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The mechanisms that link hyperandrogenism and insulin (INS) resistance (HAIR) to the increased miscarriage rate in women with polycystic ovary syndrome (PCOS) remain elusive. Previous studies demonstrate that increased uterine and placental ferroptosis is associated with oxidative stress-induced fetal loss in a pre-clinical PCOS-like rat model. Here, we investigated the efficacy and molecular mechanism of action of the antioxidant N-acetylcysteine (NAC) in reversing gravid uterine and placental ferroptosis in pregnant rats exposed to 5α-dihydrotestosterone (DHT) and INS. Molecular and histological analyses showed that NAC attenuated DHT and INS-induced uterine ferroptosis, including dose-dependent increases in anti-ferroptosis gene content. Changes in other molecular factors after NAC treatment were also observed in the placenta exposed to DHT and INS, such as increased glutathione peroxidase 4 protein level. Furthermore, increased apoptosis-inducing factor mitochondria-associated 2 mRNA expression was seen in the placenta but not in the uterus. Additionally, NAC was not sufficient to rescue DHT + INS-induced mitochondria-morphological abnormalities in the uterus, whereas the same treatment partially reversed such abnormalities in the placenta. Finally, we demonstrated that NAC selectively normalized uterine leukemia inhibitory factor, osteopontin/secreted phosphoprotein 1, progesterone receptor, homeobox A11 mRNA expression and placental estrogen-related receptor beta and trophoblast-specific protein alpha mRNA expression. Collectively, our data provide insight into how NAC exerts beneficial effects on differentially attenuating gravid uterine and placental ferroptosis in a PCOS-like rat model with fetal loss. These results indicate that exogenous administration of NAC represents a potential therapeutic strategy in the treatment of HAIR-induced uterine and placental dysfunction.

Human ectopic pregnancy (EP) remains a common cause of pregnancy-related first trimester death. Nitric oxide (NO) is synthesized from L-arginine by three NO synthases (NOS) in different tissues, including the Fallopian tube. Studies of knockout mouse models have improved our understanding of the function of NOS isoforms in reproduction, but their roles and specific mechanisms in infection-induced tubal dysfunction have not been fully elucidated. Here, we provide an overview of the expression, regulation and possible function of NOS isoforms in the Fallopian tube, highlighting the effects of infection-induced changes in the tubal cellular microenvironment (imbalance of NO production) on tubal dysfunction and the potential involvement of NOS isoforms in tubal EP after Chlamydia trachomatis genital infection. The non-equivalent regulation of tubal NOS isoforms during the menstrual cycle suggests that endogenous ovarian steroid hormones regulate NOS in an isoform-specific manner. The current literature suggests that infection with C. trachomatis induces an inflammatory response that eventually leads to tubal epithelial destruction and functional impairment, caused by a high NO output mediated by inducible NOS (iNOS). Therefore, tissue-specific therapeutic approaches to suppress iNOS expression may help to prevent ectopic implantation in patients with prior C. trachomatis infection of the Fallopian tube.

Clomiphene citrate (CC) therapy for disorders of anovulatory infertility has been linked to an increased frequency of tubal ectopic pregnancy. Although CC enhances apoptotic processes in the ovaries, villi, and decidual tissues, its effect on apoptosis in the fallopian tube is unknown. Here, we show that chronic treatment with CC induces tubal apoptosis, but not necrosis, through an intrinsic mitochondria-dependent signaling pathway in vivo. The apoptosis was specific to epithelial cells in the isthmus, and the damage was reversed with 17beta-estradiol (E2); however, pretreatment or concomitant treatment with E2 did not protect against tubal apoptosis induced by chronic treatment with CC. Chronic treatment activated estrogen receptors (ESRs), particularly cilia-localized ESR2A (formerly ERbeta2). In contrast to E2, acute treatment of superovulating rats with a high dose of CC or the ESR2-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) significantly delayed the transport of oocyte-cumulus complexes through the fallopian tube. Our findings suggest that in response to chronic CC therapy, isthmus-specific apoptosis of epithelial cells and activation of cilia-ESR2A act in parallel to block gamete and embryo passage through the fallopian tube, eventually resulting in tubal ectopic pregnancy.

In this study, it was hypothesized that progesterone (P4) acts as a survival factor primarily by actions of the classic nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with equine chorionic gonadotropin/human chorionic gonadotropin and treated in vitro with PGR antagonists. As little as 10 nM of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by cotreatment with the progestin R5020. Concurrently, P4 synthesis was decreased. Inhibition of P4 synthesis by cyanoketone similarly induced apoptosis but required greater inhibition of P4 synthesis than that seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased P4 synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of P4 synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, cotreatment with PGR antagonists protected cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for PGR in P4-mediated regulation of apoptosis. Progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor P4 or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that P4 regulates apoptosis in periovulatory granulosa cells by acting via the classic nuclear receptor.

