EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0–6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components. © 1996 Wiley-Liss, Inc.     

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.     

Caproyl esterase from rat testis: Purification and action on cumulus cells

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein. The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate. The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.     

The mechanism of cumulus cell-oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell-oocyte uncoupling could occur independently of FSH-stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell-oocyte complex. It was found that the complete suppression of FSH-stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH-stimulated cumulus cell-oocyte uncoupling. This finding showed that FSH-stimulated cumulus expansion was not required for cumulus cell-oocyte uncoupling. Since 17β-estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling-promoting action of FSH was probably not mediated by steroid hormones. A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell-oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell-oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.     

Mechanistic insights into the reduced developmental capacity of in vitro matured oocytes and importance of cumulus cells in oocyte quality determination

In vitro maturation of oocytes is a promising assisted reproductive technology (ART) for infertility treatment, although it is still not a routine technique for human ART due to reduced embryonic development. The aim of the present study was to clarify the possible reasons for reduced capacity of in vitro matured oocytes. Our results showed that the oocytes matured in vitro displayed increased abnormal mitochondrial distribution, reduced mitochondrial membrane potential, and increased reactive oxygen species levels when compared to in vivo matured oocytes. These results were not different in oocytes matured in vitro with or without cumulus cells. Notably, in vitro matured oocytes displayed increased mitochondrial DNA numbers probably due to functional compensation. In vitro matured oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca²⁺ oscillations was much lower in response to parthenogenetic activation, especially in oocytes matured in vitro without cumulus cells with nearly half of them failing to produce calcium waves upon strontium chloride stimulation. These data are important for understanding the reasons for reduced developmental potential of in vitro matured oocytes and the importance of cumulus cells for oocyte quality.     

Changes in cortical granules during porcine oocyte maturation

The structure, number, and distribution of cortical granules in porcine oocytes during maturation induced by human chorionic gonadotrophin (HCG) are reported. The number of granules remained constant for 30 hr following HCG. Thereafter, there was an approximate doubling by 50 hr. At all times examined, a dark and light form were present. Up to 40 hr the latter predominated while at 50 hr there was a marked increase in the number of the former. Movement of cortical granules to the plasma membrane took place between 20 and 30 hr post-HCG. The changes in cortical granules are correlated with the capacity of the oocytes to undergo a block to polyspermy.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

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.     

Influence of luteinizing hormone and progesterone on maturation and fertilizability of rat oocytes in vitro

The effects of luteinizing hormone (NIH-bovine LH) and progesterone on maturation in vitro of oocyte-cumulus complexes from adult proestrous rats were studied by comparing proportions of oocytes showing germinal vesicle breakdown, mucification of the cumulus oophorus, and fertilizability. Addition of either or both of the hormones to the medium in concentrations between 1.25 and 10 μg/ml during maturation had no discernible effect on germinal vesicle breakdown or on fertilization. Mucification was stimulated by LH and even more by LH plus progesterone. It was concluded that maturation in vivo is the result of concerted action of the two hormones. However, addition of LH + progesterone had no effect on the fertilizability of these oocytes. We attribute this to a relative insensitivity of the system for fertilization in vitro to subtle changes in the oocyte.     

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.     

DNA damage response during mouse oocyte maturation

Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.     

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.     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrode's albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.     

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.