Genetic Prediction of Osteoporosis by Anti-Müllerian Hormone Levels and Reproductive Factors in Women: A Mendelian Randomization Study.

Previous observational studies have suggested that anti-Müllerian hormone (AMH) and reproductive factors are linked to reduced bone mineral density (BMD) and an increased risk of osteoporosis (OP) in women. However, related studies are limited, and these traditional observational studies may be subject to residual confounders and reverse causation, while also lacking a more comprehensive observation of various reproductive factors. Univariate and multivariate two-sample Mendelian randomization analyses were conducted to determine the causal associations of AMH levels and six reproductive factors with BMD and OP, using the random-effects inverse-variance weighted method. Heterogeneity was assessed using Cochran's Q-statistic, and sensitivity analyses were performed to identify causal correlations. Age at menarche (AAM) was negatively associated with total body BMD (TB-BMD) in females aged 45-60 and over 60 years, as well as with heel bone mineral density (eBMD). Conversely, age at natural menopause (ANM) was positively associated with TB-BMD in the same age ranges and with eBMD. ANM was only causally associated with self-reported OP and showed no significant correlation with definitively diagnosed OP. Neither AMH level nor other reproductive factors were significantly associated with a genetic predisposition to BMD at any age and OP. Later AAM and earlier ANM are significantly genetically causally associated with decreased BMD but not with OP. AMH levels, length of menstrual cycle, age at first birth, age at last birth, and number of live births, in terms of genetic backgrounds, are not causally related to BMD or OP.

Epidemiologic and genetic associations of female reproductive disorders with depression or dysthymia: a Mendelian randomization study.

Observational studies have previously reported an association between depression and certain female reproductive disorders. However, the causal relationships between depression and different types of female reproductive disorders remain unclear in terms of direction and magnitude. We conducted a comprehensive investigation using a two-sample bi-directional Mendelian randomization analysis, incorporating publicly available GWAS summary statistics. Our aim was to establish a causal relationship between genetically predicted depression and the risk of various female reproductive pathological conditions, such as ovarian dysfunction, polycystic ovary syndrome(PCOS), ovarian cysts, abnormal uterine and vaginal bleeding(AUB), endometriosis, leiomyoma of the uterus, female infertility, spontaneous abortion, eclampsia, pregnancy hypertension, gestational diabetes, excessive vomiting in pregnancy, cervical cancer, and uterine/endometrial cancer. We analyzed a substantial sample size, ranging from 111,831 to 210,870 individuals, and employed robust statistical methods, including inverse variance weighted, MR-Egger, weighted median, and MR-PRESSO, to estimate causal effects. Sensitivity analyses, such as Cochran's Q test, MR-Egger intercept test, MR-PRESSO, leave-one-out analysis, and funnel plots, were also conducted to ensure the validity of our results. Furthermore, risk factor analyses were performed to investigate potential mediators associated with these observed relationships. Our results demonstrated that genetic predisposition to depression or dysthymia was associated with an increased risk of developing PCOS (OR = 1.43, 95% CI 1.28-1.59; P = 6.66 × 10-11), ovarian cysts (OR = 1.36, 95% CI 1.20-1.55; P = 1.57 × 10-6), AUB (OR = 1.41, 95% CI 1.20-1.66; P = 3.01 × 10-5), and endometriosis (OR = 1.43, 95% CI 1.27-1.70; P = 2.21 × 10-7) after Bonferroni correction, but no evidence for reverse causality. Our study did not find any evidence supporting a causal or reverse causal relationship between depression/dysthymia and other types of female reproductive disorders. In summary, our study provides evidence for a causal relationship between genetically predicted depression and specific types of female reproductive disorders. Our findings emphasize the importance of depression management in the prevention and treatment of female reproductive disorders, notably including PCOS, ovarian cysts, AUB, and endometriosis.

Generalized Parametric Contrastive Learning.

