Effect of thyroid dysfunction on NOS expression in the female rat.

Thyroid hormones (THs) are vital for normal reproductive function and dysregulation of TH impairs follicular development. Although the functions of THs on female reproduction are of great interest, the mechanisms still remain unclear. Many studies have shown that NO plays important roles in female reproduction. In the present study, we investigate the effects of TH dysregulation on nitric oxide synthase types (NOS) expression in rats. Propylthiouracil (PTU) and L-thyroxine were administered to rats to induce hypo- and hyperthyroidism, respectively. Ovarian histology was detected by immunohistochemistry (IHC) and protein or mRNA content was analyzed by Western blotting or RT-PCR, respectively. The results showed that NOS1, NOS2 and NOS3 expressions were detected in the oocyte, granulosa cell and theca cell in all follicular stages, which were up-regulated by eCG treatment. NOS1 protein content was increased in both PTU and L-thyroxine treatments. There were no significant differences in NOS2 levels between the treatment and the control group. However, NOS3 was only increased in the hyperthyroid group. These results were consistent with the IHC staining. The present study provides evidence that TH dysregulation alters NOSs profiles, which suggests that NOSs/nitric oxide (NO) is possibly involved in the regulation of female reproduction.

Roles of different n-3/n-6 PUFA ratios in ovarian cell development and steroidogenesis in PCOS rats.

Polycystic ovary syndrome (PCOS) is a complex and common endocrine disorder characterized by hyperandrogenism, which is accompanied by follicle growth arrest at the small antral stage, minimal granulosa cell proliferation, and chronic anovulation. Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Although PUFAs play an important role in the regulation of female reproduction, their role in ovarian development in PCOS is still unclear. The present study was conducted to investigate the effects of different ratios of n-3/n-6 PUFAs (omega-3/omega-6) on ovary development in PCOS rats. Serum levels of reproductive hormones and enzymes related to steroidogenesis were assessed. The results indicated that PUFAs (n-3/n-6: 1/15) significantly increased ovarian weight and improved the ovarian structure although they had no significant effect on body weight in PCOS rats. Meanwhile, apoptosis was attenuated accompanied by increased cell proliferation by PUFAs (n-3/n-6: 1/15). Moreover, serum levels of hormones (FSH and E2) were also significantly increased by PUFAs (n-3/n-6: 1/15) accompanied by decreased T levels. To investigate whether PUFAs regulate the expression of enzymes related to hormone synthesis, western blotting was used to determine the protein levels of CYP51, CYP19, StAR and 3ß-HSD. The results showed that PUFAs significantly increased the protein levels of all of these enzymes. These results indicate that PUFAs enhance the reproductive performance of PCOS by increasing the expression of steroidogenesis enzymes, which are related to hormone secretion and ovarian functions. These findings provide evidence that a balanced n-3/n-6 PUFA ratio is beneficial for PCOS reproduction.

Effect of hypothyroidism on CYP51 and FSHR expression in rat ovary.

Although thyroid hormone (TH) plays important roles in regulating ovarian development, the mechanism are still unclear. Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that involved in folliculogenesis and oocyte maturation, which is regulated by follicle stimulating hormone (FSH). However, the effect of TH on CYP51 expression in ovarian cells is unclear. The objective of this study was to determine the effects of TH on CYP51 in rat ovary. Hypothyroidism rats were induced by 6-propyl-2-thiouracil (PTU), genes expressions in ovary were analyzed by Western blot or qRT-PCR. The data showed that CYP51 was significantly decreased in hypothyroidism, which was accompanied by the down-regulation of mRNA level. Meanwhile, similar tendency was also showed in FSHR expression in hypothyroidism. To evaluate the effect of the gonadotropin on CYP51 and FSHR expression in ovarian cells in vivo, hypo rats were injected subcutaneously with equine chorionic gonadotropin (eCG) respectively. The results showed that eCG reversed CYP51 and FSHR expression in hypo group. Moreover, FSH-induced CYP51 expression was meditated by FSHR. In addition, serum concentration of FSH and E2 were also decreased in hypothyroidism, and E2 was up-regulated by eCG treatment. These results indicate that hypothyroidism changes CYP51 and FSHR expression in ovary, which are regulated by gonadotropin. Moreover, genes changes in ovary are at least partially attributed to steroids biosynthesis.

Effects of omega-3 polyunsaturated fatty acids on steroidogenesis and cellular development in PCOS rats.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, which is characterized by hyperandrogenism. Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Despite the well-known benefits of omega-3 polyunsaturated fatty acid supplementation on the modulation of PCOS ovarian function, relatively little is known about the precise regulation mechanism. The objective of this study was to determine the cellular and molecular mechanisms by which omega-3 regulates CYP51 expression and steroid biosynthesis during follicle growth in PCOS. The results indicated that the CYP51 expression was up-regulated in granulosa cells by omega-3. Moreover, the knockdown of CYP51 blocked omega-3 induced estradiol (E2) and progesterone (P4) synthesis as well as cellular viability and proliferation. These changes were accompanied by the up-regulation of the p-Akt level. Furthermore, the PI3K/Akt pathway was required for the regulation of CYP51 expression, steroidogenesis and cell development by omega-3 in PCOS granulosa cells. Our data demonstrate that omega-3 potentiates the cellular development and steroid biosynthesis via CYP51 up-regulation in PCOS, which are mediated through the activation of the PI3K/Akt pathway.

Role of OCT4 in the Regulation of FSH-Induced Granulosa Cells Growth in Female Mice.

