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PubMed2006

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth.

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.

Animals↗
PubMed2002

Bovine oocytes in early antral follicles grow in serum-free media: effect of hypoxanthine on follicular morphology and oocyte growth.

Some culture systems have been shown to support oocyte growth in mice, although there has been little success in applying these systems to other species. In the present study, we compared three culture conditions for growing bovine oocytes and examined the effect of hypoxanthine on oocyte growth. In the first experiment, early antral follicles, 0.4-0.7 mm in diameter were collected, and oocyte-cumulus-granulosa cell complexes (OCGs) and oocyte-cumulus cell complexes (OCs) were dissected from the follicles. Follicles (Fs), OCGs and OCs were embedded in collagen gels and cultured in serum-supplemented medium for 16 days. In the Fs, OCGs and OCs cultured in hypoxanthine-free medium, 21%, 9% and 4% of the oocytes showed normal morphology, respectively, and hypoxanthine (4 mM) increased the percentages in all the groups (Fs, 37%; OCGs, 29%; OCs, 10%). In the second experiment, Fs were cultured in serum-free medium with or without hypoxanthine for 16 days. Histological examination demonstrated that hypoxanthine maintained the integrity of the follicular basement membrane. After a growth culture, 91% of the oocytes showed normal morphology, and 87% of the oocytes were at the germinal vesicle stage in serum-free, hypoxanthine-supplemented medium. The mean diameters of the oocytes were significantly larger (117.6 +/- 5.7 microm) than they were in the other groups and than they had been before the culture (approximately 95 microm). After a subsequent maturation culture of the oocytes, 85% underwent germinal vesicle breakdown and 23% reached the second metaphase. These results demonstrate that growing bovine oocytes from early antral follicles grow efficiently in follicles cultured in serum-free, hypoxanthine-supplemented medium and acquire meiotic competence.

Animals↗
PubMed2004

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection.

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.

Animals↗
PubMedDate not supplied

Oocyte membrane maturation and oocyte-sperm relationship: transmission electron microscopy study.

The success of in vitro maturation (IVM) depends greatly on the acquisition of immature oocytes. Immature oocytes in prophase I (PI) and metaphase I (MI), aspirated after controlled ovarian hyperstimulation, were incapable of fertilization, leading to a lower fertilization rate. Therefore, they must be evaluated on a fine structure level for their in vitro maturation (IVM) processes and their relationship with sperm. Oocyte membrane maturation and oocyte-sperm relationship were studied using transmission electron microscopy. A total of 55 human oocytes obtained from 20 patients at various times and 83 oocytes obtained from the dissected ovarians of female Wistar rats were used for transmission electron microscopy (TEM) evaluation. Despite being in either prophase I and metaphase I or in metaphase II, the oocytes were not fertilized after 48 h of incubation. At the various stages of maturation between PI and MII, the number and the size of microvilluses on the oocyte membrane increased as MII approached and decreased after full maturation. Oocyte activation was related to oocyte membrane maturation and has an effect on the oocyte sperm penetration.

Female↗
PubMed1976

Control of chromosome behavior in amphibian oocytes. II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosome condensation in transplanted brain nuclei by oocyte cytoplasm.

We studied the effects of actinomycin D, alpha-amanitin, puromycin, and cycloheximide on the cytoplasmic activity of maturing Rana pipiens oocytes that induces chromosome condensation in transplanted brain nuclei. Treatment of oocytes with each inhibitor suppressed the chromosome condensation induced by metaphase oocytes to varying degrees depending upon the dose of inhibitor, despite the fact that untreated metaphase I oocytes already possessed chromosome condensation activity (CCA). Treatment of brain nuclei before injection completely suppressed condensation at all doses used. Chromosome condensation induced by metaphase II oocyte cytoplasm, however, was insensitive to all the inhibitors, even when the brain nuclei were pretreated. Oocytes treated with alpha-amanitin throughout maturation induced chromosome condensation when tested at metaphase II. Removal of the oocyte chromosomes after the germinal vesicle (GV) broke down did not prevent the development of CCA, whereas removal of the entire GV before initiation of maturation deprived oocytes of CCA. The results suggest that metaphase I oocyte cytoplasm stimulates synthesis of brain nuclear RNAs that are translated into proteins necessary for chromosome condensation, whereas metaphase II oocytes possess all the factors for chromosome condensation. In both cases, GV nucleoplasm appears indispensable for the development of CCA, whereas immediate activity of the oocyte genome is not required.

