Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows.

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Fine-structural cytochemical and immunocytochemical observations on nuclear bodies in the bovine 2-cell embryo.

Nuclear bodies occurring during the 2-cell stage of bovine embryos (obtained either by in vitro fertilisation of in vitro matured ovarian oocytes, or collection after fertilisation and cleavage in vivo) were studied using ultrastructural cytochemistry and immunocytochemistry to determine whether their occurrence may be linked with the onset of embryonic transcription. In addition, the species-specific ultrastructural features of the interchromatin structures of the 2-cell bovine embryo were displayed. Three different types of nuclear bodies were distinguished: (i) nucleolus precursor bodies (NPBs), (ii) loose bodies (LBs) and (iii) dense bodies (DBs). In order to determine their possible functional significance, we considered parallels between these three nuclear entities and interchromatin compartments reported in other cells. As detected by their preferential ribonucleoprotein staining, all types of nuclear bodies contained ribonucleoproteins. In contrast to the other types of nuclear bodies studied, NPBs contained argyrophilic proteins but in no case they did show morphological features of functional nucleoli. Both compact and vacuolated forms of NPBs were seen in both in vivo and in vitro embryos, sometimes simultaneously in the same nucleus. LBs and DBs reacted with antibodies to Sm antigen, indicating the presence of a group of nucleoplasmic, non-nucleolar small nuclear ribonucleoproteins (snRNPs). The immunoreactivity for Sm antigen was more intense and homogeneous in DBs than in LBs. DBs were seen in both categories of embryo. A possible kinship of DBs with the sphere organelle known from oocytes of different animal species or the prominent spherical inclusions of the early mouse embryo nuclei is suggested. The last type of intranuclear body, the LBs, showed a composite structure. Their granular component, occurring in clusters and displaying immunoreactivity for Sm antigen, was similar to interchromatin granules and was therefore named IG-like granules. Another component forming the LBs showed a much finer structure and a lower immunoreactivity with anti-Sm antibodies. We suggest that this amorphous component may be related to the IG-associated zone. All three types of intranuclear bodies were often seen close together, suggesting their possible mutual functional relationship. From these and other observations we conclude that the intranuclear bodies in 2-cell bovine embryos correspond, with the exception of the NPB, to similar structures/compartments supposed to accumulate inactive spliceosomal components in certain phases of somatic cell nucleus functions. Accordingly, the occurrence of such nuclear bodies does not represent cytological evidence for RNA synthesis. In contrast to this, an important morphological feature revealing the status of the bovine 2-cell embryo is the vacuolisation of the NPB.

Culture of bovine oocytes from small antral follicles in meiosis-inhibiting medium with butyrolactone I: RNA synthesis, nucleolar morphology and meiotic competence.

Bovine oocytes originated from follicles of two different size categories (medium (M), 3-6 mm and small (S), 1-2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100 microM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30 min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (4+,3+) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (3+,2+) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.

D-penicillamine and granulosa cells can effectively extend the fertile life span of bovine frozen-thawed spermatozoa in vitro: effect on fertilization and polyspermy.

