Effects of Short-Term Inhibition of Rho Kinase on Dromedary Camel Oocyte In Vitro Maturation.

Hammed A Tukur,Abdullah N Alowaimer,Ayman Abdel-Aziz Swelum,Islam M Saadeldin,Mutassim Abdelrahman,Riyadh S Aljumaah

doi:10.3390/ani10050750

Hammed A Tukur, Abdullah N Alowaimer + Show 4 more

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https://doi.org/10.3390/ani10050750

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Abstract

Simple SummaryOur results revealed, for the first time, that short-term inhibition of Rho-associated protein kinases (ROCK) for 4 h prior to in vitro maturation (IVM) in a biphasic IVM approach improved oocyte nuclear maturation, producing more MII oocyte, through modulating the expression of cytokinesis- and antiapoptosis-related mRNA transcripts. This positive result suggests ROCK inhibitor as a potential candidate molecule to exploit in the control of oocyte meiotic maturation.This is the first report on a biphasic in vitro maturation (IVM) approach with a meiotic inhibitor to improve dromedary camel IVM. Spontaneous meiotic resumption poses a major setback for in vitro matured oocytes. The overall objective of this study was to improve in vitro maturation of dromedary camel oocytes using ROCK inhibitor (Y-27632) in a biphasic IVM to prevent spontaneous meiotic resumption. In the first experiment, we cultured immature cumulus–oocyte complexes (COCs, n = 375) in a prematuration medium supplemented with ROCK inhibitor (RI) for 2 h, 4 h, 6 h, and 24 h before submission to normal in vitro maturation to complete 28 h. The control was cultured for 28 h in the absence of RI. In the first phase of experiment two, we cultured COCs (n = 480) in the presence or absence (control) of RI for 2 h, 4 h, 6 h, and 24 h, and conducted real-time relative quantitative PCR (qPCR) on selected mRNA transcripts. The same was done in the second phase, but qPCR was done after completion of normal IVM. Assessment of nuclear maturation showed that pre-IVM for 4 h yielded an increase in MII oocyte (54.67% vs. 26.6% of control; p < 0.05). As expected, the same group showed the highest degree (2) of cumulus expansion. In experiment 2, qPCR results showed significantly higher expression of ACTB and BCL2 in the RI group treated for 4 h when compared with the other groups. However, their relative quantification after biphasic IVM did not reveal any significant difference, except for the positive response of BCL2 and BAX/BCL2 ratio after 4 and 6 h biphasic IVM. In conclusion, RI prevents premature oocyte maturation and gave a significantly positive outcome during the 4 h treatment. This finding is a paradigm for future investigation on dromedary camel biphasic IVM and for improving the outcome of IVM in this species.

Highlights

Reproductive success in livestock ensures the continuation, survival, and conservation of species, and, by extension, food security
Rho-associated protein kinases (ROCK) inhibitor (RI) showed no impact on the cumulus morphology when cumulus–oocyte complexes (COCs) were cultured for 2, 4, 6 h when compared with control group (Figure S2)
Our studies showed the importance of ROCK in cytokinesis and regulation of dromedary camel oocyte nuclear maturation

Summary

Introduction

Reproductive success in livestock ensures the continuation, survival, and conservation of species, and, by extension, food security. One major challenge that hinders camel oocyte maturation is the spontaneous meiotic resumption that occurs when immature oocytes transfer from the natural follicular environment to be cultured in vitro. This drastic change causes oocytes to spontaneously resume meiosis due to the absence of inhibitive signals [11,12]. Oocytes will have asynchronous premature nuclear and cytoplasmic maturation, which leads to premature oocyte development. Even though these oocytes appear morphologically normal, are not competent. This issue has been the subject of numerous earlier and recent studies [15,16,17]

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