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.

Transient inhibition of CDK2 activity prevents oocyte meiosis I completion and egg activation in mouse.

Mammalian oocytes are arrested at the diplotene stage of prophase I during fetal or postnatal development. It was reported that cyclin-dependent kinases (CDK1) was the sole CDK to drive the resumption of meiosis and CDK2 was dispensable for meiosis progression in mouse oocytes according to the conditional knockout studies. However, a recent study showed that CDK2 activity is essential for meiotic division and gametogenesis by means of gene-directed mutagenesis, which avoids the compensatory activation of other CDKs. Taken the compensatory effect between CDKs after gene knockout, the physiological function of CDK2 activity in oocyte maturation remains unclear. To address this issue, we applied a specific small-molecule inhibitor to restrain CDK2 activity transiently during oocyte meiotic maturation. Surprisingly, transient inhibition of CDK2 activity severely prevented the meiosis I completion although the meiotic resumption was not affected. Then we found that CDK2 activity was required for establishment of normal spindle and chromosome dynamics. Notably, CDK2 inhibition interrupted the anaphase-promoting complex/cyclosome (APC/C)-dependent degradation pathway by maintaining the activation of spindle assembly checkpoint (SAC). Interestingly, CDK2 inhibition prevented the egg activation as well. Overall, our data demonstrate that CDK2 kinase activity is required for proper dynamics of spindle and chromosomes, whose disturbance induces the continuous SAC activation and subsequent inactivation of APC/C activity in oocyte meiosis.

The cyclin B2/CDK1 complex inhibits separase activity in mouse oocyte meiosis I.

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.

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte.

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.

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes.

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

Cyclins regulating oocyte meiotic cell cycle progression†.

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.

[Penile frenulum lengthening for premature ejaculation].

OBJECTIVE: To evaluate the effect of penile frenulum lengthening in the treatment of premature ejaculation (PE). METHODS: Thirty-four males with PE were enrolled in this study, of whom 8 had received circumcision six months before and 4 had redundant prepuce, all with short frenulum. Those with a history of circumcision underwent reconstruction and lengthening of the frenulum, and those without received frenulum lengthening only. RESULTS: Compared with the baseline, the intravaginal ejaculation latency time (IELT) was significantly increased at 1 month after operation ([1.35 ± 0.49] vs [5.71 ± 2.69] min, t = -9.42, P <0.01), (1.42 ± 0.5) vs (5.31 ± 2.74) min in the patients without circumcision (t = -7.41, P <0.01), (1.12 ± 0.35) vs (7.00 ± 2.20) min in those with circumcision (t = -7.24, P <0.01), and (1.50 ± 0.58) vs (4.75 ± 1.71) min in those with redundant prepuce (t = -3.81, P <0.05). Totally, 94% of the patients were satisfied with their sexual intercourse postoperatively. CONCLUSION: Penile frenulum plays an important role in penile erection. Reconstruction and/or lengthening of the frenulum can prolong penile erection and IELT in PE patients.

Vinorelbine administration impedes the timely progression of meiotic maturation and induces aneuploidy in mouse oocytes.

Vinorelbine is a commonly used drug to treat various malignancies, such as breast cancer, non-small cell lung cancer, and metastatic pleural mesothelioma. Its side effects include severe neutropenia, local phlebitis, gastrointestinal reactions, and neurotoxicity. In view of the scarcity of research on vinorelbine's reproductive toxicity, this study evaluated the impact of vinorelbine ditartrate, a commonly used form of vinorelbine, on oocyte maturation in vitro. Our investigation revealed that vinorelbine ditartrate had no effect on oocyte meiotic resumption. However, it did reduce the rate of first polar body extrusion, suggesting that it could significantly impede the meiotic maturation of oocytes. Vinorelbine ditartrate exposure was found to disturb the regular spindle assembly and chromosome alignment, leading to the continuous activation of the spindle assembly checkpoint (SAC) and a delayed activation of the anaphase-promoting complex/cyclosome (APC/C), ultimately causing aneuploidy in oocytes. Consequently, the administration of vinorelbine is likely to result in oocyte aneuploidy, which can be helpful in providing a drug reference and fertility guidance in a clinical context.

Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development.

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.

[Study on microRNA expression in endometrium of luteal phase and its relationship with infertility of endometriosis].

OBJECTIVE: To study the different expression of microRNA(miRNA) including mir-29c, mir-200a, mir-145 in the mid-secretary endometrium and its relationship with infertility of endometriosis. METHODS: From August 2011 to February 2013, 36 infertile cases with endometriosis confirmed by laparoscopy and pathology and excluded the other infertile factors in Department of Reproductive Medicine in Peking University Shenzhen Hospital were enrolled in this study, which were divided into 17 cases with stage I-II, 19 cases with stage III-IV according to the revised classification American Fertility Society. Forty-four healthy women with male factor infertility were chosen as control group. The relative expression levels of mir-29c, mir-200a, mir-145 in the endometrium of women in two groups were detected by using real-time quantitative polymerase chain reaction. Those women were followed up for pregnancy outcome of endometriosis group after assisted reproductive techniques (pregnancy and non-pregnancy group respectively). RESULTS: (1) The expression of miRNA between endometriosis and control groups:the average expression level of mir-29c, mir-200a, mir-145 in the endometrium of endometriosis group was 2.46 ± 1.98, 3.20 ± 2.45, 6.378 ± 3.275, which were significantly higher than 1.36 ± 1.05, 2.04 ± 1.16, 4.548 ± 1.885 in control group (P = 0.026, 0.027, 0.041, respectively). (2) The expression of miRNA between I-II stage and III-IV: the average expression level of mir-29c, mir-200a, mir-145 in the endometrium was 0.53 ± 0.51, 0.33 ± 0.26, 0.048 ± 0.021 in patients with of stage I-II, while 0.26 ± 0.18, 0.28 ± 0.12, 0.045 ± 0.016 in stage III-IV respectively, there were no statistically significant differences between the two groups (P = 0.191,0.661,0.753, respectively) . (3) The expression of miRNA between pregnancy and non-pregnancy groups:the average expression level of mir-29c, mir-200a, mir-145 in the endometrium were 0.60 ± 0.30, 1.23 ± 0.48, 0.886 ± 0.238 in pregnancy group, while 2.64 ± 1.73, 4.39 ± 2.58, 7.199 ± 3.945 in non pregnancy group, there were statistically significant differences between the two groups (P = 0.030,0.039,0.028, respectively). CONCLUSION: Up-regulation of mir-29c, mir-200a, mir-145 in the endometrial tissue might play a role in endometriosis associated infertility.

SRSF1-mediated alternative splicing is required for spermatogenesis.

Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.

Effectiveness of non-invasive chromosomal screening for normal karyotype and chromosomal rearrangements.

Purpose: To study the accuracy of non-invasive chromosomal screening (NICS) results, in normal chromosomes and chromosomal rearrangement groups and to investigate whether using trophoblast cell biopsy along with NICS, to choose embryos for transfer can improve the clinical outcomes of assisted pregnancy. Methods: We retrospectively analyzed 101 couples who underwent preimplantation genetic testing at our center from January 2019 to June 2021 and collected 492 blastocysts for trophocyte (TE) biopsy. D3-5 blastocyst culture fluid and blastocyst cavity fluid were collected for the NICS. Amongst them, 278 blastocysts (58 couples) and 214 blastocysts (43 couples) were included in the normal chromosomes and chromosomal rearrangement groups, respectively. Couples undergoing embryo transfer were divided into group A, in which both the NICS and TE biopsy results were euploid (52 embryos), and group B, in which the TE biopsy results were euploid and the NICS results were aneuploid (33 embryos). Results: In the normal karyotype group, concordance for embryo ploidy was 78.1%, sensitivity was 94.9%, specificity was 51.4%, the positive predictive value (PPV) was 75.7%, and the negative predictive value (NPV) was 86.4%. In the chromosomal rearrangement group, concordance for embryo ploidy was 73.1%, sensitivity was 93.3%, specificity was 53.3%, the PPV was 66.3%, and the NPV was 89%. In euploid TE/euploid NICS group, 52 embryos were transferred; the clinical pregnancy rate was 71.2%, miscarriage rate was 5.4%, and ongoing pregnancy rate was 67.3%. In euploid TE/aneuploid NICS group, 33 embryos were transferred; the clinic pregnancy rate was 54.5%, miscarriage rate was 5.6%, and ongoingpregnancy rate was 51.5%. The clinical pregnancy and ongoing pregnancy rates were higher in the TE and NICS euploid group. Conclusion: NICS was similarly effective in assessing both normal and abnormal populations. Identification of euploidy and aneuploidy alone may lead to the wastage of embryos due to high false positives. More suitable reporting methods for NICS and countermeasures for a high number of false positives in NICS are needed. In summary, our results suggest that combining biopsy and NICS results could improve the outcomes of assisted pregnancy.

