P-582 Novel anti-Mullerian hormone receptor 2 binding peptide (AMHR2BP) modulates murine ovarian follicle cell proliferation and oocyte function in vivo

Abstract

Abstract Study question We sought to investigate whether the inhibitory effects of the novel peptide AMHR2BP on ovarian cortex gene transcription are translated into decreased protein synthesis. Summary answer AMHR2BP, like recombinant AMH, profoundly inhibited protein transcription of FSH-R, Ki67, BMP15 and GDF9 in ovarian cortex, confirming its role in inhibition of ovarian function. What is known already We previously showed how anti-Müllerian hormone (AMH) inhibited hormone production, and ovarian cortex follicle development in in vitro, and in vivo, ovarian cortex, and in luteinized granulosa cells. We then developed an AMH receptor 2 binding peptide (AMHR2BP) that could mimic AMH functions and parallel-tested it with AMH. Using histology and real-time RT-PCR we showed that, similarly to AMH, AMHR2BP inhibited granulosa cells replication and function and preserved ovarian follicle number by minimizing the progression of follicular development throughout its administration, in a mouse model. In addition, AMHR2BP decreased follicle hormone production, cell replication and apoptosis, and inhibited oocyte function. Study design, size, duration This was a translational study where 24 18-weeks old C57BL female mice were equally divided and assigned to four treatments: Baseline (euthanized just prior to the experiment), AMHR2BP (AMHR2-BP, 50 µg /day), rAMH (recombinant AMH, 1.8 µg /day), and placebo group (normal saline), administered via intraperitoneal pumps. Mice were euthanized 3 weeks after pump placement and the ovaries were explanted for histology, real-time RT-PCR and ELISA testing. Histology and real-time RT-PCR results were previously reported. Participants/materials, setting, methods We performed ELISA on mouse ovary lysates to quantify protein concentration of FSH receptor (FSH-R), Ki67 (stimulates cell proliferation), and the two oocyte-derived hormones that stimulate mitotic proliferation in granulosa cells, BMP15 and GDF9. Both, BMP15 and GDF9 act in an additive manner and have a critical role in granulosa and theca cell growth, as well as in differentiation and maturation of the oocyte. We used Kruskall-Wallis for comparison of medians (SPSS v25; p < 0.05). Main results and the role of chance Compared with the Baseline and Placebo groups, AMHR2BP and rAMH administration caused a significant decrease in concentration of all the parameters under investigation, indicating inhibition of cellular function. Specifically, a decreased FSH-R indicated loss of pituitary stimulation of follicular growth; a decreased Ki67 showed inhibition of cellular replication; a decrease in BMP15 and GDF9 indicated stalling of granulosa and theca cell growth, as well as of oocyte differentiation and maturation. The table reports the individual groups’ data. Limitations, reasons for caution With this experiment we cannot prove a direct effect on the oocyte or the granulosa cells, as they were not individually tested. Our previous gene expression study on granulosa cells proved a direct inhibitory effect of AMHR2BP as well as recombinant AMH on granulosa cells. Wider implications of the findings We confirmed AMHR2BP’s overall inhibitory effect on ovarian function and development, for the duration of its administration. Combined with our previous findings of direct inhibition of in vitro and in vivo follicular development, we conclude that AMHR2BP could be used to inhibit age-linked loss of ovarian follicle reserve. Trial registration number Cleveland Clinic