Remarkable features of ovarian morphology and reproductive hormones in insulin-resistant Zucker fatty (fa/fa) rats.

BACKGROUND: Zucker fatty (fa/fa) rats are a well-understood model of obesity and hyperinsulinemia. It is now thought that obesity/hyperinsulinemia is an important cause of endocrinological abnormality, but to date there have been no reports on the changes in ovarian morphology or the ovarian androgen profile in rat models of obesity and insulin resistance. METHODS: In this study we investigated the effects of obesity and hyperinsulinemia on ovarian morphology and the hormone profile in insulin-resistant Zucker fatty rats (5, 8, 12 and 16 weeks of age, n = 6-7). RESULTS: Ovaries from 5-week-old fatty rats had significantly greater total and atretic follicle numbers, and higher atretic-to-total follicle ratios than those from lean rats. Ovaries from 12- and 16-week-old fatty rats showed interstitial cell hyperplasia and numerous cysts with features of advanced follicular atresia. In addition, serum testosterone and androstenedione levels significantly declined in fatty rats from age 8 to 16 weeks, so that fatty rats showed significantly lower levels of serum testosterone (12 and 16 weeks) and androstenedione (all weeks) than lean rats. This may reflect a reduction of androgen synthesis during follicular atresia. Serum adiponectin levels were high in immature fatty rats, and although the levels declined significantly as they matured, it remained significantly higher in fatty rats than in lean rats. On the other hand, levels of ovarian adiponectin and its receptors were significantly lower in mature fatty rats than in lean mature rats or immature fatty rats. CONCLUSIONS: Our findings indicate that ovarian morphology and hormone profiles are significantly altered by the continuous insulin resistance in Zucker fatty rats. Simultaneously, abrupt reductions in serum and ovarian adiponectin also likely contribute to the infertility seen in fatty rats.

The contributions of resistin and adiponectin gene single nucleotide polymorphisms to the genetic risk for polycystic ovary syndrome in a Japanese population.

Polycystic ovary syndrome (PCOS) is a heterogeneous group of disorders that occur fairly commonly in women of reproductive age and are characterized by a variety of clinical manifestations, including insulin resistance that is independent of obesity. Recent studies suggest that altered adipocytokine gene expression is closely associated with insulin resistance and that single nucleotide polymorphisms (SNPs) modulate the expression and/or function of these genes, thereby affecting insulin sensitivity. With that in mind, we investigated whether SNPs at position -420 of the resistin gene (RETN) and/or -11377 of the adiponectin gene (ADIPOQ) modulate the susceptibility to PCOS. We evaluated the genotypes of 117 women with PCOS and 380 healthy fertile controls and measured the index of insulin resistance and hormonal profiles in the PCOS women. The RETN-420G/G homozygous variant genotype occurred significantly more frequently among the PCOS group than among the control group (15.4% vs. 8.4%, p = 0.035). PCOS women with the RETN-420G/G genotype also showed significantly higher BMIs and greater insulin resistance than those with RETN-420 C/C or C/G genotypes. The ADIPOQ SNP at -11377 showed no association with PCOS. We conclude that the RETN G/G at -420 genotype is associated with PCOS in Japanese women.

Polycystic ovary syndrome is associated with genetic polymorphism in the insulin signaling gene IRS-1 but not ENPP1 in a Japanese population.

Recent studies indicate that insulin resistance resulting from altered post-receptor signaling is associated with polycystic ovary syndrome (PCOS). We hypothesized that insulin receptor substrate-1 (IRS-1) Gly972Arg polymorphism and/or ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) Lys121Gln polymorphism predisposes women to PCOS and that these polymorphisms also affect anthropometric variables, glucose metabolism and androgen synthesis. To test those ideas, we studied the genotypes, indexes of insulin resistance, and hormone profiles in 123 Japanese women with PCOS and 380 healthy Japanese controls. We found that there were significantly more IRS-1 972Arg carriers among the PCOS patients than among the healthy controls (10.6% vs. 4.8%, p=0.029), which is consistent with our finding that women carrying the IRS-1 972Arg allele had a significantly increased risk of developing PCOS (odds ratio: 3.31, 95% confidence interval: 1.49-7.35). By contrast, the ENPP1 Lys121Arg polymorphism was distributed equally among PCOS patients and controls. In addition, neither of these polymorphisms studied affected the anthropometric variables, metabolic parameters or androgen levels of women with PCOS. We conclude that the IRS-1 Gly972Arg polymorphism is associated with PCOS in the Japanese population.

