Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.     

Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH,and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine whether they would be useful markers of oocyte competence. In serum-free media, only 1000 ng/ml pFSH induced marked cumulus expansion, but the effect of 100 ng/ml pFSH was amplified in the presence of 10% serum. In contrast, cumulus expansion occurred with 1 ng/ml rhFSH in the absence of serum. FSHr mRNA was highest at 0–6 h of maturation, then abundance decreased. Similarly, Cx43 mRNA expression was highest from 0–6 h but decreased by 24 h of maturation. However, the relative abundance of LHr mRNA did not change from 6–24 h of maturation. Decreased levels of FSHr, LHr and Cx43 mRNAs were detected in COCs of poorer quality. In conclusion, expansion of bovine cumulus occurred at low doses of rhFSH in serum-free media. In summary, FSHr, LHr and Cx43 mRNA abundance reflects COC quality and FSHr and Cx43 mRNA expression changes during in vitro maturation; these genes may be useful markers of oocyte developmental competence.     

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte-cumulus cells functional coupling and intracellular concentration of glutathione

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus-oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.     

Connexin 43 coupling in bovine cumulus cells,during the follicular growth phase,and its relationship to in vitro embryo outcomes

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus?oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two‐cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.     

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus‐oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3‐isobutyl 1‐methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. © 1999 Wiley‐Liss, Inc.     

Growth hormone-related effects on apoptosis,mitosis, and expression of connexin 43 in bovine in vitro maturation cumulus-oocyte complexes

Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. Therefore, the aim of our study was to elucidate the mechanisms of GH action during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). As shown by detection of the nuclear cell proliferation-associated antigen Ki-67, COCs matured in vitro in the presence of GH revealed a significantly (P < 0.05) higher proportion of proliferating cumulus cells (12.6%) compared with the COCs matured in the control medium TCM 199 (9.9%). In contrast, the percentage of proliferating cells was not increased by supplementation of the medium with a combination of GH and insulin-like-growth factor I (IGF-I). Apoptosis as determined by TUNEL (terminal doxynucleotidyl transferase mediated dUTP nick-end labeling) was significantly (P < 0.05) reduced in the cumulus cells by GH treatment. COCs matured with a combination of GH and IGF-I revealed the lowest percentage of apoptotic cells (11%). The localization and quantification of the gap junction protein connexin 43 (Cx 43) demonstrated that GH induced a significant decrease in the synthesis of the Cx 43 protein in the cumulus cells. Our results imply that GH increases cumulus expansion by promotion of cell proliferation and inhibition of apoptosis. Whereas the increase in cell proliferation is a direct effect of GH, the antiapoptotic effects of GH during in vitro maturation are modulated by IGF-I. Stimulatory effects of GH on oocyte maturation are correlated with changes in the synthesis of gap junction proteins.     

The influence of cAMP before or during bovine oocyte maturation on embryonic developmental competence

This study was designed to evaluate the effects of pretreatment with various forms of cAMP before or during bovine oocyte maturation on the acquisition of embryonic developmental competence. The objective of the 4 experiments was to induce differentiation of the early maturing oocyte in conditions of maintained meiotic arrest or normal maturation. To promote differentiation, different forms of cyclic AMP-dependent protein kinase (PKA) pathways were investigated. The factors studied included follicular fluid, invasive adenylate cyclase (iAC), dibutyryl-cAMP (dbcAMP) and 3-isobutyl-1-methyl-xanthine (IBMX) with or without cycloheximide (CHX). High concentrations of iAC pretreatment were beneficial to the oocyte competence in BSA-iAC maturation while harmful in normal maturation. Also, after 2 to 3 h IBMX-iAC pretreatment, another 6 h of CHX treatment with or without iAC was harmful to the embryonic developmental competence of fertilized oocytes even though it did not have any effect on cleavage rate. Experiment 4 was to assess the role of cAMP in acquisition of oocyte developmental competence before meiotic resumption. Results supported that the intracellular cAMP concentration during the interval between oocyte isolation from the follicle and the beginning of in vitro maturation is critical for requiring optimal developmental competence.     

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.     

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.     

All‐trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes

In developing follicles, cellular coupling within cumulus–oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All‐trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.     

Apoptosis in porcine cumulus‐oocyte complexes: Relationship with their morphology and the developmental competence

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus‐oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin‐V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin‐V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin‐V (+) oocytes within immature and post‐IVM COCs, but TUNEL (+) oocytes were only observed in post‐IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post‐IVM COCs. However, the difference was only significant for annexin‐V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.     

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.     

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus‐oocyte complexes: A biological and spectroscopic approach

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus‐oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ?‐aminocaproic acid (?‐ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε‐ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε‐ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ?‐ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.     

Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification

In most mammalian ovaries, the cumulus cell-oocyte complex (COC) expands at the time of ovulation by depositing an extensive extracellular matrix between the cumulus cells. This phenomenon can be reproduced in vitro by culturing COCs with follicle-stimulating hormone (FSH) and serum. Biosynthesis of hyaluronic acid (HA) and proteoglycans by mouse COCs in vitro was studied using [3H]glucosamine and [35S]sulfate as metabolic precursors. Radiolabeled complex carbohydrates were analyzed by ion exchange chromatography, specific enzyme digestion followed by high performance liquid chromatography, and gel filtration. The specific activities of [3H]hexosamines in the labeled molecules were determined by measuring the incorporation of 3H and 35S into chondroitin 4-sulfate disaccharides. When COCs were stimulated with FSH, HA biosynthesis increased 20-30-fold between 3-12 h later when expansion occurs, reaching a maximum rate of approximately 780 pmol (as glucosamine)/COC/h compared with the unstimulated rate of approximately 26 pmol/COC/h. The final concentration of HA in the expanded COC was calculated to be approximately 250 micrograms/ml. The effects of dibutyryl cyclic AMP (Bt2cAMP) on COC expansion and HA synthesis were similar to those of FSH, suggesting that the effects of FSH are mediated by cAMP. However, FSH significantly decreased the specific activity of the incorporated hexosamines while Bt2cAMP did not. Serum is necessary for the accumulation of HA in the COC matrix. HA synthesis in FSH-stimulated COCs was as high or higher in the absence of serum, but most was recovered in the medium and not in the COC matrix. The molecular size of the HA was greater than 2 million dalton in either case, suggesting that the serum did not alter physical properties of HA. Stimulation of proteoglycan biosynthesis by either FSH or Bt2cAMP was less pronounced (three to four times control) than for HA and was sustained throughout an 18-h culture period. A reduction of 80% in the deposition of newly synthesized PGs in the COC matrix by 0.5 mM beta-xyloside treatment did not affect the expansion of the cumulus.     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.