Molecular Reproduction and Development最新文献

Ovarian tumor ubiquitinating 6A (OTUD6A) is a deubiquitinating enzyme whose aberrant expression has been linked to various diseases, including inflammation and prostate cancer. Research indicates that deubiquitinating enzymes (DUBs) play a significant role in spermatogenesis in mice. However, the role of OTUD6A in spermatogenesis remains unclear. To investigate the function of OTUD6A in mouse spermatogenesis, we generated Otud6a-knockout mice using the CRISPR/Cas9 system. Our results showed that OTUD6A is predominantly expressed in the testis and localized to the cytoplasm of spermatogonia and spermatocytes. Although no significant differences were observed in testicular size or morphology between Otud6a-knockout and wild-type mice, the knockout mice exhibited increased germ cell apoptosis, decreased epididymal sperm counts, abnormalities in sperm motility and subfertility. These findings indicate that Otud6a-knockout leads to male subfertility in mice.

Intergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Such intergenic transcription has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation. Using Automatic Readthrough Transcription Detection (ARTDeco) and poly(A)-selected RNA-seq libraries from in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb upstream of reference genes, extending up to 15 kb upstream). Read-throughs (continued transcription from TES of expressed reference genes, 4–15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3084 to 6565 or 33.36% to 66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (p < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1504 read-outs, 1045 read-ins, and 1021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log₂ fold change ≥ 2, p < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and downstream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing. In summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or downstream.

Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a key reader of N6-methyladenosine modifications that regulate target mRNA stability in eukaryotic cells; however, its role in germ cells has never been explored. Here, we analyzed the spatiotemporal expression of IGF2BP1 and revealed that it was present not only in oocytes of the mouse ovary but also in ZBTB16-positive undifferentiated spermatogonia in the mouse testis. Coimmunoprecipitation and fluorescence staining revealed that IGF2BP1 interacted with TRIM71, a regulator of spermatogonia differentiation, but that its expression was unaffected in the testes of Trim71 knockout mice. We also show that IGF2BP1 colocalized with components of the mRNA processing body (P-body), including DDX6 and EDC4. However, contrary to our expectations, using VASA (DDX4)-Cre-mediated conditional knockout mice, we found that germ cell-specific knockout of Igf2bp1 did not seem to affect the fertility of male or female mice. Further analysis revealed that spermatogenesis and ZBTB16-positive undifferentiated spermatogonia numbers in the testes of mutant mice remained unchanged and that there were no obvious changes in testicular morphology or cell subpopulations. In summary, although IGF2BP1 is preferentially expressed in germ cells, its function in germ cells may be dispensable.

Ovarian tissue transplantation (OTT) has been suggested as an alternative to preserving female fertility in livestock species, as currently performed in women. The OTT technique has been tested as xenografting or autografting in different body sites and animal species. Currently, there are no reports available regarding the autotransplantation of ovarian tissue using the bovine model and also testing the effect of vascular endothelial growth factor (VEGF) on graft survival. This study evaluated the effects of ovarian tissue short-term exposure to VEGF before heterotopic autotransplantation into a subcutaneous site (flank region) for 7 days in cattle. The initial finding was that after OTT and without pre-exposure to VEGF, the ovarian grafts had 42% of normal preantral follicles, which indicates a substantial step forward for this technique in cattle. Furthermore, VEGF exposure actively facilitated the process of neoangiogenesis, the proliferation capability of the stromal cells for activation of the cell cycle, maintained the balance between types I and III collagen fibers, and reduced the total collagen of the grafted tissue. Moreover, ovarian fragments previously exposed to VEGF tended to have greater follicular density; however, a detrimental effect of VEGF on follicular morphology was noticed. In conclusion, this study marks a significant step forward in bovine OTT and provides a foundation for further investigations into the specific pathways, stages, and durations of VEGF exposure to unveil strategies for refining ovarian transplantation techniques in cattle and other species.

In vitro maturation (IVM) is a form of assisted reproductive technology (ART) applied to obtain mature oocytes in culture. Decline in IVM success rates by age has led consideration of novel approaches based on cellular dynamics. Our aim was to achieve proteostasis in old bovine oocytes from 13 to 16-year-old bovine with a lower potential for fertilization. Lysosomal activation was achieved through increasing concentrations of proton pump activators PIP2 (0.1, 0.5, 1, and 5 μM), PMA (0.1, 1, 10, and 50 μM), and DOG (0.1, 1, 10, and 50 μM) at 6, 12, 18, and 24 h of IVM in old bovine oocytes. Morphological analysis was performed and IVM rates were determined. DQ-Red BSA was applied to live oocytes to determine proteolytic activation while lysosome density was determined by Lysotracker probe. Protein carbonylation was detected through oxyblot analysis. Polar body extrusion (PBE), through which a haploid nonfunctional polar body is released in the perivitelline space after completion of the first meiotic division, was observed in PIP2-0.1 μM, -0.5μM-6h; PIP2-5μM-12h; PMA-0.1μM-18h; PIP2-0.1μM, -0.5μM-24h groups. Oocyte diameter was the highest in DOG-1μM-6h, PMA-0.1μM-12h, PIP2-1μM-18h, and PIP2-0.5μM-24h groups. Morphological scores of oocytes were higher in young and old control groups. PIP2, PMA, and DOG affected oocyte quality positively after 6 h of IVM yielding in oocyte scores similar to the control group oocytes. However, they had a negative impact on the oocyte scores in longer periods of IVM, except for lower doses PMA (0.1 and 1 μM) at 12 h and PIP2 (0.5 μM) and PMA (0.1 μM) at 18 h, which were able to maintain the scores relatively closer to the control oocytes. Proteolytic activation was achieved in all groups at 6 h of culture. At all other time points PIP2 and PMA groups showed a better response to proteolytic activation. Lysosome density was increased in PIP2-5μM-6h; PIP2-0.1μM, -1μM-12h; PIP2-1μM, -5μM-18h as well as PMA-0.1μM-6h; PMA-1μM, -10μM-12h; PMA-1μM-18h; DOG-50μM-6h and DOG-0.1μM-12h. Protein carbonylation was the lowest in PIP2-0.1 μM groups at 12, 18, and 24 h. This study suggests that proton pump activators PIP2 and PMA was found to have a positive impact on IVM in terms of both morphological scores and proteolytic activation in a time and dose dependant manner.