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Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in the seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

Oocyte meiotic maturation is a vital and final process in oogenesis. Unlike somatic cells， the oocyte needs to undergo two continuous meiotic divisions (meiosis I and meiosis II) to become a haploid gamete. Notably, oocyte meiotic progression includes two rounds of unique meiotic arrest and resumption. The first arrest occurs at the G2 (germinal vesicle) stage and meiosis resumption is stimulated by a gonadotropin surge; the second arrest takes place at the metaphase II stage, the stage from which it is released when fertilization takes place. The maturation-promoting factor, which consists of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDK1), is responsible for regulating meiotic resumption and progression, while CDK1 is the unique CDK that acts as the catalytic subunit of maturation-promoting factor. Recent studies showed that except for cyclin B1, multiple cyclins interact with CDK1 to form complexes, which are involved in the regulation of meiotic progression at different stages. Here, we review and discuss the control of oocyte meiotic progression by cyclins A1, A2, B1, B2, B3, and O.

The quality of oocytes is a vital factor for embryo development. Meiotic progression through metaphase I usually takes a relatively long time to ensure correct chromosome separation, a process that is critical for determining oocyte quality. Here, we report that cell division cycle 5-like (Cdc5L) plays a critical role in regulating metaphase-to-anaphase I transition during mouse oocyte meiotic maturation. Knockdown of Cdc5L by small interfering RNA injection did not affect spindle assembly but caused metaphase I arrest and subsequent reduced first polar body extrusion due to insufficient anaphase-promoting complex/cyclosome activity. We further showed that Cdc5L could also directly interact with securin, and Cdc5L knockdown led to a continuous high expression level of securin, causing severely compromised meiotic progression. The metaphase-to-anaphase I arrest caused by Cdc5L knockdown could be rescued by knockdown of endogenous securin. In summary, we reveal a novel role for Cdc5L in regulating mouse oocyte meiotic progression by interacting with securin.

AimsTo characterize the genetic landscape and mutation spectrum of patients with corneal dystrophies (CDs) in a large Han ethnic Chinese Cohort with inherited eye diseases (IEDs).MethodsRetrospective study. A large IED cohort was recruited in this study, including 69 clinically diagnosed CD patients, as well as other types of eye diseases patients and healthy family members as controls. The 792 genes on the Target_Eye_792_V2 chip were used to screen all common IEDs in our studies, including 22 CD-related genes.ResultsWe identified 2334 distinct high-quality variants on 22 CD-related genes in a large IEDs cohort. A total of 21 distinct pathogenic or likely pathogenic mutations were identified, and the remaining 2313 variants in our IED cohort had no evidence of CD-related pathogenicity. Overall, 81.16% (n = 56/69) of CD patients received definite molecular diagnoses, and transforming growth factor-beta-induced protein (TGFBI), CHTS6, and SLC4A11 genes covered 91.07, 7.14, and 1.79% of the diagnosed cases, respectively. Twelve distinct disease-associated mutations in the TGFBI gene were identified, 11 of which were previously reported and one is novel. Four of these TGFBI mutations (p.D123H, p.M502V, p.P501T, and p.P501A) were redefined as likely benign in our Han ethnic Chinese IED cohort after performing clinical variant interpretation. These four TGFBI mutations were detected in asymptomatic individuals but not in CD patients, especially the previously reported disease-causing mutation p.P501T. Among 56 CD patients with positive detected mutations, the recurrent TGFBI mutations were p.R124H, p.R555W, p.R124C, p.R555Q, and p.R124L, and the proportions were 32.14, 19.64, 14.29, 10.71, and 3.57%, respectively. Twelve distinct pathogenic or likely pathogenic mutations of CHTS6 were detected in 28 individuals. The recurrent mutations were p.Y358H, p.R140X, and p.R205W, and the proportions were 25.00, 21.43, and 14.29%, respectively. All individuals associated with TGFBI were missense mutations; 74.19% associated with CHTS6 mutations were missense mutations, and 25.81% were non-sense mutations. Hot regions were located in exons 4 and 12 of TGFBI individuals and located in exon 3 of CHTS6 individuals. No de novo mutations were identified.ConclusionFor the first time, our large cohort study systematically described the variation spectrum of 22 CD-related genes and evaluated the frequency and pathogenicity of all 2334 distinct high-quality variants in our IED cohort. Our research will provide East Asia and other populations with baseline data from a Han ethnic population-specific level.

Background To evaluate optimal thawing time, the early thawing or the routine thawing time, for transferring vitrified-warmed, and cultured overnight cleavage stage of the slow-growing embryos on Day 3 in frozen embryo transfer (FET) cycle. Methods This was a retrospective cohort study from January 2017 to July 2018, a total of 705 slow-growing embryos FET cycles in which the patients were aged p = 0.01), while there was no statistically significant difference in pregnancy loss in early thawing group (39/170 (22.9%)) versus in routine thawing group (16/62 (25.8%)) (OR 0.89 (CI 0.53–1.47), p = 0.65). Conclusion For slow-growing embryos, higher pregnancy outcomes were shown in early thawing strategy as compared to the routine thawing, which suggested that the improvement of endometrium-embryo synchronism may correct the time difference brought by the slow-growing embryos.

PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.

Cyclin B1 and its interaction with CDK1 are thought to be critical for meiosis I progression in oocytes. However, using oocyte-specific conditional knockouts, Li et al. show that Cyclin B2 activity can compensate for Cyclin B1 to trigger meiosis resumption., Mammalian oocytes are arrested at the prophase of the first meiotic division for months and even years, depending on species. Meiotic resumption of fully grown oocytes requires activation of M-phase–promoting factor (MPF), which is composed of Cyclin B1 and cyclin-dependent kinase 1 (CDK1). It has long been believed that Cyclin B1 synthesis/accumulation and its interaction with CDK1 is a prerequisite for MPF activation in oocytes. In this study, we revealed that oocyte meiotic resumption occurred in the absence of Cyclin B1. Ccnb1-null oocytes resumed meiosis and extruded the first polar body. Without Cyclin B1, CDK1 could be activated by up-regulated Cyclin B2. Ccnb1 and Ccnb2 double knockout permanently arrested the oocytes at the prophase of the first meiotic division. Oocyte-specific Ccnb1-null female mice were infertile due to failed MPF activity elevation and thus premature interphase-like stage entry in the second meiotic division. These results have revealed a hidden compensatory mechanism between Cyclin B1 and Cyclin B2 in regulating MPF and oocyte meiotic resumption.

After ovulation, metaphase II oocytes undergo a time-dependent deterioration in vivo or in vitro, which is referred to as postovulatory oocyte aging, a process during which a series of deleterious molecular and cellular changes occur. In this study, we found that short-term injection of resveratrol (3,5,4'-trihydroxystilbene) effectively ameliorated oxidative stress-induced damage in postovulatory oocyte aging of middle-aged mice in vivo. Resveratrol induced changes that delayed the aging-induced oocyte deterioration including the elevated expression of the anti-aging molecule Sirtuin 1 (SIRT1); it reduced intracellular reactive oxygen species (ROS) level, and improved mitochondria function. In addition, these beneficial changes may also help to prevent apoptosis. Taken together, our data suggest that resveratrol can effectively protect against postovulatory oocyte aging in vivo primarily by preventing ROS production.

Chromosome segregation is driven by separase, activity of which is inhibited by binding to securin and cyclin B1/CDK1. In meiosis, premature separase activity will induce aneuploidy or abolish chromosome segregation owing to the untimely destruction of cohesin. Recently, we have proved that cyclin B2 can compensate for cyclin B1 in CDK1 activation for the oocyte meiosis G2/M transition. In the present study, we identify an interaction between cyclin B2/CDK1 and separase in mouse oocytes. We find that cyclin B2 degradation is required for separase activation during the metaphase I-anaphase I transition because the presence of stable cyclin B2 leads to failure of homologous chromosome separation and to metaphase I arrest, especially in the simultaneous absence of securin and cyclin B1. Moreover, non-phosphorylatable separase rescues the separation of homologous chromosomes in stable cyclin B2-arrested cyclin B1-null oocytes. Our results indicate that cyclin B2/CDK1 is also responsible for separase inhibition via inhibitory phosphorylation to regulate chromosome separation in oocyte meiosis, which may not occur in other cell types.

Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2β in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2β deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2β-deficient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our findings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF.

The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150 mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6 h, 12 h and 18 h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10 mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6 h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18 h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation.

Bone homeostasis is regulated by a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclastogenesis is controlled by the ratio of receptor activator of NF-κB ligand (RANKL) relative to its decoy receptor, osteoprotegerin (OPG). The source of OPG has historically been attributed to osteoblasts (OBs). While activated lymphocytes play established roles in pathological bone destruction, no role for lymphocytes in basal bone homeostasis in vivo has been described. Using immunomagnetic isolation of bone marrow (BM) B cells and B-cell precursor populations and quantitation of their OPG production by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), cells of the B lineage were found to be responsible for 64% of total BM OPG production, with 45% derived from mature B cells. Consistently B-cell knockout (KO) mice were found to be osteoporotic and deficient in BM OPG, phenomena rescued by B-cell reconstitution. Furthermore, T cells, through CD40 ligand (CD40L) to CD40 costimulation, promote OPG production by B cells in vivo. Consequently, T-cell–deficient nude mice, CD40 KO mice, and CD40L KO mice display osteoporosis and diminished BM OPG production. Our data suggest that lymphocytes are essential stabilizers of basal bone turnover and critical regulators of peak bone mass in vivo.

