Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro.

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3ß-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3rd, 7th, 14th, and 21st day after induction. Relative expression levels of 3ß-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.

Long non-coding RNA small nucleolar RNA host gene 1 alleviates the progression of recurrent spontaneous abortion via the microRNA-183-5p/ZEB2 axis.

Long non-coding RNAs (lncRNAs) have been elucidated to play vital roles in the phenotype of trophoblast cells. Nevertheless, the effect of SNHG1 has not been investigated on trophoblast cells in recurrent spontaneous abortion (RSA). We aim to investigate the effect of SNHG1 on the phenotype of trophoblast cells during RSA. The RSA mice were established by mating female CBA/J mice with male DBA/2 mice. Microarray analysis was applied in RSA mice, and SNHG1 was identified as a significantly downregulated lncRNA. SNHG1 improved pregnancy outcome and reduced embryo resorption in RSA mice. Trophoblast cell proliferation, apoptosis, migration, and invasion were investigated by CCK8, EdU, TUNEL, wound healing, and Transwell assays. SNHG1 promoted proliferation, migration, and invasion of trophoblast cells, and reduced apoptosis. Mechanistically, SNHG1 bound to miR-183-5p in trophoblast cells. Moreover, miR-183-5p directly targeted ZEB2. Rescue experiment showed that ZEB2 silencing reversed the ameliorative effect of SNHG1 on pregnancy outcome and the promotion of trophoblast activity in RSA mice by impaired the Wnt/ß-catenin pathway. In conclusion, we found that SNHG1 plays a critical role in the progression of RSA via miR-183-5p/ZEB2 and Wnt/ß-catenin signaling. It has potential to be a therapeutic marker of RSA.

Does ICSI for in vitro fertilization cause more aneuploid embryos?

BACKGROUND: High proportion of human embryos produced by in vitro fertilization (IVF) is aneuploidy. Many factors are related to the prevalence of embryonic aneuploidies, such as maternal age, sperm quality, and in vitro manipulation of oocytes. Oocytes are usually inseminated by intracytoplasmic sperm injection (ICSI) procedures for preimplantation genetic testing. There is still no available information whether insemination procedures, regular IVF or ICSI, affect embryonic aneuploidies. METHODS: In this case report, a patient at her age of 47 years old received donated oocytes from a young donor for infertility treatment. Half of oocytes were inseminated by regular IVF and other half of oocytes were inseminated by ICSI. Fertilized oocytes were cultured to blastocyst stage and then biopsied for preimplantation genetic testing for aneuploidies (PGT-A). The proportions of aneuploidies were compared between two insemination procedures. RESULTS: Forty-seven oocytes were retrieved, 23 were inseminated by regular IVF and 24 were removed from enclosed cumulus cells for ICSI. Out of 24 oocytes, 21 oocytes at metaphase II were inseminated by ICSI. After fertilization assessment, it was found that 12 oocytes from regular IVF fertilized normally. Nine blastocysts (75%) were biopsied and 1 (11.1%) was aneuploidy. By contrast, 19 out of 21 oocytes inseminated by ICSI fertilized normally, 14 blastocysts (73.7%) were obtained and 7 (50.0%) were aneuploidy. Transfer of a euploid blastocyst from regular IVF resulted in a healthy baby delivery. CONCLUSION: These results indicate that more embryos produced by ICSI are aneuploidy as compared with embryos produced by regular IVF. The results indicate that in vitro manipulation of oocytes for ICSI procedure may have adverse effect on human oocytes, and it may be one of the reasons causing aneuploid embryos in human IVF.

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos.

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 µM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4-0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.