WDR87 interacts with CFAP47 protein in the middle piece of spermatozoa flagella to participate in sperm tail assembly.

Spermatogenesis is a complex process that includes spermatogonia self-renewal, spermatocyte meiosis and spermatozoa assembly. Recent studies have revealed that WD40-repeat domain-containing (WDR) proteins play important roles in spermatocyte division, spermatozoa flagella assembly and head shaping. In this study, we investigated the expression pattern of WDR87 and found that it was highly expressed in the testis of both humans and mice. Immunofluorescence staining revealed that mouse WDR87 was distributed in the perinuclear cytoplasm of primary spermatocytes, secondary spermatocytes and round spermatids. In the spermiogenesis stage, with extension of the nucleus, WDR87 migrated to the manchette and finally localized to the middle piece of the spermatozoa tail. Furthermore, we identified a cilia- and flagella-associated protein, CFAP47, which interacted with WDR87 in the flagellar midpiece of the spermatozoa, suggesting that WDR87 may be associated with multiple morphological abnormalities of the flagella (MMAF). Subsequently, we screened gene mutations in seven MMAF individuals and found two novel mutations in CFAP47 (c.706G>A, Val236Met; c.1337C>T, Thr446Met) in one case. Immunoblotting and immunofluorescence revealed that CFAP47 was dramatically reduced in spermatozoa from the CFAP47-mutated man. Meanwhile, the expression of WDR87 was also significantly decreased, and weak signals were detected adjacent to the spermatozoa nuclei, indicating that CFAP47 was necessary for WDR87 transportation during spermatozoa flagella biogenesis. These data indicate that WDR87 is located in the middle piece of the sperm tail and interacts with CFAP47 to form a complex which is involved in spermatozoa tail assembly.

CircRNA circUSP36 impairs the stability of NEDD4L mRNA through recruiting PTBP1 to enhance ULK1-mediated autophagic granulosa cell death.

BACKGROUND: Hypercholesterolemia is defined as a high risk factor for causing female infertility by changing the cholesterol level in granulosa cells to impair the microenvironment of oocyte development and maturation. High blood levels of oxidized low-density lipoprotein (ox-LDL) undergoes an increase of autophagic granulosa cell death. Unfortunately, this underlying molecular mechanism remains largely elusive. We aim to uncover the role of circ-ubiquitin specific peptidase 36 (USP36) in autophagic granulosa cell death. METHODS: Exposure of ox-LDL on the ovarian granulosa cell-like human granulosa (KGN) cells line was established for simulating the situation of hypercholesterolemia in vitro. Levels of circUSP36 and ULK1 were detected using real-time polymerase chain reaction (RT-PCR). Cell viability and apoptosis were assessed using (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunofluorescence staining of LC3 was performed to evaluate activity of autophagy. Western blot was employed to determine expression of apoptosis and autophagy-associated markers. RNA immunoprecipitation (RIP) and RNA pull-down assays were subjected to verify the circUSP36-PTBP1-NEDD4L regulatory axis. RESULTS: Treatment of ox-LDL induced aberrantly up-regulated circUSP36. Knockdown of circUSP36 alleviated cell apoptosis and excessive autophagy of granulosa cells triggered by ox-LDL. Mechanistically, reinforced expression of circUSP36 guided and facilitated PTBP1 binding to the coding region (CDS) of NEDD4L, resulting in NEDD4L mRNA decay. ULK1 was regulated by the circUSP36-PTBP1-NEDD4L axis in granulosa cells, thereby contributing to autophagic granulosa cell death. CONCLUSIONS: In summary, ox-LDL fostered autophagic granulosa cell death through circUSP36-mediated NEDD4L mRNA decay, thus elevating ULK1 expression.

The KIDScore™ D3 scoring system contributes to the prediction of embryonic development potential: A promising tool for screening high-quality embryos.

