人参皂甙Rb1对环磷酰胺所致卵巢早衰大鼠的保护作用

目的探讨人参皂甙Rb1对化疗所致卵巢早衰(Premature ovarian failure,POF)大鼠的Bcl-2及Bax基因表达的影响。方法选用30只2-3月龄具有正常动情周期雌性Wistar大鼠随机分3组,分别为对照组,治疗组和模型组。采用免疫组化和免疫印迹方法分别观察和半定量检测POF大鼠卵巢凋亡调节基因Bcl-2和Bax的蛋白表达情况,同时对比观察人参皂甙Rb1对其表达的影响。结果模型组卵巢颗粒细胞Bax蛋白的平均光密度值较对照组明显增高(P0.05);模型组Bcl-2蛋白的表达量较对照组明显下调(P0.05)。Rb1治疗后Bcl-2蛋白表达明显升高,Bax蛋白表达明显下调(P0.05)。结论 Rb1可能是通过下调bax蛋白水平减少卵巢颗粒细胞的凋亡,对化疗所致卵巢早衰起到治疗的作用,进而延缓卵巢衰老。     

Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Amniotic Fluid Stem Cells Prevent Follicle Atresia and Rescue Fertility of Mice with Premature Ovarian Failure Induced by Chemotherapy

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.     

Transplantation of UC-MSCs on collagen scaffold activates follicles in dormant ovaries of POF patients with long history of infertility

Premature ovarian failure (POF) is a refractory disease for clinical treatment with the goal of restoring fertility. In this study, umbilical cord mesenchymal stem cells on a collagen scaffold (collagen/UC-MSCs) can activate primordial follicles in vitro via phosphorylation of FOXO3a and FOXO1. Transplantation of collagen/UC-MSCs to the ovaries of POF patients rescued overall ovarian function, evidenced by elevated estradiol concentrations, improved follicular development, and increased number of antral follicles. Successful clinical pregnancy was achieved in women with POF after transplantation of collagen/UC-MSCs or UC-MSCs. In summary, collagen/UC-MSC transplantation may provide an effective treatment for POF.     

Chemotherapy-induced premature ovarian failure: mechanisms and prevention

Significant advances have been made in the previously unexplored areas of the mechanisms involved in cyclophosphamide (CTX)-induced ovarian toxicity and the protective effects of luteinizing hormone-releasing hormone (LHRH agonists. The structure and function of granulosa cells and oocytes are affected by the chemotherapeutic agent, CTX. Results of experiments in female rats indicate that LHRH agonists may protect the ovaries from the toxic effects of chemotherapy. The protective effect may be related to the inhibition of ovarian mitotic activity during LHRH agonist administration. This inhibition is much more pronounced in female compared to male rats. This may be related to the observed better gonadal protective effects in females compared to males. Further experiments are underway to determine whether similar protective effects occur in female primates.     

Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin

Purpose

Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined.

Experimental design

Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect.

Results

Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment.

Conclusion

Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to.     

Peroxiredoxin 4 protects against ovarian ageing by ameliorating d-galactose-induced oxidative damage in mice

Peroxiredoxin 4 (Prdx4), a member of the Prdx family, is a vital ER-resident antioxidant in cells. As revealed in our previous study, Prdx4 expression was detected in ovarian granulosa cells and was closely related to ovarian function. This research aimed to explore the effect and underlying molecular mechanism of the protective role of Prdx4 against d-gal-induced ovarian ageing in mice. The d-gal-induced ovarian ageing model has been extensively used to study the mechanisms of premature ovarian failure (POF). In this study, adult Prdx4^−/− and wild-type mice were intraperitoneally injected with d-gal (150 mg/kg/day) daily for 6 weeks. Ovarian function, granulosa cell apoptosis, oxidative damage and ER stress in the ovaries were evaluated in the two groups. Ovarian weight was significantly lower, the HPO axis was more strongly disrupted, and the numbers of atretic follicles and apoptotic granulosa cells were obviously higher in Prdx4^−/− mice. In addition, Prdx4^−/− mice showed increased expression of oxidative damage-related factors and the ovarian senescence-related protein P16. Moreover, the levels of the proapoptotic factors CHOP and activated caspase-12 protein, which are involved in the ER stress pathway, and the level of the apoptosis-related BAX protein were elevated in the ovaries of Prdx4^−/− mice. Thus, d-gal-induced ovarian ageing is accelerated in Prdx4^−/− mice due to granulosa cell apoptosis via oxidative damage and ER stress-related pathways, suggesting that Prdx4 is a protective agent against POF.Subject terms: Infertility, Experimental models of disease     

