Although a number of studies describe the in vitro co-culture model, still the positioning of embryos to the uterine lumen and examination of blastocysts attached to the endometrial tissue was not so easy due to the small size of blastocysts compared to the endometrial tissues. The current study was aimed to develop an in vitro co-culture system to study the early implantation. Rat uterine explants (1–2 mm) were isolated, cultured and further characterized. Then from uterine horns morphologically normal embryos were flushed and hatching was induced by Acidic Tyrode’s solution (pH-2.5) for 15–30 second to remove the zona pellucida. Individual hatched blastocyst and cultured explant was placed in a 96U (U shaped round bottom) well plate. Results showed that stable attachments were observed after 48 hours of co-culture, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. Furthermore, steroid hormones are critical for endometrial receptivity and further implantation process. The steroid hormone treatment revealed that the rate of attachment of embryos to the explants were significantly increased in P4 treated group (63.63%) compared to the control or non-treated group (35.48%). On the other hand, attachments of embryos to the explants were significantly reduced in E2 treated group compared to the control group, where no stable attachments were observed in E2 treated group (0.0%). The study suggests that the co-culture model is suitable for the study of early implantation and steroid hormones influence the rate of attachment in this system.

Vitrification of mammalian oocytes is an indispensible approach for assisted reproductive technologies. However, the underlying mechanisms responsible for low developmental competence of vitrified germinal vesicle (GV) oocytes remain unknown. We show here that vitrified oocytes had a significant increase of chromosomal misalignment and abnormal spindle either at Metaphase (Met) I or II. Using monastrol assay, vitrified GV oocytes showed an increased incidence of aneuploidy at Met II. The high incidence of aneuploidy suggests that Spindle Assembly Checkpoint (SAC) is perturbed in vitrified oocytes. To investigate our hypothesis, we conducted nocodazole (NOC) and DNA damage assays. In contrast to control oocytes, vitrified oocytes extruded first polar bodies (PBs) in the presence of NOC or etoposide which are known to induce SAC activation. We then examined the localization of Mad2, an essential regulator of SAC signaling. Under NOC-induced Met I arrest status, in contrast to Mad2 localization at kinetochores in control oocytes, vitrified oocytes showed diffused localization across chromosomal arms. These results indicate that SAC is no longer functional in vitrified oocytes. Importantly, we found that vitrification stimulated the activities of cathepsin B and caspase 3 which are the putative pathways of impaired SAC function. This hypothesis was confirmed by restoring Mad2 localization and SAC function in vitrified oocytes treated with E-64, a cathepsin B inhibitor. These results provide the first demonstration that vitrification perturbs faithful chromosome segregation by impairing SAC function through activation of cathepsin B in mouse oocytes.

This experiment was conducted to assess the effect of cumulus cell removal after 20 h of in vitro maturation (IVM) in porcine oocytes from small follicles when they are supplemented or not with vascular endothelial growth factor (VEGF). Cumulus-oocyte complexes from small follicles (< 3 mm in diameter) were cultured in the absence or presence of 200 ng/ml of VEGF during the first 20 h of IVM. The oocytes were then denuded from cumulus cells and continued the IVM culture for further 24 h. At the end of IVM, the meiotic progression of the oocytes was examined and only mature oocytes were cultured for 5 days after parthenogenetic activation. On days 2 and 5, cleavage and blastocyst rates were assessed respectively. The cell number in blastocysts was also counted by DAPI staining. Data from 6 replicated trials were analyzed by 2-way ANOVA. Denuding the oocytes after 20 h significantly increased the maturation rate regardless of the presence or not of VEGF when compared with the control (63.9%, 65.1% vs 45.6%, respectively). However, the removal of cumulus cells did not affect the percentages of cleavage and blastocyst formation following parthenogenetic activation. The cell number of the blastocysts was also similar amongst the groups. From these results, we conclude that removing cumulus cells 20 h after the start of IVM improves maturation rates of oocytes from small follicles, but does not affect the developmental competence, regardless of the presence of VEGF during the first 20 h of culture.