Sepsis is defined as a systemic inflammatory response to infection. This study is aimed to evaluate the effects of experimental sepsis on the proliferation and apoptosis of granulosa and theca cells in the rat ovary. 28-day-old immature Wistar-Albino female rats were treated with pregnant mare serum gonadotrophin to develop the first generation of preovulatory follicles. Sepsis was induced by cecal ligation and puncture (CLP). Following in vivo 5-Bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. Besides electron microscopic evaluation, BrdU, cleaved caspase-3, p27 immunostaining, and TUNEL labeling were performed. In CLP-operated animals, cleaved caspase-3 immunoreactivity was significantly increased in Graafian follicles. TUNEL and BrdU labeling in the ovarian follicles were not statistically different between CLP and sham-operated rats. In septic animals, p27 immunoreactivity was increased significantly in the nuclei of oocytes and decreased in the cytoplasm of granulosa and theca cells in multilaminar primary follicles compared to the sham group. In ultrastructural evaluation, increased apoptosis was observed in theca interna and granulosa cells in both the early and late stages of follicles in the CLP group. In conclusion, experimentally-induced sepsis leads to apoptosis in ovarian follicles at advanced stages of development. Our data suggest that although sepsis may not cause a potential threat to developing follicles at least in the short term, more severe damage may occur during advanced stages of follicle development.

Tributyltin (TBT), an organotin chemical used as a catalyst and biocide, can stimulate cholesterol efflux in non-steroidogenic cells. Since cholesterol is the first limiting step for sex hormone production, we hypothesized that TBT disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. We investigated TBT\'s effect on cholesterol trafficking, luteinization, and steroidogenesis in theca cells of five species (human, sheep, cow, pig, and mice). Primary theca cells were exposed to an environmentally relevant dose of TBT (1 or 10 ng/ml) and/or retinoid X receptor (RXR) antagonist. The expression of RXRα in sheep theca cells was knocked down using shRNA. Steroidogenic enzymes, cholesterol transport factors, and nuclear receptors were measured by RT-qPCR and Western blotting, and intracellular cholesterol, progesterone, and testosterone secretion by ELISA. TBT upregulated StAR and ABCA1 in ovine cells, and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression but did not alter testosterone or progesterone production. RXR antagonist and RXRα knockdown demonstrates that TBT\'s effect is partially through RXR. TBT\'s effect on ABCA1 and StAR expression was recapitulated in all five species. TBT, at an environmentally relevant dose, stimulates theca cell cholesterol extracellular efflux via the RXR pathway, triggers a compensatory upregulation of StAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all five species evaluated (human, sheep, cow, pig, and mice) and are supportive of TBT\'s conserved mechanism of action across mammalian species.

With granulosa and theca cells, the ovaries are responsible for producing oocytes and secreting sex steroids such as estrogen and progesterone. Endoplasmic reticulum stress (ERS) plays an important role in follicle atresia and embryo implantation. In this study, goat granulosa cells were isolated from medium-sized (4-6 mm) healthy follicles. Primary granulosa cells were immortalized by transfection with human telomerase reverse transcriptase (hTERT) to establish a goat granulosa cell line (hTERT-GGCs). These hTERT-GGCs expressed hTERT and had relatively long telomeres at passage 50. Furthermore, hTERT-GGCs expressed the gonadotropin receptor genes CYP11A1, StAR, and CYP19A1, which are involved in steroidogenesis. Additionally, progesterone was detectable in hTERT-GGCs. Although the proliferation potential of hTERT-GGCs significantly improved, there was no evidence to suggest that the hTERT-GGCs are tumorigenic. In addition, thapsigargin (Tg) treatment led to a significant dose-dependent decrease in progesterone concentration and steroidogenic enzyme expression. In summary, we successfully generated a stable goat granulosa cell line. We found that Tg induced ERS in hTERT-GGCs, which reduced progesterone production and steroidogenic enzyme expression. Future studies may benefit from using this cell line as a model to explore the molecular mechanisms regulating steroidogenesis and apoptosis in goat granulosa cells.

