Anti-Müllerian hormone and its receptor are detected in most gonadotropin-releasing-hormone cell bodies and fibers in heifer brains

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Highlights

  • AMH and AMHR2 are detected in the bovine POA, ARC, and ME, including the external zone of the ME.

  • Most cell bodies or fibers of GnRH neurons are positive for both AMH and AMHR2 in the POA, ARC, and ME.

  • There are also GnRH-negative AMHR2-positive cell bodies or fibers of neurons, and GnRH-negative AMH-positive cell bodies or fibers of neurons.

Abstract

Circulating concentrations of Anti-Müllerian hormone (AMH) can indicate fertility in various animals, but the physiological mechanisms underlying the effect of AMH on fertility remain unknown. We recently discovered that AMH has extragonadal functions via its main receptor, AMH receptor type 2 (AMHR2). Specifically, AMH stimulates the secretion of luteinizing hormone and follicle-stimulating hormone from bovine gonadotrophs. Moreover, gonadotrophs themselves express AMH to exert paracrine/autocrine functions, and AMH can activate gonadotropin-releasing-hormone (GnRH) neurons in mice. This study aimed to evaluate whether AMH and AMHR2 are detected in areas of the brain relevant to neuroendocrine control of reproduction: the preoptic area (POA), arcuate nucleus (ARC), and median eminence (ME), and in particular within GnRH neurons. Reverse transcription-polymerase chain reaction detected both AMH and AMHR2 mRNA in tissues containing POA, as well as in those containing both ARC and ME, collected from postpubertal heifers. Western blotting detected AMH and AMHR2 protein in the collected tissues. Triple fluorescence immunohistochemistry revealed that most cell bodies or fibers of GnRH neurons were AMHR2-positive and AMH-positive, although some were negative. Immunohistochemistry revealed that 75% to 85% of cell bodies and fibers of GnRH neurons were positive for both AMH and AMHR2 in the POA, ARC, and both the internal and external zones of the ME. The cell bodies of GnRH neurons were situated around other AMH-positive cell bodies or fibers of GnRH and non-GNRH neurons. Our findings thus indicate that AMH and AMHR2 are detected in most cell bodies or fibers of GnRH neurons in the POA, ARC, and ME of heifer brains. These data support the need for further study as to how AMH and AMHR2 act within the hypothalamus to influence GnRH and gonadotropin secretion.

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.

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