The Journal of Reproduction and Development最新文献

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52-53% or 48-51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

Unlike sex steroids, mineralocorticoids have attracted limited attention in ovarian physiology. Recent studies on primates have indicated possible local synthesis and action of mineralocorticoids in the ovary. Here, we examined developmental changes in the levels of mineralocorticoids and expression of genes encoding their biosynthetic enzymes and receptor in the bovine ovary. The follicles and corpora lutea (CL) were collected from F1 heifers. Expression levels of 21α-hydroxylase (CYP21A2), 11β-hydroxylase-1 (CYP11B1), and the mineralocorticoid receptor (NR3C2) in granulosa cells (GC), thecal layers (TL), and CL tissues were quantified by real-time PCR, whereas mineralocorticoids in the follicular fluid were measured by enzyme immunoassay (EIA). TL and GC expressed CYP21A2 and NR3C2, whereas CYP11B1 was expressed at very low or undetectable levels. The expression levels of these genes were not significantly different among small/large and healthy/atretic follicles but were higher in TL than in GC. CYP21A2 and NR3C2 were expressed in all CL stages with higher expression observed in the mid-stage. CYP11B1 expression was only apparent in the mid-stage CL. Aldosterone was detected in all follicles, and its concentration was not significantly different among the follicular groups. In paired large-healthy/atretic follicles, the concentration of deoxycorticosterone, a precursor of aldosterone, was approximately ten-fold higher than that of aldosterone and not significantly different between healthy and atretic follicles. In conclusion, the presence of mineralocorticoids and expression of NR3C2 in the bovine follicle together with the developmental change in the expression of CYP21A2, CYP11B1, and NR3C2 in the CL suggest possible endocrine/paracrine/autocrine roles of mineralocorticoids in the bovine ovary.