|Spicer, Leon -|
Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: March 21, 2011
Publication Date: July 30, 2011
Citation: Echternkamp, S.E., Eborn, D.R., Spicer, L.J. 2011. Hedgehog signaling pathway in small bovine ovarian follicles [abstract]. Biology of Reproduction. 85 (1 Supplement):206 (Abstract # 673). Technical Abstract: The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and adult tissues including bovine ovarian granulosa and theca cells. Binding of hedgehog to the patch receptor derepresses smoothened resulting in transcription activation or repression. Cattle genetically selected for twin ovulations (Twinner) exhibit a greater number of antral follicles during the estrous cycle than cattle not selected for twins (Control), suggestive of enhanced folliculogenesis in Twinners. The objective was to further assess the relationship among gene expression for Indian hedgehog (IHH), its patch 1 receptor (PTCH1), and aromatase (CYP19A1) during development of small bovine follicles and their contribution to increased follicle numbers in Twinners. Ovaries were collected at 3, 6, or 9 days after estrus by ovariectomy from cyclic Twinner (n = 12) and Control (n = 12) cows. Pieces of cortical tissue were fixed in 4% formaldehyde, dehydrated in ethanol and then xylene, embedded in paraffin, and subsequently sectioned. Abundance of mRNA for IHH, PTCH1, and CYP19A1 was analyzed within individual small (<= 5 mm) antral follicles by in situ hybridization using 35S-UTP-labelled bovine-specific antisense and sense probes. Slides were exposed to nuclear track emulsion for 4 wk followed by quantification of silver grain density in 4 areas within the intact granulosa and theca layer of each follicle (2 to 7 follicles/cow) using image analysis system. Antisense minus sense density measurements were averaged for the 4 replicates/follicle, and data were expressed as proportion of measured area occupied by specific pixels. Follicles were categorized as estrogen inactive (E-I) when area occupied by specific pixels for CYP19A1 mRNA was < 10%. Expression data were analyzed by analysis of variance; independent variables were genetic line (Twinner vs. Control), estrogen status (E-A vs. E-I), follicle size, (<= 1, 2-3, or 4-5 mm), and day of cycle (3, 6, or 9). Detection of IHH mRNA was localized to granulosa and cumulus cells; whereas, PTCH1 mRNA was detected primarily in theca cells of the same follicle. PTCH1 expression was also detected in granulosa cells of some E-I follicles. Abundance of mRNA for IHH was 37-fold greater in E-A compared with E-I follicles; whereas, PTCH1 mRNA was abundant in both E-A and E-I but twofold greater in E-A verses E-I follicles. In E-A follicles, CYP19A1 expression was correlated positively with IHH (r = 0.65) and PTCH1 (r = 0.37) expression. Expression of IHH or PTCH1 mRNA in E-A follicles did not differ between Twinner and Control cows or among days. Abundance of IHH, but not PTCH1, mRNA was twofold greater in E-A follicles > 1 mm compared with <= 1 mm (E status x size); however, a greater proportion of the 4-5 mm follicles were E-I compared with follicles < 4 mm. Observed patterns for IHH and PTCH1 mRNA in granulosa and theca cells further indicate a potential paracrine role for the hedgehog signaling pathway in ovarian follicular development in cattle. Hedgehog signaling does not appear to contribute to increased folliculogenesis in adult Twinner ovaries but may have a role in germ cell proliferation during embryogenesis. USDA is an equal opportunity provider and employer.