Amanitins↗
PubMed1990

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.

Animals↗
PubMed1997

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Animals↗
PubMed2002

Inositol 1,4,5-trisphosphate receptor function in human oocytes: calcium responses and oocyte activation-related phenomena induced by photolytic release of InsP(3) are blocked by a specific antibody to the type I receptor.

Type I inositol 1,4,5-trisphosphate-sensitive receptors (InsP(3)R) are expressed in human oocytes and may be involved in operating the Ca(2+) release triggered by the fertilizing sperm. This study examines the contribution of type I InsP(3)R in operating Ca(2+) release in human oocytes secondary to InsP(3) itself, using a specific function-blocking antibody in conjunction with photolytic release of microinjected InsP(3). Intracellular Ca(2+) responses were assessed in oocytes microinjected with only caged InsP(3) in experiment set A, while in experiment sets B and C, sibling oocytes were injected with caged InsP(3) and the blocking antibody or a corresponding volume of medium, prior to flash photolysis. In experiment set C, certain fertilization-related phenomena (cortical granule exocytosis and chromatin configurations) were assessed using optical sections and three-dimensional image reconstructions obtained from a confocal laser scanning microscope. In experiment set A, photolytic release of InsP(3) triggered a Ca(2+) response (increase from approximately 100 to 220 nmol/l followed by an exponential recovery, n = 8) and a wave in the oocytes that spread from the stimulation point to the opposite pole. In set B, photolytic InsP(3) release generated Ca(2+) responses in control oocytes (n = 9), but not in the antibody-injected oocytes (n = 7). In set C, cortical granule exocytosis and anaphase chromosome configurations were noted in the control oocytes after flash photolysis (n = 6). These changes were completely absent in antibody injected oocytes as their cortical granules were intact and the chromosomes were in metaphase. These oocytes had also lacked Ca(2+) responses as in set B (n = 5). This study demonstrates the functional presence of type I InsP(3)R-operated Ca(2+) channels in human oocytes and further suggests an active role of InsP(3) in triggering the Ca(2+) rise and secondary activation phenomena at fertilization.

Antibodies, Monoclonal↗
PubMed1995

Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa.

When intracytoplasmic sperm injection (ICSI) is performed, it is important to know the capacity of sperm cells to activate the oocytes, although knowledge of their ability to fuse with the oocytes is not vital. Hamster oocytes are not suitable for this purpose because they are easily activated by the injection procedure Itself. We therefore investigated whether mouse oocytes could be used to assess the activation properties of human spermatozoa. Mouse oocytes were randomized for injection with initially motile spermatozoa, medium, heat-treated or salt-extracted spermatozoa, and the survival and activation rates were examined. About half of the mouse oocytes survived the intracytoplasmic injection of a human sperm cell. Unlike hamster oocytes, the rate of activation provoked by the injection procedure itself was acceptably low (20%), resembling in this respect the behaviour of human oocytes. Following the injection of initially motile human spermatozoa, all mouse oocytes were activated. The injection of heat-treated or salt-extracted human spermatozoa resulted in activation rates of 14 and 15% respectively, comparable with the results following sham ICSI. These data support the hypothesis of a sperm-associated oocyte activation factor. In most activated oocytes, the human sperm nucleus decondensed to form a male pronucleus. Cytogenetic analysis at the first metaphase revealed that human sperm chromosomes were able to undergo replication in a heterologous environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

An analysis of human oocytes and follicles from stimulated cycles: oocyte morphology and associated follicular fluid characteristics.

The aim of this study was to determine whether certain characteristics of the follicular biochemistry, previously shown by us to be associated with oocyte developmental capacity, also reflected differences in oocyte appearance, and to determine the range of oocyte characteristics induced by ovarian stimulation. A representative sample of 33 human oocytes and associated follicular fluids was obtained after a follicular growth period considered suitable for IVF. Individual follicular fluid protein and proteoglycan levels, and follicular volume were compared with the morphological characteristics of each oocyte. Oocytes which retained the germinal vesicle nuclear status after exposure to human chorionic gonadotrophin tended to occur in small follicles (less than or equal to 2 ml) and to be highly vacuolated and with characteristic predicted a low potential for cleavage. Among those oocytes which had progressed to MII nuclear maturity [in the medium (2.5-6.5 ml) and large (greater than or equal to 7 ml) volume follicles] the degree of oocyte vacuolation was negatively correlated with alpha 1-antitrypsin level, while the degree of organelle clumping was correlated with proteoglycan and immunoglobulin levels. Only five of the oocytes (15%) in this sample had follicular characteristics consistent with a normal potential for pregnancy. These MII oocytes occurred within the medium volume range, had low vacuolation levels, and a low degree of organelle clumping. In contrast, those oocytes with a low potential for cleavage based on their follicular biochemistry, showed high cytoplasmic vacuolation levels.