Direct ovarian effects of a GnRH agonist in hypophysectomized immature rats: inhibition of theca-interstitial luteinizing hormone receptor mRNA expression and induction of interstitial cell differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.     

Regulation of prostaglandin endoperoxide synthase by cyclic adenosine 3',5'-monophosphate in the in vitro-perfused rat ovary

The preovulatory regulation of two enzymes in the prostaglandin biosynthetic pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (ISN), was examined in granulosa cells and residual tissue of rat ovaries perfused in vitro. Ovaries from rats primed with pregnant mare's serum gonadotropin (20 IU) were perfused for up to 20 h starting the morning of induced proestrus. The amounts of PGS and ISN present were analyzed with immunoblotting techniques. Soluble extracts from granulosa cells and residual ovarian tissues were obtained at different times (0 h, 3 h, 7 h, 12 h) after treatment in vitro with luteinizing hormone (LH, 0.1 microgram/ml) and 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) and at 7 h in untreated control ovaries or after treatment with forskolin (30 microM) or LH (0.1 microgram/ml). The levels in the perfusion medium of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone, testosterone, and estradiol were measured and the number of ovulations were examined. The levels of PGS after treatment with LH + IBMX increased up to 7 h and remained high at 12 h, a time that is close to the time of ovulation. The increase was more pronounced in the granulosa cells than in the residual tissue. Treatment with forskolin induced synthesis of PGS in granulosa cells, and the levels at 7 h were similar to those after stimulation with LH + IBMX. The levels of PGS were lower in granulosa cells of the group stimulated with LH alone than in granulosa cells from ovaries stimulated with LH + IBMX or forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orthotopic Transplantation of Cryopreserved Mouse Ovaries and Gonadotrophin Releasing Hormone Analogues in the Restoration of Function following Chemotherapy-Induced Ovarian Damage

Therapy advances are constantly improving survival rates of cancer patients, however the toxic effects of chemotherapy drugs can seriously affect patients’ quality of life. In women, fertility and premature ovarian endocrine dysfunction are of particular concern. It is urgently we find methods to preserve or reconstruct ovarian function for these women. This study compares GnRHa treatment with ovarian tissue cryopreservation and orthotopic transplantation in a chemotherapy-induced ovarian damage murine model. 56 inbred Lewis rats were divided into 4 treatment groups: Saline control (group I); cyclophosphamide only (group II); cyclophosphamide plus GnRHa (group III); cyclophosphamide and grafting of thawed cryopreserved ovaries (group IV). Body weight, estrous cycle recovery time, ovarian weight, morphology and follicle count, as well as breeding and fertility were compared among groups. Only group IV was able to restore to normal body weight by the end of the observation period and resumed normal estrous cycles in a shorter time compared to other treatment groups. There was a decrease in primordial follicles in all treatment groups, but group III had the greatest reduction. Although, there was no difference in pregnancy, only one animal littered normal pups in group II, none littered in group III and four littered in group IV. Thus, cryopreservation and orthotopic transplantation of ovarian tissue can restore the fertility of rats subjected to chemotherapy in a manner that is superior to GnRHa treatment. We also observed increased rates of hepatic, splenic and pulmonary haemorrhage in group III, suggesting there may be synergistic toxicity of GnRHa and cyclophosphamide.     

Low Expression of Mfn2 Is Associated with Mitochondrial Damage and Apoptosis of Ovarian Tissues in the Premature Ovarian Failure Model

Background

This study aimed to construct a working model for detecting the mitochondrial damage and expression of Mfn2. It furthermore explored the pathogenesis of premature ovarian failure (POF) induced by cisplatin.

