Role of cumulus cells for rat oocyte maturation and metabolism

Oocytes collected from immature PMSG-treated rats on the morning of proestrus were allowed to mature in culture either surrounded by their cumulus cells or after denudation. It was found that the time course of oocyte nuclear maturation was similar whether the cumulus cells were present or not. The oxygen consumption of noncultured oocytes was 0.12 nl/hr/oocyte and increased by 40% after four to eight hours in culture with intact cumulus. Respiration of oocytes cultured without cumulus remained constant throughout the culture, except for a transient decrease after four hours. It is concluted that the cumulus cells do not affect the spontaneous nuclear maturation in vitro, but that the metabolism in oocytes cultured with intact cumulus is different from that of cultured denuded oocytes. Furthermore, it appears that the rise in oocyte oxygen consumption is not a prerequisite for nucler maturation.     

Cyclooxygenase-2 is expressed by cumulus cells during oocyte maturation in cattle.

Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.     

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.     

Adenylyl cyclases in oocyte maturation: a characterization of AC isoforms in bovine cumulus cells

Mammalian oocytes are arrested at the G(2)/M transition in the meiotic cell cycle. It is well known that a decrease in intraoocyte cAMP concentrations accompanies resumption of meiosis, but the precise trigger of this decrease remains a mystery. Follicular somatic cells are intimately coupled to the oocyte and are thought to transmit maturation signals to the oocyte in response to hormonal stimulation. Here, we investigate the nature of the follicular somatic cell response to hormonal stimulation by identifying and characterizing the adenylate cyclase isoforms present in bovine cumulus cells. RT-PCR and Western blot analysis revealed the presence of multiple adenylyl cyclase isoforms in bovine granulosa and cumulus cells. Pharmacological manipulation of the AC isoforms showed that multiple isoforms were indeed active. Our data indicate that the PKC inhibited adenylate cyclases IV and VI and the calcium-stimulated isoform I predominate in bovine cumulus cells.     

The relationship between cumulus cell-oocyte coupling,oocyte meiotic maturation,and cumulus expansion

The timing of the reduction of cumulus cell-oocyte coupling was correlated with oocyte meiotic maturation and the expansion (mucification) of the cumulus oophorus using immature mice treated with gonadotropins. Three hours after the injection of an ovulatory dose of human chorionic gonadotropin (hCG), more than 90% of the oocytes isolated from large Graafian follicles had undergone germinal vesicle breakdown, indicating that oocyte meiotic maturation had been initiated. However, no cumulus expansion or reduction of intercellular coupling was detected at this time. By 6 hr after hCG injection, the index of oocyte-cumulus cell coupling was still not less than that found in oocyte-cumulus cell complexes isolated from control mice not receiving hCG. Cumulus expansion at 6 hr post-hCG was limited to the outer cumulus cells while those adjacent to the oocyte were still tightly packed. Cumulus expansion appeared complete by 9 hr after hCG injection and the cumulus cell-oocyte coupling index was greatly reduced. These results show that oocyte meiotic maturation in the mouse is not initiated by a reduction in cumulus cell-oocyte coupling or by cumulus expansion. However, the results suggest that the reduction of intercellular coupling in vivo may be a result of cumulus expansion.     

Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro

It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0h group) were cultured for 24h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20h without supplements (second culture step; 44h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24h group) or partly (H-DO 24h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24h (DO 24h+CC group). The maturation rates of all the cumulus-removed groups (DO 0h, DO 24h, H-DO 24h and DO 24h+CC groups) were lower (34.3-45.0%) than that of the control group (64.5%; P<0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0h, DO 24h and DO 24h+CC groups) were lower (4.03-5.26pmol/oocyte) than that of the control group (9.60pmol/oocyte; P<0.05); however, the H-DO 24h group had an intermediate value (7.0pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4-59.3%) than that of the control group (89.4%; P<0.05), whereas the H-DO 24h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0-4.5%) than that of the control group (19.9%; P<0.05), and the H-DO 24h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation (P<0.01) and blastocyst formation (P<0.01), and also with the number of cells per blastocyst (P<0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.     

Identification of molecular markers for oocyte competence in bovine cumulus cells

Cumulus cells (CCs) have an important role during oocyte growth, competence acquisition, maturation, ovulation and fertilization. In an attempt to isolate potential biomarkers for bovine in vitro fertilization, we identified genes differentially expressed in bovine CCs from oocytes with different competence statuses, through microarray analysis. The model of follicle size, in which competent cumulus–oocyte complexes (COCs) were recovered from bigger follicles (≥8.0 mm in diameter) and less competent ones from smaller follicles (1–3 mm), was used. We identified 4178 genes that were differentially expressed (P < 0.05) in the two categories of CCs. The list was further enriched, through the use of a 2.5‐fold change in gene expression as a cutoff value, to include 143 up‐regulated and 80 down‐regulated genes in CCs of competent COCs compared to incompetent COCs. These genes were screened according to their cellular roles, most of which were related to cell cycle, DNA repair, energy metabolism, metabolism of amino acids, cell signaling, meiosis, ovulation and inflammation. Three candidate genes up‐regulated (FGF11, IGFBP4, SPRY1) and three down‐regulated (ARHGAP22, COL18A1 and GPC4) in CCs from COCs of big follicles (≥8.1 mm) were selected for qPCR analysis. The selected genes showed the same expression patterns by qPCR and microarray analysis. These genes may be potential genetic markers that predict oocyte competence in in vitro fertilization routines.     

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.     

The effects of plasma-derived extracellular vesicles on cumulus expansion and oocyte maturation in mice

Cumulus cell expansion is required for the ovulation of a fertilizable oocyte. Extracellular vesicles (EVs) are bilayer-lipid membrane vesicles that may be found in a variety of bodily fluids and play an important role in biological processes. This study aimed to examine the effects of plasma-derived EVs on cumulus expansion and in vitro maturation (IVM) of the oocyte. EVswere isolated using ultracentrifugation from the plasma of female mice. The morphology and size of EVs were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Western blotting allowed us to identify CD63, CD81, CD9, and HSP70 protein markers of EVs; the expression of the genes related to cumulus cell expansion, including hyaluronan synthase 2 (Has2) and prostaglandinendoperoxide synthase 2 (Ptgs2), were assessed using real-time polymerase chain reaction. Plasma-derived EVs labeled with Dil dye were successfully incorporated with cumulus cells during IVM. Plasma-derived EVs significantly induced cumulus expansion and maturation of oocytes. The percentage of oocytes that reached the MII stage was significantly greater in the EVs treatment group compared with other groups. Although treatment with epidermal growth factor (EGF) significantly increased cumulus expansion in cumulus-oocyte complexes (COCs), the impact was less than that seen with plasma-derived EVs. Furthermore, EVs generated from plasma substantially enhanced Has2 and Ptgs2 mRNA expression in the cumulus-oocyte complex. This research indicates that EVs derived from plasma are capable of promoting cumulus expansion and oocyte maturation.     

Regulation of mouse oocyte maturation: effect of elevating cumulus cell cAMP on oocyte cAMP levels

We have reexamined the possibility that cumulus cell cAMP can enter the oocyte via the gap junctions connecting the two cell types (Schultz et al., 1983a). Since our recent results indicate that the mouse oocyte possesses a very active cyclic nucleotide phosphodiesterase (PDE) (Bornslaeger et al., 1984), we have altered our experimental protocol to ensure that mouse oocyte PDE activity is inhibited throughout the duration of an experiment. Our results demonstrate the apparent transfer of cAMP from cumulus cells to the oocyte; these results are discussed in terms of current models for regulation of mammalian oocyte maturation.