An endometrial receptivity scoring system basing on the endometrial thickness, volume, echo, peristalsis, and blood flow evaluated by ultrasonography.

Background: Establishing a successful pregnancy depends on the endometrium and the embryo. It is estimated that suboptimal endometrial receptivity account for one-third of implantation failures. Despite the indepth understanding of the processes associated with embryo-endometrial cross-talk, little progress has been achieved for diagnosis and treatments for suboptimal endometrial receptivity. Methods: This retrospective study included women undergoing their first frozen-thawed embryo transfer (FET) cycles at our reproductive medicine center from March 2021 to August 2021. Transvaginal three-dimensional (3D) ultrasound was performed in the morning on the day of embryo transfer for all the thawed embryo transfer patients, to evaluate endometrial receptivity, including endometrial thickness, echogenicity, volume, movement and blood flow. Results: A total number of 562 patients of FET with 315 pregnancies (56.0%) was analyzed. It was found that only the echo of the endometrial central line was different between the pregnant group and non-pregnant group. Other parameters, such as endometrial thickness, volume, endometrial peristalsis, or the endometrial blood flow were not statistically different between the two groups. Then, according to the relationship between the different groups and the clinical pregnancy rate, a score of 0 to 2 was respectively scored. The sum of the scores for the six items was the patient's endometrial receptivity score. It showed that the clinical pregnancy rate increased as the endometrial receptivity score increased, and when the receptivity score reaches at least 5, the clinical pregnancy rate is significantly improved (63.7% versus 49.5%, P=0.001). Conclusion: We developed an endometrial receptivity scoring system and demonstrated its validity. It may aid clinicians in choosing the useful marker in clinical practice and for informing further research.

Inhibition of NADPH Oxidase-ROS Signal using Hyaluronic Acid Nanoparticles for Overcoming Radioresistance in Cancer Therapy.

Upregulation of NADPH oxidases (NOXs) in cancer cells leads to chronic increase in intracellular reactive oxygen species (ROS) and adaptation to a high ROS level for cell survival and, thereby, low sensitivity to radiotherapy. To overcome resistance to radiotherapy, we have developed a bioactive and CD44 targeted hyaluronic acid nanoparticle encapsulated with an NOX inhibitor, GKT831 (HANP/GKT831). We found that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 alone in cancer cells. Systemic delivery of HANP/GKT831 led to the targeted accumulation in breast cancer patient derived xenograft (PDX) tumors in nude mice. Importantly, the combination of systemic delivery of HANP/GKT831 with a low dose of local radiotherapy significantly enhanced tumor growth inhibition in breast cancer PDX models. Our results showed that HANP/GKT831 primed tumor cells to radiation-induced DNA damage and cell death by downregulation of DNA repair function and oncogenic signal pathways.

[Abnormal expression and significance of MIR-184 in human renal carcinoma].

