Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine

Abstract

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 M roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine

“…Later, maximal efficiency of meiosis inhibition was reached by Crocomo et al. (,b) with 86.7% of oocytes kept at GV stage after treatment for 6 h with 75 μ m roscovitine in serum‐supplemented medium without oil overlay. According to these same authors, the meiotic inhibition was completely reversible and the gene expression pattern in oocytes and cumulus cells was not affected.…”

“…In sheep, the inhibitory potential of roscovitine as well as its effects on ultrastructure of cumulus– oocyte complexes has been recently investigated (Crocomo et al. , ,b). Irreversible alterations such as degeneration of cumulus cells, swelling of mitochondria and reduction of cortical granules were reported by Crocomo et al.…”

Roscovitine use for the delay of meiotic progression in prepubertal sheep oocytes

“…In each replicate, about 20-25 COCs were randomly allocated to each 650 µL droplet of the medium, in four-well Petri dishes (Nunclon, Roskilde, Denmark), and cultured in an incubator at 38.5ºC and 5% CO 2 in air. The inhibitor concentration, inhibition time, and culture conditions were established according to Crocomo et al (2015aCrocomo et al ( , 2015bCrocomo et al ( , 2015cCrocomo et al ( , 2015d. The stock solution of roscovitine (1 mg mL -1 ) was prepared in dimethylsulphoxide, aliquoted, and stored at -20ºC until use.…”

“…According to the chromatin configuration observed under a fluorescence-inverted microscope (Olympus IX 70, Olympus, Tokyo, Japan), oocytes were classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase-I (MI), anaphase-I (AI), telophase-I (TI), and metaphase-II (MII). Those with altered nuclear structure were considered degenerated (DEG) (Le Beux et al, 2003;Crocomo et al, 2015a).…”