The effect of D-penicillamine on the fertile life span of frozen-thawed bull spermatozoa was studied in Experiment 1. After thawing, the washed spermatozoa were incubated for 8 h in fertilization medium with 1 mg/mL polyvinyl alcohol (PVA) and 0.5 mg/mL D-penicillamine. The addition of cumulus-free oocytes together with 10% of bovine serum (BOS) + 4 IU/mL heparin to the 8-h incubated spermatozoa resulted in high fertilization and polyspermy rates (97/103, 94.2% and 49/97, 50.5%, respectively). When BOS was substituted with 3 mg/mL of BSA, the fertilization and polyspermy rates decreased to 33/91 (36.3%) and 3/33 (9.1%), respectively. The total absence of fertilization was observed after substitution of proteins with PVA. The 8-h sperm incubation in fertilization medium without D-penicillamine and following fertilization in medium + different capacitation supplements resulted in the total absence of or a very low fertilization rate. In Experiment 2, the first set of cumulus-oocyte complexes (COC) and cumulus-free oocytes were fertilized for 8 h with nonincubated spermatozoa. The second set of COC and cumulus-free oocytes were fertilized in the same wells with spermatozoa after removal of the first set. The fertilization rate for the first set of COC and cumulus-free oocytes was 65/67 (97%) and 73/73 (100%), respectively, with 21/65 (32.3%) and 32/73 (43.8%) polyspermy, respectively. In the second set, the high penetration rate (67/73, 91.8%) was observed only for COC, while that for cumulus-free oocytes (19/76, 25%) was significantly lower (P < 0.01). In Experiment 3, the fertilization rate of COC in medium + PVA + 10 or 100 IU/mL heparin was 69/92 (75%) and 52/74 (70.2%), respectively, with no polyspermy. In medium + BSA + 10 or 100 IU/mL heparin, the fertilization rate of COC was 64/72 (88.9%) and 70/79 (88.6%), respectively, with polyspermy at 2/64 (3.1%) and 7/70 (10%), respectively. In medium + BOS + 10 or 100 IU/mL heparin, the fertilization rate was 79/82 (96.3%) and 79/80 (98.7%), respectively, with a significantly (P < 0.05) higher polyspermy rate (34/79, 43% and 30/79, 38%, respectively). The vitality and capacitation changes of frozen-thawed and 8-h incubated spermatozoa were assessed by Hoechst and chlortetracycline (CTC) staining.

Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes,Without influencing chromosome condensation activity.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes.

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

Fertilisation competence of bovine normally matured or aged oocytes derived from different antral follicles: morphology, protein synthesis, H1 and MBP kinase activity.

We have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M--medium, 2.5-5.0 mm; S--small, 1.5-2.5 mm; T--tiny, 1.0-1.5 mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24 h, the fertilisation of M oocytes cultured for 40 h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24 h or 40 h were mostly arrested at defective late diakinesis-metaphase I (77.5-100%) stage. After fertilisation of S and T oocytes cultured for 24 h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40 h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24 h and decreased during the 40 h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24 h of culture and remained stable until 40 h. The fertilisation after 24 h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40 h culture resulted in complete inactivation of both these kinases to their basal levels.

The cell nucleus in early bovine and caprine preimplantation embryos: fine structural cytochemistry and immunoelectron microscopy.

Fine structural cytochemistry and immunocytochemistry were used to study nucleic acids and nuclear proteins in nuclear bodies (NB) of pronuclear and 2-cell bovine and caprine embryos on ultrathin sections of paraformaldehyde fixed and Lowicryl K4M or LR White embedded specimens. The most striking feature detected in some of these nuclear bodies (NBs) was the presence of non-nucleolar proteins known to be involved in pre-mRNA splicing. One category of such intranuclear bodies (showing a rather dense finely fibrillar composition and named here dense body-DB) contained the Sm-antigen (an antigen common to a major group of nucleoplasmic spliceosomal snRNPs). Another, more numerous category of NBs differed morphologically from the former one by a much looser composition of fibrillogranular elements (loose body-LB). Moreover, it showed the presence of the non-snRNP splicing factor SC-35, in addition to the Sm-antigen. Both categories of these nuclear bodies were distinguished clearly from the nucleolar precursor bodies (NPBs) by an absence of immunolabeling of NPB with antibodies against nuclear proteins involved in splicing. Moreover, the former NBs are not stained with silver, while NPBs already in pronuclei exhibit strong affinity to silver. In addition to the immunolabeling in prominent (approx. 0.2-2.0 microns) NBs, regularly occurring high concentration of snRNP was revealed in very small (approx. 0.05 micron), morphologically poorly defined areas (named here small snRNP-enriched areas-SSA), harboring moreover a set of nuclear proteins similar to that of the coiled body. Numerous observations of the presence of these small areas in nuclear bodies and in their close vicinity, in nucleoplasm, in proximity of the nuclear envelope and also in ooplasm suggested that they are possible carriers of certain nuclear proteins moving between nuclear bodies, nucleoplasm and cytoplasm. A functional relationship of all these embryonic subnuclear elements has not been elucidated so far but their mutual relation is suggested, since the NPBs and other nuclear bodies usually occur in a close association. Fine structural and immunoelectron microscopic observations further suggest a similarity of the nuclear bodies in the early ruminant embryo with specific intranuclear bodies ("snurposomes") known from Xenopus laevis oocytes. A new and striking feature emerging from these observations is a possible involvement of a group of nucleoplasmic proteins in a yet unknown way in the differentiation processes concomitant with early embryonic nucleologenesis.

