Antigonadotropic activity of pineal gland of the Indian palm squirrel Funambulus pennanti

Bioassay of the pineal extract of F. pennanti was performed in immature female mice which was previously sensitized with human chorionic gonadotrophin. Reduction of ovarian and uterine weights indicated an antigonadotropic nature of the pineal gland of this animal.     

Loss of abnormal spindle-like,microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm^-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15–20 weeks, 40–50 weeks and 70–80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10–15 weeks, 40–50 weeks and 70–80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging.     

Long-term exposure of male and female mice to 50 Hz magnetic field: effects on fertility

The effect of an extremely low frequency (ELF) magnetic field on the fertility of adult male and female Swiss mice was investigated. Adult male and female mice were exposed to a 50 Hz sinusoidal magnetic field at approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. There were no exposure related effects on the fertility of male or female mice. The number of implantation sites, viable fetuses, and the total number of resorptions were not significantly affected in females impregnated by males exposed to the 50 Hz magnetic field as compared with the control group. The number of implantation sites, viable fetuses and the total number of resorptions in exposed females were also not statistically different from the control group. There were no significant effects on the weights of the testes, seminal vesicles, preputial gland or body weights of males exposed to 50 Hz magnetic field. Furthermore, body and uterine weights were not affected in females exposed to 50 Hz field; however, ovarian weight was significantly increased in females exposed to the same field. These results suggest that exposure of male and female mice to low frequency magnetic field had no adverse effects on fertility and reproduction in mice.     

Hyal-1 but not Hyal-3 deficiency has an impact on ovarian folliculogenesis and female fertility by altering the follistatin/activin/Smad3 pathway and the apoptotic process

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.     

Anti-steroidogenic activity of floral extract of Thespesia populnea Corr. in mouse ovary

Anti-steroidogenic activity of various extracts of T. populnea was screened in female albino mice. The weight of the uterus and ovaries were reduced significantly and the cholesterol and ascorbic acid content in ovaries were significantly elevated due to the treatment with extract of T. populnea. The significant inhibition of delta 5, 3 beta hydroxy steroid dehydrogenase and glucose-6-phosphate dehydrogenase, the two key enzymes involved in ovarian steroidogenesis were also observed in mouse ovaries after 15 days of treatment.     

实验卵巢瘤肿的形态研究

瘤肿发生的原因至今还不甚明瞭,但几十年东由于在动物方面的研究以及临床方面的观察知其原因是多方面的,例如:病毒,物理的和化学的刺激,遗传的因素等等,对某些瘤肿的发生来说,都有重要的系关。实验证明,某些激素在一定的情况下也能诱导瘤肿的发生。动情素有刺激组织生长的能力,所以在它的长期作用下,很多器官例如:乳腺,子宫,脑垂腺等,都能从善性增生的基础上形成瘤肿。Burrows and Horning(1952)和 Lipschutz(1950)的专著对固醇激素,特别是动情素,与瘤肿的系关有了详尽的讨论。同样,脑垂腺激素也有类似的作用。近年来某些品系的小鼠在阉割后可发生肾上腺皮质瘤的发现[Woolley 和 Little(1945)],和抗     

Impaired ovulation in mice with targeted deletion of the neuronal isoform of nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) plays an important role in numerous reproductive processes. To date, most studies have assessed the role of NO by using nonspecific pharmacological inhibitors of the precursor to NO, nitric oxide synthase (NOS). These pharmacological NOS inhibitors suppress all isoforms of NOS; thus, the precise contribution of each isoform to female reproductive physiology is unknown. The purpose of this study was to determine the specific role of neuronal NOS (nNOS) in the regulation of ovulation in female mice lacking the gene that encodes for nNOS (nNOS-/-). MATERIALS AND METHODS: Ovulation was assessed in wild-type (WT) and nNOS-/- female mice by examining the number of ovarian rupture sites and number of oocytes recovered from the oviducts following mating or exposure to exogenous gonadotropins (i.e., 5 IU pregnant mares serum gonadotropin [PMSG] and 5 IU human chorionic gonadotropin [hCG]). Ovulatory efficiency was determined as the number of ovulated oocytes per number of ovarian rupture sites. To examine whether ovulatory deficits in nNOS-/- mice were due to alternations in central mechanisms, plasma luteinizing hormone (LH) concentrations were assessed in WT and nNOS-/- mice that were challenged with 25 ng of gonadotropin-releasing hormone (GnRH). To determine whether ovulatory deficits in nNOS-/- mice were due to local ovulation processes, nerves innervating the reproductive tract of WT and nNOS-/- females were examined for the presence of nNOS protein. RESULTS: There were substantial fertility deficits in nNOS-/- female mice; the nNOS-/- mice had fewer oocytes in their oviducts following spontaneous and gonadotropin-stimulated ovulation. Pituitary responsiveness to exogenous GnRH challenge was intact in nNOS-/- mice. Dense nNOS protein staining was observed in nerves innervating the reproductive tracts of WT mice. CONCLUSIONS: The reproductive deficits in nNOS-/- females are most likely due to alternations in the transfer of oocytes from the ovaries to the oviducts during ovulation. These results suggest that defects in neuronally derived NO production may contribute to female infertility.     