Method

Forty young female mice were divided randomly into two groups. The first was the treatment group intraperitoneally administered cisplatin (1.5mg/kg). The untreated control group was likewise injected with physiological saline for 10 days. One month later, we observed the ovarian weight and morphological changes, particularly the development of follicles and concentration of sex hormones. Immunohistochemistry and western blotting were used to measure the two groups. We later evaluated ovarian cell apoptosis with TUNEL and analyzed Bcl-2 and Bax levels. We used transmission electron microscopy in order to observe the ultrastructure of ovarian cells. The phosphomolybdic acid colorimetric method was used to measure the ATP content in the ovarian tissue. Finally, the mitochondrial membrane potential of ovarian cells was detected with JC-1 dye.

Results

The cisplatin resulted in a decline of body weight, reduced ovarian weight significantly, and resulted in disorders of the extrous cycle. The follicles’ number decreased within the tissue’s stromal hyperplasia. Moreover, E₂ levels were reduced, and elevated gonadotropin levels were observed. However, Mfn2 was present in the cell’s cytoplasm in both groups. Nevertheless, the Mfn2 levels and the expression of Bcl-2 were significantly decreased (p<0.05), but the expression of Bax and the apoptosis index (AI) was increased. In addition, the ATP levels (35.2 ±5.7μmol/g) of the control group were significantly higher (13.5 ± 3.8 μmol/g). Lastly, an obvious impairment of mitochondrial function and structure was observed.

Conclusion

The intreperitoneal injection of cisplatin, when administered for 10 days, establishes a POF model. Thus, the above results suggest that lower expression of Mfn2 may be involved in the mechanism of premature ovarian failure by affecting both the mitochondria’s energy metabolism and its apoptosis. This decides the termination of the follicles’ development.     

Postnatal androgenization induces premature aging of rat ovaries

In the present paper, we report that ovaries of adult rats treated with testosterone propionate (TP) on a critical postnatal Day 5 exhibit histologic and immunohistochemical findings which resemble those of the anovulatory ovaries in middle-aged female rats. The sterile rat model has been long known whereas ovarian failure seems to be a reason for anovulation with normal hypothalamo-pituitary-gonadotropin background. Appropriate function of ovarian steroidogenic cells is also regulated by mesenchymal cells. To characterize the ovarian failure, we studied the histology, luteinizing hormone receptor (LHR) expression, and characterized changes of vascular pericytes, T cells, and dendritic cells in ovarian steroidogenic compartments consisting of interstitial cells (ISC) of ovarian interstitial glands, and granulosa and theca interna cells of ovarian follicles. Normal adult ovaries contained 63% of mature interstitial glands. The mature ISC exhibited moderate cytoplasmic and strong surface LHR expression and fine (<5 micrometer) cytoplasmic vacuoles (ISC of 'luteal type'). They originated from young ISC of 'thecal type,' which exhibited strong cytoplasmic LHR expression. Remaining 37% were aged interstitial glands, which consisted of aged ISC (increased cytoplasmic vacuolization, nuclear pyknosis, and reduced surface LHR expression) and regressing ISC (weak cytoplasmic and no surface LHR expression). However, no mature ISC of 'luteal type' were detected in anovulatory ovaries of adult rats (45- and 60-day-old) injected with TP (100 or 500 microgram) on postnatal Day 5 (TP rats). Their ovaries contained 96% of aged interstitial glands with aged and regressing ISC. Remaining 4% were abnormal interstitial glands with direct transition of young ISC of 'thecal type' into aged ISC (young/aged glands). Lack of mature ISC, and similar amount of aged (96%) and young/aged interstitial glands (4%) was also detected in anovulatory ovaries of untreated persistently estrous middle-aged (10-month-old) females (aging PE rats). The aging process in TP and aging PE rats was accompanied by regression of vascular pericytes, T cells, and dendritic cells within the interstitial glands. In addition, anovulatory ovaries of TP rats and aging PE females contained mature follicles exhibiting LHR overexpression by granulosa cells, and aged (cystic) follicles with reduced layers of granulosa cells lacking LHR expression. In contrast, when the rats were injected with 500 microgram of TP later, on postnatal Day 10, the adult females exhibited estrous cycles and normal ovaries with corpora lutea. These results show that injection of TP during the critical postnatal period causes a lack of mature and preponderance of aged ISC in adult ovaries, accompanied by degeneration of mesenchymal cells. We suggest that mesenchymal cells regulate qualitative aspects of tissue-specific cells, and this function of mesenchymal cells is programmed during the critical period of development.     