Using the KIDScoreTM D3 (KID3) scoring system, day 3 embryos observed by time-lapse imaging (TLI) were scored to explore the predictive value of the KID scoring system on the developmental potential of embryos. The kinetic parameters of 477 normal fertilized embryos from 77 patients who underwent TLI in our hospital from January 2019 to June 2020 were evaluated by KID3, and the embryos were divided into five groups according to the scores for retrospective analysis of blastocyst formation. Additionally, the high-quality blastocyst formation rate, pregnancy rate and early abortion rate were analyzed via KID3 and traditional morphological assessments, and comparisons of differences among different ages were also performed. In the KID3 estimate, the blastocyst or high-quality blastocyst formation rate in the score 5 group was markedly higher than that in the score 1-4 groups. Blastocyst or high-quality blastocyst formation rates in the A group (the results of two evaluation tools indicated they were excellent embryos) and the B group (KID3: excellent embryos, traditional evaluation: not excellent embryos) were evidently increased in comparison with the C or D group (KID3: not excellent embryos, traditional evaluation: excellent embryo or not, respectively). Furthermore, the percentages of score 5 embryos, blastocyst and high-quality blastocyst formation rates for patients ≥ 35 years old were markedly decreased compared with those for patients < 34 years old, while the trends of nondiploid cleavage, multinucleation and asymmetric division were the opposite. Collectively, the KID3 scoring system may be a promising predictive tool for screening embryos with better developmental potential.

Vigilin interacts with ER-ß to protect against palmitic acid-induced granulosa cells apoptosis via inhibiting calcineurin-mediated Drp1 signaling pathway.

BACKGROUND: Hypercholesterolemia is one of the causes of female infertility, and as a common fatty acid in follicular fluid, palmitic acid (PA) level plays a vital role in granule cell which is closely related to the developmental potential of follicle. METHODS: The ovarian granulosa cell-like human granulosa (KGN) cell line and the immortalized normal ovarian surface epithelial cell line (IOSE80) were used to verify the effect of PA on cell viability and apoptosis by MTT and flow cytometry assay, respectively. Then mitochondria damage was confirmed by mitochondrial membrane potential, mitochondrial ROS detection assay and western blot in KGN cells. Thorough luciferase reporter assay and RIP-qPCR, the relationship between vigilin and ER-ß was investigated. RESULTS: In our study, PA induced mitochondrial damage-mediated cell apoptosis of KGN cells was dose-dependently, while PA shown no effects on in IOSE80 cells. Then the role of calcineurin (CnA)-mediated Drp1 signaling pathway on KGN cells was confirmed by treating with Mdivi-1 or FK506T. In addition, the changed level of vigilin and ER-ß was observed in cell apoptosis of KGN cells induced by PA. By transfecting vigilin vector or ER-ß vector into KGN cells, respectively, vigilin and ER-ß were demonstrated to regulate the apoptosis of KGN cells. And vigilin was a binding protein of ER-ß mRNA. CONCLUSION: Vigilin could interact with ER-ß mRNA to promote ER-ß expression. And Vigilin/ ER-ß relieve the mitochondrial damage and cell apoptosis induced by PA through regulating CnA-mediated Drp1 signaling pathway, which revealed the mechanism and strategy of hypercholesterolemia in female infertility.

The role of mTOR in ovarian Neoplasms, polycystic ovary syndrome, and ovarian aging.

The mammalian target of rapamycin, mTOR, is a serine-threonine protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-AKT axis. The pathway can regulate cell growth, proliferation, and survival by activating ribosomal kinases. Recent studies have implicated the mTOR signaling pathway in ovarian neoplasms, polycystic ovary syndrome (PCOS) and premature ovarian failure (POF). Preclinical investigations have demonstrated that the PI3K/AKT/mTOR pathway is frequently activated in the control of various ovarian functions. mTOR allows cancer cells to escape the normal biochemical system and regulates the balance between apoptosis and survival. Some recent studies have suggested that involvement of the mTOR signaling system is an important pathophysiological basis of PCOS. Overexpression of the mTOR pathway can impair the interaction of cumulus cells, lead to insulin resistance, and affect the growth of follicles directly. The roles of mTOR signaling in follicular development have been extensively studied in recent years; abnormalities in this process lead to a series of pathologies such as POF and infertility. To improve understanding of the role of the mTOR signaling pathway in the pathogenesis and development of ovarian diseases, here we review the roles of mTOR signaling in such diseases and discuss the corresponding therapeutic strategies that target this pathway. Clin. Anat. 31:891-898, 2018. © 2018 Wiley Periodicals, Inc.