In this paper, we propose the Generalized Parametric Contrastive Learning (GPaCo/PaCo) which works well on both imbalanced and balanced data. Based on theoretical analysis, we observe supervised contrastive loss tends to bias on high-frequency classes and thus increases the difficulty of imbalanced learning. We introduce a set of parametric class-wise learnable centers to rebalance from an optimization perspective. Further, we analyze our GPaCo/PaCo loss under a balanced setting. Our analysis demonstrates that GPaCo/PaCo can adaptively enhance the intensity of pushing samples of the same class close as more samples are pulled together with their corresponding centers and benefit hard example learning. Experiments on long-tailed benchmarks manifest the new state-of-the-art for long-tailed recognition. On full ImageNet, models from CNNs to vision transformers trained with GPaCo loss show better generalization performance and stronger robustness compared with MAE models. Moreover, GPaCo can be applied to semantic segmentation task and obvious improvements are observed on 4 most popular benchmarks. Our code is available at https://github.com/dvlab-research/Parametric-Contrastive-Learning.

Immunity and reproduction protective effects of Chitosan Oligosaccharides in Cyclophosphamide/Busulfan-induced premature ovarian failure model mice.

Introduction: Premature ovarian failure (POF) is a major cause of infertility among women of reproductive age. Unfortunately, there is no effective treatment available currently. Researchers have shown that immune disorders play a significant role in the development of POF. Moreover, growing evidence suggest that Chitosan Oligosaccharides (COS), which act as critical immunomodulators, may have a key role in preventing and treating a range of immune related reproductive diseases. Methods: KM mice (6-8 weeks) received a single intraperitoneal injection of cyclophosphamide (CY, 120mg/kg) and busulfan (BUS, 30mg/kg) to establish POF model. After completing the COS pre-treatment or post-treatment procedures, peritoneal resident macrophages (PRMs) were collected for neutral erythrophagocytosis assay to detect phagocytic activity. The thymus, spleen and ovary tissues were collected and weighed to calculate the organ indexes. Hematoxylin-eosin (HE) staining was performed to observe the histopathologic structure of those organs. The serum levels of estrogen (E2) and progesterone (P) were measured via the enzyme-linked immunosorbent assay (ELISA). The expression levels of immune factors including interleukin 2 (IL-2), interleukin 4 (IL-4), and tumor necrosis factor α (TNF-α), as well as germ cell markers Mouse Vasa Homologue (MVH) and Fragilis in ovarian tissue, were analyzed by Western blotting and qRT-PCR. In addition, ovarian cell senescence via p53/p21/p16 signaling was also detected. Results: The phagocytic function of PRMs and the structural integrity of thymus and spleen were preserved by COS treatment. The levels of certain immune factors in the ovaries of CY/BUS- induced POF mice were found to be altered, manifested as IL-2 and TNF-α experiencing a significant decline, and IL-4 presenting a notable increase. Both pre-treatment and post-treatment with COS were shown to be protective effects against the damage to ovarian structure caused by CY/BUS. Senescence-associated ß-galactosidase (SA-ß-Gal) staining results showed that COS prevents CY/BUS-induced ovarian cell senescence. Additionally, COS regulated estrogen and progesterone levels, enhanced follicular development, and blocked ovarian cellular p53/p21/p16 signaling which participating in cell senescence. Conclusion: COS is a potent preventative and therapeutic medicine for premature ovarian failure by enhancing both the ovarian local and systemic immune response as well as inhibiting germ cell senescence.

Chito-oligosaccharides and macrophages have synergistic effects on improving ovarian stem cells function by regulating inflammatory factors.

