|Aad, Pauline - OK STATE UNIV STILLWATER|
|Spicer, Leon - OK STATE UNIV STILLWATER|
Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2008
Publication Date: September 1, 2008
Citation: Aad, P., Spicer, L., Echternkamp, S. 2008. The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: Evidence of a link between the IGF and hedgehog systems [abstract]. Biology of Reproduction. Special Issue:109-110 (Abstract #242). Technical Abstract: The hedgehog system is involved in the regulation of ovarian function in drosophila, but its role in regulating ovarian follicular function in mammals is unclear. Therefore, gene expression of Indian hedgehog ligand (Ihh), its type 1 receptor (patched 1; Patch1), and IGF type 2 receptor (IGF2R) were quantified in bovine granulosa (GC) and/or theca (TC) cells. In experiment (exp) 1, cows selected (Twinner) and unselected (Control) for twin births and ovulations were slaughtered between day 3 and 6 of an estrous cycle. Ovaries were collected and antral follicles (>5 mm) were collected and snap frozen individually in liquid nitrogen; follicular fluid (FF), GC, and TC were subsequently isolated separately. In exp 2 through 6, GC and TC were collected separately from ovaries obtained at a local abattoir. Cells were cultured for 48 h in 10% FCS and then for either 5 or 24 h in serum-free medium containing Sonic hedgehog (Shh), IGF-1, or IGF-2 in exp 2 to 5 or starved for 12 h before 3-H thymidine treatment in the presence of Shh in exp 6. Cell numbers were determined using the coulter counter. Total RNA was extracted from GC and TC separately, and levels of Ihh, Patch1, and IGF2R mRNA were quantified using multiplex real-time RT-PCR and expressed as relative mRNA abundance normalized to constitutively expressed 18S ribosomal RNA. In exp 1, follicles were classified as medium (4 to <8 mm; n = 84) or large (8 to 22 mm; n = 78). Abundance of thecal Patch1 and IGF2R mRNA was less (P < 0.05) in Twinner versus Control cows. Thecal IGF2R and Patch1 mRNA and granulosa Ihh mRNA decreased with increased follicular size; thecal IGF2R and Patch1 mRNA were correlated positively (r = 0.43; P < 0.001). Treatment of GC with IGF-1 (exp 2) decreased (P < 0.05) the abundance of Ihh mRNA; treatment with Shh (exp 3) had no effect on IGF2R mRNA. In cultured TC, abundance of Patch1 mRNA was increased (P < 0.05) by Shh (exp 4) but unaffected by IGF-1 (exp 5). Shh increased (P < 0.05) cell proliferation as measured by 3-H thymidine incorporation into DNA (exp 6). For the first time, we have shown a possible control of the hedgehog system by IGF-1. Because IGF2R sequesters free IGF-II and, thus, reduces its binding to the IGF type I receptor, we hypothesize that reduced thecal IGF2R levels in follicles of Twinner cows may increase the amount of free or bioavailable IGF-II, which may act in a paracrine fashion to regulate the expression patterns of the hedgehog system, leading to up regulation of follicular development.