Inhibition of autophagy in theca cells induces CYP17A1 and PAI-1 expression via ROS/p38 and JNK signalling during the development of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a clinical syndrome characterized by hyperandrogenism, oligo/anovulation, and polycystic ovary. Autophagy is an intracellular system that degrades cytosolic proteins and organelles. The relationship between autophagy and PCOS has not been clarified. We found that p62 and ubiquitin were significantly increased in theca cells of women with PCOS using immunohistochemistry. Autophagy inhibition by palmitic acid and chloroquine in bovine theca cells increased p62 and ubiquitin and induced the expression of cytochrome P450 17A1 (CYP17A1) and plasminogen activator inhibitor-1 (PAI-1) mRNA. Furthermore, palmitic acid and chloroquine exposure significantly increased reactive oxygen species (ROS) and activated p38 and c-Jun N-terminal kinase (JNK). Inhibition of p38 and JNK significantly reduced CYP17A1 and PAI-1 mRNA expression. We showed that inhibition of autophagy in theca cells may have contributed to the pathogenesis of PCOS, based on CYP17A1 and PAI-1 mRNA expression via the ROS/p38 and JNK signalling pathways.

Luteinizing Hormone Facilitates Antral Follicular Maturation and Survival via Thecal Paracrine Signaling in Cattle.

LH supplementation in assisted reproductive technology cycles improves the ongoing pregnancy rate in women with poor ovarian response (POR). However, our knowledge of the precise role of LH during the follicular phase of the menstrual cycle is incomplete. To explore the role of LH in the maturation of small antral follicles, we used an in vitro two-cell system that involved coculturing bovine granulosa cells (GCs) and theca cells (TCs) on a collagen membrane. Treatment of TCs with LH stimulated androgen production in TCs by inducing the expression of androgenic factors, subsequently increasing estrogen biosynthesis in GCs by providing androgen substrates, and inducing aromatase expression. LH stimulation of TCs induced functional LH receptor expression in GCs, a response modulated by the synthesis and action of estrogen. In the presence of TCs, LH stimulation of TCs and FSH stimulation of GCs increased the expression of IGF-1, IGF-2, and IGF-1 receptor in GCs. LH-induced expression of thecal IGF-1 protected GCs from apoptosis and promoted GC survival. Furthermore, LH stimulation of TCs increased FSH sensitivity in GCs. Thus, the LH-TC axis may be involved in the acquisition of LH dependence and the survival of small antral follicles by upregulating androgen/estrogen biosynthesis and activating the IGF system. The use of LH supplementation in ovarian stimulation may increase gonadotropin sensitivity in small antral follicles and promote follicular growth and survival by suppressing GC apoptosis and follicular atresia, resulting in multiple follicular development, even in patients with POR.

Clinical significance of atypical squamous cells of undetermined significance after treatment for cervical intraepithelial grade 3 neoplasia: A retrospective single-center cohort study.

The aim of the present study was to evaluate the clinical significance of atypical squamous cells of undetermined significance (ASC-US) following cervical conization for cervical intraepithelial neoplasia (CIN) grade 3. This study was a retrospective cohort analysis. The medical records of women treated with conization for CIN 2-3 were reviewed and 142 patients with CIN 3 who had been diagnosed using the conization specimens were selected. The mean follow-up period after conization was 41.8 months. Cytological abnormalities after conization were observed in 19.0% of the patients and consisted of ASC-US (13.4%) and worse than low-grade squamous intraepithelial lesion (LSIL; 5.6%). Recurrence was defined as a diagnosis worse than CIN 2, and the recurrence rate was 29.6% among patients with abnormal cytology. The recurrence rate was 15.7% in the ASC-US group and 71.4% in the worse than LSIL group. There was no significant difference in the time of initial identification of abnormal cytology after treatment between the worse than LSIL and the ASC-US groups (P=0.054). However, the ASC-US group had a significantly better cumulative recurrence-free rate compared with the worse than LSIL group (P<0.05). Women with ASC-US following treatment for CIN appear to be at a relatively high risk. Regarding the risk stratification of women following treatment for CIN, if surveillance cytology shows ASC-US, immediate colposcopy is recommended, along with long-term follow-up.

Protective effect of dienogest on chemotherapy-induced reduced fertility in female rats.

Reduced fertility is one of the main long-term consequences of chemotherapy given for lymphoma, leukemia, and other malignancies in young women. We examined with a female rat model whether and how dienogest, a fourth-generation progestin, modulates reduced fertility following exposure to gonadotoxic chemotherapy. Female rats were administered cyclophosphamide with or without GnRH agonist and different concentrations of dienogest for 20 days. Animals were sacrificed on Day 29, and the numbers of follicle at primordial, preantral and antral stage in the ovaries were counted histologically. Rats treated with sterile saline solution (as control), cyclophosphamide, cyclophosphamide plus GnRH agonist, and cyclophosphamide plus dienogest were also mated with male rats to evaluate their fertility and pregnancy outcomes. Cyclophosphamide significantly reduced the number of primordial follicles, whereas dienogest suppressed depletion of primordial follicle pool induced by chemotherapy. Although the rats exposed to cyclophosphamide alone failed to deliver live births, co-treatment with dienogest improved the pregnancy outcomes of treated rats. The protective effect of dienogest on chemotherapy-induced ovarian damage and reduced fertility was comparable to that of GnRH agonist. The present results suggest that the co-administration of dienogest and chemotherapy may be a useful strategy in preserving ovarian function and fertility in premenopausal women facing gonadotoxic chemotherapy.

Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition.

Several clinical studies have shown a correlation of hypersecretion of LH and polycystic ovary syndrome (PCOS), infertility, and miscarriage in women, suggesting that chronically elevated LH impairs fertility. Growth arrest of small antral follicles in PCOS is also assumed to be associated with an abnormal endocrine environment involving increased LH stimulation, a hyperandrogenic milieu, and subsequent dysregulated FSH action in the ovarian follicles. In this study, we examined whether and how LH modulates follicular development and steroid production during preantral-early antral follicle transition by using a rat preantral follicle culture system. LH augments testosterone and estradiol production in preantral follicles via up-regulating mRNA abundance of CYP17A1 and CYP19A1. LH promotes rat preantral follicle growth, and the follicular size reaches that of early antral follicles in vitro, a response attenuated by the specific androgen receptor antagonist and a targeted disruption of androgen receptor gene. Sustained follicle stimulation by LH, but not by androgen, decreases FSH receptor mRNA levels and FSH receptor signaling and inhibits FSH-induced follicular growth. The data suggest that LH promotes preantral-early antral transition via the increased synthesis and growth-promoting action of androgen. However, chronic LH stimulation impairs FSH-dependent antral follicle growth by suppressing granulosa cell FSHR expression via the modulation of intraovarian regulators, including LH-induced thecal factors.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells.

BACKGROUND: Theca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK). METHODS: Ovarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells. RESULTS: Akt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA) does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor) suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca cells. CONCLUSION: These results indicate that LH stimulates CYP17 mRNA expression and androgen production in theca cells via activation of the PI3K/Akt pathway. The LH-induced Akt phosphorylation and androgen production are modulated by the MAPK signaling in bovine theca cells.