Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch.

Male factor infertility is a problem in today's society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GCspecific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., Dmrtb1, Sohlh1). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., Amh, Fshr). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation.

Dimethylsulfoxide and conjugated linoleic acids affect bovine embryo development in vitro.

Conjugated linoleic acids (CLA) are employed to overcome the bovine periparturitional negative energy balance. Especially of interest are trans10,cis12 -linoleic acid (t10c12-CLA) and cis9,trans11-linoleic acid (c9t11-CLA). Their impact on embryonic development, though, is not clear. Here, effects of both above-mentioned CLA on bovine in vitro-produced embryos were assessed. Zygotes (n=2098) were allocated to one of seven groups: cultured with 50 or 100µM of either c9t11-CLA or t10c12-CLA, with 14 or 28mM DMSO or without supplement (control). Messenger RNA analysis of target gene transcripts (IGF1R, IGFBP2, IGFBP4, CPT2, ACAA1, ACAA2, FASN, SCD) via RT-qPCR was performed in single blastocysts. Cleavage rates did not differ, whereas development rates were decreased in both t10c12-supplemented groups in comparison to the unsupplemented group (31.7% ±2.2 control vs 20.2% ±2.0 50µM t10c12 vs 21.0% ±2.8 100µM t10c12). Compared with the unsupplemented group, SCD was expressed at a lower level in embryos cultured with 50µM c9t11-CLA. The relative amount of several transcripts was increased in embryos cultured with 14mM DMSO in comparison to those that developed in the presence of 50µM t10c12-CLA (IGFBP2, ACAA1, CPT2, FASN, SCD) or 50µM c9t11-CLA (IGF1R, IGFBP2, ACAA1, CPT2, FASN, SCD). The molecular analyses show that CLA influence embryonic fat metabolism.

Influence of selected (pre-)maturational parameters on in vitro development and sex distribution of bovine embryos.

The objectives of this research were to study the influence of a reduced oxygen concentration during in vitro maturation (IVM) and examine the effect of follicular glucose concentration on bovine in vitro development and sex distribution. In the first experiment, abattoir-derived cumulus-oocyte complexes (COC) were matured under 5% O2 or 20% O2. Secondly, COC were isolated and the glucose (G) concentration of each follicle was determined. COC were pooled in groups (G (< 1.1 mMol) or G (≥ 1.1 mMol)) according to the glucose content before being subjected to in vitro production (IVP). Cleavage and development rates were assessed on days 3, 7 and 8 post insemination. Blastocysts of each group were sexed by polymerase chain reaction (PCR). Expanded blastocysts were stained to assess total cell numbers and live-dead cell ratio. Cleavage and development rates stayed similar after reducing the O2 concentration during IVM. The sex ratio of embryos generated from oocytes matured under 5% O2 was shifted in favour of the female (♀: 61.9%), whereas the sex ratio of embryos belonging to the IVM 20% O2 group did not differ significantly from the expected 50:50 ratio. Neither a 'higher' nor a 'lower' intrafollicular glucose concentration influenced cleavage and development rates, cell numbers or live-dead cell ratio. Eighty five per cent (G (<1.1)) and 63.6% (G (≥ 1.1)) of the analysed embryos were female. In summary, neither a reduced O2 concentration during IVM nor selection based on follicular glucose concentrations affected the morphological quality of embryos. Although the sex distribution was shifted in favour of female embryos in all three experimental groups, more male embryos could be seen in the G (≥ 1.1) group compared with the G(<1.1) group.

Expression of mRNA, before and after freezing, in bovine blastocysts cultured under different conditions.

Production methods and culture systems have been shown to affect blastocyst mRNA expression and cryopreservability, which may serve as sensitive indicators of embryo quality and developmental competence. In the present study, the impact of four established culture conditions for producing bovine blastocysts (in vitro production, IVP; gamete intra-fallopian transfer, GIFT; transfer of cleaved stages into the oviduct, CLVT; multiple ovulation embryo transfer, MOET) was assessed, in terms of both cryosurvival and levels of mRNA expression of several selected genes (occludin, desmocollin 2, solute carrier family 2 member 3, BAX, BCL-XL, heat shock protein 1A, aquaporin 3, DNA methyltransferase 1a) detected with RT-qPCR. At 24 hours post-thawing, blastocysts derived from in vitro production showed a significantly higher re-expansion rate compared to the other groups. At later times, this difference was no longer significant. Before freezing, embryos of the MOET group showed significantly more desmocollin 2 mRNA compared to embryos produced using other culture methods. After freezing, significant upregulation was found in transcripts of heat shock protein 1A in embryos of all groups; of solute carrier family 2 member 3, only in IVP derived embryos; of BAX, BCL-XL, occludin, desmocollin 2, only in the MOET and IVP groups. Aquaporin 3 and DNA methyltransferase 1a were neither up- nor downregulated in blastocysts of any group. In conclusion, these findings suggest that, after freezing, embryos seem to have switched on mRNA synthesis, an active metabolism, operational cell connections, and are prepared for hatching and beyond.