Cells responding to hedgehog signaling contribute to the theca of ovarian follicles.

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.

Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility.

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 (Amhr2cre ). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+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.

In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2(cre/+)SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

Role of the cell cycle in regression of the corpus luteum.

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 F(2)(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.

Overactivation of hedgehog signaling alters development of the ovarian vasculature in mice.

The hedgehog (HH) signaling pathway is critical for ovarian function in Drosophila, but its role in the mammalian ovary has not been defined. Previously, expression of a dominant active allele of the HH signal transducer protein smoothened (SMO) in Amhr2(cre/+)SmoM2 mice caused anovulation in association with a lack of smooth muscle in the theca of developing follicles. The current study examined events during the first 2 wk of life in Amhr2(cre/+)SmoM2 mice to gain insight into the cause of anovulation. Expression of transcriptional targets of HH signaling, Gli1, Ptch1, and Hhip, which are used as measures of pathway activity, were elevated during the first several days of life in Amhr2(cre/+)SmoM2 mice compared to controls but were similar to controls in older mice. Microarray analysis showed that genes with increased expression in 2-day-old mutants compared to controls were enriched for the processes of vascular and tube development and steroidogenesis. The density of platelet endothelial cell adhesion molecule (PECAM)-labeled endothelial tubes was increased in the cortex of newborn ovaries of mutant mice. Costaining of preovulatory follicles for PECAM and smooth muscle actin showed that muscle-type vascular support cells are deficient in theca of mutant mice. Expression of genes for steroidogenic enzymes that are normally expressed in the fetal adrenal gland were elevated in newborn ovaries of mutant mice. In summary, overactivation of HH signaling during early life alters gene expression and vascular development and this is associated with the lifelong development of anovulatory follicles in which the thecal vasculature fails to mature appropriately.

Dominant activation of the hedgehog signaling pathway alters development of the female reproductive tract.

The role of hedgehog (HH) signaling in reproductive tract development was studied in mice in which a dominant active allele of the signal transducer smoothened (SmoM2) was conditionally expressed in the Müllerian duct and ovary. Mutant females are infertile, primarily because they fail to ovulate. Levels of mRNA for targets of HH signaling, Gli1, Ptch1, and Hhip, were elevated in reproductive tracts of 24-day-old mutant mice, confirming overactivation of HH signaling. The tracts of mutant mice developed abnormally. The uterine luminal epithelium had a simple columnar morphology in control mice, but in mutants contained stratified squamous cells typical of the cervix and vagina. In mutant mice, the number of uterine glands were reduced and the oviducts were not coiled. Expression of genes within the Hox and Wnt families that regulate patterning of the reproductive tract were altered. Hoxa13, which is normally expressed primarily in the vagina and cervix, was expressed at 12-fold higher levels in the uterus of mutant mice compared with controls. Wnt5a, which is required for development of the cervix and vagina and postnatal differentiation of the uterus, was expressed at higher levels in the oviduct and uterus of mutant mice compared with controls. Mating mutant females with fertile or vasectomized males induced a severe inflammatory response in the tract. In summary, overactivation of HH signaling causes aberrant development of the reproductive tract. The phenotype observed could be mediated by ectopic expression of Hoxa13 in the uterus and elevated levels of Wnt5a in the oviducts and uterus.

Reduced signaling through the hedgehog pathway in the uterine stroma causes deferred implantation and embryonic loss.

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.

WNT4 is required for normal ovarian follicle development and female fertility.

To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2(tm3(cre)Bhr) strain to target deletion of Wnt4 to granulosa cells. Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis.

Dominant activation of the hedgehog signaling pathway in the ovary alters theca development and prevents ovulation.

The role of the hedgehog (HH) signaling pathway in ovarian function was examined in transgenic mice in which expression of a dominant active allele of the signal transducer smoothened (SmoM2) was directed to the ovary and Müllerian duct by cre-mediated recombination (Amhr2(cre/+)SmoM2). Mutant mice were infertile and had ovarian and reproductive tract defects. Ovaries contained follicles of all sizes and corpora lutea (CL), but oocytes were rarely recovered from the oviducts of superovulated mice and remained trapped in preovulatory follicles. Measures of luteinization did not differ. Cumulus expansion appeared disorganized, and in vitro analyses confirmed a reduced expansion index. Microarray analysis indicated that expression levels of genes typical of smooth muscle were reduced in mutant mice, and RT-PCR showed that levels of expression of muscle genes were reduced in the nongranulosa, theca-interstitial cell-enriched fraction. Whereas a layer of cells in the outer theca was positively stained for smooth muscle actin in control ovaries, this staining was reduced or absent in mutant ovaries. Expression of a number of genes in granulosa cells that are known to be important for ovulation did not differ in mutants and controls. Expression of components of the HH pathway was observed in both granulosa cells and in the nongranulosa, residual ovarian tissue and changed in response to treatment with equine chorionic gonadotropin/human gonadotropin. The results show that appropriate signaling through the HH pathway is required for development of muscle cells within the theca and that impaired muscle development is associated with failure to release the oocyte at ovulation.

The hedgehog signaling pathway in the mouse ovary.