Altered expression of Fas/Fas ligand/caspase 8 and membrane type 1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats.

One of the characteristics of polycystic ovary syndrome (PCOS) is the presence of cystic follicles in various stages of growth and atresia, the latter of which is known to be the result of apoptosis and tissue remodeling. To further investigate the process of follicular atresia, we compared ovarian expression and localization of Fas, Fas ligand (FasL), casapse-8 and membrane-type1 matrix metalloproteinase (MT1-MMP) in rats treated with dehydroepiandrosterone (DHEA) as a model of PCOS, and in control rats. We found that the numbers of TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive follicles were significantly higher in ovaries from PCOS rats than in those from control rats (P < 0.05), as were ovarian levels of FasL mRNA and protein, processed caspase-8 protein and MT1-MMP mRNA. Correspondingly, we also observed an increase in the level of MTI-MMP catalytic activity and a decrease in the level of pro-caspase-8 protein. In addition, immunohistochemical analyses showed that MT1-MMP and FasL co-localize with TUNEL-positive apoptotic granulosa cells within atretic follicles of PCOS ovaries. Our results suggest that under the PCOS-like conditions induced by DHEA, the Fas/FasL/Caspase-8 (death receptor dependent) pathway is pivotal for follicular atresia, and that increased levels of MT1-MMP likely play an important role in tissue remodeling during structural luteolysis.

Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution.

BACKGROUND: The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution. METHODS: We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20). RESULTS: We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2. CONCLUSION: These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.

Hyperstimulation and a gonadotropin-releasing hormone agonist modulate ovarian vascular permeability by altering expression of the tight junction protein claudin-5.

We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 microg/kg.d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm(2)) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.

Significance of matrix metalloproteinases in the pathophysiology of the ovary and uterus.

Matrix metalloproteinases (MMP) are capable of degrading a variety of extracellular matrix (ECM) proteins and are also involved in the processing of a number of bioactive molecules. Our findings indicate that the functions of MMP in the ovary and uterus are organ-specific and time-dependently vary during the reproductive cycle. Prolactin induces structural luteolysis indicated by loss of luteal weight, protein and DNA within 36 h after pretreatment with ergot alkaloid. MMP activation appears crucial for the selective depletion of protein during luteal involution, which entails loss of ECM accompanied by apoptosis. During GnRHagonist-induced luteolysis, this response was also associated with marked increases in MMP-2, which degraded collagen type IV, and MT1-MMP, which in addition to activating MMP-2 also degrades collagen type I, III and V. We also found that the level of MT1-MMP and MMP-2 expression in the human CL is greater during the late luteal phase than during either the early mid luteal phases or during gestation, respectively. That dehydroepiandrosterone (DHEA) treatment caused the formation of cysts from antral follicles in the ovaries of immature rats while depressing MMP-2 collagenolytic activity and enhancing lysyl oxidase expression highlights the importance of collagen degradation in the process of ovulation and suggests that changes in the activities of these enzymes play a key role in ovarian cystogenesis in polycystic ovary syndrome patients. Furthermore, immunohistochemical analyses showed that MT1-MMP and FasL co-localize with TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive apoptotic granulosa cells in rats treated with DHEA, that the Fas/FasL/Caspase-8 (death receptor-dependent) pathway is pivotal for follicular atresia and that increased levels of MT1-MMP likely play an important role in tissue remodeling during follicular atresia. After parturition, the uterus undergoes involution, a conspicuous feature characterized by a rapid reduction in the collagen content mediated by degradation of extracellular collagen bundles. Our findings strongly suggest that MT1-MMP, MMP-2 and MMP-9 are each time-dependently regulated and play important roles in tissue remodeling during postpartum uterine involution. (Reprod Med Biol 2006; 5: 235-243).