As a member of the POU (Pit-Oct-Unc) transcription factor family, OCT4 (Octamer-binding transcription factor 4) is associated with the cellular proliferative. However, the roles of OCT4 in regulating the transition from preantral follicle to early antral follicle are still remains unclear. To evaluate the effect of OCT4 on cellular development in ovary, mice were injected with eCG in vivo or granulosa cells were co-cultured with FSH in vitro. The results showed that eCG up-regulated ovarian OCT4 expression. Meanwhile, OCT4 expression in granulosa cells was also up-regulated by FSH, and knockdown of OCT4 by siRNA significantly decreased FSH-induced cellular viability. Moreover, gonadotropin increased p-GSK3ß (Glycogen synthase kinase 3-beta) level, ß-catenin expression and its translocation to nuclear in ovarian cells. In addition, the inhibition of GSK3ß activity by CT99021 significantly increased the expression of ß-catenin and OCT4 in granulosa cells. And knockdown ß-catenin by siRNA dramatically abolished FSH-induced OCT4 expression and cellular development. Furthermore, FSH-induced the phosphorylation of GSK3ß, expression of ß-catenin and OCT4, and translocation of ß-catenin were mediated by the PI3K/Akt pathway. Taken together, the present study demonstrates that FSH regulated OCT4 expression via GSK3ß/ß-catenin pathway, which was mediated by the PI3K/Akt pathway. And these regulations are involved in ovarian cell development.

Effects of Thyroid Dysfunction on Reproductive Hormones in Female Rats.

Thyroid hormones (THs) play a critical role in the development of ovarian cells. Although the effects of THs on female reproduction are of great interest, the mechanism remains unclear. We investigated the effects of TH dysregulation on reproductive hormones in rats. Propylthiouracil (PTU) and L-thyroxine were administered to rats to induce hypo- and hyperthyroidism, respectively, and the reproductive hormone profiles were analyzed by radioimmunoassay (RIA). Ovarian histology was evaluated with hematoxylin and eosin (H&E) staining, and gene protein level or mRNA content was analyzed by western blotting or reverse transcription polymerase chain reaction (RT-PCR). The serum levels of gonadotropin releasing hormone (GnRH) and follicle stimulating hormone (FSH) in both rat models were significantly decreased on day 21, although there were no significant changes at earlier time points. There were no significant differences in luteinizing hormone (LH) or progesterone (P4) levels between the treatment and the control groups. Both PTU and L-thyroxine treatments downregulated estradiol (E2) concentrations; however, the serum testosterone (T) level was increased only in hypothyroid rats at day 21. In addition, the expression levels of FSH receptor, cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were decreased in both rat models. Moreover, the onset of puberty was significantly delayed in the hypothyroid group. These results provide evidence that TH dysregulation alters reproductive hormone profiles, and that the initiation of the estrous cycle is postponed in hypothyroidism.

Regulation by 3,5,3'-tri-iodothyronine and FSH of cytochrome P450 family 19 (CYP19) expression in mouse granulosa cells.

Cytochrome P450 family 19 (CYP19) plays an important role in follicular development, which is regulated by FSH. Although 3,5,3'-tri-iodothyronine (T3) combines with FSH to induce preantral follicle growth and granulosa cell development, the mechanism involved remains unclear. The aim of the present study was to determine the cellular and molecular mechanisms by which thyroid hormone (TH) and FSH regulate CYP19 expression and sterol biosynthesis during preantral follicle growth. Mice were injected subcutaneously (s.c.) with eCG (Equine chorionic gonadotropin). The results showed that eCG increased CYP19 expression in ovarian cells. CYP19 expression in granulosa cells was increased after FSH treatment, and this response was enhanced by T3. Knockdown of CYP19 significantly decreased granulosa cell viability and hormone-stimulated proliferation. In addition, CYP19 knockdown also blocked T3- and FSH-induced oestradiol (E2) synthesis in granulosa cells. Furthermore, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was required for T3 and FSH regulation of CYP19 expression. In conclusion, the results of the present study indicate that CYP19 is important for T3- and FSH-induced granulosa cell development in the early stages. CYP19 could be a downstream effector of the PI3K/Akt pathway in regulating TH and FSH during follicular development and sterol biosynthesis. The findings suggest that CYP19 is a novel mediator of T3- and FSH-induced follicular development.

cGMP/PKG-I Pathway-Mediated GLUT1/4 Regulation by NO in Female Rat Granulosa Cells.

Nitric oxide (NO) is a multifunctional gaseous molecule that plays important roles in mammalian reproductive functions, including follicular growth and development. Although our previous study showed that NO mediated 3,5,3'-triiodothyronine and follicle-stimulating hormone-induced granulosa cell development via upregulation of glucose transporter protein (GLUT)1 and GLUT4 in granulosa cells, little is known about the precise mechanisms regulating ovarian development via glucose. The objective of the present study was to determine the cellular and molecular mechanism by which NO regulates GLUT expression and glucose uptake in granulosa cells. Our results indicated that NO increased GLUT1/GLUT4 expression and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of cyclic guanosine monophosphate (cGMP) level and cGMP-dependent protein kinase (PKG)-I protein content. The results of small interfering RNA (siRNA) analysis showed that knockdown of PKG-I significantly attenuated gene expression, translocation, and glucose uptake. Moreover, the PKG-I inhibitor also blocked the above processes. Furthermore, NO induced cyclic adenosine monophosphate response element binding factor (CREB) phosphorylation, and CREB siRNA attenuated NO-induced GLUT expression, translocation, and glucose uptake in granulosa cells. These findings suggest that NO increases cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the cGMP/PKG pathway. Meanwhile, the activated CREB is also involved in the regulation. These findings indicate that NO has an important influence on the glucose uptake of granulosa cells.