Nonhuman primates exhibit reproductive physiology that is similar to humans. For example, female rhesus macaques have 28-day mono-ovular menstrual cycles that closely resemble women's reproductive cycles, and utilize the LH-chorionic gonadotropin (CG) endocrine system unique to higher primates and absent in lower primates and nonprimates . Thus, nonhuman primates are the most relevant models for studies of human reproduction. Previous studies have shown that INSL3 is more likely to have a functional endocrine role in males than in females as the blood level of INSL3 in males is significantly higher than that in females . Our data demonstrates that INSL3 is also relatively enriched in the ovary of macaques, and the transcript of its full-length receptor can be detected in the ovary. The localization of the ligand to the theca layer and receptor to the granulosa cells suggests a potential local regulatory (paracrine) role of INSL3 in follicle maturation in nonhuman primates.
The structure and expression of INSL3-RXFP2 have been studied in primates in a limited manner. Previously an alternative splicing of INSL3, which possesses an extra exon (exon 1A) between exons 1 and 2, has been reported in marmoset monkeys  and humans (AY082014.1). Our data suggest that this particular splice variant of INSL3 does not exist in rhesus macaques for the primers spanning both exons consistently amplified one single transcript from different tissues derived from several monkeys. INSL3 mRNA has been detected in various mammalian tissues, mostly only by highly sensitive RT-PCR. In the marmoset monkey, INSL3 transcript was only identified in the testis by Northern blot and the ovarian corpora lutea by RT-PCR . Besides the ovary and testis, we also detected INSL3 mRNA in the hypothalamus and pituitary in the macaque. The hypothalamic expression of INSL3 transcript is consistent with a previous report in the cow, but has not been observed in rodents . However, an antibody recognizing INSL3 precursor failed to detect the protein in the hypothalamus despite evident INSL3 expression in the gonad and pituitary, suggesting a low-level or perhaps even no translation in this tissue. A recent report in rats demonstrated that Leydig cells in the testis could be the only source of circulating INSL3 . It remains to be elucidated whether INSL3 produced in the pituitary contributes to circulating INSL3 in the rhesus macaque. It is noteworthy that the relative intensities of INSL3 bands in the Western blot described in the current study do not represent the relative abundance of INSL3 in each tissue analyzed, because the selective cell types expressing INSL3 may not distribute evenly throughout the entire tissue (e.g., theca cells surrounding antral follicles in the ovary) and only part of each macaque tissue was used for protein extraction. No specific antibodies against monkey INSL3 were available, so we tested several anti-human INSL3 antibodies and eventually selected two different antibodies for their optimal performance in Western blot and IHC. Human INSL3 shares ~90% and ~60% identical amino acids with macaque and mouse INSL3, respectively. The antibody used for Western blot was raised against a short human INSL3 peptide that only differs in 2 amino acids from mouse INSL3 precursor; thus, it is not surprising that the antibody reacts with both monkey and mouse INSL3.
Full-length macaque RXFP2 is transcribed in several tissues including the hypothalamus, ovary and uterus. A previous study showed that RXFP2 was expressed in the testis but not in the ovary in humans . The contradiction could be, at least in part, attributed to the selection of primers, i.e., while only full-length RXFP2 transcript was amplified by primers spanning between exons 11 and 15 in our experiment, the amplification of 3'-UTR region of human RXFP2 would include various spliced transcripts. Indeed, the identification of three RXFP2 splice variants was previously reported in the human uterus and adrenal glands , although only one of the sequences, namely LGR8.1 which lacks exon 11 of RXFP2, can be found in the GenBank (AY899851.1). None of the three splice variants identified from the macaque corresponds exactly to LGR8.1, although two of them (i.e., RXFP2-sv2 and RXFP2-sv3) are missing exon 11. We noticed that the primers used to amplify LGR8.1 span exons 7-13 of human RXFP2, which could explain why the primer pair failed to identify the missing exon 15 that was found in all three splice variants reported in the current study. Unlike LGR8.1 that can be translated into a truncated RXFP2 protein with partial leucine-rich repeat (LRR) region, translation of all three RXFP2 splice variants identified in the current study can cause early termination of the full-length open reading frame (ORF) prior to the seven-transmembrane (7TM) domain-coding region (Figure 1b).
Within the ovary, INSL3 transcript and peptide are predominantly expressed in the theca cells surrounding antral follicles and weakly detectable in the corpus luteum. This finding is similar to previous observations in the cow  as well as in two other primate species, marmoset monkeys and humans [16, 17]. INSL3 was originally demonstrated as a male hormone required for testis positioning during fetal development [2, 32, 33]. A previous finding showing that INSL3 was expressed in the ruminant ovary at a much higher level than other species was attributed to the natural loss of the gene for relaxin . Here we show that INSL3 is also highly expressed in the primate ovary, even though relaxin gene still exists in this species . The function of INSL3 in the female reproductive system has not been well characterized. Besides the potential anti-apoptotic role of INSL3 in the mouse ovary , Hsueh's group  reported that INSL3 could function as a local mediator for the mid-cycle LH surge and promote the oocyte meiotic resumption through binding to RXFP2, an inhibitory G (Gi) protein-coupled receptor (GPCR). Although some recent evidence shows that the oocyte maturation in response to LH may be independent of Gi proteins , the highly enriched local INSL3 and the presence of its receptor imply a possible local regulatory role of the ligand-receptor in the ovary, especially during preovulatory period. Notably, we did not detect RXFP2 transcript or protein in the germinal vesicle intact oocyte, so any effect of INSL3 on maturation must be mediated through the cumulus granulosa cells. More functional studies in higher species will be required to solve this puzzle. The failure of RXFP2 detection in the ovary could be due to very low-level expression of GPCRs in individual cells, the specificity of antibodies applied, and/or an unknown nature of RXFP2 gene transcription and translation. The presence of three differentially spliced transcripts of RXFP2 in the ovary, and the localization of full-length RXFP2 mRNA in granulosa cells has not been previously reported. The majority of primate genes produce splice variant transcripts ; it is impossible to predict whether these splice variants can actually be translated into proteins, whether these proteins are functional, or whether these alternatively spliced transcripts have a function independent of protein synthesis. However, since the reported splice variants of RXFP2 encode truncated protein isoforms without the seven-transmembrane domain, these protein products are unlikely to function as GPCRs even if they do exist [39, 40]. It also remains to be determined whether the cellular localization of full-length RXFP2 transcript in the ovary is specific to rhesus monkeys.