BACKGROUND: Chronic low-grade inflammation and ovarian germline stem cells (OGSCs) aging are important reasons for the decline of ovarian reserve function, resulting in ovarian aging and infertility. Regulation of chronic inflammation is expected to promote the proliferation and differentiation of OGSCs, which will become a key means for maintaining and remodeling ovarian function. Our previous study demonstrated that Chitosan Oligosaccharides (Cos) promoted the OGSCs proliferation and remodelled the ovarian function through improving the secretion of immune related factors,but the mechanism remains unclear, and the role of macrophages, the important source of various inflammatory mediators in the ovary needs to be further studied. In this study, we used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore what contribution macrophages give during this process. Our finding provides new drug treatment options and methods for the prevention and treatment of premature ovarian failure and infertility. METHODS: We used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore the important contribution of macrophages in it. The immunohistochemical staining was used to locate the OGSCs in the mouse ovary. Immunofluorescent staining, RT-qPCR and ALP staining were used to identify the OGSCs. CCK-8 and western blot were used to evaluate the OGSCs proliferation. ß-galactosidase(SA-ß-Gal) staining and western blot were used to detect the changing of cyclin-dependent kinase inhibitor 1A(P21), P53, Recombinant Sirtuin 1(SIRT1) and Recombinant Sirtuin 3(SIRT3). The levels of immune factors IL-2, IL-10, TNF-α and TGF-ß were explored by using Western blot and ELISA. RESULTS: We found that Cos promoted OGSCs proliferation in a dose-and time-dependent manner, accompanied by IL-2, TNF-α increase and IL-10, TGF-ß decrease. Mouse monocyte-macrophages Leukemia cells(RAW) can also produce the same effect as Cos. When combined with Cos, it can enhance the proliferative effect of Cos in OGSCs, and further increase IL-2, TNF-α and further decrease IL-10, TGF-ß. The macrophages can enhance the proliferative effect of Cos in OGSCs is also associated with the further increase in IL-2, TNF-α and the further decrease in IL-10, TGF-ß. In this study, we determined that the anti-aging genes SIRT-1 and SIRT-3 protein levels were increased by Cos and RAW respectively, whereas the senescence-associated SA-ß-Gal and aging genes P21 and P53 were decreased. Cos and RAW had a protective effect on OGSCs delaying aging. Furthermore, RAW can further decrease the SA-ß-Gal and aging genes P21 and P53 by Cos, and further increase SIRT1 and SIRT3 protein levels in OGSCs by Cos. CONCLUSION: In conclusion, Cos and macrophages have synergistic effects on improving OGSCs function and delaying ovarian aging by regulating inflammatory factors.

Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome.

Introduction: Polycystic Ovary Syndrome (PCOS) is the most common reproductive endocrine disorder among women of reproductive age, which is one of the main causes of anovulatory infertility. Even though the rapidly developed assisted reproductive technology (ART) could effectively solve fertility problems, some PCOS patients still have not obtained satisfactory clinical outcomes. The poor quality of oocytes caused by the abnormal follicular development of PCOS may directly contribute to the failure of ART treatment. Ovarian granulosa cells (GCs) are the most closely related cells to oocytes, and changes in their functional status have a direct impact on oocyte formation. Previous studies have shown that changes in the ovarian microenvironment, like oxidative stress and inflammation, may cause PCOS-related aberrant follicular development by impairing the physiological state of the GCs. Therefore, optimizing the ovarian microenvironment is a feasible method for enhancing the development potential of PCOS oocytes. Methods: In this study, we first detected the expression of inflammatory-related factors (TGF-ß1, IL-10, TNFα, IL-6) and oxidative stress-related factors (HIF-1α and VEGFA), as well as the proliferation ability and apoptosis level of GCs, which were collected from control patients (non-PCOS) and PCOS patients, respectively. Subsequently, human ovarian granulosa cell line (KGN) cells were used to verify the anti-inflammatory and anti-oxidative stress effects of chitosan oligosaccharide (COS) on GCs, as well as to investigate the optimal culture time and concentration of COS. The optimal culture conditions were then used to culture GCs from PCOS patients and control patients. Results: The results showed that GCs from PCOS patients exhibited obvious inflammation and oxidative stress and significantly reduced proliferation and increased apoptosis. Furthermore, COS can increase the expression of anti-inflammatory factors (TGF-ß1 and IL-10) and decrease the expression of pro-inflammatory factors (TNFα and IL-6), as well as promote the proliferation of GCs. Moreover, we found that COS can reduce the level of reactive oxygen species in GCs under oxidative stress by inhibiting the expression of HIF-1α and VEGFA and by suppressing the apoptosis of GCs induced by oxidative stress. Conclusion: We find that inflammation and oxidative stress exist in the GCs of PCOS patients, and COS can reduce these factors, thereby improving the function of GCs.

