Spatiotemporal localization of meiotic regulators in the macaque ovary

Abstract

OBJECTIVE: To identify novel oocyte meiotic regulators that can potentially be used as nonhormonal contraceptive targets, we examined the spatial and temporal localization of three candidate genes in rhesus macaques: insulin-like peptide 3 (INSL3), LGR8, and WEE2. INSL3-LGR8 signaling pathway was shown to promote oocyte maturation by decreasing intra-oocyte cAMP levels in rodents. WEE2 is a key inhibitory kinase maintaining the inactive state of Metaphase Promotion Factor (MPF) in mouse oocytes. The expression and functions of these genes are unknown in primates.DESIGN: Nonhuman primate descriptive study.MATERIALS AND METHODS: Total RNA was extracted from adult rhesus monkey tissues using Trizol reagent (Invitrogen). Oocytes were collected from 6-10 y.o.f undergone controlled ovarian stimulation protocols and follicle aspiration was performed prior to (germinal vesicle, GV) or 27-34 hours after (metaphase (M)1 and M2) an ovulatory stimulus (hCG); RNA extraction was performed with the Absolutely RNA Nanoprep kit (Stratagene). First-strand cDNA was synthesized with Superscript III Reverse Transcriptase and Oligo(dT)12-18 primers (Invitrogen). Polymerase Chain Reaction (PCR) was carried out with macaque gene-specific primers. Paraffin sections of macaque ovary and testis were used for in situ hybridization (ISH) and immunohistochemistry (IHC). ISH was carried out with 35S-labled gene-specific RNA probes. IHC was performed with specific antibodies against INSL3 and LGR8 (Phoenix Pharmaceuticals, INC).RESULTS: Consistent with previous findings in rodents, macaque INSL3 was specifically detected in theca cells in antral follicles in the ovary and Leydig cells in the testis. macaque LGR8 is expressed in fully-grown GV-intact, maturing (MI), and mature (MII) oocytes in the ovary, and spermatocytes and spermatids in the testis. Macaque WEE2 is specifically expressed in the ovary, within the ovary, WEE2 is exclusively localized in oocytes in primary follicles and beyond. Transcripts of all three genes are highly enriched in preovulatory follicles and/or postovulatory oocytes, coinciding with the timing of oocyte meiotic maturation.CONCLUSIONS: We demonstrated the spatiotemporal localization of three candidate oocyte meiotic regulators, INSL3, LGR8 and WEE2, in rhesus macaque ovaries. Though the functions of these factors remain to be further elucidated, their expression pattern implies roles in oocyte maturation, similar to those observed in rodents, in nonhuman primates. OBJECTIVE: To identify novel oocyte meiotic regulators that can potentially be used as nonhormonal contraceptive targets, we examined the spatial and temporal localization of three candidate genes in rhesus macaques: insulin-like peptide 3 (INSL3), LGR8, and WEE2. INSL3-LGR8 signaling pathway was shown to promote oocyte maturation by decreasing intra-oocyte cAMP levels in rodents. WEE2 is a key inhibitory kinase maintaining the inactive state of Metaphase Promotion Factor (MPF) in mouse oocytes. The expression and functions of these genes are unknown in primates. DESIGN: Nonhuman primate descriptive study. MATERIALS AND METHODS: Total RNA was extracted from adult rhesus monkey tissues using Trizol reagent (Invitrogen). Oocytes were collected from 6-10 y.o.f undergone controlled ovarian stimulation protocols and follicle aspiration was performed prior to (germinal vesicle, GV) or 27-34 hours after (metaphase (M)1 and M2) an ovulatory stimulus (hCG); RNA extraction was performed with the Absolutely RNA Nanoprep kit (Stratagene). First-strand cDNA was synthesized with Superscript III Reverse Transcriptase and Oligo(dT)12-18 primers (Invitrogen). Polymerase Chain Reaction (PCR) was carried out with macaque gene-specific primers. Paraffin sections of macaque ovary and testis were used for in situ hybridization (ISH) and immunohistochemistry (IHC). ISH was carried out with 35S-labled gene-specific RNA probes. IHC was performed with specific antibodies against INSL3 and LGR8 (Phoenix Pharmaceuticals, INC). RESULTS: Consistent with previous findings in rodents, macaque INSL3 was specifically detected in theca cells in antral follicles in the ovary and Leydig cells in the testis. macaque LGR8 is expressed in fully-grown GV-intact, maturing (MI), and mature (MII) oocytes in the ovary, and spermatocytes and spermatids in the testis. Macaque WEE2 is specifically expressed in the ovary, within the ovary, WEE2 is exclusively localized in oocytes in primary follicles and beyond. Transcripts of all three genes are highly enriched in preovulatory follicles and/or postovulatory oocytes, coinciding with the timing of oocyte meiotic maturation. CONCLUSIONS: We demonstrated the spatiotemporal localization of three candidate oocyte meiotic regulators, INSL3, LGR8 and WEE2, in rhesus macaque ovaries. Though the functions of these factors remain to be further elucidated, their expression pattern implies roles in oocyte maturation, similar to those observed in rodents, in nonhuman primates.