OBJECTIVE: To evaluate the histomorphometry of ovarian interstitial cells, as well as the blood sex steroid concentrations of female rats with polycystic ovaries induced by continuous light.
METHODS: Twenty female rats were divided into two groups: Control Group - in the estrous phase (CtrlG), and a group of rats with polycystic ovaries induced by continuous illumination (POG). CtrlG animals were maintained on a light period from 07:00 a.m. to 07:00 p.m., and POG animals with continuous illumination (400 Lux) for 60 days. After this period all animals were anesthetized and blood was collected for the determination of serum estradiol (E2), progesterone (P4), and testosterone (T), followed by removal of the ovaries that were fixed in 10% formalin and processed for paraffin embedding. Five-µm histological sections were stained with hematoxylin and eosin and used for histomorphometric analysis. Morphological analyses, cyst count, determination of concentration and of the nuclear volume of interstitial cells were performed with the aid of a light microscope adapted to a high resolution camera (AxioCam), whose images were transmitted to and analyzed by the computer using AxioVision Rel 4.8 software (Carl Zeiss). Data were analyzed statistically by the Student\'s t-test (p<0.05).
RESULTS: Morphological analysis showed the presence of ovarian cysts in POG animals and corpora lutea in CtrlG animals, as well as evidence of the origin of interstitial cells from the internal theca of these cysts. POG animals presented increased serum estradiol levels (pg/mL) compared to CtrlG animals (POG=124.9 ± 4.2>CtrlG=73.2 ± 6.5, p<0.05), the same occurring with testosterone levels (pg/mL) (POG=116.9 ± 4.6>CtrlG=80.6 ± 3.9, p<0.05). However, progesterone levels (ng/mL) were higher in CtrlG than in POG animals (CtrlG=16.3 ± 2.0>POG=4.2 ± 1.5, p<0.05). Morphometry showed a significant increase in nuclear volume in POG animals (POG=102.1 ± 5.2>CtrlG=63.6 ± 16.5, p<0.05), as well as in the area occupied (%) by interstitial cells (POG=24.4 ± 6.9>CtrlG=6.9 ± 3.2, p<0.05) compared to CtrlG animals.
CONCLUSIONS: The interstitial cells of the rat polycystic ovary probably originate from ovarian cysts due to the degeneration of granulosa cells and differentiation of the internal theca cells. The elevations of serum testosterone and estradiol were probably due to the significant increase in cell activity and in the area occupied by interstitial cells.

The early post-hatch development of immunoreactivity to vimentin, desmin, smooth muscle actin (SMA) and laminin, in relation to follicle histogenesis, was described in this study. Ovigerous cords in day old quails contained pre-granulosa cells and oocytes. Pre-granulosa cells at the cortico-medullary junction were vimentin immunopositive. A laminin immunopositive basement membrane and desmin immunopositive mesenchymal cells lined the ovigerous cords. Ovigerous cords in 3-day-old quails contained developing primordial follicles, the vimentin immunopositive pre-granulosa cells of which were partially encircled by a basement membrane and desmin immunopositive mesenchymal cells. In 5- to 7-day-old quails, ovigerous cords formed an outer cortical region, while primordial follicles formed the inner cortical region. Early pre-vitellogenic follicles were present in 9- to 13-day-old quails. Underlying the granulosa cells of these follicles was a laminin immunopositive basement membrane and a layer of desmin immunopositive thecal cells. Early and late pre-vitellogenic follicles dominated the ovary in 15- to 17-day-old quails. The thecal layer in these follicles was desmin immunopositive, but SMA immunonegative. The results of the study have shown that the process of primordial follicle development in the Japanese quail is similar to that reported in mammals. The study suggests that in the quail pre-granulosa cells originate predominantly from the medulla. The study has shown that, in the Japanese quail, thecal cells are derived from desmin immunopositive mesenchymal cells lining the ovigerous cords.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by an adverse metabolic profile. Although dietary changes are advocated, optimal nutritional management remains uncertain. Polyunsaturated fatty acids (PUFAs), particularly long-chain (LC) n-3 (omega-3) PUFAs, improve metabolic health, but their therapeutic potential in PCOS is unknown.
OBJECTIVE: We aimed to determine the associations between plasma PUFAs and metabolic and hormonal aspects of PCOS to investigate the efficacy of LC n-3 PUFA supplementation and to support the findings with mechanistic cellular studies.
METHODS: We selected a cross-sectional PCOS cohort (n = 104) and conducted a principal component analysis on plasma fatty acid profiles. Effects of LC n-3 PUFA supplementation on fasting and postprandial metabolic and hormonal markers were determined in PCOS subjects (n = 22) by a randomized, crossover, placebo-controlled intervention. Direct effects of n-6 (omega-6) compared with n-3 PUFAs on steroidogenesis were investigated in primary bovine theca cells.
RESULTS: Cross-sectional data showed that a greater plasma n-6 PUFA concentration and n-6:n-3 PUFA ratio were associated with higher circulating androgens and that plasma LC n-3 PUFA status was associated with a less atherogenic lipid profile. LC n-3 PUFA supplementation reduced plasma bioavailable testosterone concentrations (P < 0.05), with the greatest reductions in subjects who exhibited greater reductions in plasma n-6:n-3 PUFA ratios. The treatment of bovine theca cells with n-6 rather than with n-3 PUFAs up-regulated androstenedione secretion (P < 0.05).
CONCLUSIONS: Cross-sectional data suggest that PUFAs modulated hormonal and lipid profiles and that supplementation with LC n-3 PUFAs improves androgenic profiles in PCOS. In bovine theca cells, arachidonic acid modulated androstenedione secretion, which suggests an indirect effect of n-3 PUFAs through the displacement of or increased competition with n-6 PUFAs. This trial was registered at clinicaltrials.gov as NCT01189669.