The bone wasting induced by estrogen deficiency is, in part, a consequence of increased T cell production of the osteoclastogenic cytokine TNF-α. This phenomenon is due to an expansion of T cells, but the responsible mechanism is unknown. We now show that ovariectomy (ovx) disregulates T lymphopoiesis and induces bone loss by stimulating, through a rise in IL-7 levels, both thymic-dependent differentiation of bone marrow-derived progenitors and thymic-independent, peripheral expansion of mature T cells. Attesting to the relevance of the thymic effects, thymectomy decreases by ≈50% the bone loss and the stimulation of T lymphopoiesis induced by ovx. In contrast,in vivoattenuation of the elevated IL-7 completely prevents the stimulation of T lymphopoiesis and the bone loss that follow ovx. Thus, the disruption of both T cell and bone homeostasis induced by ovx is mediated by IL-7 and due to both the thymic and extrathymic mechanisms. We conclude that IL-7 is a pivotal upstream target through which estrogen regulates hematopoietic and immune functions that are critical for bone homeostasis.

Estrogen (E) deficiency leads to an expansion of the pool of tumor necrosis factor (TNF)-producing T cells through an IFN-γ-dependent pathway that results in increased levels of the osteoclastogenic cytokine TNF in the bone marrow. Disregulated IFN-γ production is instrumental for the bone loss induced by ovariectomy (ovx), but the responsible mechanism is unknown. We now show that mice with T cell-specific blockade of type β transforming growth factor (TGFβ) signaling are completely insensitive to the bone-sparing effect of E. This phenotype results from a failure of E to repress IFN-γ production, which, in turn, leads to increased T cell activation and T cell TNF production. Furthermore, ovx blunts TGFβ levels in the bone marrow, and overexpression of TGFβin vivoprevents ovx-induced bone loss. These findings demonstrate that E prevents bone loss through a TGFβ-dependent mechanism, and that TGFβ signaling in T cells preserves bone homeostasis by blunting T cell activation. Thus, stimulation of TGFβ production in the bone marrow is a critical “upstream” mechanism by which E prevents bone loss, and enhancement of TGFβ levelsin vivomay constitute a previously undescribed therapeutic approach for preventing bone loss.

Background The adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported. Methods In this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER’s structural dynamics during oocyte maturation, fertilization and early embryo development. Results We report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice. Conclusions These results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development.

Oocyte aging severely decreases the quality of oocytes, which hampers fertilization and subsequent embryo development. In the present study, age-dependent molecular changes in goat oocytes were investigated. First, the quality of goat oocytes with various in vitro culture times (24, 30, 36, 48, and 60 hours) was evaluated on the basis of developmental rates of parthenogenetically activated embryos and apoptosis of cumulus cells (CCs). Second, relative gene expression of six genes (mitochondrial genes: PGC-1α and NRF-1; epigenetic modification genes: SNRPN and HAT1; mitotic spindle checkpoint protein: SMAD2; and hyaluronan synthase gene: HAS3) were analyzed during oocyte aging. Third, we further studied the changes of seven genes (PGC-1α and NRF-1; apoptotic-related genes: BAX and BCL2; hyaluronan synthase gene: HAS2; metabolism-related gene: STAR; and superoxide dismutase gene: SOD1) in CCs during oocyte aging. In these studies, the blastocyst rate gradually decreased and the number of apoptotic cells significantly increased as the culture time increased (P < 0.05). Moreover, relative gene expressions of PGC-1α, NRF-1 and SMAD2 significantly decreased from 24 to 36 hours (P < 0.05), whereas the levels of HAT1 and HAS3 slowly increased as culture was prolonged. Furthermore, the levels of PGC-1α, BCL2, HAS2 and SOD1 quickly reduced, and BAX significantly increased from 24 to 36 hours in aged CCs (P < 0.05). In conclusion, goat oocytes started to age at 30 hours in vitro culture, and gene expression patterns of oocytes and CCs significantly changed as the oocytes aged. Gene expression pattern changes in CCs may provide a convenient and effective way to detect oocyte aging without compromising oocyte integrity.

Expansion of the pool of tumor necrosis factor (TNF)-α-producing T cells is instrumental for the bone loss induced by estrogen deficiency, but the responsible mechanism is unknown. Here we show that ovariectomy up-regulates IFN-γ-induced class II transactivator, a multitarget immune modulator, resulting in increased antigen presentation by macrophages, enhanced T cell activation, and prolonged lifespan of active T cells. Up-regulation of class II transactivator derives from increased production of IFN-γ by T helper 1 cells, resulting from enhanced secretion of IL-12 and IL-18 by macrophages. The resulting T cell expansion and bone loss are preventedin vivoby both blockade of antigen presenting cell-induced T cell activation, and silencing of IFN-γ receptor signaling. Thus, increased IFN-γ-induced class II transactivator expression and the resulting enhanced T cell proliferation and lifespan are critical to the bone wasting effect of estrogen deficiency.

AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen. METHODS: Hepatitis B viral DNA was isolated from HBV carriers’ semen and sera using phenol extraction method and QIAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5 × 107 and 1.67 × 107 copies of HBV DNA per mL in two HBV infected patients’ sera, while 2.14 × 105 and 3.02 × 105 copies of HBV DNA per mL in the semen. CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.