The hedgehog (HH) signaling pathway plays an essential role in the Drosophila ovary, regulating cell proliferation and differentiation, but a role in the mammalian ovary has not been defined. Expression of components of the HH pathway in the mouse ovary and effects of altering HH signaling in vitro were determined. RT-PCR analyses show developmentally regulated expression of sonic (Shh), indian (Ihh) and desert (Dhh) HH in the ovary. Expression is detected in whole ovary, granulosa cells, and corpora lutea. The mRNAs for the two receptors, patched homolog 1 and 2 (Ptch1, Ptch2), and the signal transducer, smoothened (Smo), are also expressed. Immunohistochemistry using an antibody that detects all three HH ligands demonstrated HH protein primarily in granulosa cells of follicles from primary to antral stages of development. Follicles also stained for PTCH1 and SMO in both granulosa and theca cells. Treatment of cultured preantral follicles and granulosa cells with recombinant SHH increased growth and proliferation while treatment with the HH pathway inhibitor, cyclopamine, had no effect. Therefore, activation of HH signaling can increase cell proliferation and follicle growth but is not essential for these processes in vitro. Treatment of granulosa cells with SHH increased levels of mRNA for Gli1, a transcriptional target of HH signaling, while cyclopamine decreased expression. SHH had no effect on production of progesterone by cultured granulosa cells, while cyclopamine increased progesterone production. The results demonstrate a functional HH pathway in the follicle and identify granulosa cells as at least one of the potential targets of HH signaling.

The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle.

Experiments were conducted to test whether oestradiol (E2) protects granulosa cells from Fas ligand (FasL)-induced apoptosis and whether protection involves modulation of the cell cycle of proliferation. Treatment of cultured bovine granulosa cells with E2 decreased susceptibility to FasL-induced apoptosis. The effects of E2 were mediated through oestrogen receptor and were not mediated by stimulation of IGF production. E2 also increased the percentage of cells progressing from G1 to S phase of the cell cycle, and increased expression of cyclin D2 protein and the cell proliferation marker Ki67. Progression from G1 to S phase of the cell cycle was necessary for the protective effect of E2; blocking progression from G1 to S phase with the cdk2 inhibitor roscovitine, or blocking cells in S phase with hydroxyurea, prevented protection by E2. The stages of the cell cycle during which granulosa cells are susceptible to apoptosis were assessed. First, treatment with the G1 phase blocker, mimosine, protected cells from FasL-induced apoptosis, indicating that cells in G0 or early- to mid-G1 phase are relatively resistant to apoptosis. Secondly, examination of recent DNA synthesis by cells that became apoptotic indicated that apoptosis did not occur in S, G2 or M phases. Taken together, the experiments indicate that cells may be most susceptible to apoptosis at the transition from G1 to S phase. E2 stimulates transition from G1 to S phase and protects against apoptosis only when cell cycle progression is unperturbed.

Apoptosis of bovine ovarian surface epithelial cells by Fas antigen/Fas ligand signaling.

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-gamma, 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.

Progesterone receptor and the cell cycle modulate apoptosis in granulosa cells.

Our previous studies showed that exposure of bovine preovulatory follicles to the LH surge-induced resistance of granulosa cells, but not theca cells, to apoptosis. Here, the temporal development of resistance to apoptosis and potential roles of progesterone receptor (PR) and alterations in the cell cycle in mediating this effect were examined. Injection of cows with GnRH induced an LH surge within 2 h. Granulosa cells isolated 0, 6, and 10 h after GnRH were sensitive to Fas ligand-induced apoptosis, but cells isolated at 14 h were resistant. PR was first detectable in granulosa cells at 10 and 14 h and was not detectable in theca. Treatment of granulosa cells isolated 14 h after GnRH with the PR antagonist, RU486, induced susceptibility to apoptosis, an effect mediated by PR and not glucocorticoid receptor. After GnRH treatment, granulosa cells, but not theca cells, exited the cell cycle, expression of cyclin D2 was reduced, and p27(Kip1) was elevated. Treatment of granulosa cells isolated from small antral follicles with the G1 phase blocker, mimosine, reduced Fas ligand-induced killing, suggesting that nonproliferating cells are resistant to apoptosis. Treatment of granulosa cells isolated 14 h after GnRH with RU486 induced reentry of some cells into the cell cycle and reversed resistance to apoptosis, suggesting that cycling cells became susceptible to apoptosis. Treatment with mimosine prevented the ability of RU486 to promote susceptibility to apoptosis. In summary, the LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle, and these events promote resistance to apoptosis.

Cell cycle progression and activation of Akt kinase are required for insulin-like growth factor I-mediated suppression of apoptosis in granulosa cells.

Ovarian follicle development is dependent on growth factors that stimulate cell proliferation and act as survival factors to prevent apoptosis of follicle cells. We examined the mechanism of the protective effect of IGF-I against Fas ligand-induced apoptosis of granulosa cells and its relationship to cell proliferation. IGF-I activated both the phosphoinositide 3'-OH kinase (PI3K) and the MAPK pathways. Experiments using specific inhibitors of these pathways showed that protection by IGF-I was mediated by the PI3K pathway and not the MAPK pathway. Recombinant adenoviruses were used to test whether the downstream target of PI3K activation, Akt kinase, was required for protection against apoptosis. Expression of dominant negative Akt prevented protection by IGF-I whereas expression of constitutively active Akt (myrAkt) mimicked the effect of IGF-I. Treatment with IGF-I, or expression of myrAkt, increased progression from G(0)/G(1) to S phase of the cell cycle whereas expression of dominant negative Akt inhibited G(0)/G(1) to S phase progression and prevented the stimulatory effect of IGF-I. We tested whether cell cycle progression was required for protection from apoptosis using the cyclin-dependent kinase-2 inhibitor roscovitine, which blocks cells at the G(1)/S transition. Roscovitine prevented the protective effect of IGF-I and myrAkt expression against apoptosis. Therefore, activation of Akt is not sufficient to protect granulosa cells from apoptosis in the absence of cell cycle progression. In summary, IGF-I protects granulosa cells from apoptosis by activation of the PI3K/Akt pathway. This protective effect can occur only when progression from G(1) to S phase of the cell cycle regulated by the PI3K/Akt pathway is unperturbed.