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.

Progesterone-receptor (PR) stimulation promotes survival in rat and human periovulatory granulosa cells. To investigate the mechanisms involved, periovulatory rat granulosa cells were incubated in vitro with or without the PR-antagonist Org 31710. Org 31710 caused the expected increase in apoptosis, and expression profiling using cDNA microarray analysis revealed regulation of several groups of genes with functional and/or metabolic connections. This regulation included decreased expression of genes involved in follicular rupture, increased stress responses, decreased angiogenesis, and decreased cholesterol synthesis. A decreased cholesterol synthesis was verified in experiments with both rat and human periovulatory granulosa cells treated with the PR-antagonists Org 31710 or RU 486 by measuring incorporation of [14C]acetate into cholesterol, cholesterol ester, and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory rat granulosa cells using 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (lovastatin, mevastatin, or simvastatin) increased apoptosis, measured as DNA fragmentation and caspase-3/7 activity. The increase in apoptosis caused by simvastatin was reversed by addition of the cholesterol synthesis-intermediary mevalonic acid. These results show that PR antagonists reduce cholesterol synthesis in periovulatory granulosa cells and that cholesterol synthesis is important for granulosa cell survival.

The intracellular progesterone receptor (PR) in the mammalian ovary is a part of the physiological pathway that facilitates ovulation. Two PR isoforms (A and B) exist, with different molecular and biological functions. Previous studies have revealed that the cellular ratio of the PR isoforms is important for progesterone-responsive tissues and is under developmental control in different species. However, the relative expression of PR isoforms in the ovary is unknown. In this study we have demonstrated first that the expression of both PR isoforms in mouse granulosa cells was rapidly upregulated by hCG treatment and dramatically down-regulated when the granulosa cells were undergoing luteinization. The relative level of protein expression of the A and B forms was 2:1 and the highest total PR protein expression was found after hCG stimulation. Second, we demonstrated that the expression of PR protein was specific to granulosa cells of periovulatory follicles and was absent in undifferentiated granulosa cells of growing follicles. It was not detected in other cell types (i.e., corpora lutea or any stage of follicles with features of apoptosis). Third, we demonstrated that treatment with the PR antagonist RU 486 in vivo resulted in down-regulation of both isoforms in parallel with increased activation of caspase-3, a decreased level of proliferating cell nuclear antigen, and a reduced rate of ovulation. Fourth, we demonstrated, in vitro, that the PR antagonists RU 486 and Org 31710 increased internucleosomal DNA fragmentation parallel with a decrease in DNA synthesis in granulosa cells, which express PR. These results indicate that PR and its isoforms participate in regulation of ovulation, along with suppression of granulosa cell apoptosis and promotion of cell survival in the mouse ovary. apoptosis, granulosa cells, progesterone receptor

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.

It has become evident that apoptosis, an active form of cell 'suicide', plays an important role in the normal function of all tissues. A balance of cell proliferation and apoptosis is maintained in a healthy individual and any imbalance of the two processes could lead to pathological changes. In both sexes, massive apoptosis accounts for the demise of a majority of gonadal cells (ovarian granulosa cells and male germ cells) during reproductive life. Recent studies have indicated the important role of gonadotrophins as survival factors in both the ovary and the testis. Furthermore, intra-gonadal survival. factors in the ovary (oestrogens, insulin-like growth factor I, epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta, nitric oxide, etc.) and testis (androgens) have been shown to act in concert with the gonadotrophins. In contrast, several apoptotic factors (androgens, gonadotrophin-releasing hormone-like peptide and interleukin-6) may be important in inducing the demise of ovarian follicles. Understanding of the hormonal and cellular mechanisms responsible for gonadal cell apoptosis will provide new approaches for the treatment of gonadal degenerative conditions such as premature ovarian failure and cryptorchidism, as well as for the design of new contraceptive approaches.

A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF-I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

Ectopic pregnancy (EP) is an enigmatic reproductive disorder. Although tubal EP is difficult to predict, several hypotheses about its etiology have been proposed. In retrospective case-control studies, smoking is associated with an increased rate of EPs in the fallopian tube. Studies of experimental animals in vivo and human fallopian tubal tissues in vitro have suggested mechanisms of fallopian tubal damage and dysfunction induced by nicotine and other smoking-related chemicals that may explain this association. However, the pathogenesis of smoking-induced modulation of implantation leading to tubal EP is largely unknown. Because cigarette/tobacco smoke adversely affects the success of intrauterine implantation, there is a great need to determine how embryo implantation occurs in the fallopian tube in female smokers of reproductive age.

