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Susan M Quirk, Robert G Cowan, and Rebecca M Harman

The corpus luteum contains differentiated steroidogenic cells that have exited the cell cycle of proliferation. In some tissues, deletion of quiescent, differentiated cells by apoptosis in response to injury or pathology is preceded by reentry into the cell cycle. We tested whether luteal cells reenter the cell cycle during the physiological process of luteolysis. Ovaries were obtained after injection of cows with a luteolytic dose of prostaglandin F2 α (PGF). In luteal sections, cells co-staining for markers of cell proliferation (MKI67) and apoptosis (cPARP1) increased 24 h after PGF, indicating that cells that reenter the cell cycle undergo apoptosis. The percent of steroidogenic cells (CYP11A1-positive) co-staining for MKI67 increased after PGF, while co-staining of non-steroidogenic cells did not change. Dispersed luteal cells were stained with Nile Red to distinguish lipid-rich steroidogenic cells from nonsteroidogenic cells and co-stained for DNA. Flow cytometry showed that the percent of steroidogenic cells progressing through the cell cycle and undergoing apoptosis increased after PGF. Culturing luteal cells induced reentry of steroidogenic cells into the cell cycle, providing a model to test the influence of the cell cycle on susceptibility to apoptosis. Blocking cells early in the cell cycle using inhibitors reduced cell death in response to treatment with the apoptosis-inducing protein, Fas ligand (FASL). Progesterone treatment reduced progression through the cell cycle and decreased FASL-induced apoptosis. In summary, steroidogenic cells reenter the cell cycle upon induction of luteal regression. While quiescent cells are resistant to apoptosis, entry into the cell cycle promotes susceptibility to apoptosis.

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Robert G Cowan and Susan M Quirk

Cell-fate mapping was used to identify cells that respond to the hedgehog (HH) signaling pathway and that are incorporated into the theca cell layer during ovarian follicle development. Expression of Gli1 is increased by HH signaling and can be used as a marker of cells responsive to HH in reporter mice. In transgenic Gli1ERcre/tdT mice, injection of tamoxifen (TAM) induces cre-mediated recombination and expression of td tomato (tdT) which leads to permanent fluorescent marking of cells expressing Gli1 and their progeny. The identity of tdT-positive cells was determined by co-staining ovaries for endothelial cells (CD31), pericytes (CSPG4), vascular smooth muscle cells (VSMC; smooth muscle actin) and steroidogenic cells (cytochrome P450 17A1). Gli1ERcre/tdT mice were injected with TAM on the day of birth. Cells positive for tdT in 2-day-old mice were identified as pericytes, located primarily in the medulla of the ovary in close proximity to endothelial cells. In both prepubertal mice and adult mice treated with equine chorionic gonadotropin to induce the formation of preovulatory follicles, tdT-positive cells were located within the theca cell layer and were identified as pericytes, VSMC and steroidogenic theca cells. Granulosa cells are known to express two HH ligands, Indian HH and desert HH (DHH). In DHHcre/tdT reporter mice, endothelial cells were marked as tdT-positive indicating that endothelial cells, in addition to granulosa cells, express Dhh in the ovary. These findings suggest that HH signaling may stimulate the development of the vasculature along with steroidogenic capacity of the theca layer during follicle development.

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Rebecca M Harman, Robert G Cowan, Yi Ren, and Susan M Quirk

The role of the hedgehog (HH) signaling pathway in implantation was studied in mice in which the HH signal transducer, smoothened (SMO), was conditionally deleted in the stromal compartment of the uterus, using CRE recombinase expressed through the Amhr2 cre allele. In Amhr2 cre/ + Smo null/flox-mutant mice, Smo mRNA in uterine stroma was reduced 49% compared to that in Amhr2 + / + Smo null/flox control mice, while levels in the luminal epithelium were not different. Litter size was reduced 60% in mutants compared with controls, but ovulation rate and the number of implantation sites on day 7 of pregnancy did not differ. The number of corpora lutea was equivalent to the number of implantation sites, indicating that most ovulations resulted in implanted embryos. However, on days 13 to 15, the rate of embryo resorption was elevated in mutants. In control mice, on day 5, implantation sites were present and blastocysts were well-attached. In contrast, blastocysts were readily flushed from uteri of mutant mice on day 5 and implantation sites were rare. On days 5.5 and 6, implantation sites were present in mutant mice, and by day 6 embryos could not be flushed from the uterus. The weight of implantation sites on day 7 was decreased by 42% in mutant mice, consistent with delayed development. Signaling through SMO in the endometrial stroma is required for optimal timing of implantation, and deferred implantation leads to defective embryo development and subsequent pregnancy loss.

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Kate A Margalit, Robert G Cowan, Rebecca M Harman, and Susan M Quirk

Ovarian surface epithelial cells (OSEs), a single layer of cells that cover the surface of the ovary, undergo turnover at the site of follicular rupture at ovulation. Greater than 90% of ovarian cancers arise from the OSEs. The objective of this study was to determine whether OSEs have the capacity to regulate their own demise through expression of Fas antigen (Fas) and Fas ligand (FasL) and activation of Fas-mediated apoptosis. In initial experiments, primary cultures of bovine OSEs responded to treatment with recombinant FasL by undergoing apoptosis. The percentage of cell death was not affected by the presence or absence of serum in the media or by co-treatment with interferon-γ, a treatment shown to potentiate Fas-mediated apoptosis in a number of cell types. Subsequent experiments tested the ability of stress-inducing drugs, anisomycin and daunorubicin, to promote apoptosis by stimulating an endogenous Fas–FasL pathway in OSEs. Treatment with FasL, anisomycin or daunorubicin induced cell death and this was suppressed by co-treatment with a peptide inhibitor of caspases, ZVAD. Treatment with anisomycin or daunorubicin in the presence of ZVAD increased expression of FasL mRNA and protein but did not alter expression of Fas mRNA or protein. Treatment of OSEs with a recombinant protein that blocks interaction of FasL with Fas (Fas:Fc) reduced apoptosis in response to anisomycin and daunorubicin, indicating that drug-induced apoptosis was mediated at least partially through endogenous Fas–FasL interactions. In summary, OSEs undergo apoptosis in response to stress-inducing drugs through activation of an endogenous Fas pathway.

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Fernando F Migone, Pei-hsuan Hung, Robert G Cowan, Vimal Selvaraj, Susan S Suarez, and Susan M Quirk

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2 cre). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2 cre/+ SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.