OBJECTIVE: To explore the role of microRNA-184(MIR-184) in the development of renal cell carcinoma(RCC). METHODS: The expressions of MIR-184 in 51 patients with RCC Investigated, normal adjacent tissues (ADTs) matched by fluorescence quantitative PCR technology (RT-qPCR) and the correlations analyzed between MIR-184 expression and the age, gender and clinical stage of RCC patients. RESULTS: The average expression of MIR-184 in RCC was -14.664 6 ± 5.362 4, while that in ADTs was -10.408 7 ± 3.482 7(P<0.01). Bounded with the MIR-184 expression in RCC, patients were divided into lower-expression group and higher-expression group. Meanwhile, the RCC patients were divided into three groups according to the age, gender and clinical stage of the patients. Chi-square statistical analysis showed that the expression level of MIR-184 was not significantly correlated with the patient's age, gender and clinical stage (respectively: P>0.03, P>0.99, P>0.03). CONCLUSION: MIR-184 in RCC was significantly lower than that in ADTs, which may have potential significance in the occurrence and development of RCC.

Critical Functions of PP2A-Like Protein Phosphotases in Regulating Meiotic Progression.

Meiosis is essential to the continuity of life in sexually-reproducing organisms through the formation of haploid gametes. Unlike somatic cells, the germ cells undergo two successive rounds of meiotic divisions after a single cycle of DNA replication, resulting in the decrease in ploidy. In humans, errors in meiotic progression can cause infertility and birth defects. Post-translational modifications, such as phosphorylation, ubiquitylation and sumoylation have emerged as important regulatory events in meiosis. There are dynamic equilibrium of protein phosphorylation and protein dephosphorylation in meiotic cell cycle process, regulated by a conservative series of protein kinases and protein phosphatases. Among these protein phosphatases, PP2A, PP4, and PP6 constitute the PP2A-like subfamily within the serine/threonine protein phosphatase family. Herein, we review recent discoveries and explore the role of PP2A-like protein phosphatases during meiotic progression.

The Cyclin B2/CDK1 Complex Conservatively Inhibits Separase Activity in Oocyte Meiosis II.

Recently, we have reported that the cyclin B2/CDK1 complex regulates homologous chromosome segregation through inhibiting separase activity in oocyte meiosis I, which further elucidates the compensation of cyclin B2 on cyclin B1's function in meiosis I. However, whether cyclin B2/CDK1 complex also negatively regulates separase activity during oocyte meiosis II remains unknown. In the present study, we investigated the function of cyclin B2 in meiosis II of oocyte. We found that stable cyclin B2 expression impeded segregation of sister chromatids after oocyte parthenogenetic activation. Consistently, stable cyclin B2 inhibited separase activation, while introduction of non-phosphorylatable separase mutant rescued chromatid separation in the stable cyclin B2-expressed oocytes. Therefore, the cyclin B2/CDK1 complex conservatively regulates separase activity via inhibitory phosphorylation of separase in both meiosis I and meiosis II of mouse oocyte.

Measurement of Bone Metastatic Tumor Growth by a Tibial Tumorigenesis Assay.

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

Toxic effects of patulin on mouse oocytes and its possible mechanisms.

Patulin is a secondary metabolite mainly secreted by fungi and is the most common mycotoxin found in apples and apple-based products. For the past few years, numerous studies suggested the wide distribution and toxicity of patulin. In this study, we investigated the toxic effect of patulin on mouse oocytes and its possible mechanisms. The results showed that patulin treatment did not affect meiotic resumption, but inhibited oocyte maturation as indicated by failure of first polar body extrusion. Further mechanistic study showed that patulin treatment disturbed normal spindle assembly, chromosome alignment and morphology. We also found increased oxidative stress by testing the level of ROS and decreased mitochondrial membrane potential, indicating mitochondria dysfunction. In summary, our results suggest that patulin treatment causes oocyte meiotic arrest by disturbing normal spindle assembly and chromosome alignment, which may be caused by dysfunctions of mitochondria.

Cell Division Cycle 5-Like Regulates Metaphase-to-Anaphase Transition in Meiotic Oocyte.

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.