ResLT: Residual Learning for Long-Tailed Recognition.

Deep learning algorithms face great challenges with long-tailed data distribution which, however, is quite a common case in real-world scenarios. Previous methods tackle the problem from either the aspect of input space (re-sampling classes with different frequencies) or loss space (re-weighting classes with different weights), suffering from heavy over-fitting to tail classes or hard optimization during training. To alleviate these issues, we propose a more fundamental perspective for long-tailed recognition, i.e., from the aspect of parameter space, and aims to preserve specific capacity for classes with low frequencies. From this perspective, the trivial solution utilizes different branches for the head, medium, tail classes respectively, and then sums their outputs as the final results is not feasible. Instead, we design the effective residual fusion mechanism - with one main branch optimized to recognize images from all classes, another two residual branches are gradually fused and optimized to enhance images from medium+tail classes and tail classes respectively. Then the branches are aggregated into final results by additive shortcuts. We test our method on several benchmarks, i.e., long-tailed version of CIFAR-10, CIFAR-100, Places, ImageNet, and iNaturalist 2018. Experimental results manifest the effectiveness of our method. Our code is available at https://github.com/jiequancui/ResLT.

Female germline stem cells: aging and anti-aging.

The delay of ovarian aging and the fertility preservation of cancer patients are the eternal themes in the field of reproductive medicine. Acting as the pacemaker of female physiological aging, ovary is also considered as the principle player of cancer, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases and etc. However, its aging mechanism and preventive measures are still unclear. Some researchers attempt to activate endogenous ovarian female germline stem cells (FGSCs) to restore ovarian function, as the most promising approach. FGSCs are stem cells in the adult ovaries that can be infinitely self-renewing and have the potential of committed differention. This review aims to elucidate FGSCs aging mechanism from multiple perspectives such as niches, immune disorder, chronic inflammation and oxidative stress. Therefore, the rebuilding nichs of FGSCs, regulation of immune dysfunction, anti-inflammation and oxidative stress remission are expected to restore or replenish FGSCs, ultimately to delay ovarian aging.

Chitosan Oligosaccharides Alleviate H2O2-stimulated Granulosa Cell Damage via HIF-1α Signaling Pathway.

Oocyte maturation disorder and decreased quality are the main causes of infertility in women, and granulosa cells (GCs) provide the only microenvironment for oocyte maturation through autocrine and paracrine signaling by steroid hormones and growth factors. However, chronic inflammation and oxidative stress caused by ovarian hypoxia are the largest contributors to ovarian aging and GC dysfunction. Therefore, the amelioration of chronic inflammation and oxidative stress is expected to be a pivotal method to improve GC function and oocyte quality. In this study, we detected the protective effect of chitosan oligosaccharides (COS), on hydrogen peroxide- (H2O2-) stimulated oxidative damage in a human ovarian granulosa cell line (KGN). COS significantly increased cell viability, mitochondrial function, and the cellular glutathione (GSH) content and reduced apoptosis, reactive oxygen species (ROS) content, and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial-derived growth factor (VEGF) in H2O2-stimulated KGN cells. COS treatment significantly increased levels of the TGF-ß1 and IL-10 proteins and decreased levels of the IL-6 protein. Compared with H2O2-stimulated KGN cells, COS significantly increased the levels of E2 and P4 and decreased SA-ß-gal protein expression. Furthermore, COS caused significant inactivation of the HIF-1α-VEGF pathway in H2O2-stimulated KGN cells. Moreover, inhibition of this pathway enhanced the inhibitory effects of COS on H2O2-stimulated oxidative injury and apoptosis in GCs. Thus, COS protected GCs from H2O2-stimulated oxidative damage and apoptosis by inactivating the HIF-1α-VEGF signaling pathway. In the future, COS might represent a therapeutic approach for ameliorating disrupted follicle development.