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid.

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.

Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: an autoradiographic study.

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either "maternal" or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-(3)H]uridine radioactivity accumulated (after a 2-hr pulse in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-(3)H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-(3)H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte-embryo system may explain the specific morphology of both oocyte and early embryo "nucleoli".

F-actin is involved in control of bovine cumulus expansion.

Previously, we showed that the gonadotropin-induced expansion of bovine cumulus oophorus occurs concomitantly with the rearrangement of microfilaments (MFs) inside cumulus cell cytoplasm (Sutovský et al., 1993: Biol Reprod 49:1277-1287; Sutovský et al., 1994: Reprod Nutr Dev 34:415-425) and that cumulus expansion in cattle is accompanied by the increased expression of extracellular matrix (ECM) glycoproteins laminin and type IV collagen as well as of their actin-linked membrane receptors, integrin subunits alpha-6 and beta-1 (Sutovský and Motlík: 1994). The present study was undertaken to determine the spatial and temporal relationship between cytoskeletal rearrangement and ECM synthesis during cumulus expansion. Using electron microscopy and confocal (LSCM) and conventional fluorescence microscopy, we compared the expression of the above integrins and ECM proteins and the rearrangement of cytoskeleton in the gonadotropin-stimulated bovine oocyte cumulus complexes (OCCs) with those exposed to gonadotropin stimulation and to ECM synthesis inhibitor 6-diazo-5-oxo-L-norleucin (DON), or MF-disorganizing drug cytochalasin B (CB). In control OCCs, the 24-hr culture in the presence of follicle stimulating hormone/luteinizing hormone (FSH/LH) caused the expansion of cumuli oophori and an extensive rearrangement of MFs in the cytoplasm of cumulus cells. Concomitantly, we observed an increased deposition of laminin and type IV collagen in the intercellular spaces among cumulus cells. The redistribution of microtubules (MTs), intermediate filaments (IFs), and integrin chains alpha-6 and beta-1 also occurred at this time. The addition of 20 micrograms/ml of CB prevented cumulus expansion and accumulation of laminin and type IV collagen in the OCCs. Moreover, cytochalasin treatment blocked the redistribution of MTs and IFs, and caused the disorganization of MFs and dispersion of integrins in cumulus cells. In contrast, the distribution of integrins and cytoskeletal elements was not affected when we blocked cumulus expansion and ECM protein accumulation by DON. These data suggest that F-actin acts upstream of ECM synthesis in the cascade of events leading to the expansion of bovine cumulus ooophorus.

Microfilaments, microtubules and intermediate filaments fulfil differential roles during gonadotropin-induced expansion of bovine cumulus oophorus.

The relationship between cytoskeleton and morphology of cumulus granulosa cells in expanding bovine oocyte-cumulus complexes (OCCs) cultured in vitro has been investigated by the means of indirect immunofluorescence and transmission electron microscopy. The round-shaped cells in unstimulated control OCCs displayed a homogeneous distribution of cytoskeletal networks and cytoplasmic organelles. Luteinizing hormone (LH) stimulation caused the redistribution of microfilaments (MFs), accelerated the development of Golgi apparatus, and led to the generation of lipid droplets in cumulus cells. These changes culminated in the elongation and polarization of cumulus cells and in the extension of the cytoplasmic networks of microtubules (MTs) and intermediate filaments (IFs) into the newly formed cytoplasmic projections. The culture of OCCs in the presence of microfilament disruptor cytochalasin B prevented cumulus expansion, formation of cellular projections and cell elongation and suppressed the development of the Golgi apparatus. On the contrary, cytochalasin had no effect on the abundance and distribution of lipid droplets and on the integrity of IFs and MTs. The present data support the hypothesis that the response of cumulus granulosa cells to LH is partially mediated by F-actin.

Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles.