The distribution of insulin-like growth factor I (JGF-J; somatomedin C) was mapped in testes of different aged rats by using isninunohistochemical techniques. The antiserum used, K 624, has been demonstrated to be specific for human IGF-I, as defined by several criteria. Antibodies to the MI subunit of ribonucleotide reductase, a key enzyme in DNA synthesis, were used to visualize rneiotic and mitotic cells. Cytoplasmic IGF-I-like immunoreactivity as demonstrable during thefirst two postnatal weeks in spermatogenic cells, in Sertoli cells, and in Leydig cells. The IGF-I-like immunoreactivity decreased in the Sertoli and Leydig cells during the third and fourth postnatal weeks, and in adult rats, only sperinatogenic cells showed IGF-I-like immunoreactivity. In mature rat testes, the spermatocyteswere strongly immunoreactive. During puberty and adulthood, the spermatogonia expressedsubunit Ml ribonucleotide reducta.se immunoreactivily, whereas no IGF-I-like i,nmunoreactivity could be detected. No e.xu’acel!ular immunoreactivity was observed. We propose that IGF-1 and/or IGF-1-like substances, possiblyformed by primary spermatocytes,are likely to be involved in differentiation processes, but not in the initiation of cell proliferation in adult testes. The autocrine and/or paracrine action of IGF-I and/or IGF-1-like substances may thus have different action in developing testes than in adult testes. Our results do, however, not allow firm statementsabout whether IGF-I and related substances exert their actions on Sertoli cells or spermatogenic cells.

The influence of adenosine analogs on adenylate cyclase activity was investigated in membrane preparations of luteinizedovariesand in cellhomogenates of isolatedlutealcells.The adenosine receptor agonist 5’-(Nethyl)-carboxamido adenosine (NECA) dose-dependently stimulated adenylate cyclase activityin membrane preparations of 5-day-old luteinizedovarieswith an apparent EC50 of 0.58 pM. The other adenosine analogs testedwere lesspotent in stimulating the adenylate cyclase activity with the following rank order of potency: NECA N6 -(R -phenyl-isopropyl)-adenosine< N6 -(S-phenyl-isopropyl)-adenosine. In homogenates of isolated cells from 5-day-old corpora lutea, NECA stimulated adenylate cyclase with the same EC50 as in the membranes from luteinized ovaries. The effect of NECA was antagonized by the adenosine receptor antagonist 8-phenyltheophylline. In incubated luteal cells of both 2- and 5- to 6-day-old luteinized ovaries, NECA stimulated cyclic adenosine 3’, 5’-monophosphate (cAMP) accumulation and markedly potentiated luteinizing hormone-stimulated cAMP accumulation. Progesterone synthesis was also stimulated by NECA in incubated cells. The study demonstrates effects of adenosine analogs on adenylate cyclase and cAMP accumulation that fulfill the criteria for adenosine A2 receptor-mediated effects in luteal cells and membranes. These data suggest that adenosine may have a local regulatory action in luteal tissue through adenosine receptor activation.

In the cumulus-oocyte complex (COC), as well as in the whole ovarian follicle, gonadotropins stimulate glycolysis, measured as lactate accumulation. In contrast to this, COC oxygen consumption is decreased by gonadotropins. One possible explanation for this inhibition is a competition for a limited supply of cofactors, common for both the glycolytic pathway and the respiratory chain. In the present study, addition of adenosine to COCs cultured with gonadotropins restored the oxygen consumption to basal levels. This effect was specific for metabolizable adenosine. Other nucleosides or non-metabolizable 2-chloro-adenosine had no effect. Adenosine also increased ATP and decreased lactate accumulation by the COC. The nucleoside transport inhibitor dipyridamol abolished the effects of adenosine on oxygen consumption and ATP accumulation. Taken together, these results support the hypothesis that a relative lack of adenosine-derived cofactors is probably involved in the decrease of oxygen consumption in vitro by gonadotropin-stimulated cumulus cells.

Oocyte-cumulus complexes and mural granulosa cells, respectively, isolated from pre-ovulatory rat follicles and cultured in lactate-free medium showed a continuous accumulation of lactate during a 1 to 7 h incubation. Lactate production was stimulated by gonadotrophins, both when administered in vivo and in vitro, with the exception that LH given in vivo did not affect granulosa cell lactate production. Since the oocyte is known to have specific demands on energy substrate, it is suggested that the lactate produced is important for the growing and maturing oocyte.