BACKGROUND: 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS) was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary.
METHODS: Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement.
RESULTS: In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC) in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly.
CONCLUSIONS: The present study proves for the first time that ovarian androgen biosynthesis can be inhibited by silencing CYP17 expression. It may provide a novel strategy for therapy of hyperandrogenism diseases, and also set an example for the use of RNAi technology in endocrine diseases.

OBJECTIVE: To explore an optimal laser dose of transvaginal ultrasound-guided ovarian interstitial laser coagulation in management of anovulation in patients with polycystic ovary syndrome (PCOS).
METHODS: Randomized, controlled trial.
METHODS: A reproductive medical center.
METHODS: Eighty women with PCOS and clomiphene citrate-resistant infertility underwent ultrasound-guided transvaginal ovarian interstitial yttrium aluminum garnet laser treatment. All subjects were divided randomly into four groups of A, B, C, and D.
METHODS: Group A, one coagulation point per ovary; group B, two points; group C, three points; group D, four to five points.
METHODS: Postoperative ovulation rate, pregnancy rate, and some biochemical parameters.
RESULTS: The rates of ovulation in groups C (75.00%, 95% confidence interval [CI]: 51%-91%) and D (80.00%, 95% CI: 56%-94%) within 6 postoperative months were significantly higher than in groups A (5.00%, 95% CI: 0%-25%) and B (15.00%, 95% CI: 3%-38%). The pregnancy rates in groups C (45.00%, 95% CI: 23%-69%) and D (40.00%, 95% CI: 19%-64%) also were significantly higher than in groups A (5.00%, 95% CI: 0-25%) and B (10.00%, 95% CI: 1%-32%). The mean serum T levels were significantly lower in groups C (2.08 +/- 0.62 nmol/L) and D (2.07 +/- 0.42 nmol/L) compared with groups A (3.10 +/- 0.63 nmol/L) and B (2.95 +/- 0.63 nmol/L).
CONCLUSIONS: One and two intraovarian laser coagulation points per ovary are associated with poor outcomes. Three points per ovary seem to represent the plateau of effective dose for the ovarian interstitial laser treatment in PCOS.

OBJECTIVE: To evaluate the effects of tubal ligation on ovarian and tubal tissues by means of immunohistochemical evaluation of two hypoxia related mediators: vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS).
METHODS: Fourteen Sprague-Dawley female rats were divided into two groups: a tubal ligation (Pomeroy technique) was carried out on rats in group 1 (n = 7) whereas those in group 2 served as controls (n = 7). Salpingo-oophorectomy was performed in group 1 during the second oestrous period following tubal ligation. Rats in group 2 were submitted to a salpingo-oophorectomy, as well. VEGF and iNOS immunoreactivities in ovarian and tubal tissues were evaluated by means of immunohistochemistry. Immunohistochemical scores and number of antral follicles were compared.
RESULTS: In the ovary, VEGF immunoreactivity was significantly more intense in the granulosa (p = 0.002) and the theca cells (p = 0.001) of rats in group 1 but, in ovarian medulla (p = 0.259) and germinal epithelium (p = 0.209), it was not significantly different from that of rats in group 2. The iNOS immunoreactivity in ovarian granulosa cells (p = 0.073) and germinal epithelial cells (p = 0.805) did not differ between the two groups. The cytoplasmic VEGF (p = 0.001) and iNOS (p = 0.017) immunoreactivities in the uterine tube, were significantly more intense in group 1. However, VEGF immunoreactivity in the lamina propria of the uterine tube (p = 0.209) was of similar intensity in both groups.
CONCLUSIONS: Tubal ligation may lead to supraphysiological hypoxia as evidenced by increased VEGF and iNOS immunoreactivities in ovarian and tubal tissues.

The morphological and endocrine aspects of the ovarian interstitial tissue of adult female viscachas were investigated to establish the probable function and the biological significance of this compartment in this rodent. Pregnant and nonpregnant adult female viscachas were used. The histological characteristics, histochemical properties, and ultrastructural features of the interstitial tissue were studied. A morphometric study was carried out to measure the relative area of lipid droplets. The progesterone and androstenedione levels in ovarian tissue as well as in serum were determined by radioimmunoassay. In this species, the histological observations showed an abundant interstitial tissue that contained a large amount of lipids. The cholesterol and its esters were present in nonpregnant females and were scarce in pregnant animals. The most ultrastructural differences were observed at mid-pregnancy. At this stage, the interstitial cells showed features that suggested higher steroidogenic activity. Furthermore, during mid-pregnancy, the relative area of lipid droplets was smaller. Both progesterone and androstenedione levels in ovarian tissue and serum were higher during pregnancy. Our results suggest that the interstitial tissue may be storage of precursor substances for the steroidogenesis via. These precursors are probably used when the endocrine requirements are high, that is, during the pregnancy. Thus, this compartment may contribute to the normal gestation of Lagostomus. However, the relation between the interstitial tissue and the pregnancy is complex, and further studies are needed to clearly establish it.