Ovarian Expression of Insulin-Like Peptide 3 (INSL3) and Its Receptor (RXFP2) During Development of Bovine Antral Follicles and Corpora Lutea and Measurement of Circulating INSL3 Levels During Synchronized Estrous Cycles

Abstract

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

Functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

“…In mouse ovary, INSL3 expression is higher in the follicular phase than in the luteal phase, further suggesting a positive correlation of INSL3 with follicular maturation (44) intrabursal injection of INSL3 promoted oocyte maturation in pregnant mare serum gonadotropin-primed rats but, unlike LH or chorionic gonadotropin, did not induce ovulation (38). In the bovine ovary, expression of RXFP2 mRNA is most abundant in TCs, consistent with an autocrine/paracrine action of TC-derived INSL3 on the same cell type (28,45). RXFP2 is also expressed to a lesser degree in GC and corpus luteum tissue.…”

“…Notable among these are BMPs, activins, and inhibins. As discussed here, another peptide unrelated to the TGF-β family, INSL3, is known to be expressed abundantly in ovarian TC and, to a lesser extent, corpus luteum (20,22,27,28), but its functional role in the ovary has remained obscure.…”

“…Having confirmed that the BMP6-induced reduction in INSL3 mRNA expression was accompanied by a corresponding inhibition of INSL3 protein secretion by the cells, we showed that three other BMPs (BMP2, BMP4, and BMP7) known to be expressed in bovine follicles (14,17,29) exerted a similar suppressive action on INSL3 expression, at both the mRNA and protein level. Moreover, BMP treatment suppressed thecal expression of RXFP2, the cognate signaling receptor for INSL3 (30) that is coexpressed with INSL3 in follicular theca cells (28,31). On the basis of these findings, we hypothesized that the BMPinduced suppression of thecal androgen production might actually be mediated via an inhibition of thecal INSL3-RXFP2 signaling acting in an autocrine/paracrine manner.…”

“…TC expression of INSL3 is acutely up-regulated by LH/CG treatment in rats (38), whereas longer-term treatment of bovine TCs with a high dose level of LH or with serum (to promote luteinization) down-regulates INSL3 expression (28,39,40). In this study, involving nonluteinized TCs, no consistent stimulatory effect of LH on INSL3 mRNA expression or secretion of INSL3 protein was seen, despite a clear-cut stimulation of A4 secretion.…”

Regulation of the reproductive cycle and early pregnancy by relaxin family peptides

“…0-100 pg/ ml) and appears to reflect the growth of antral follicles (Anand- Ivell et al, 2013). In cows, blood concentrations are a little higher Satchell et al, 2013), and in in heifers whose ovarian cycle has been synchronized by a luteolytic dose of prostaglandin F2alpha, INSL3 rises to a coordinated peak reflecting the growth of an antral follicle wave . Such experiments also confirm that antral follicles are indeed the major, if not exclusive, source of circulating INSL3 within the non-pregnant female mammal.…”