No abstract

Small ubiquitin-related modifier-1 (SUMO-1)-dependent modifications of many target proteins are involved in a range of intracellular processes. Previous studies reported the localization of SUMO-1 during oocyte meiosis, and that overexpression of Sentrin/SUMO-specific protease 2 (SENP2), a de-SUMOylation protease, altered SUMO-modified proteins, and caused defects in metaphase-II spindle organization. In this study, we detailed the consequences of SUMO-1-mediated SUMOylation by either inhibition of SUMO-1 or UBC9 with a specific antibody or their depletion by specific siRNA microinjection. Inhibition or depletion of SUMO-1 or UBC9 in germinal vesicle (GV)-stage oocytes decreased the rates of germinal vesicle breakdown and first polar body (PB1) extrusion; caused defective spindle organization and misaligned chromosomes; and led to aneuploidy in matured oocytes. Stage-specific antibody injections suggested that SUMO-1 functions before anaphase I during PB1 extrusion. Further experiments indicated that the localization of γ-tubulin was disordered after SUMO-1 inhibition, and that SUMO-1 depletion disrupted kinetochore-microtubule attachment at metaphase I. Moreover, SUMO-1 inhibition resulted in less-condensed chromosomes, altered localization of REC8 and securin, and reduced BUBR1 accumulation at the centromere. On the other hand, overexpression of SUMO-1 in GV-stage oocytes had no significant effect on oocyte maturation. In conclusion, our results implied that SUMO-1 plays crucial roles during oocyte meiotic maturation, specifically involving spindle assembly and chromosome behavior, by regulating kinetochore-microtubule attachment and the localization of γ-tubulin, BUBR1, REC8, and securin.

Inhibin, a member of the transforming growth factor-β [TGF-β] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary–gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of β-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-β concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-β in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinβ which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

The objective of this study was to examine the efficacy and safety of a novel inhibin vaccine containing inhibin α (1–32) fragments in mice. A recombinant plasmid pVAX‐asd‐IS was constructed by inserting recombinant inhibin α (1–32) and the hepatitis B surface antigen S into the plasmid in which the asd gene, rather than the kanamycin gene, was a selection marker. Ninety Kuming mice were divided into six groups consisting of 15 mice each. First group was (C1) injected with 200 µl of PBS, second (C2) received 1 × 1010 CFU of crp−/asd− C500/pVAX‐asd and served as vector control, third did not receive any treatment (C3), while fourth, fifth, and sixth group received 1 × 1010, 1 × 109, 1 × 108 CFU of the recombinant inhibin vaccine crp−/asd− C500/pVAX‐asd‐IS (group T1, T2, T3), respectively. Western blotting demonstrated that recombinant expressed inhibin protein possessed immune function and that this plasmid could replicate for up to 40 generations stably. Vaccination with this strain at a dose of 1 × 1010 CFU/200 µl per mouse induced high anti‐inhibin antibody levels, significantly increased large‐follicle production in T1 group (p < 0.05) and average litter size (p > 0.05) compared with control groups. Integration studies showed no evidence of inhibin fusion gene integrated into mice's genome 2‐month after immunization. These results suggest that the vaccine described in the present study may provide a safe method to improve reproductive traits in animals. A trend towards increased litter size and significant increase in large follicle population depict that this vaccine may have direct application in large animal industry.

Bora is the binding partner of Aurora A, which is required for its activation and phosphorylation of Polo like kinase 1 (Plk1), and is involved in the spindle assembly and progress of the cell cycle during mitosis. In this study, we examined the expression, localization, and function of Bora during mouse oocyte meiosis. The expression level of Bora was increased during oocyte meiotic maturation, with an elevated level at metaphase. Immunofluorescence analysis showed that Bora was concentrated as a dot shortly after germinal vesicle breakdown (GVBD), associating first with the surrounding chromosomes and then with the spindle throughout oocyte meiotic maturation. Further experiments confirmed that Bora co-localized with α-tubulin at prometaphase/metaphase, but dissociated from α-tubulin at anaphase/telophase. In metaphase-II-arrested oocytes, Bora was evenly distributed in the cytoplasm after treatment with a microtubule-depolymerizing agent, or recruited to the spindle after treatment with a microtubule-polymerizing agent, indicating that Bora was physically connected to the meiotic spindle and α-tubulin at metaphase. Furthermore, inhibition or depletion of Bora by either anti-Bora antibody or Bora siRNA microinjection significantly reduced the rates of GVBD and inhibited first polar body extrusion; caused morphologically defective spindles and misaligned chromosomes; arrested maturing oocytes at prometaphase/metaphase-I stage, or left oocytes and their first polar bodies with severely misaligned chromosomes and defective spindles; and/or caused the disappearance of Aurora A and Plk1 at the spindle. These results indicated that Bora acts as a critical regulator of Aurora A and Plk1, and is involved in microtubule organization during oocyte meiosis.