Molecular Reproduction and Development最新文献

Echinoderms produce functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes remains a mystery. Here, we present the first single cell RNA sequencing data sets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus [Sp] and Lytechinus variegatus [Lv]), and one sea star species (Patiria miniata [Pm]). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum.

We have investigated whether inflammasomes and pyroptosis are activated in maternal nicotine exposure (MNE) offspring mice and whether they are involved in MNE-promoted metabolic associated fatty liver disease (MAFLD) in adult offspring. We injected pregnant mice subcutaneously with saline vehicle or nicotine twice a day on gestational days 11–21. Offspring mice from both groups were fed with a normal diet (ND) or a high-fat diet (HFD) for 6 months at postnatal day 21 to develop the MAFLD model. Serum biochemical indices were analyzed, and liver histology was performed. The expression levels of inflammasome and pyroptosis proteins were detected by western blot. We found MNE significantly aggravated the injury of MAFLD in adult offspring mice. MNE activated inflammasomes and pyroptosis in both infant and adult offspring mice. HFD treatment activated inflammasomes but not pyroptosis at 3 months, while it showed no effect at 6 months. However, pyroptosis was more severe in MNE-HFD mice than in MNE-ND mice at 6 months. Taken together, our data suggest MNE promotes MAFLD progression in adult offspring mice. MNE also induces NLRP3 and NLRP6 inflammasome activation and pyroptosis in both infant and adult offspring mice, which may be involved in MNE-promoted progression of MAFLD.

The process of aging refers to physiological changes that occur to an organism as time progresses and involves changes to DNA, proteins, metabolism, cells, and organs. Like the rest of the cells in the body, gametes age, and it is well established that there is a decline in reproductive capabilities in females and males with aging. One of the major pathways known to be involved in aging is epigenetic changes. The epigenome is the multitude of chemical modifications performed on DNA and chromatin that affect the ability of chromatin to be transcribed. In this review, we explore the effects of aging on female and male gametes with a focus on the epigenetic changes that occur in gametes throughout aging. Quality decline in oocytes occurs at a relatively early age. Epigenetic changes constitute an important part of oocyte aging. DNA methylation is reduced with age, along with reduced expression of DNA methyltransferases (DNMTs). Histone deacetylases (HDAC) expression is also reduced, and a loss of heterochromatin marks occurs with age. As a consequence of heterochromatin loss, retrotransposon expression is elevated, and aged oocytes suffer from DNA damage. In sperm, aging affects sperm number, motility and fecundity, and epigenetic changes may constitute a part of this process. 5 methyl-cytosine (5mC) methylation is elevated in sperm from aged men, but methylation on Long interspersed nuclear elements (LINE) elements is reduced. Di and trimethylation of histone 3 lysine 9 (H3K9me2/3) is reduced in sperm from aged men and trimethylation of histone 3 lysine 27 (H3K27me3) is elevated. The protamine makeup of sperm from aged men is also changed, with reduced protamine expression and a misbalanced ratio between protamine proteins protamine P1 and protamine P2. The study of epigenetic reproductive aging is recently gaining interest. The current status of the field suggests that many aspects of gamete epigenetic aging are still open for investigation. The clinical applications of these investigations have far-reaching consequences for fertility and sociological human behavior.

In mammals, the generation of sperm cells capable of fertilization is a highly complex process including spermatogenesis in the testis and maturation in the epididymis. In our previous study, we have demonstrated that FAM71D (Family with sequence similarity 71, member D), which could interact with calmodulin, was highly expressed in human and mouse testis. To investigate the physiological role of FAM71D in spermatogenesis, we next generate Fam71d loss-of-function mouse model using CRISPR/Cas9 technology. We performed immunofluorescence and RT-qPCR to examine the protein and mRNA expression in testicular cells. We found that FAM71D was predominantly localized in the round and elongated spermatids. And FAM71D KO mice displayed normal development of germ cell and fertility. Furthermore, testicular histology and sperm concentration showed no significant difference between WT and KO mice. These data demonstrate that FAM71D is dispensable for mouse spermatogenesis and male fertility.

Controlling ovarian hyperstimulation syndrome (OHSS) in the controlled ovarian hyperstimulation treatment is necessary to increase the implantation success rate. This study aimed to explore the effect of naringin on the endometrial receptivity of OHSS rats. Female rats were randomly assigned to six groups: Blank, model, low-dose naringin (100 mg/kg/day), medium-dose naringin (200 mg/kg/day), high-dose naringin (400 mg/kg/day), and positive (0.18 mg/kg/day estradiol valerate) groups. Except for the blank group, rats established the OHSS model on Day 7, and their treatments were from Day 0 to 14, separately. Hematoxylin and eosin, immunohistochemical, and scanning electron microscopy were performed to detect the naringin effects on the endometrial receptivity of the OHSS model. Next, circRNAs transcriptome analysis was performed to screen circRNAs. Western blot analysis and real-time quantitative PCR were used to verify it. Our study showed that naringin treatments increased embryo number, endometrial thickness, pinopodes number, and Ki67 expression in the OHSS rats. Moreover, the result of circRNAs transcriptome sequencing showed that naringin significantly inhibited the rnocirc_008140 expression in the OHSS rats and significantly inhibited the changes of 28 gene ontology terms and three Kyoto Encyclopedia of Genes and Genomes pathways which were induced by OHSS. Abcc4 and Rps6ka5 genes were the enriched genes of those pathways. Finally, 24 miRNA target genes of rnocirc_008140 were predicted. Our study showed that naringin significantly improved the endometrial receptivity of OHSS rats to increase the embryo implantation success by reducing rnocirc_008140-adsorbed miRNAs to regulate Abcc4 and Rps6ka5 expression.

