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The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities., (© 2016 by the Society for the Study of Reproduction, Inc.)

The Hippo signaling pathway is essential for regulating proliferation and apoptosis in mammalian cells. The LATS1 kinase is a core member of the Hippo signaling pathway that phosphorylates and inactivates the transcriptional co-activators YAP1 and WWTR1. Deletion of Lats1 results in low neonate survival and ovarian stromal tumors in surviving adults, but the effects of Lats1 on early follicular development are not understood. Here, the expression of Hippo pathway components including Wwtr1, Stk4, Stk3, Lats2, and Yap1 transcripts were decreased by 50% in mouse ovaries between 2 and 8 days of age while expression was maintained from 8 days to 21 days and after priming with eCG. LATS1, LATS2, and MOB1B were localized to both germ and somatic cells of primordial to antral follicles. Interestingly, YAP1 was predominantly cytoplasmic, whereas WWTR1 was nuclear in oocytes and somatic cells. Deletion of Lats1 caused an increase in germ cell apoptosis from 1.7% in control ovaries to 3.6% in Lats1 mutant ovaries and a 58% and 32% decrease in primordial and activated follicle numbers in cultured mutant ovaries. Surprisingly, there was an increase in Bmp15 but not Gdf9, Figla, Nobox transcripts or the somatic-specific transcripts Amh and Wnt4 in cultured Lats1 mutant ovaries. Last, Lats1 mutant ovaries developed ovarian cysts at a higher frequency (43%) than heterozygous (24%) and control ovaries (8%). Results showed that the Hippo pathway is active in ovarian follicles and that LATS1 is required to maintain the pool of germ cells and primordial follicles., (© 2015 by the Society for the Study of Reproduction, Inc.)

Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (> or =10 mm) and cysts were negatively correlated (r = -0.45, P < 0.01). Smaller follicles (<10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles <10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.

This study was designed to investigate the expression of insulin-like growth factor-1 (IGF-1) during cystogenesis in the dehydroepiandrosterone (DHEA)-induced rat polycystic ovarian syndrome (PCO) model. IGF-1 expression patterns in DHEA-treated rat ovaries were compared with those in control ovaries. In situ hybridization revealed a similar distribution of IGF-1 mRNA in DHEA-treated and control ovaries: in both, IGF-1 mRNA expression was confined to the granulosa cells of preantral and small antral follicles. Some hybridization signals for IGF-1 mRNA were also found in theca and infrequently in the interstitial cells. No signal was observed in larger antral follicles, atretic follicles, or cysts. This similarity indicates that there might be a shared mechanism in the early follicular development of normal folliculogenesis and DHEA-induced cystogenesis. The effects of DHEA on granulosa cells were analyzed in vitro in their quiescent, proliferative, differentiative, and preovulatory stages. Northern analysis revealed three transcripts for IGF-1 (7.5 kilobases [kb], 1.6 kb, and a group of signals between 0.4 and 0.9 kb) in cells at all stages except the preovulatory. The strongest signal was observed in cells of the proliferative stage of control cultures, while expression of IGF-1 increased only in the DHEA-treated cells cultured in the differentiative stage (when they secrete estrogen). Increase in IGF-1 expression may contribute to the hypersteroidogenism observed in the DHEA-treated rat PCO model.

Twice-daily treatments with subovulatory doses of hCG result in the development of large ovarian cysts that possess the capacity to produce preovulatory amounts of estradiol (E2) in the presence of exogenous substrate (Biol Reprod 1991; 45:34-42). To determine the effects of prolonged stimulation by subovulatory doses of LH-like activity on the ability of ovarian follicles to produce aromatizable androgens and their metabolites and on the capacity of similar follicles to metabolize exogenous androstenedione (A4) and testosterone, pregnant rats were treated with either 0 (control), 1, or 3 IU hCG twice daily for 9 days, beginning on Day 13 of pregnancy. The largest follicles or cysts in the ovaries of these animals on Days 15, 17, 19, and 22 were incubated for 4 h in the presence of 1) medium alone, 2) 1 mM cAMP, 3) 800 ng/ml A4 with or without cAMP, or 4) 800 ng/ml testosterone with or without cAMP. In the presence or absence of cAMP, follicular incubates from controls displayed limited amounts of A4, testosterone, E2, estrone (E1), and 5alpha-reduced androgen accumulation compared to incubates from rats treated with hCG. By Day 22, follicular incubates from rats treated with 3 IU hCG contained more A4 than incubates from animals treated with 1 IU hCG. However, on each day tested, incubates from rats treated with 1 IU hCG displayed at least as much, and usually more, testosterone, E2, E1, and 5alpha-reduced androgens as did incubates from rats treated with 3 IU hCG. Follicles from rats treated with 3 IU hCG lost their ability to respond to cAMP with increased steroidogenesis. The capacity of follicles from controls and from rats treated with 3 IU hCG to metabolize exogenous A4 to testosterone, E2, and 5alpha-reduced androgens was maintained between 32 and 37 ng of steroid/4 h from Day 15 to Day 22, whereas the capacity of follicles from rats treated with 1 IU hCG to metabolize A4 ranged from 68 to 92 ng of steroid/4 h. Similar patterns for E2 and 5alpha-reduced steroid production were observed when exogenous testosterone was used as substrate. Follicles from all in vivo treatment groups displayed similar capacities to reverse-metabolize exogenous testosterone to A4 and E1 on Day 15 of pregnancy. This capacity increased dramatically, from 29 to 75 ng of steroid/4 h, between Days 15 and 17 for follicles from rats treated with 3 IU hCG. A similar increase was not observed for follicular incubates from rats treated with 1 IU hCG until Day 22 of pregnancy, and was never observed for follicles from controls. In summary, prolonged exposure to stimulation by subovulatory doses of LH-like activity differentially affects the forward and reverse metabolism of aromatizable androgens by ovarian cysts that are developing in the pregnant rat. The limited ability of large, hCG-induced cysts to forward-metabolize A4 and the apparent increased capacity of these follicles to reverse-metabolize testosterone to A4 indirectly support the notion that follicular 17-ketosteroid reductase and 17beta-hydroxysteroid dehydrogenase activities may be differentially regulated during the induction of ovarian cysts in response to prolonged stimulation by gonadotropins in the anovulatory environment of the pregnant rat.

The daily administration of human chorionic gonadotropin (hCG) to rats with thiouracil-induced hypothyroidism results in the development of cystic ovaries. This study was undertaken to delineate hormonal changes during the first 48 h of hCG treatment. Groups of euthyroid and hypothyroid rats were injected daily with hCG or saline for up to two days and killed at 0, 12, 24, or 48 h after the initial hCG injection. Sera were analyzed for progesterone (P), testosterone (T), 17 beta-estradiol (E2), and prolactin (Prl) by specific radioimmunoassay (RIA). Serum levels of these hormones were not significantly different in the euthyroid and hypothyroid rats. However, P was significantly elevated at 12, 24, and 48 h in the hypothyroid/hCG rats. T and Prl were significantly elevated at 12 and 48 h in the hypothyroid/hCG rats. T levels were also elevated at 12 and 48 h in the euthyroid rats receiving hCG. In contrast, hCG had no effect on P and Prl levels in the euthyroid rats. E2 levels were undetectable in the euthyroid and hypothyroid rats. The administration of hCG increased E2 in both the euthyroid and hypothyroid rats at 48 h with significantly more E2 detected in the hypothyroid rats. These results show that ovarian steroids and Prl levels increase during the early stages of cyst induction and suggest they may be important in triggering ovarian cyst formation.