Fangchinoline inhibits mouse oocyte meiosis by disturbing MPF activity.

Fangchinoline (FA) is an alkaloid derived from the traditional Chinese medicine Fangji. Numerous studies have shown that FA has a toxic effect on various cancer cells, but little is known about its toxic effects on germ cells, especially oocytes. In this study, we investigated the effects of FA on mouse oocyte maturation and its potential mechanisms. Our results showed that FA did not affect meiosis resumption but inhibited the first polar body extrusion. This inhibition is not due to abnormalities at the organelle level, such as chromosomes and mitochondrial, which was proved by detection of DNA damage and reactive oxygen species. Further studies revealed that FA arrested the oocyte at the metaphase I stage, and this arrest was not caused by abnormal kinetochore-microtubule attachment or spindle assembly checkpoint activation. Instead, FA inhibits the activity of anaphase-promoting complexes (APC/C), as evidenced by the inhibition of CCNB1 degeneration. The decreased activity of APC/C may be due to a reduction in CDC25B activity as indicated by the high phosphorylation level of CDC25B (Ser323). This may further enhance Maturation-Promoting Factor (MPF) activity, which plays a critical role in meiosis. In conclusion, our study suggests that the metaphase I arrest caused by FA may be due to abnormalities in MPF and APC/C activity.

Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Insights into the adverse effects of prepubertal chronic ethanol exposure on adult female reproduction.

Heavy drinking in women is known to adversely affect pregnancy and fertility. However, pregnancy is a complex process, and the adverse effects of ethanol on pregnancy does not mean that ethanol will have adverse effects on all stages from gamete to fetal formation. Similarly, the adverse effects of ethanol before and after adolescence cannot be generalized. To focus on the effects of prepubertal ethanol on female reproductive ability, we established a mouse model of prepubertal ethanol exposure by changing drinking water to 20% v/v ethanol. Some routine detections were performed on the model mice, and details such as mating, fertility, reproductive organ and fetal weights were recorded day by day after discontinuation of ethanol exposure. Prepubertal ethanol exposure resulted in decreased ovarian weight and significantly reduced oocyte maturation and ovulation after sexual maturation, however, normal morphology oocytes with discharged polar body showed normal chromosomes and spindle morphology. Strikingly, oocytes with normal morphology from ethanol exposed mice showed reduced fertilization rate, but once fertilized they had the ability to develop to blastocysts. RNA-seq analysis showed that the gene expression of the ethanol exposed oocytes with normal morphology had been altered. These results show the adverse effects of prepubertal alcohol exposure on adult female reproductive health.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

MAPRE2 regulates the first meiotic progression in mouse oocytes.

Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, cell migration and morphogenesis. Microtubule-associated RP/EB family member 2 (MAPRE2/EB2) is a highly conserved core component of +TIPs networks, but whether this molecule is required for mammalian meiotic progression is unknown. In this study, we investigated the expression and function of MAPRE2 during oocyte maturation. Our results showed that MAPRE2 was consistently expressed from germinal vesicle (GV) to metaphase II (MII) stages and that MAPRE2 was distributed in the cytoplasm of oocytes at GV stage and along the spindle at metaphase I (MI) and MII stages. Small interfering RNA-mediated knockdown of Mapre2 severely impaired microtubule stability, kinetochore-microtubule attachment, and chromosome alignment and subsequently caused spindle assembly checkpoint (SAC) activation and cyclin B1 nondegradation, leading to failure of chromosome segregation and first polar body extrusion. This study demonstrates for the first time that MAPRE2 plays an important role during mouse oocyte meiosis.

Perinatal outcomes of singleton live births after preimplantation genetic testing during single frozen-thawed blastocyst transfer cycles: a propensity score-matched study.

OBJECTIVE: To determine whether singleton pregnancy achieved after preimplantation genetic testing (PGT) is associated with a higher risk of adverse perinatal outcomes than in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) singleton pregnancy. DESIGN: A retrospective cohort study. SETTING: A university-affiliated fertility center. PATIENT(S): This cohort study included singleton live births resulting from PGT (n = 232) and IVF/ICSI singleton pregnancies (n = 2,829) with single frozen-thawed blastocyst transfer. Multiple baseline covariates were used for propensity score matching, yielding 214 PGT singleton pregnancies matched to 617 IVF/ICSI singleton pregnancies. INTERVENTION(S): Trophectoderm biopsy. MAIN OUTCOME MEASURE(S): The primary outcome was gestational hypertension, and various clinical perinatal secondary outcomes related to maternal and neonatal health were measured. RESULT(S): Compared with IVF/ICSI singleton pregnancy, PGT singleton pregnancy was associated with a significantly higher risk of gestational hypertension (adjusted odds ratio, 2.58; 95% confidence interval, 1.32, 5.05). In the matched sample, the risk of gestational hypertension remained higher with PGT singleton pregnancy (odds ratio, 2.33; 95% confidence interval, 1.04, 5.22) than with IVF/ICSI singleton pregnancy. No statistical differences were noted in any other measured outcomes between the groups. CONCLUSION(S): The perinatal outcomes of PGT and IVF/ICSI singleton pregnancies were similar except for the observed potentially higher risk of gestational hypertension with PGT singleton pregnancy. However, because the data on PGT singleton pregnancies are limited, this conclusion warrants further investigation.

Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis.

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 ß-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal ß-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 ß-catenin at S552.

FBXO34 Regulates the G2/M Transition and Anaphase Entry in Meiotic Oocytes.

There are two important events in oocyte meiotic maturation, the G2/M transition and metaphase I progression. Thousands of proteins participate in regulating oocyte maturation, which highlights the importance of the ubiquitin proteasome system (UPS) in regulating protein synthesis and degradation. Skp1-Cullin-F-box (SCF) complexes, as the best characterized ubiquitin E3 ligases in the UPS, specifically recognize their substrates. F-box proteins, as the variable adaptors of SCF, can bind substrates specifically. Little is known about the functions of the F-box proteins in oocyte maturation. In this study, we found that depletion of FBXO34, an F-box protein, led to failure of oocyte meiotic resumption due to a low activity of MPF, and this phenotype could be rescued by exogenous overexpression of CCNB1. Strikingly, overexpression of FBXO34 promoted germinal vesicle breakdown (GVBD), but caused continuous activation of spindle assembly checkpoint (SAC) and MI arrest of oocytes. Here, we demonstrated that FBXO34 regulated both the G2/M transition and anaphase entry in meiotic oocytes.

The methylation status in GNAS clusters May Be an epigenetic marker for oocyte quality.

During follicle growth, DNA methylation is gradually established, which is important for oocyte developmental competence. Due to the facts that oocytes from prepubertal individuals show reduced developmental outcomes when compared to those from sexually mature individuals, and the fact that oocytes derived from in vitro follicle culture have much lower developmental competence, it is worth exploring whether prepubertal superovulation and in vitro follicle culture will cause changes in DNA methylation imprinting status in oocytes. In this study, we found that the CpG island in maternally imprinted GNAS clusters was hypermethylated in the MII-stage oocytes from sexually mature mice, but was hypomethylated in oocytes from prepuberty individuals. The GNAS clusters in the MII-stage oocytes obtained by in vitro follicle culture showed heterogeneous methylation levels, indicating different qualities of oocytes, however, three other maternally imprinted genes, Peg1, Lot1 and Impact, were all hypermethylated in the MII-stage oocytes derived from both prepubertal superovulation and in vitro follicle culture. Taken together, the findings suggest that the methylation status in GNAS clusters may potentially represent a novel epigenetic marker for oocyte quality detection.