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats.

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis-related molecules (Fas, Fas ligand (FasL), Bcl-2, Bax). We gave 4-day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG-hCG-treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom+PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom+PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom+PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.

Gonadotropin-releasing hormone agonist administration reduced vascular endothelial growth factor (VEGF), VEGF receptors, and vascular permeability of the ovaries of hyperstimulated rats.

OBJECTIVE: Using hyperstimulated rats, to elucidate the mechanisms of gonadotropin-releasing hormone agonist (GnRH-a) treatment to prevent early ovarian hyperstimulation syndrome (OHSS). DESIGN: Descriptive study of hyperstimulated rats as an early OHSS model with Western blot analysis, Northern blot hybridization, and vascular permeability assay. SETTING: Experimental laboratory research. ANIMAL(S): Sprague-Dawley female rats were used for collecting ovarian samples. INTERVENTION(S): Hyperstimulated rats received consecutive GnRH-a treatment from the start of pregnant mare serum gonadotropin (PMSG) treatment through 2 days after hCG administration. MAIN OUTCOME MEASURE(S): Expressions of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1: Flt-1), VEGF receptor-2 (VEGFR-2: KDR/Flk-1), and vascular permeability by Evans blue leakage. RESULT(S): GnRH-a treatment significantly reduced expressions of VEGF, VEGFR-1, and VEGFR-2 both in mRNA and protein levels in the ovaries of hyperstimulated rats. GnRH-a treatment also reduced vascular permeability in the ovaries of hyperstimulated rats. CONCLUSION(S): It is speculated that GnRH-a treatment may prevent early OHSS by reducing vascular permeability through the decrease in VEGF and its receptors.

The significance of membrane type 1 metalloproteinase in structural involution of human corpora lutea.

The expression of membrane type 1 (MT1) matrix metalloproteinase (MMP), MMP-2, and tissue inhibitors of MMP-1 and -2 during structural involution of the human corpus luteum was examined using immunohistochemistry, Northern blotting, Western blotting, gelatin zymography and in-situ hybridization techniques. The corpora lutea of 20 patients were investigated at the time of total hysterectomy and were obtained from five patients each in the early, mid- and late luteal phases and during gestation. Immunohistochemistry for MT1-MMP in corpus luteum showed that the protein appeared in granulosa lutein cells in the late luteal phase. Both the expression of MT1-MMP mRNA and the amount of the protein increased in the late luteal phase. In-situ hybridization showed that MT1-MMP mRNA was localized mainly in the cytoplasm of granulosa lutein cells. These results suggest that increased expression of MT1-MMP may be a major factor in remodelling of the human corpus luteum during structural luteolysis.

Anti-invasive effect of MMI-166, a new selective matrix metalloproteinase inhibitor, in cervical carcinoma cell lines.

OBJECTIVES: The aim of our study was to evaluate the anti-invasive effect of MMI-166, a new matrix metalloproteinase (MMP) inhibitor in cervical carcinoma cell lines. METHODS: We analyzed the invasive activities of cervical carcinoma cell lines (CAC-1, CaSki, and SiHa) and the gene expression of various matrix proteinases (matrix metalloproteinase-1 [MMP-1], MMP-2, MMP-9, membrane-type MMP type 1 [MT1-MMP], MT2-MMP, and MT3-MMP) and their inhibitors (tissue inhibitor of metalloproteinase type 1 [TIMP-1] and TIMP-2). The effect of MMI-166 was analyzed by in vitro invasion assay. The cytotoxicity of MMI-166 was determined by MTT assay. The gelatinase activity was analyzed by gelatin zymography. RESULTS: Cervical carcinoma cell lines, which produced both MMP-2 and MT1-MMP, showed invasive capacity in the in vitro invasion assay. The invasion of cervical carcinoma cells was suppressed by MMI-166. No remarkable suppression of the proliferation by MMI-166 was observed in the MTT assay. Gelatin zymography revealed complete suppression of MMP-2 activity by MMI-166. CONCLUSIONS: MMI-166 inhibited the MMP-2 activity in cervical carcinoma cells and it is useful for the regulation of cervical carcinoma cell invasion.