Nuclear and cytoplasmic quality of oocytes derived from serum-free culture of secondary follicles in vitro.

In vitro culture of follicles is a promising technology to generate large quantities of mature oocytes and it could offer a novel option of assisted reproductive technologies. Here we described a 2-dimensional follicular serum-free culture system with 3-dimensional effect that can make secondary follicles develop into antral follicles (78.52%), generating developmentally mature oocytes in vitro (66.45%). The oocytes in this serum-free system completed the first meiosis; spindle assembly and chromosome congression in most oocytes matured from follicular culture were normal. However, these oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca2+ oscillations was also lower in response to parthenogenetic activation, after which a 2-cell embryonic developmental block occurred. Oocytes matured from follicular culture displayed increased abnormal mitochondrial distribution and increased reactive oxygen species levels when compared to in vivo matured oocytes. These data are important for understanding the reasons for reduced developmental potential of oocytes matured from follicular culture, and for further improving the cultivation system.

Mechanistic insights into the reduced developmental capacity of in vitro matured oocytes and importance of cumulus cells in oocyte quality determination.

In vitro maturation of oocytes is a promising assisted reproductive technology (ART) for infertility treatment, although it is still not a routine technique for human ART due to reduced embryonic development. The aim of the present study was to clarify the possible reasons for reduced capacity of in vitro matured oocytes. Our results showed that the oocytes matured in vitro displayed increased abnormal mitochondrial distribution, reduced mitochondrial membrane potential, and increased reactive oxygen species levels when compared to in vivo matured oocytes. These results were not different in oocytes matured in vitro with or without cumulus cells. Notably, in vitro matured oocytes displayed increased mitochondrial DNA numbers probably due to functional compensation. In vitro matured oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca2+ oscillations was much lower in response to parthenogenetic activation, especially in oocytes matured in vitro without cumulus cells with nearly half of them failing to produce calcium waves upon strontium chloride stimulation. These data are important for understanding the reasons for reduced developmental potential of in vitro matured oocytes and the importance of cumulus cells for oocyte quality.

[In vitro culture of the testis tissue of BALB/c mice with spermatogenic dysfunction].

OBJECTIVE: To explore the pathways of development and maturation of the testis tissue in mice with spermatogenic dysfunction in vitro. METHODS: Sixty-eight 8-week-old BALB/c male mice were randomly divided into four groups of equal number, normal control, Sertoli cell only syndrome (SCOS), severe hypo-spermatogenesis (H-S1), and mild hypo-spermatogenesis (H-S2), and the models were established in the latter three groups by intraperitoneal injection of busulfan at 40 mg/kg for 4, 6 and 8 weeks, respectively. The testis tissues of the mice were cultured with the agarose gel method in vitro till the 4th week, followed by determination of the expressions of the marker proteins STRA8 at meiotic initiation, SCP3 during meiosis, and TNP1 after meiosis by immunohistochemistry. The development and maturation of the germ cells during the in vitro culture were evaluated, and their apoptosis detected by TUNEL. RESULTS: The more severe the testicular tissue injury, the lower the expression of the STRA8 protein in the SCOS, H-S1 and H-S2 groups as compared with the normal control before in vitro culture on agarose gel (P < 0.05), and the STRA8 expression was significantly upregulated in the former three groups after 4 weeks of culturing (P < 0.05). The expression of SCP3 was the lowest in the SCOS but the highest in the H-S2 group before culturing (P < 0.05), and was not as high as that in the control, though increased after 4 weeks of culturing. TNP1 was positively expressed in all the mice of the control, some individuals of the H-S1 and H-S2 groups (P< 0.05), but not in the SCOS group at 4 weeks. The apoptosis of germ cells was significantly increased in the SCOS but decreased in the H-S groups compared with that in the normal control after 4 weeks of culturing (P< 0.05). CONCLUSIONS: In vitro culture on agarose gel induces the meiosis of the testis tissue in BALB/c mice with spermatogenic dysfunction, and the effect is even better in those with mild spermatogenic dysfunction.