Anti-Müllerian hormone and its receptor are detected in most gonadotropin-releasing-hormone cell bodies and fibers in heifer brains
Introduction
The hypothalamic-pituitary-gonadal axis drives reproduction and one of the most important components of the axis are the gonadotropin-releasing-hormone (GnRH) neurons [1,2]. GnRH neurons originate in the preoptic area (POA) and arcuate nucleus (ARC) and project to the median eminence (ME), the interface between the neural and peripheral endocrine systems, and secrete GnRH into the pituitary portal blood vessels [3,4]. The secreted GnRH binds to GnRH receptors on the lipid raft portion of the plasma membrane of gonadotrophs to stimulate the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [5]. Given their central role in reproduction, it is important to clarify mechanisms controlling GnRH neurons in the hypothalamus.
Anti-Mullerian hormone (AMH) is a glycoprotein that belongs to the transforming growth factor-β superfamily, which includes inhibin and activin. The best-studied tissue that secretes AMH is the immature granulosa cells in the ovaries of adult humans and animals [6] and AMH reportedly plays various important roles therein [7,8]. Interestingly, plasma AMH concentrations can predict the fertility of adult female goats, ewes, cows, and women [[9], [10], [11]]. Recent studies have revealed that AMH exerts extragonadal functions in the gonadotrophs of the anterior pituitary. The main AMH receptor, AMH receptor type 2 (AMHR2), colocalizes with GnRH receptors on the lipid raft of gonadotrophs [12]. Furthermore, AMH activates AMHR2 and thereby stimulates the synthesis and secretion of LH and FSH in the gonadotrophs of bovines and rodents [[12], [13], [14]]. However, it remains unknown whether AMH and AMHR2 play any significant roles in the hypothalamus with regard to regulating GnRH release.
Little is known concerning the relationship between AMH and the brain. Although the brains of adult tilapia express AMH, the specific localization of AMH expression in the brain remains unclarified [15]. Another recent study found that GnRH neurons contain AMHR2 in various regions of female human and rodent brains, including the POA, ARC, and ME [16]. Furthermore, both in vivo and in vitro studies have demonstrated that AMH potently activates GnRH neurons, and consequently GnRH-dependent LH secretion in adult female mice [16]. However, it remains unknown as to whether female mammalian brains express AMH. Therefore, this study evaluated whether AMH and AMHR2 are detected in the POA, ARC, and ME of heifers, and especially within GnRH neurons.
Section snippets
Brain and ovary sample collection
All experiments were performed in accordance with the Guiding Principles for the Care and Use of Experimental Animals in the Field of Physiological Sciences (Physiological Society of Japan) and were approved by the Committee on Animal Experiments of Yamaguchi University.
We obtained brain samples from healthy, postpubertal (26 mo of age) nonlactating Japanese black heifers managed by our contracted farmers in western Japan. The farms had open free stall barns with free access to water. The
Detection of AMH and AMHR2 mRNA in POA and ARC&ME tissues
The agarose gel electrophoresis yielded PCR products of the expected sizes, indicating that AMH (328 bp; Fig. 1A) and AMHR2 (320 bp; Fig. 1B) were amplified from the POA and ARC&ME tissues. The same was found for the PCR products obtained from ovary tissues. Homology searching for the obtained sequences of amplified products in the gene databases revealed that the best match alignment was bovine AMH (NM_173890.1) or bovine AMHR2 (NM_001205328.1). Both had a query coverage of 100%, an e-value of
Discussion
The present study detected AMH and AMHR2 in the bovine POA, ARC, and ME. To the best of our knowledge, this study is the first to report AMH in the brains of mammals and AMHR2 in the brains of ruminants. The discovery of AMH and AMHR2 in the POA, ARC, and ME warrants further exploration because their localization may have significant implications for reproduction.
In the POA, ARC, and ME, we observed that most cell bodies or fibers of GnRH neurons were AMHR2-positive and AMH-positive. Little is
Ethical considerations
The study was approved by the Committee on Animal Experiments of Yamaguchi University, and complied with relevant legislation.
CRediT authorship contribution statement
O. Kereilwe: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Visualization. H. Kadokawa: Conceptualization, Supervision, Methodology, Formal analysis, Investigation, Writing - original draft, Funding acquisition.
Acknowledgments
O. Kereilwe was supported by MEXT, Japan (Ministry of Education, Culture, Sports, Science, and Technology) with the provision of a scholarship.
This research was partly supported by a Grant-in Aid for Scientific Research (JSPS Kakenhi Grant Number 18H02329) from the Japan Society for the Promotion of Science (Tokyo, Japan) to H. Kadokawa.
The authors declare no conflicts of interest.
References (39)
- et al.
GnRH-associated peptide (GAP) is cosecreted with GnRH into the hypophyseal portal blood of ovariectomized sheep
Biochem Biophys Res Commun
(1987) - et al.
Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations
Anim Reprod Sci
(2014) - et al.
Anti-Müllerian hormone and polycystic ovary syndrome
Best Pract Res Clin Obstet Gynaecol
(2016) - et al.
Anti-Müllerian hormone (AMH) receptor type II expression and AMH activity in bovine granulosa cells
Theriogenology
(2016) - et al.
Mutant isoforms of the anti-Müllerian hormone type II receptor are not expressed at the cell membrane
J Biol Chem
(1996) - et al.
Tissue-specific glycosylation at the glycopeptide level
Mol Cell Proteomics
(2015) Control of puberty onset and fertility by gonadotropin-releasing hormone neurons
Nat Rev Endocrinol
(2016)- et al.
Regulation of GnRH pulsatility in ewes
Reproduction
(2018) - et al.
Identification and distribution of neuroendocrine gonadotropin-releasing hormone neurons in the Ewe
Biol Reprod
(1997) - et al.
Nutritional influences on folliculogenesis
Reprod Domest Anim Suppl
(2012)