Controlling chronic low-grade inflammation to improve follicle development and survival.

Chronic low-grade inflammation is one cause of follicle development disturbance. Chronic inflammation exists in pathological conditions such as premature ovarian failure, physiological aging of the ovaries, and polycystic ovary syndrome. Inflammation of the whole body can affect oocytes via the follicle microenvironment, oxidative stress, and GM-CSF. Many substances without toxic side-effects extracted from natural organisms have gradually gained researchers' attention. Recently, chitosan oligosaccharide, resveratrol, anthocyanin, and melatonin have been found to contribute to an improvement in inflammation. This review discusses the interrelationships between chronic low-grade inflammation and follicle development, the underlying mechanisms, and methods that may improve follicle development by controlling the level of chronic low-grade inflammation.

ADA-Tucker: Compressing deep neural networks via adaptive dimension adjustment tucker decomposition.

Despite recent success of deep learning models in numerous applications, their widespread use on mobile devices is seriously impeded by storage and computational requirements. In this paper, we propose a novel network compression method called Adaptive Dimension Adjustment Tucker decomposition (ADA-Tucker). With learnable core tensors and transformation matrices, ADA-Tucker performs Tucker decomposition of arbitrary-order tensors. Furthermore, we propose that weight tensors in networks with proper order and balanced dimension are easier to be compressed. Therefore, the high flexibility in decomposition choice distinguishes ADA-Tucker from all previous low-rank models. To compress more, we further extend the model to Shared Core ADA-Tucker (SCADA-Tucker) by defining a shared core tensor for all layers. Our methods require no overhead of recording indices of non-zero elements. Without loss of accuracy, our methods reduce the storage of LeNet-5 and LeNet-300 by ratios of 691× and 233 ×, respectively, significantly outperforming state of the art. The effectiveness of our methods is also evaluated on other three benchmarks (CIFAR-10, SVHN, ILSVRC12) and modern newly deep networks (ResNet, Wide-ResNet).

Tri-ortho-cresyl phosphate (TOCP) induced ovarian failure in mice is related to the Hippo signaling pathway disruption.

As a plasticizer widely used in society, tri-ortho-cresyl phosphate (TOCP) is reported to inhibit spermatogenesis and growth of spermatogonial stem cells. However, its effects on female reproductive system are virtually unknown. The present study investigated the effects of TOCP on ovarian follicle development by using mouse model of chronic TOCP exposure, and examined the expression of the core components of the Hippo pathway, which had been proven to be crucial for ovarian follicle development. Furthermore, through up-regulation of Hippo-yes-associated protein 1 (Yap1) in ovaries, the potential protective effects of Yap1 over-expression on TOCP-induced ovarian dysfunction were observed. The results showed that TOCP impaired ovarian function in a dose-dependent manner, and the expression of the Hippo pathway changed significantly in TOCP-exposed ovaries. Further, YAP1 over-expression partially reversed the TOCP-induced ovarian impairment. Collectively, these data indicate that the Hippo pathway is involved in the mechanism by which TOCP elicits ovarian function impairment.

Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.

Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes-associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P-MST/MST1 and upregulated the ratio of P-YAP1/YAP1 significantly, whereas Yap1-treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo-YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development.

The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals.

The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years.