With the widespread application of embryo cryopreservation in assisted reproductive techniques, it is necessary to assess the safety of long-term cryopreservation of human embryos and it is unclear whether storage time has an impact on the DNA methylation profiles of human embryos. Nine women who received IVF treatment were recruited for this study. The retrieved eight-cell human embryos were classified into three groups including fresh embryos, cryopreserved embryos stored for 3 years, and cryopreserved embryos stored for 8 years. Single-cell whole-genome bisulfite sequencing (scWGBS) was conducted. The genome-wide methylation pattern of the fresh and two cryopreserved groups were similar. In addition, the methylation level in different genomic regions showed comparable patterns and no significant differences were observed in the methylation level of imprinted genes among the three groups. A total of 587 differentially methylated regions (DMRs) in the 3-year group and 540 DMRs in the 8-year group were identified comparing to fresh group. However, they were not enriched in promoters and had a similar genome-wide distributions, suggesting that these DMRs may not contribute to the changes in corresponding gene expressions. Our study illustrated that long-term cryopreservation will not affect the DNA methylation profiles of human eight-cell embryos at single-cell level.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) system is the most efficient and widely used technology for genome editing in all sorts of organisms, including livestock animals. Here, we examined the feasibility of CRISPR/Cas9-derived genome editing (GE) in vitrified porcine zygotes, where the flexible planning of experiments in time and space is expected. OCT4 and CD46 genes were targeted, and the Cas9/sgRNA ribonucleoprotein complexes (RNP) were electroporated into zygotes at 2 h after warming. Vitrification or GE alone did not significantly reduce the developmental rates to the blastocyst stage. However, vitrification followed by GE significantly reduced blastocyst development. Sequencing analysis of the resultant blastocysts revealed efficient GE for both OCT4 (nonvitrified: 91.0%, vitrified: 95.1%) and CD46 (nonvitrified: 94.5%, vitrified: 93.2%), with no significant difference among them. Immunocytochemical analysis showed that GE-blastocysts lacked detectable proteins. They were smaller in size, and the cell numbers were significantly reduced compared with the control (p < 0.01). Finally, we demonstrated that double GE efficiently occurs (100%) when the OCT4-RNP and CD46-RNP are simultaneously introduced into zygotes after vitrification/warming. This is the first demonstration that vitrified porcine zygotes can be used in GE as efficiently as nonvitrified ones.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.

Independent cell volume regulation is first acquired by the oocyte in two steps that occur during meiotic maturation: (1) activation of the glycine transporter GLYT1 (Slc6a9) that mediates the intracellular accumulation of glycine to provide osmotic support in the mature egg and early preimplantation embryo, and (2) release of the oocyte from the strong attachment to its rigid extracellular matrix shell, the zona pellucida (ZP). It was recently shown that oocyte-ZP detachment requires metallopeptidase activity that is proposed to cleave transmembrane ZP proteins connecting the oocyte to the ZP. It is unknown, however, how GLYT1 is activated. We hypothesized that oocyte-ZP detachment precedes and may be required for GLYT1 activation. In identically treated pools of oocytes, oocyte-ZP detachment occurred ~20 min before GLYT1 activation. In individual oocytes, GLYT1 activity was detected only in those that were mostly or fully detached. Blocking detachment using previously validated small molecule metallopeptidase inhibitors partly suppressed GLYT1 activation. However, removal of the ZP did not accelerate GLYT1 activation. This indicates that oocyte-ZP detachment or cleavage of transmembrane ZP proteins may be required for GLYT1 to become fully activated, or alternatively that metallopeptidase activity independently affects both detachment and GLYT1 activation.

During pregnancy, it is necessary to create appropriate conditions for the development of the placenta and the fetus. However, during parturition, the placenta must be separated and subsequently removed as soon as possible to not expose the female to the possibility of infection. In this study, the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFβ1) concentrations was described during bovine pregnancy (second, fourth, and sixth months; n = 3/each month), at normal parturition (NR) and parturition with fetal membrane retention (R). The presence of THBS1 and TGFβ1 was confirmed in bovine placental tissues of both maternal and fetal parts. Enzyme-linked immunosorbent assay showed statistically significant differences (p < 0.05) in THBS1 concentrations (pg/mg protein) between examined parturient samples (maternal part: 5.76 ± 1.61 in R vs. 2.26 ± 1.58 in NR; fetal part: 2.62 ± 1.94 in R vs. 1.70 ± 0.23 in NR). TGFβ1 concentrations (pg/mg protein) were significantly lower (p < 0.05) in the retained fetal membranes compared to the released fetal membranes in the maternal part of the placenta (26.22 ± 7.53 in NR vs. 17.80 ± 5.01 in R). The participation of THBS1 in the activation of TGFβ1 in parturient bovine placental tissues leading to the normal release of fetal membranes may be suggested.