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.     

Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte

In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring ³H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24–32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24?32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17β-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei. These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.     

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.     

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.     

The potential role of gap junction communication between cumulus cells and bovine oocytes during in vitro maturation

Preliminary studies in our laboratory have indicated that modulating cumulus expansion early or late during culture has a profound influence on the subsequent development of cumulus-enclosed oocytes. Our objectives were to evaluate the effect of short term exposure to recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on cumulus expansion and developmental competence of bovine oocytes. A highly significant (P < 0.0001) improvement in blastocyst development rate as a proportion of cleaved oocytes after IVM of oocytes was observed in the presence of r-hFSH for the first 6 hr of culture. To demonstrate the importance of the functional coupling between the oocyte and the cumulus compartment during that period of 6 hr, cumulus-oocyte complexes (COCs) were matured with r-hFSH for the first 6 hr followed by 18 hr in presence of 1-heptanol or 1-octanol (gap junction inhibitors) to block the communication between the two. With the coupling inhibitors, the blastocyst yield was significantly decreased (P < 0.05). A brief treatment (30 min) with the weak base methylamine, known to reverse the gap junction inhibitors effect, significantly (P < 0.05) reversed the inhibitory action of these agents on the blastocyst rate. Gap junction communication between the oocyte and surrounding cumulus cells was further studied using microinjection of the fluorescent dye Lucifer Yellow. Morphological evidences (dye transfer) were obtained that support the presence of functional coupling for a longer period with the FSH short exposure. In conclusion, high developmental rates of bovine oocytes can be achieved with a short exposure to r-hFSH. This effect is believed to be mediated through gap junctions as developmental competence of oocytes is compromised by the inhibition of their function.     

Development of microsatellite markers and comparative mapping for bovine chromosome 19

Previous research has mapped an ovulation rate quantitative trait locus (QTL) to bovine chromosome 19. In an effort to enhance comparative mapping information and develop additional markers for refined QTL mapping, microsatellite markers were developed in a targeted approach. A bovine bacterial artificial chromosome (BAC) library was screened for loci with either known or predicted locations on bovine chromosome 19. An average of 6.4 positive BAC were identified per screened locus. A total of 10 microsatellite markers were developed for five targeted loci with heterozygosity of 7-83% in a sample of reference family parents. The newly developed markers were typed on reference families along with four previously mapped marker loci and used to create a linkage map. Comparison of locus order between human and cattle provides support for previously observed rearrangement. One of the mapped loci myotubularin related protein 4 (MTMR4) potentially extends the proximal boundary of a conserved linkage group.     

Maintenance of meiotic arrest in bovine oocytes in vitro using butyrolactone I: effects on oocyte ultrastructure and nucleolus function

In this study, we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, affects oocyte cytoplasmic morphology and nuclear function in terms of nucleolar ultrastructure and immunocytochemistry. Bovine oocytes were recovered from three classes of follicle size: 1.5-2, 2-3, and 3-6 mm. The oocytes were incubated for 40 hr with BL-I, and subsequently processed for transmission electron microscopy or immunocytochemistry. A control group of oocytes were processed immediately upon recovery (0 hr). In general, incubation with BL-I for 40 hr disrupted contact between cells of the cumulus oophorous and the oocyte, caused degeneration of the cortical granules and the peripheral migration of all cytoplasmic organelles. At the level of the nucleus, it induced convolution of the nuclear membrane and caused acceleration of nucleolar compaction in oocytes from follicles < 3 mm and fragmentation of nucleoli, particularly evidenced by immunocytochemistry, in oocytes from follicles > 3 mm. Furthermore, the effects appear to be more profound in fully-grown oocytes.     

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.     

Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22h in vitro , 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro , only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.