Cell Nucleus↗
PubMed2005

Neonatal genistein treatment alters ovarian differentiation in the mouse: inhibition of oocyte nest breakdown and increased oocyte survival.

Early in ovarian differentiation, female mouse germ cells develop in clusters called oocyte nests or germline cysts. After birth, mouse germ cell nests break down into individual oocytes that are surrounded by somatic pregranulosa cells to form primordial follicles. Previously, we have shown that mice treated neonatally with genistein, the primary soy phytoestrogen, have multi-oocyte follicles (MOFs), an effect apparently mediated by estrogen receptor 2 (ESR2, more commonly known as ERbeta). To determine if genistein treatment leads to MOFs by inhibiting breakdown of oocyte nests, mice were treated neonatally with genistein (50 mg/kg per day) on Days 1-5, and the differentiation of the ovary was compared with untreated controls. Mice treated with genistein had fewer single oocytes and a higher percentage of oocytes not enclosed in follicles. Oocytes from genistein-treated mice exhibited intercellular bridges at 4 days of age, long after disappearing in controls by 2 days of age. There was also an increase in the number of oocytes that survived during the nest breakdown period and fewer oocytes undergoing apoptosis on Neonatal Day 3 in genistein-treated mice as determined by poly (ADP-ribose) polymerase (PARP1) and deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL). These data taken together suggest that genistein exposure during development alters ovarian differentiation by inhibiting oocyte nest breakdown and attenuating oocyte cell death.

Animals↗
PubMed1997

Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse.

Horse oocytes with expanded (EX) cumuli appear to have greater meiotic competence than do horse oocytes with compact (CP) cumuli but are thought to come from atretic follicles. We evaluated the relationships among cumulus expansion, follicle viability, initial chromatin configuration, and meiotic competence of horse oocytes. Follicle walls were sectioned for histological examination, and the follicles were scraped to obtain the oocytes. Half of the oocytes were evaluated immediately and half were matured for 24 h in vitro. Cumulus expansion was significantly associated with follicle atresia. Initially, significantly more EX than CP oocytes had chromatin condensed into one mass within the germinal vesicle (CC configuration; 61% vs. 32%). After culture, significantly more EX than CP oocytes had matured (74% vs. 30%). The proportion of oocytes with the CC configuration was lowest in viable follicles and increased in follicles with slight to moderate atresia. The maturation rate of oocytes from viable follicles was significantly lower than for oocytes from follicles with slight or moderate atresia. The CC chromatin configuration appears to be associated with meiotic competence in horse oocytes. The association of follicle atresia with increased meiotic competence suggests that acquisition of meiotic competence is related to a loss of suppressive activity by the degenerating follicle.

Animals↗
PubMed2000

Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis.

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 + newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO(2), 7% O(2), 88% N(2) culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Amino Acids↗
PubMed2006

Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes.

BACKGROUND: Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. METHODS: An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. RESULTS: The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the present study. CONCLUSION: The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for a full oocyte activation response. This study may contribute to the understanding of possible negative effects of skipping gamete interaction in IVF techniques.

Animals↗
PubMed2004

Follicle-stimulating hormone regulates oocyte growth by modulation of expression of oocyte and granulosa cell factors.

Oocyte-granulosa cell communication is essential for oocyte development. The aims of this study were: 1) to determine the effect of FSH on expression of Kit ligand (KL), growth/differentiation factor-9, bone morphogenetic protein (BMP)-15, and Kit during growth of oocyte-granulosa cell complexes (OGCs) in vitro; 2) to investigate the role of BMP-15 in regulation of KL expression; and 3) to correlate mRNA expression with oocyte growth. OGCs from 12-d-old mice were cultured for up to 7 d in the presence of FSH [0.05 ng/ml (low), 5 ng/ml (high)] or BMP-15 (10 or 100 ng/ml). Transcripts were quantified using real-time RT-PCR, and oocyte and OGC diameters were measured. FSH regulated KL expression in a biphasic manner, with low FSH decreasing the KL-1/KL-2 ratio, and high FSH increasing the KL-1/KL-2 ratio, compared with controls (P < 0.05). The decrease in KL-1/KL-2 ratio with low FSH was due to increased KL-2 mRNA expression. Both FSH concentrations increased OGC diameter (P < 0.05), but only low FSH promoted oocyte growth (P < 0.05). High FSH also decreased BMP-15 expression (P < 0.05). FSH-stimulated oocyte growth was inhibited by Gleevec, an inhibitor of Kit activity. BMP-15 increased both KL-1 and KL-2 mRNA levels in a dose-dependent manner (P < 0.05) but did not alter the KL-1/KL-2 ratio or promote oocyte growth. When the KL-1/KL-2 ratio was increased by exogenous KL-1, FSH-stimulated oocyte growth was suppressed (P < 0.05), suggesting that lowered KL-1/KL-2 ratio is important for oocyte growth. In summary, the correct concentration of FSH is crucial for appropriate modulation of KL and BMP-15 to promote oocyte growth.