The normalcy of nuclear differentiation in 8-cell bovine embryos derived by in vitro procedures from oocytes isolated from antral follicles of three different size categories was studied using autoradiographic localization of RNA synthesis and fine-structure nuclear morphology. In the few embryos derived from the oocytes from the small category of follicles (1-2 mm) the unlabeled nuclei prevailed. In contrast, the oocytes isolated from the more progressed follicles (medium and large size: 2-8 mm) yielded embryos with only slight, if at all detectable, differences to the picture expected in in vivo developed normal embryos. The diverging features probably concerned a slower onset of gene transcription, its consecutive pattern of localization as well as less pronounced progress of chromatin condensation and nucleolar differentiation. The assessment of these morphological changes and characteristics for individual embryos was obscured by marked differences in the status of the nuclei of particular blastomeres forming an embryo. Here the differences were seen in the fragmentation of nuclei, in the degree of chromatin condensation and in the absence of the nucleolus-associated chromatin in some of the blastomeres. It is suggested that most of the embryos from medium and large follicles have a comparable differentiation pattern of nuclear function development as embryos developing normally in vivo.

Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles.

This study investigated the capacity of healthy oocytes derived from follicles of different size to undergo normal fertilization and early embryonic development in vitro and full-term development in vivo. Ovaries were collected from a local abattoir and dissected and classified as follows: group A, greater than 4-8 mm (large); group B, greater than 2-4 mm (medium); and group C, greater than 1-2 mm (small). Oocytes were isolated by puncturing the follicular wall and pressing of the follicle. Only healthy-looking cumulus-oocyte complexes (COC) were used for in vitro maturation. Oocytes were fertilized in vitro by frozen/thawed semen from one bull. Approximately one-fourth of all oocytes was fixed and stained 15-20 h after fertilization, to determine penetration rates. The remaining eggs were transferred to culture medium and were cultivated for up to 9.5 days. Cleavage was observed 65 h and 7 days after fertilization. Expanded, hatching, and hatched blastocysts were fixed and stained after 9.5 days of culture. A total of 86 blastocysts derived from group A and B oocytes was nonsurgically transferred to synchronized recipients 7-8 days after onset of culture. A total of 6.624 follicles were dissected from 265 ovaries, and 1,485 oocytes were isolated from 1,671 group A follicles, 3,509 oocytes from 3,862 group B follicles, and 965 oocytes from 1,091 group C follicles. The fertilization rate, rate of normal fertilization, rate of polyspermy, and rate of other abnormal fertilization features were as follows: group A, 84.9%, 43.2%, 34.1%, 7.6%; group B, 83.6%, 44.8%, 31.1%, 7.8%; and group C, 61.7%, 13.1%, 33.7%, 19.1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopic localization of small nuclear ribonucleoproteins during bovine early embryogenesis.

Small nuclear ribonucleoproteins (snRNPs) were localized using human autoimmune or monoclonal anti-snRNP antibodies and ultrastructural immunocytochemistry in early preimplantation bovine embryos before and at transcription onset. Bovine cleavage stages up to 16-cell embryos were obtained either by culture of in vitro-fertilized ovarian oocytes or by isolation at slaughter of embryos fertilized and developed in vivo. Before transcription onset, up to the four-cell stage, diffuse labeling of nucleoplasm was detected, whereas cytoplasm labeling remained low. At the transcription onset, labeling of all eight-cell embryo nuclei was markedly concentrated at the borderline of already formed, condensed chromatin aggregates, where it was associated mainly with perichromatin fibrils. The condensed chromatin blocks revealed by several different staining methods were more prominent than is the case in most somatic cells. The degree of chromatin condensation and the pattern of its distribution differed between in vivo- and in vitro-produced eight-cell embryos: the in vitro embryos showed a higher degree of chromatin condensation and a peripheral distribution of chromatin blocks. A relation of this observation to the developmental potential of cow embryos is suggested. In two- and four-cell embryos, intensive labeling was seen in interchromatin granules, which, in their turn, were often seen in close proximity to the nucleolus precursor bodies, or in the four-cell stage were interconnected to them. No labeling was ever seen, using antibodies specific for the snRNP Sm antigen, in nucleolar precursor bodies during embryonic nucleologenesis nor in the resulting nucleoli. There was some incidental labeling of the large central vacuole appearing at the beginning of the nucleolus precursor body transformation, testifying the nucleoplasmic origin of this entity.

Penetration of intact bovine ova with ram sperm in vitro.

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf.

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.