The role of FTO variants in the susceptibility of polycystic ovary syndrome and in vitro fertilization outcomes in Chinese women.

We investigated the association between single nucleotide polymorphisms (SNPs) in the fat mass and obesity associated (FTO) gene (rs9926289 A/G, rs79206939 A/G, rs9930506 A/G, rs8050136 A/C, and rs1588413 C/T) and polycystic ovary syndrome (PCOS), as well as outcomes of in vitro fertilization (IVF). A case-control study consisting of 147 PCOS patients and 120 healthy controls was conducted. FTO SNPs were genotyped by PCR to determine allelic frequencies, and IVF outcomes were analyzed. The results showed that FTO rs8050136 (p = .025) and rs1588413 (p = .042) were significantly associated with PCOS susceptibility, and women with risk alleles were often found to be obese (p < .05). For SNP rs8050136, women with AA + AC genotypes had higher body mass indexes (BMIs), oral glucose tolerance test/2 h (OGTT) levels and implantation rates but lower follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) day progesterone levels and ovulation numbers (all p < .05) than those with the CC genotype. For SNP rs1588413, women carrying risk alleles exhibited higher BMIs, implantation rate, and levels of luteinizing hormone (LH), estradiol, and OGTT/2 h (all p < .05) compared with those with non-risk genotypes. Therefore, these findings suggest that rs8050136 and rs1588413 are associated with PCOS susceptibility, and that women with risk alleles have less ovulation numbers but higher implantation rates than those with other genotypes.

Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells.

PURPOSE: This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes. METHODS: After sequential culture for 5-6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis. RESULTS: The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P < 0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes. CONCLUSIONS: Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.

New Insights into mTOR Signal Pathways in Ovarian-Related Diseases: Polycystic Ovary Syndrome and Ovarian Cancer

mTOR, the mammalian target of rapamycin, is a conserved serine/threonine kinase which belongs to the phosphatidyl-linositol kinase-related kinase (PIKK) family. It has two complexes called mTORC1 and mTORC2. It is well established that mTOR plays important roles in cell growth, proliferation and differentiation. Over-activation of the mTOR pathway is considered to have a relationship with the development of many types of diseases, including polycystic ovary syndrome (PCOS) and ovarian cancer (OC). mTOR pathway inhibitors, such as rapamycin and its derivatives, can directly or indirectly treat or relieve the symptoms of patients suffering from PCOS or OC. Moreover, mTOR inhibitors in combination with other chemical-molecular agents may have extraordinary efficacy. This paper will discuss links between mTOR signaling and PCOS and OC, and explore the mechanisms of mTOR inhibitors in treating these two diseases, with conclusions regarding the most effective therapeutic approaches.

[Research on the selection of embryos for transfer using a combined grading for embryo growth rate and morphology and zygote pronuclear morphology].

OBJECTIVE: To evaluate the value of using a combined grading for embryo growth rate and morphology and zygote pronuclear morphology, and to select embryos for transfer in clinical in-vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI). METHODS: In this retrospective study, a total of 2714 normal zygotes (2 pronuclei) from 434 treatment cycles with IVF/ICSI was analyzed between May 2003 and December 2003. These zygotes were divided into two groups (synchronous group and nonsynchronous group) according to the developmental synchrony of pronuclei. The developmental potential of zygotes from these two groups was observed. Embryos for transfer were selected initially by embryo growth rate and morphology, secondarily by zygote grade. The clinical pregnancy rates and implantation rates were compared according to whether the embryos from synchronous group were transferred. RESULTS: There were 2714 normal zygotes, the good-quality embryos (> or = 6 cells, grade I - II) rate of the synchronous group (41.88%, 743/1774) was significantly higher (P < 0.001) than the nonsynchronous group (33.94%, 319/940). Totally 395 cycles transferred at least one embryo from synchronous group, the clinical pregnancy rate was 47.85% (189/395) and implantation rate was 27.49% (273/993). There was no significant difference from 39 cycles which did not transfer embryos from nonsynchronous group. The clinical pregnancy rate was 43.59% (17/39) and implantation rate was 25.00% (21/84). CONCLUSION: The data indicate that there are no significant differences in pregnancy rates and implantation rates between different zygote grade when selecting embryos for transfer by combined grading.