Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro

Abstract

Purpose The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. Methods The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro-and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. Results AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo-and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. Conclusions AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Molecular evidence that follicle development is accelerated in vitro compared to in vivo

“…In the present study, AMH expression was indeed highly upregulated (>5 fold) in vivo from the small preantral to the small antral stage; however, AMH mRNA was twofold lower at the small antral stage in vitro than that in vivo, accompanied by decreased AMH secretion per granulosa cell during culture. These observations are consistent with those reported by Xu and coworkers (Xu et al 2016) who showed a decreased rate of AMH secretion during the final stages of antral follicular growth in vitro. This decrease may alleviate the inhibitory action of AMH on follicular differentiation in vitro, described elsewhere (Campbell et al 2012).…”

“…Studies that have focused on the in vitro development of preantral follicles in large non-rodent species have evaluated the quality of the follicle and its enclosed oocyte in culture by studying their survival and growth capacity, associated with the secretion of steroids or inhibins (sheep (Cecconi et al 2004), goat (Chaves et al 2012 and human (Xu et al 2009)) or anti-Müllerian hormone (primate (Xu et al 2016)) and/or oocyte competence to development (buffalo (Gupta et al 2008), sheep (Barboni et al 2011) and goat (Magalhaes et al 2011)). The transcriptome of small follicles developed in vivo has been established for sheep (Bonnet et al 2011) and goat (Magalhaes-Padilha et al 2013).…”

Rapid actions of anti‐Müllerian hormone in regulating synaptic transmission and long‐term synaptic plasticity in the hippocampus

“…Plasma was analyzed for Amh protein concentrations using an AMH (Rat and Mouse) ELISA kit (AnshLabs, Webster, TX, USA) by the Endocrine Technologies Core in the Oregon National Primate Research Center (ONPRC) at OHSU based on the manufacturers' instructions. 19 Positive staining was only detected in granulosa cells of growing follicles expressing Amh. Because only 10-15 µL of CSF could be collected without blood contamination from individual mouse, 17 Western blot was used to evaluate Amh levels in the CSF, as previously described.…”

“…19 Mitochondrial ribosomal protein S10 (Mrps10) served as the internal control. 19 Mitochondrial ribosomal protein S10 (Mrps10) served as the internal control.…”

“…20 RNA was reverse-transcribed into cDNA using a GoScript Reverse Transcription System (Promega Corporation, Madison, WI, USA). 19 PCR primers were designed using NCBI/ Primer-BLAST (https ://www.ncbi.nlm.nih.gov/tools/ primer-blast/ ) for Amh, Amhr2 and Alk2/3/6 ( Table 1). PCR was conducted using GoTaq Green Master Mix (Promega Corporation) with the following parameters: initial denaturation 95°C/1 minutes, followed by 36 cycles of denaturation 95°C/30 seconds, annealing 58°C/45 seconds, and extension 68°C/45 seconds.…”