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

Abstract

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.

Rocking the Boat: The Decisive Roles of Rho Kinases During Oocyte, Blastocyst, and Stem Cell Development

“…The distribution and function of ROCK in oocyte and embryo varies as development progresses suggesting a spatiotemporal role of ROCK at different stages of oocyte and embryo development ( Figure 2 ). Northern blot analysis of Rho kinase protein, real-time PCR (rtPCR) analysis of Rho-kinase mRNA, and inhibition of ROCK has been employed to show the presence and involvement of Rho-kinase in the oocyte and embryo of rabbits ( Stankova et al, 2014 ), mice ( Duan et al, 2014a , b ), cows ( Hwang et al, 2013 ; Lee et al, 2015 ), felines ( Arayatham et al, 2017 ), swine ( Zhang et al, 2014 ), and camels ( Tukur et al, 2020 ). In the bovine oocyte, ROCK was localized around the nucleus at the germinal vesicle (GV) stage but spread to the rest of the cytoplasm in later developmental stages ( Lee et al, 2015 ).…”

“…However, prolonged inhibition of ROCK can have profound negative effects on oocyte and embryo development. In the camel, the prolonged inhibition resulted in either abnormal polar bodies or polar body failure ( Tukur et al, 2020 ). Duan et al (2014b) found that prolonged inhibition of ROCK activity during mouse oocyte maturation interfered with meiotic spindle migration and polar body extrusion.…”

“…There is compelling evidence for the roles of ROCK signaling in oocyte meiotic and cytoplasmic maturation ( Liu et al, 2018 ; Tukur et al, 2020 ), including those in embryo cleavage, blastocysts, and segregation of ICM ( Kawagishi et al, 2004 ; Laeno et al, 2013 ). Moreover, ROCK signaling is pivotal in pluripotent and embryonic stem cells, where it is implicated in proliferation, differentiation, and survival ( Zhang et al, 2011 ; Kamishibahara et al, 2016 ; Vernardis et al, 2017 ).…”

“…Consequently, the biotechnology industry has developed several isoform-selective and non-selective ROCK inhibitors to determine the role of ROCK in a variety of cells and tissues ( Liao et al, 2007 ; Defert and Boland, 2017 ; Saadeldin et al, 2020 ). Notably, ROCK inhibitors including fasudil and Y-27632 have been widely used in oocyte, embryo, and stem cell research ( Laeno et al, 2013 ; Duan et al, 2014a ; Defert and Boland, 2017 ; So et al, 2020b ; Tukur et al, 2020 ). Interestingly, the selective efficacy of ROCK inhibitors is both dose and time dependent.…”

“…With a primary role in the spatiotemporal organization of actin cytoskeletal-related cellular events, ROCK proteins act as the key that enables the interaction of actin cytoskeleton with molecular motors, that convert signals into forces essential for the regulation of cellular functions such as contraction, adhesion, migration, proliferation, and apoptosis ( Riento and Ridley, 2003 ; Jaffe and Hall, 2005 ; Ohgushi et al, 2010 ). Ever since the discovery of the pleiotropic nature of ROCK, it has sparked the interest of various research and medical fields, including the pharmaceutical ( Hahmann and Schroeter, 2009 ; Feng et al, 2015 ; Honjo and Tanihara, 2018 ; Knipe et al, 2018 ; Zhou, 2018 ), neuroregeneration ( Wang et al, 2015 ), stem cell, and regenerative medicine ( Krawetz et al, 2011 ; Li et al, 2015 ; Vernardis et al, 2017 ), cancer research ( Street and Bryan, 2011 ; Kümper et al, 2016 ; Wei et al, 2016 ), and reproductive and developmental biology fields ( Kawagishi et al, 2004 ; Laeno et al, 2013 ; Duan et al, 2014b ; Miranda-Rodríguez et al, 2017 ; Tukur et al, 2020 ).…”