Animals↗
PubMed1993

Porcine oocytes release cumulus expansion-enabling activity even though porcine cumulus expansion in vitro is independent of the oocyte.

In the mouse, the oocyte secretes a factor that enables cumulus cells to undergo expansion in response to epidermal growth factor (EGF) or FSH while expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. This study was undertaken to confirm independence of the porcine cumulus oophorus of the oocyte for its expansion and to determine if the porcine oocyte secretes the putative cumulus expansion-enabling factor that is required for FSH-stimulated mouse cumulus expansion. Porcine oocyte-cumulus cell complexes (P-OCC), oocytectomized oocyte-cumulus cell complexes (P-OOX) and intact clumps of cumulus cells (P-CCC) were cultured at 39 C in TCM-199 medium containing EGF (1 ng/ml) or FSH (1.5 micrograms/ml). After 24h culture periods, cumulus expansion was scored on an arbitrary scale of 0 to +4. EGF stimulated similar cumulus expansion (expansion score +3 compared to 0 for controls) in all the three groups. FSH stimulated cumulus expansion in significantly higher number of complexes in all groups compared to EGF. However, there was no difference in cumulus expansion among the three groups with EGF or FSH. To determine the production of the factor by the porcine oocyte, isolated clumps of cumulus cells of the mouse (M-CCC) were cocultured with porcine denuded oocytes (P-DO) in TCM-199 with or without 1.5 micrograms/ml FSH at 37 C for 24h. No expansion was observed when M-CCC were cultured alone in the presence of FSH or cocultured with P-DO in the absence of FSH. However, coculture of M-CCC with P-DO in the presence of FSH resulted in expansion of M-CCC similar to that observed in intact mouse oocyte-cumulus cell complexes (M-OCC) in response to FSH. These studies indicate that even though porcine cumulus expansion in vitro is not dependent on the oocyte, the porcine oocyte is capable of secreting cumulus expansion-enabling factor(s).

Animals↗
PubMed2001

A mammalian oocyte-specific linker histone gene H1oo: homology with the genes for the oocyte-specific cleavage stage histone (cs-H1) of sea urchin and the B4/H1M histone of the frog.

Oocytes and early embryos of multiple (non-mammalian) species lack the somatic form of the linker histone H1. To the best of our knowledge, a mammalian oocyte-specific linker (H1) histone(s) has not, as yet, been reported. We have uncovered the cDNA in question in the course of a differential screening (suppression subtractive hybridization (SSH)) project. Elucidation of the full-length sequence of this novel 1.2 kb cDNA led to the identification of a 912 bp open reading frame. The latter encoded a novel 34 kDa linker histone protein comprised of 304 amino acids, tentatively named H1oo. Amino acid BLAST analysis revealed that H1oo displayed the highest sequence homology to the oocyte-specific B4 histone of the frog, the respective central globular (putative DNA binding) domains displaying 54% identity. Substantial homology to the cs-H1 protein of the sea urchin oocyte was also apparent. While most oocytic mRNAs corresponding to somatic linker histones are not polyadenylated (and remain untranslated), the mRNAs of (non-mammalian) oocyte-specific linker histones and of mammalian H1oo, are polyadenylated, a process driven by the consensus signal sequence, AAUAAA, detected in the 3'-untranslated region of the H1oo cDNA. Our data suggest that the mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homolog of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the GV (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the two-cell embryo, extinction becoming apparent at the four- to eight-cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.

Amino Acid Sequence↗
PubMed2006

Effect of exposure duration of ovaries and oocytes at ambient temperature on parthenogenetic development of porcine follicular oocytes.

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.

Animals↗