleavage of Kiss1 (
In summary, we have systematically assessed the change in expression of genes crucial to GnRH-(1–5) signaling in response to aging and different estradiol replacement regimens designed to model clinical hormone replacement in women. Examining expression of GnRH-(1–5) signaling genes in the mPOA, ARC, and PIT is crucial as these regions are associated with regulation of GnRH (as well as LH and FSH)
This study was carried out in accordance with the recommendations of The Guide for the Care and Use of Experimental Animals. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin.
Conception or design of the study: WY, AG, and TW. Data collection: BB, WY, and AG. Data analysis and interpretation: BB, WY, AG, and TW. Drafting and critical revision of the article: BB, WY, AG, and TW. Final approval of the version to be published: WY, AG, and TW.
The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
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.
The authors thank Brian Pham for technical expertise on this project and Dr. Cara Olsen of the USUHS Biostatistics Consulting Center for her support in statistical analyses. The authors also thank Dr. Zhao Zhang Li of the USUHS Biomedical Instrumentation Center for her support in sequencing qPCR amplicons for primer verification.
The Supplementary Material for this article can be found online at