In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

Antioxidant requirements for bovine oocytes varies during in vitro maturation,fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.     

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

Changes in protein synthesis pattern during in vitro maturation of goat oocytes.

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis.     

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.     

In vitro maturation of cattle oocytes taken from ovaries with different morphofunctional states

We studied the capacity of cattle oocytes taken from ovaries with different morphofunctional state for development to metaphase 2 in vitro. A classification of ovaries has been proposed according to their morphofunctional state: (1) ovaries with a yellow body from the last cycle, without dominating follicle, with many follicles of varying diameter; (2) ovaries with a yellow body from the last cycle, with dominating follicle (from 10 mm in diameter); (3) ovaries with a large functioning yellow body and follicles of varying diameter; (4) ovaries with a follicular cystoid formation (more than 25 mm in diameter); (5) ovaries with a yellow body from past cycles and small (1-2 mm) follicles, supposedly with a weakened hormonal function. It was shown that the morphofunctional state of ovaries determined the total number of oocytes isolated from an ovary and number of morphologically normal oocytes feasible for cultivation. At the same time, no reliable differences in the capacity for extrusion of the first polar body between the oocytes from the ovaries of different types were found in the experiments on in vitro oocytes maturation. Since the coefficient of correlation between the extrusion of the first polar body and maturation to metaphase 2 was in 0.95, there is every reason to believe that the capacity for development to metaphase 2 does not depend on the morphofunctional state of ovaries.     

Protein patterns of pig oocytes during in vitro maturation

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.     

Changes in protein synthesis during maturation of sheep oocytes in vivo and in vitro.

The qualitative profiles of the proteins synthesized by sheep oocytes at various stages of maturation were determined by electrophoretic separation in one dimension on polyacrylamide SDS gels. No change in protein synthetic pattern was observed in ooce changes had taken place in at least 12 separate protein bands. Marked alterations in the synthesis of some proteins were apparent 15 h after LH; formation of proteins in 5 of the original bands was either reduced or not detectable, while new synthesis was evident from the appearance of 7 additional bands. The pattern of proteins produced by oocytes cultured within the follicle corresponded closely with that observed in vivo: changes in synthesis were initiated about 9 h after addition of gonadotrophin and were completed by 15 h. Oocytes cultured outside the follicle in a gonadotrophin-containing medium did not exhibit a change in protein synthesis and at 15 h only those proteins produced during the early stages of maturation were being synthesized.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes

Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro.     

Cumulus cell‐enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in vitro maturation in pigs

We investigated (i) follicle stimulating hormone (FSH)‐modulated changes in the expression of glutathione (GSH) and its rate‐limiting enzyme, glutamate cysteine ligase (GCL), in porcine oocytes and cumulus cells, and (ii) the contribution of gap‐junctional communications (GJCs) in cumulus‐oocyte complexes (COCs) to intraoocyte GSH accumulation. In experiment (i), COCs were cultured for 48 h with (+FSH group) or without FSH (−FSH group). The GSH content of oocytes increased with cultivation time in the +FSH group, but decreased in the −FSH group. The GSH content of cumulus cells at 48 h was also higher in the +FSH group than that in the −FSH group. Expression of GCL subunit mRNAs in oocytes and cumulus cells was increased by FSH stimulation until 12 h, and then fell to the baseline level. On the other hand, the amount of GCL subunit proteins in oocytes and cumulus cells increased gradually throughout the period of culture with FSH. In experiment (ii), blocking of GJCs in COCs during 0–24 h of culture led to a decrease in the GSH content of oocytes at 24 h of culture, whereas the GSH content at 48 h of culture did not differ even after blocking of the GJCs during 24–48 h of culture. These findings indicate that FSH initiates GSH synthesis in cumulus cells and oocytes by modulating the expression of GCL, and that porcine oocytes are able to synthesize GSH without GJC‐mediated support from cumulus cells, at least in the later half of maturation culture. J. Cell. Physiol. 222: 294–301, 2010. © 2009 Wiley‐Liss, Inc.     

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of bovine oocytes

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4–24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [³⁵S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation. © 1996 Wiley-Liss, Inc.     

Changes in histone modifications during in vitro maturation of porcine oocytes

Nuclear core histone modifications influence chromosome structures and functions. Recently, the involvement of histone acetylations in the cell memory of gene expression has been suggested in mouse oocyte maturation. At present, there is little available data on histone modifications in mammalian oocyte maturation. In the present study, we examined changes in the acetylation of histone H3 lysines 9 (H3K9) and 14 (H3K14), and histone H4 lysines 5 (H4K5), 8 (H4K8) and 12 (H4K12), and trimethylation of H3K9 during in vitro maturation of porcine oocytes. Immunocytochemical analyses revealed that the all of the lysines examined were highly acetylated in the germinal vesicle stage, and this level of acetylation was maintained until the first prometaphase. In the first metaphase, the lysines near the N-terminal end, H3K9 and H4K5, were completely deacetylated. The acetylation of the lysines far from the N-terminal end, H3K14, H4K8, and H4K12, was markedly decreased but still present. The acetylations were increased transiently at the first anaphase and telophase, and then decreased again at the second metaphase to the same level as the first metaphase. Since effective concentrations of trichostatin A (TSA) to inhibit the deacetylation were different in various lysine residues, multiple histone deacetylases (HDACs) were suggested to function during meiotic maturation. The trimethylation of H3K9 was maintained in a high level throughout maturation. These results suggest that the histone acetylation during porcine oocyte maturation is precisely controlled by the cell cycle.     

Quality of vitrified porcine immature oocytes is improved by coculture with fresh oocytes during in vitro maturation

It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two‐chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker? Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.     

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes

The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final blastocyst yields were reduced.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.