PKC-ßII is downregulated in the premature ovarian failure SD rat model.

We investigated the role of protein kinase c (PKC) -α and -ß during the ovarian follicular dynamics using estrous cycle, gonadotropin-induced ovulation, and antral follicle culture, 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure (POF) in the SD rat models. We found the higher activity of PKC during the proestrus stage along with expression of PKC-α during the estrus and metestrus stages of the estrous cycle while PKC-ß expression was increased during the diestrus, proestrus, and estrus stages. In response to pregnant mare gonadotropin (PMSG)-induced follicular recruitment and ovulation, the phosphorylated (Thr-642) PKC-ß was increased. PKC activity inhibition by hispidin during the proestrus stage resulted in decreased antral follicles and corpus luteum. Treatment with hispidin resulted in the downregulation of granulosa cell (GC) biomarker, follicle stimulating hormone receptor (FSHR) expression in the cultured pre-antral follicle. During the forskolin-induced luteinization of human granulosa cells, the expression level of PKC-α and ß (I and II) was decreased. In the POF condition, the activity of total PKC and the expression levels of PKC-α and ß (I and II) were increased. Immunostaining depicted ubiquitous expression of PKC-α in the ovary during the estrous cycle and POF conditions. Taken together, we conclude the association of PKC-α and -ß (I and II) during ovarian follicular dynamics where the expression level of PKC-α is increased, but the expression level of PKC-ß (I and II) is suppressed in the POF condition in the SD rat model.

Electroporation of Mouse Follicles, Oocytes and Embryos without Manipulating Zona Pellucida.

Electroporation is an effective technique of transfection, but its efficiency depends on the optimization of various parameters. In this study, a simplified and efficient method of gene manipulation was standardized through electroporation to introduce a recombinant green fluorescent protein (GFP) construct as well as RNA-inhibitors in intact mouse follicles, oocytes and early embryos, where various electroporation parameters like voltage, pulse number and pulse duration were standardized. Electroporated preantral follicles were cultured further in vitro to obtain mature oocytes and their viability was confirmed through the localization of a known oocyte maturation marker, ovastacin, which appeared to be similar to the in vivo-derived mature oocytes and thus proved the viability of the in vitro matured oocytes after electroporation. Standardized electroporation parameters, i.e., three pulses of 30 V for 1 millisecond at an interval of 10 s, were applied to manipulate the expression of mmu-miR-26a in preantral follicles through the electroporation of miR inhibitors and mimics. The TUNEL apoptosis assay confirmed the normal development of the electroporated embryos when compared to the normal embryos. Conclusively, for the first time, this study demonstrated the delivery of exogenous oligonucleotides into intact mouse follicles, oocytes and embryos without hampering their zona pellucida (ZP) and further development.

Mitochondrial membrane potential (MMP) regulates sperm motility.

Sperm motility is the major decisive factor in determining male fertility. The objective of the present study was to analyse the effect of mitochondrial membrane potential (MMP) on the temporal regulation of sperm motility. Observations were recorded in various rodent species and among differentially motile sperm fractions including swim up and leftover layer of human semen sample using JC-1 stain (a marker of the MMP) through FACS. Swim-up sperms having highest motility showed significantly higher MMP as compared to leftover sperms, which had the least motility. Interestingly, infertile patients with compromised motility showed low MMP as compared to the healthy individuals. Further, as per the time lapse, sperm motility goes down, at the same time, it was observed that MMP also decreases in human as well as in rodent sperms. Treatment of known spermicides on human sperms reduced their motility drastically which in turn also reduced its MMP significantly. Treatment of human sperms with oxidative uncoupler also impeded their motility by reducing MMP, indicating a definitive role on MMP on sperm motility and fertility. Based on the results of the study, MMP can be considered as a potential regulator and indicator of sperm motility and hence could be directly related to male fertility.

Effect of insulin supplementation on in vitro maturation of pre-antral follicles from adult and pre-pubertal mice.

This study was aimed to determine the impact of insulin concentrations on in vitro pre-antral follicle growth, survival, antrum formation rate, and retrieval of mature oocytes in mice. Mice pre-antral follicle growth were recorded on days 2, 4, 6, 8, 10, and 12 in α-modified essential media (α-MEM) supplemented with insulin concentrations of 6, 8, and 10 µg/ml along with 10% FBS, 100 mIU/ml follicle stimulating hormone, 10 mIU/ml luteinizing hormone, 100 µg/ml penicillin, and 50 µg/ml streptomycin. After 12 d of growth in vitro, follicles were allowed to mature for 16-18 h in α-MEM supplemented with 1.5 IU/ml human chorionic gonadotrophin (hCG) and 5 ng/ml epidermal growth factor (EGF). The initial diameter (54.86 ± 2.5 µm) of mice oocyte progressively increased in all the three insulin concentration groups and attained a maximum size on day 12 (71.90 ± 2.8 µm). Supplementation with higher concentrations of insulin (both 8 and 10 µg/ml) significantly enhanced antrum formation without effecting the oocyte diameter and percent retrieval of mature oocyte in all the three concentration groups. Both in vitro cultured as well as in vivo collected follicles and oocytes showed similar localization and expression of oocyte maturation markers SAS1B and GDF9. Insulin concentration of 8 µg/ml was found to be optimal for in vitro follicle culture of adult mice (42-49 d). Optimized follicle culture conditions were also assessed successfully with pre-pubertal mice (12-14 d); however, adult mice showed higher follicle survival, antrum formation, and more mature oocytes production in comparison to pre-pubertal mice.