海参花抗氧化作用研究

目的：近年来关于天然活性成分的抗氧化特性研究越来越受到人们的关注,大量研究表明海参能通过抗氧化作用,减轻自由基对组织的损伤,提高机体免疫力。环磷酰胺可引起组织氧化损伤,导致卵巢早衰。为此,本文着重研究海参花对卵巢早衰（POF）小鼠的抗氧化作用。方法：选取健康雌性昆明小鼠80只,随机分为8组,每组10只,分别为正常组,模型组,海参花水提物低、高剂量组,海参花醇提物低、高剂量组,海参花冻干粉组和阳性药组。除正常组每天注射生理盐水外,其余各组每天注射环磷酰胺（28mg/kg）,连续5天,造成小鼠卵巢早衰、生殖系统紊乱。造模成功后,治疗组分别灌服海参花提取物和阳性药,正常组和模型组给予同体积蒸馏水。末次灌胃禁食,处死迅速解剖取出卵巢和子宫,低温下匀浆组织。测定各组小鼠卵巢及子宫组织中总蛋白含量、超氧化物歧化酶（SOD）活性、谷胱甘肽过氧化物酶（GSH-Px）活性以及丙二醛（MDA）含量,并测定各组小鼠间子宫、卵巢、脾、胸腺脏器系数。结果：与模型组相比,海参花提取物能显著增加小鼠的子宫、卵巢、胸腺等脏器重量,升高卵巢及子宫组织中总蛋白含量、SOD活性、GSH-Px活性,并能降低MDA含量。结论：海参花提取物能改善环磷酰胺引起的卵巢和子宫氧化损伤,具有良好的抗氧化作用,为海参花的研究开发提供理论依据。     

Hepatic lipase deficiency attenuates mouse ovarian progesterone production leading to decreased ovulation and reduced litter size

The lipolytic enzyme hepatic lipase (HL) may facilitate mobilization of cholesterol substrate for ovarian steroidogenesis. We investigated whether HL was necessary for optimum reproduction in the female mouse by analyzing breeding performance and ovarian responses to gonadotropins in HL-/- mice. HL-/- female mice bred with HL-/- males had the same pregnancy success rate and pup survival rate as did wild-type (WT) mice but had significantly smaller litters, producing 1.7 fewer pups per litter. Mice were primed with eCG/hCG, and at 6 h post-hCG the HL-/- mice had smaller ovaries than did the WT mice. HL deficiency specifically affected ovarian weight; adrenal gland weights did not differ between WT and HL-/- mice. HL-/- mice weighed more than age-matched WT mice. Between the two mouse genotypes, uterine weights were the same, indicating that estrogen production was equivalent. However, the HL-/- ovaries produced significantly less progesterone than did the WT ovaries within 6 h of hCG stimulation. HL-/- ovaries had the same number of large antral follicles as did the WT ovaries but had fewer hemorrhagic sites, which represent ovulations, fewer corpora lutea, and more oocytes trapped in corpora lutea. We suggest that reduced progesterone synthesis following hCG stimulation attenuated the final maturation of preovulatory follicles, resulting in smaller ovaries. Furthermore, reduced progesterone production limited the expression of proteolytic enzymes needed for tissue remodeling, resulting in fewer ovulations with a corresponding increase in trapped or unovulated oocytes and providing a possible explanation for the smaller litter size observed in spontaneously ovulating HL-/- mice.     

Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-alpha knockout mice

Estrogen receptor-alpha (ERalpha) knockout (ERalphaKO) female mice are infertile. Initially, they exhibit normal follicular development, but by 4-5 wk of age, they begin to develop hemorrhagic ovarian cysts. Follicles in adult ERalphaKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERalpha is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERalphaKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERalphaKO and WT mice. Gonadotropin-treated ERalphaKO females that ovulated produced 7.09 +/- 0.77 oocytes per mouse, whereas gonadotropin-treated WT female mice had 16.17 +/- 0.84 oocytes. Surprisingly, ruptured ERalphaKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERalphaKO mice had 3-fold higher concentrations of serum progesterone than did control ERalphaKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERalphaKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERalpha, although to a lesser extent than in WT mice.     

Increased female fertility in aquaporin 8-deficient mice

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.     

Mouse ovarian factors with angiogenic activities: partial characterization.

Extracts of nonluteal mouse ovaries (Jcl: ICR strain) were assayed for neovascularization by implanting Elvax films, impregnated with test samples, on the lateral wall of the sheath of m. rectus abdominis in young adult mice of the same strain. Neovascularization occurred in a dose-dependent manner. Angiogenic activity was increased in extracts of ovaries from mice treated with follicle-stimulating hormone (FSH). The angiogenic activity was retained when the extract was heated to 100 degrees C for 15 min or adsorbed with activated charcoal. The ovarian extract was purified by affinity chromatography on a Con A-Sepharose 4B column. The adsorbed fraction possessed higher angiogenic activity. The ovarian extract was fractionated by ammonium sulfate precipitation. High angiogenic activity was found in the fraction collected between 20 to 50% saturation.     

Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.     

Bortezomib Prevents Acute Doxorubicin Ovarian Insult and Follicle Demise,Improving the Fertility Window and Pup Birth Weight in Mice

Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment.     

Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse

This study aims to compare the effect of early and late onset administration of oral antioxidants on number and quality of oocytes retrieved from aged mice after exogenous ovarian stimulation. Control hybrid females were fed a standard diet supplemented or not supplemented with pharmacological doses of vitamins C and E either from the first day of weaning or from the age of 32 weeks until they were autopsied at the age 40-42, 50-52, or 57-62 weeks after exogenous ovarian stimulation. Analysis of chromosomal distribution, DNA organization and cellular morphology was performed in ovulated cumulus-enclosed and -free oocytes, ovarian non-germinal vesicle oocytes enclosed by or free of mucous cumulus cells and in vitro-matured ovarian germinal-vesicle oocytes. Both early and late onset administration of oral antioxidants counteracted the negative effects of female aging on number of ovarian oocytes and total percentage of oocytes retrieved from oviducts and ovaries exhibiting a normal distribution of chromosomes in the metaphase-II plate and/or morphological traits of apoptosis. Although both early and late onset administration of oral antioxidants can counteract the negative effects of female aging on number and quality of oocytes, transference of these results to human beings should be made with caution because of the potential side effects of high doses of vitamins on reproductive function as well as many other undesirable systemic disorders.     

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.     

Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse.

Xenogeneic transplantation of ovarian cortex into an immunodeficient animal host may be an approach toward fertility preservation for young female patients undergoing cancer therapy. Our objective was to evaluate the development of follicles in human ovarian cortex placed s.c. in non-obese diabetic-severe combined immune deficiency (NOD-SCID) mice (n = 54). The following variables were compared: 1) male versus female mice as hosts, 2) intact versus pituitary down-regulated mice, and 3) warm versus cold tissue transport. After 2 wk, 37 of 50 (74%) of the human xenografts contained follicles. At 12 wk after transplantation, exogenous gonadotropin stimulation resulted in follicle growth in 19 of 37 (51%) of the grafts, including the development of antral follicles, which could be palpated and visualized through the mouse skin. Significantly more developing follicles were identified in male versus female mice (13 of 17 vs. 6 of 20, respectively; p = 0.013) after stimulation. No difference was found between intact and pituitary down-regulated mice as hosts. Follicular survival was significantly increased by warm versus cold tissue transport. Our results suggest that s.c. ovarian cortex xenografting into NOD-SCID mice is feasible. Primordial follicles in ovarian xenografts retain their developmental potential and form antral follicles following gonadotropin stimulation.     

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.