Bovine gonadotrophs express anti-Müllerian hormone (AMH): comparison of AMH mRNA and protein expression levels between old Holsteins and young and old Japanese Black females

Abstract

Anti-Müllerian hormone (AMH) is secreted from ovaries and stimulates gonadotrophin secretion from bovine gonadotroph cells. Other important hormones for endocrinological gonadotroph regulation (e.g. gonadotrophin-releasing hormone, inhibin and activin) have paracrine and autocrine roles. Therefore, in this study, AMH expression in bovine gonadotroph cells and the relationships between AMH expression in the bovine anterior pituitary (AP) and oestrous stage, age and breed were evaluated. AMH mRNA expression was detected in APs of postpubertal heifers (26 months old) by reverse transcription-polymerase chain reaction. Based on western blotting using an antibody to mature C-terminal AMH, AMH protein expression was detected in APs. Immunofluorescence microscopy utilising the same antibody indicated that AMH is expressed in gonadotrophs. The expression of AMH mRNA and protein in APs did not differ between oestrous phases (P>0.1). We compared expression levels between old Holsteins (79.2±10.3 months old) and young (25.9±0.6 months old) and old Japanese Black females (89.7±20.3 months old). The APs of old Holsteins exhibited lower AMH mRNA levels (P<0.05) but higher AMH protein levels than those of young Japanese Black females (P<0.05). In conclusion, bovine gonadotrophs express AMH and this AMH expression may be breed-dependent.

Anti‐Müllerian hormone is expressed and secreted by bovine oviductal and endometrial epithelial cells

“…We utilized the same method of immunohistochemistry to detect ovarian AMH and AMHR2 as reported previously (Kereilwe & Kadokawa, 2019;Kereilwe et al, 2018)…”

Decreased Anti-Müllerian hormone and Anti-Müllerian hormone receptor type 2 in hypothalami of old Japanese Black cows

“…We recently discovered the extragonadal functions of AMH mediated by its primary receptor, AMH receptor type 2 (AMHR2). Specifically, AMHR2 colocalizes with gonadotropin-releasing hormone (GnRH) receptors on the lipid rafts of gonadotrophs [ 7 , 8 ], and AMH stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from bovine gonadotrophs. Moreover, recently, GnRH neurons in the areas of the brain relevant to neuroendocrine control of reproduction [the preoptic area (POA), arcuate nucleus (ARC), and median eminence (ME)] in humans and rodents were reported to express AMHR2 [ 2 ].…”

“…We used a previously reported method of western blotting [ 7 , 8 ] using the same anti-AMH rabbit polyclonal antibody (ARP54312_P050; Aviva Systems Biology, San Diego, CA, USA) and the same anti-bovine AMHR2 antibody. Both antibodies had been verified using immunofluorescence analysis and western blotting in bovine small follicles [ 7 , 8 ]. The anti-AMH antibody recognizes the mature C-terminal form of human AMH (corresponding to amino acids 468–517; SVDLRAERSVLIPETYQANNCQGVCGWPQSDRNPRYGNHVVLLLKMQARG).…”

“…We performed western blotting as previously described [ 6 , 7 , 8 ]. Briefly, proteins were extracted from frozen POA ( n=5 for each group) and ARC&ME ( n= 5, for each group) tissues using a mortar, liquid nitrogen, and a tissue protein extraction reagent (T-PER; Thermo Fisher Scientific; Waltham, MA, USA) with protease inhibitors (Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific).…”

“…The ME is the interface between the neural and peripheral endocrine systems through which GnRH is secreted into the pituitary portal blood vessels [ 4 ]. Using three-dimensional immunofluorescence, our recent study suggested that bovine GnRH neurons secrete AMH as well as GnRH into the pituitary portal blood [ 6 ], which may lead to AMH stimulation of LH and FSH secretion from gonadotrophs in the pituitary [ 7 , 8 ]. However, it should be noted that less than 30% of AMH- and AMHR2-positive neurons are non-GnRH neurons in the POA, ARC, and ME according to our previous study [ 6 ].…”