Hippo signaling pathway reveals a spatio-temporal correlation with the size of primordial follicle pool in mice.

BACKGROUND: The Hippo signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms and regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Hippo signaling are expressed in mammalian ovaries, but the exact functions of this pathway in primordial follicle development remains unclear. METHODS: To analyze the spatio-temporal correlation between the core component of Hippo pathway and the size of primordial follicle pool, Western blot, Real-time PCR and immunohistochemistry were used, and the expression and localization of MST1, LATS2 and YAP1 mRNA and protein were examined in 3 d, 1 m, 5 m, 16 m postnatal mice ovary and the culture model of mice primordial follicle in vitro. RESULTS: Both the protein and mRNA expression of the MST1 and LATS2 were decreased significantly as mouse age increased (p < 0.05), however, the mRNA expression of them increased significantly in 16 m compared with 5 m as well as the protein expression of LATS2.The expression of YAP showed the opposite trend, and the significant protein expression of pYAP was increased before 1 m, after which no significant change was observed. Moreover, the ratio of pYAP/YAP decreased significantly. Culturing ovaries for 8 d in vitro resulted in the activation of primordial follicles in 3 d postnatal mice ovaries, and these developed into primary follicles with the expression of PCNA increasing significantly (p < 0.05). The mRNA and protein expression of MST and LATS decreased significantly (p < 0.05), and the expression of YAP increased significantly (p < 0.05, p < 0.01), whereas the ratio of pYAP/YAP decreased significantly (p < 0.05). CONCLUSION: The above results reveal that the expression of the core components of Hippo pathway changed during mouse follicular development, especially before and after primordial follicle activation in vitro. The primordial follicle activation may be related to the significant decrease of the ratio of pYAP1/YAP1. In conclusion, Hippo signaling pathway expressed in mice ovaries and have spatio-temporal correlation with the size of primordial follicle pool.

The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development.

Imprinted genes play important roles in placenta development and function. Parthenogenetic embryos, deficient in paternally expressed imprinted genes, lack extra-embryonic tissues of the trophoblast lineage. Parthenogenetic trophoblast stem cells (TSCs) are extremely difficult to derive, suggesting that an imprinted gene(s) is necessary for TSC establishment or maintenance. In a candidate study, we were able to narrow the list to one known paternally expressed gene, Sfmbt2. We show that mouse embryos inheriting a paternal Sfmbt2 gene trap null allele have severely reduced placentae and die before E12.5 due to reduction of all trophoblast cell types. We infected early embryos with lentivirus vectors expressing anti-Sfmbt2 shRNAs and found that TSC derivation was significantly reduced. Together, these observations support the hypothesis that loss of SFMBT2 results in defects in maintenance of trophoblast cell types necessary for development of the extra-embryonic tissues, the placenta in particular.

Strain-specific spontaneous activation during mouse oocyte maturation.

OBJECTIVE: To examine whether spontaneous oocyte activation is determined by genetic differences and interacts with culture environment. DESIGN: Experimental study. SETTING: Temple University School of Medicine. ANIMAL(S): C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5-6 weeks). INTERVENTION(S): Immature oocytes from different mouse strains collected and cultured in different maturation conditions, including different serum, serum replacement, bovine serum albumin (BSA), and follicle-stimulation hormone (FSH). MAIN OUTCOME MEASURE(S): The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators. RESULT(S): Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiotic division with in vivo maturation (VVM) after superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes that spontaneously activated during the first meiotic division with IVM have unusual replicated sister chromatid pairs with slight connections at centromeres at first mitosis, whereas oocytes that activated in vivo display haploidization from the second meiotic division. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cyclin B and securin expression. CONCLUSION(S): Both mouse strain and culture environment have a statistically significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.

Essential role of maternal UCHL1 and UCHL3 in fertilization and preimplantation embryo development.

Post-translational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1(gad-/-) mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1(gad-/-) mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex.

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos.

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.