Expression of Follicle-Stimulating Hormone Receptor Variants During the Estrous Cycle of the Ewe.

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Several splice-variants of the follicle stimulating hormone receptor (FSHR) have been identified in sheep. These include a G protein-coupled (FSHR-1), a putative dominant negative (FSHR-2), and a growth factor type-I (FSHR-3) form. These variants likely couple to different signaling pathways and may be involved in FSH responsiveness at different stages of follicular development. However, with the exception of FSHR-1, the roles of variant forms of the FSHR during follicular development in the ewe are unknown. Thus, our objective was to examine expression patterns of variant forms of the FSHR at different times during the estrous cycle and in different sized follicles in the ewe. In Exp. 1, 8 cycling Suffolk-cross ewes were euthanized 24 (n=3), 36 (n=3) and 48 (n=2) hr after the onset of estrus. In Exp. 2, 7 anestrous Suffolk-cross ewes received CIDRs for 14d, followed by treatment (n=3) with PMSG (500IU), or no PMSG (n=4; CIDR-only). Ewes were euthanized 24 (n=4; 2 CIDR-only, 2 PMSG-treated) or 36 (n=3; 2 CIDR-only, 1 PMSG-treated) hr after CIDR removal. Ovaries were harvested from each ewe and all visible follicles were aspirated. Granulosa cells (GC) were isolated from follicular fluid; each isolate was pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm) or preovulatory (≥ 6.1 mm). Total RNA was extracted from GC and reverse-transcribed. The resulting cDNA was subjected to qPCR using primers designed to amplify each FSHR variant specifically. For Exp. 1, FSHR-3 was the most highly expressed (P≤0.05) variant in small and medium follicles. Expression of FSHR-2 and FSHR-3 was higher (P≤0.05) in medium follicles compared to small and preovulatory follicles. Expression of FSHR-1 was not significantly different between follicle size classes, but was higher (P≤0.05) than FSHR-2 in small follicles. No differences in FSHR variant expression were found at different times after onset of estrus, nor were FSHR variants expressed at significantly different levels in preovulatory follicles. For Exp. 2, application of CIDRs during anestrous appeared to cause slight differences in expression patterns of FSHR variants. The most highly expressed variant (P≤0.05) in small and medium follicles was still FSHR-3; however, FSHR-1 expression was higher (P≤0.05) in large versus small and medium follicles, while no differences were found in experiment 1 for FSHR-1 in different sized follicles. Thus, progesterone treatment during anestrous may enhance expression of the G protein-coupled form of the FSHR as follicles develop. FSHR-2 was expressed at the lowest (P≤0.05) levels of any variant, but was higher (P≤0.05) in large compared to medium and small follicles. No significant differences in FSHR variant expression were found within large follicles. This is the first study to examine expression patterns of FSHR-1, FSHR-2, and FSHR-3 during development of follicles in the mature sheep ovary. Our results indicate that FSHR-3 is the predominant variant of the FSHR expressed by GC during the ewe estrous cycle, and thus FSH may have important effects on follicular development through other forms of its receptor in addition to the G protein-coupled form. (poster)