Reactive oxygen species‐induced SIAH1 promotes granulosa cells' senescence in premature ovarian failure

Reactive oxygen species (ROS) exposure triggers granulosa cells'' (GCs) senescence, which is an important causal factor for premature ovarian failure (POF). However, underlying mechanism in this process remains unknown. In our study, we observed increased ROS levels in POF ovarian tissues, POF patient follicular GCs and cyclophosphamide (CTX) pretreated GCs. Correspondingly, increased SIAH1, reduced TRF2 and GC senescence were also found in these cases. Silencing of SIAH1 rescued ROS‐induced TRF2 reduction and cell senescence in GCs. Moreover, SIAH1 co‐localized with TRF2 in the cytoplasm, facilitating its ubiquitination degradation, further leading to telomere abnormalities in GCs. In conclusion, our findings indicate that ROS induces telomere abnormalities by augmenting SIAH1‐mediated TRF2 degradation, leading to cell senescence in GCs in POF processing.     

Rapamycin preserves the primordial follicle pool during cisplatin treatment in vitro and in vivo

Rapamycin has been proven to effectively inhibit the activation of primordial follicles while cisplatin‐induced the loss of primordial follicles due to the over‐activation of the primordial follicle stockpile. Whether rapamycin could inhibit the loss of primordial follicles induced by cisplatin is still unknown. The ovaries of neonatal Sprague Dawley rats were cultured in vitro in different doses of rapamycin (0.08, 0.16, and 0.32 μg/ml) and cisplatin (0.1, 0.4, and 0.8 μg/ml). The immature BALB/c mice were administered cisplatin with or without rapamycin by intraperitoneal injection. Ovaries were collected to analyze the histomorphology, the messenger RNA (mRNA) expression of anti‐Mullerian hormone (AMH), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) and the expression of key proteins of mammalian target of rapamycin (mTOR) pathway. Growing follicle counts of ovaries cultured in vitro in the R0.16 and R0.32 groups were decreased and the ratio of growing to primordial follicles was also decreased in a dose‐dependent manner. In the C0.8 group, growing follicles were decreased compared with the other groups while the ratio was substantially increased in the C0.4 and C0.8 group. Co‐treatment attenuated primordial follicle loss and reduced the upregulated ratio induced by cisplatin. Ovarian follicle dynamics in vivo was consistent with the in vitro results. Primordial follicles counts were statistically increased and the ratio was reduced in the rapamycin group compared with the control group. Primordial follicle counts were dramatically reduced in the cisplatin group whereas co‐treatment with rapamycin slightly recovered its counts. There was no obvious difference in the number of growing follicles between the cisplatin group and other groups. The ratio was significantly increased in cisplatin‐treated mice whereas decreased in the co‐treatment group. The apoptosis rate of antral follicles in cisplatin‐treated mice was higher than the other groups while the apoptosis rate was decreased in the co‐treatment group in vivo. Compared with the control and rapamycin group, the mRNA expression of AMH, GDF9, and BMP15 were downregulated in the cisplatin group. The co‐treatment group recovered the mRNA expression of BMP15. In addition, the expression of key protein of mTOR pathway rpS6 and its phosphorylated forms were increased in the cisplatin‐treated group while co‐treatment decreased their expression. Rapamycin attenuated the loss of primordial follicles induced by cisplatin through the inhibitory effect of rapamycin on the mTOR pathway. These results suggest that rapamycin may be an effective drug for the protection of ovarian function during chemotherapy.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice

Background

Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs.

Methods

Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H₂O₂), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment.

Results

Mangafodipir attenuated apoptosis induced by H₂O₂ and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects.

Conclusions

Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid

Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17β-HSD I, and 17β-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats.