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Biomedical subjects

N Gérard

Publications and source records attributed to N Gérard.

At least 19 recordsLinked to original sources
PubMed2004

The interleukin-1 system and female reproduction.

Interleukins (ILs) are known best for their involvement in the immune system and their role during inflammation. In the ovary, a growing body of evidence suggests that the ovarian follicle is a site of inflammatory reactions. Thus ovarian cells could represent sources and targets of ILs. Since then, the IL-1 system components (IL-1alpha, IL-1beta, IL-1 receptor antagonist, IL-1 receptors) have been demonstrated to have several sites of synthesis in the ovary. These factors have been localized in the various ovarian cell types, such as the oocyte, granulosa and theca cells, in several mammalian species. IL-1-like bioactivity has been reported in human and porcine follicular fluid at the time of ovulation. The role of IL-1 in local processes is still poorly known, although there is evidence for involvement in the ovulation process, and in oocyte maturation. More precisely, IL-1 may be involved in several ovulation-associated events such as the synthesis of proteases, regulation of plasminogen activator activity, prostaglandin and nitric oxide production. IL-1 also regulates ovarian steroidogenesis. These different aspects of the involvement of the IL-1 system in important aspects of female reproduction are discussed.

Animals↗
PubMed2004

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Animals↗
PubMed2003

Inhibition of in vitro maturation of equine oocytes by interleukin 1 beta via specific IL-1 receptors.

Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured for 30 h and nuclear maturation of oocytes was evaluated by DNA staining. In Expt 1, seven different media were tested: medium 1 (TCM199+BSA); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); medium 3 (medium 1+eLH); and media 4, 5, 6 and 7 (medium 3 containing 0.1, 1.0, 10.0 and 50.0 ng IL-1 beta ml(-1), respectively). In Expt 2, four different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and media 3 and 4 (medium 2+IL-1RA at 50 and 100 ng ml(-1), respectively). In Expt 3, three different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1RA ml(-1)); and medium 3 (medium 2+50 ng IL-1 beta ml(-1)). In Expt 4, four different media were tested: medium 1 (TCM199+BSA+eLH); and media 2, 3 and 4 (medium 1+IL-1RA at 50, 100 and 150 ng ml(-1), respectively). In Expt 5, three different media were tested: medium 1 (TCM199+BSA+EGF); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and medium 3 (medium 2+50 ng IL-1RA ml(-1)). In Expt 1, LH alone induced an increase in the rate of in vitro maturation (IVM) of equine oocytes (P<0.05), whereas IL-1 beta alone did not have any effect compared with medium 1. IL-1 beta (50 ng ml(-1)) significantly inhibited the eLH-induced IVM of oocytes (P<0.05) compared with medium 3. A decrease in rate of maturation was observed from a concentration of 10 ng IL-1 beta ml(-1) onwards. In Expt 2, the presence of IL-1RA in the culture medium inhibited the effect of IL-1 beta and restored the rate of oocyte maturation (P<0.05) observed in the presence of LH alone. In Expts 3 and 4 it was demonstrated that IL-1RA alone had no positive effect on the eLH-induced rate of maturation. In Expt 5, IL-1 beta inhibited the EGF-induced resumption of meiosis (P<0.05). The addition of IL-1RA inhibited this effect and restored the rate of oocyte maturation (P<0.05) observed with EGF alone. In conclusion, the present data confirm the inhibitory effect of IL-1 beta on IVM of equine oocytes induced by eLH and demonstrate its inhibitory effect on EGF-induced oocyte maturation. The rate of maturation decreased in a dose-dependent way and the lowest rate of maturation was observed at 50 ng IL-1 beta ml(-1) (P<0.05). The use of IL-1RA inhibited these effects, demonstrating that the action of IL-1 beta is receptor-mediated. Moreover, the results clearly show that, in equine species, IL-1 beta is involved in the physiology of COCs by regulating resumption of meiosis.

Animals↗
PubMed2002

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes.

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

Animals↗
PubMed2002

Analysis of the variations of follicular fluid composition during follicular growth and maturation in the mare using proton nuclear magnetic resonance (1H NMR).

Follicular development and ovulatory processes in mammals involve local biochemical changes as a result of substantial modifications in cellular metabolism, the most well known of which is steroid variation. In the present study, the intrafollicular variation of several other components was studied using proton nuclear magnetic resonance ((1)H NMR). This approach made it possible to demonstrate that the intrafollicular biochemical content changes during follicular growth and maturation. Follicular fluid was aspirated by ovarian puncture of the dominant follicle at various physiological stages of its development: early dominant, late dominant and preovulatory. Serum samples were collected during each puncture session. (1)H NMR was used to evaluate intrafollicular and circulating glycoconjugates (sugar chains and N-acetyl groups), lipoproteins (CH(3) and CH(2) groups), glucose metabolites (trimethylamines, acetate and lactate), amino acids (glutamine/glutamate and alanine), creatine/creatinine and polyamines. Follicular fluids were assayed by radioimmunoassay for oestradiol and progesterone contents. The intrafollicular contents of alanine and lipoproteins (CH(3) groups) decreased in the dominant follicle during growth, whereas concentrations of progesterone and oestradiol increased significantly. After injection of gonadotrophin to induce ovulation, follicular maturation was characterized by a decrease in glycoconjugates (sugar chains), trimethylamines and acetate, a decrease in oestradiol concentration, and a further increase in CH(3) groups of lipoproteins and progesterone. The results from the present study showed a clear correlation between the intrafollicular content of alanine and that of oestradiol. A correlation between progesterone and glycoconjugates (sugar chains) was also observed. Therefore, (1)H NMR was shown to be effective for studying specific changes in the biochemical composition of the follicular fluid that occur during follicular development. For the first time, the variation of several compounds (glycoconjugates, lipoproteins, glucose metabolites, amino acids and polyamines) in relation to growth and maturation was demonstrated. Some of these changes could be of crucial importance for follicular maturation and ovulation as well as for oocyte maturation and further fertilization.

Alanine↗
PubMed2001

North African genes in Iberia studied by Y-chromosome DNA haplotype V.

Haplotype V at the Y-chromosome specific DNA polymorphism (p49/TaqI) was reported in a study concerning 487 males originating from five different geographic locations in Iberia and North Africa. The highest frequency of haplotype V (68.9%) was previously observed in Berbers from Morocco, and it was previously established that this haplotype is a characteristic Berberian haplotype in North Africa. Percentages of haplotype V geographic distribution reveal a gradient of decreasing frequencies with latitude in Iberia: 40.8% in Andalusia, 36.2% in Portugal, 12.1% in Catalonia, and 11.3% in Basques; such a cline of decreasing haplotype V frequencies from the South to the North in Iberia clearly establishes a North African toward Iberian gene flow.

Adult↗
PubMed2001

North African genes in Iberia studied by Y-chromosome DNA haplotype 5.

The frequency of haplotype 5 at the Y-chromosome-specific DNA polymorphism (p49/TaqI) was reported in a study of 487 males originating from five different geographic locations in Iberia and North Africa. The highest frequency of haplotype 5 (68.9%) was previously observed in Berbers from Morocco, and it has been established that this haplotype is a characteristic Berber haplotype in North Africa. The relative frequencies of haplotype 5 distribution show a geographical gradient of decreasing frequency according to latitude in Iberia: 40.8% in Andalusia, 36.2% in Portugal, 12.1% in Catalonia, and 11.3% in the Basque Country; such a cline of decreasing frequency of haplotype 5 from the south to the north in Iberia clearly establishes a gene flow from North Africa towards Iberia.

Adult↗
PubMed2000

[Pharmacogenetics or "farm-ecogenetics"?].

Pharmacogenetics and ecogenetics constitute the study of the contribution of genetic factors in interindividual variations in drugs and xenobiotics metabolism respectively and also its physiopathology (toxicity, therapeutic failure and pejorative effect). However, the frontiers between these two fields are interpenetrant with no defined limits. Among the enzymes involved in transformation of drugs/xenobiotics, the cytochrome P450 superfamily plays a major role. Most of the "poor metabolisers" either homozygotes or compound heterozygotes for mutant cytochrome P450 alleles have no detectable enzyme activity. Although phenotyping using a marker drug is usefull, it suffers from several constraints and is influenced by several environmental factors and physiopathological state of the individual. To overcome not only these difficulties but also to improve the overall predictiveness of a drug biotransformation, genotype technology offers an advantage and pharmaco-economic technology further expands the horizon. These fast moving fields must, in near future, allow tailoring of drug prescription on a personalized basis and should, in principle, help in the design and development of novel drug molecules.

Biotransformation↗
PubMed2000

The course of rheumatoid arthritis and predictors of psychological, physical and radiographic outcome after 5 years of follow-up.

OBJECTIVE: : To examine the course of RA over 5 yr and identify predictors of psychological, physical and radiographic outcome. PATIENTS AND METHODS: Out of 238 patients with rheumatoid arthritis of no more than 4 (mean 2.2) yr duration, 182 (76%) completed a clinical examination with follow-up at 1, 2, and 5 yr. The course of the disease was assessed by measures of psychological and physical health status, disease process and radiographic damage. RESULTS: : Over 5 yr we observed at a group level a stable disease course for measures of disease process, psychological and physical health status. Radiographic damage progressed. Health status and radiographic damage after 5 yr were predicted by the baseline measures for the respective outcome. Physical function was also predicted by age and by psychological status when the physical dimension of the Arthritis Impact Measurement Scales was chosen as outcome variable. Erythrocyte sedimentation rate and presence of rheumatoid factor predicted radiographic progression. CONCLUSIONS: : The 5 yr course of RA was characterized by preserved health status measures and clinically preserved disease process measures, whereas joint damage progressed steadily. Outcomes after 5 yr can be predicted partly by certain measures at baseline.

Analysis of Variance↗
PubMed2000

Intrafollicular concentrations of steroids and steroidogenic enzymes in relation to follicular development in the mare.

The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17beta concentrations by RIA. Intrafollicular concentrations of progesterone and estradiol-17beta significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17beta concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450(scc), 3betaHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450(scc), 3betaHSD, and aromatase, as well as progesterone and estradiol-17beta, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Localization of the gene for fibrodysplasia ossificans progressiva (FOP) to chromosome 17q21-22.

Fibrodysplasia ossificans progressiva (FOP) is a very rare disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of muscles. To identify the chromosomal localization of the FOP gene, we conducted a genomewide linkage analysis using seven affected families. The FOP phenotype is linked to markers located in the 17q21-22 region (LOD score of 3.41 at the recombination fraction theta = 0). Crossover events localize the putative FOP gene within a 12cM interval, bordered proximally by D17S809 and distally by D17S1838. Noggin (NOG) gene, located in 17q22, is an excellent candidate gene for FOP.

Chromosome Mapping↗
PubMed1999

CYP2D6 polymorphism in a Gabonese population: contribution of the CYP2D6*2 and CYP2D6*17 alleles to the high prevalence of the intermediate metabolic phenotype.

AIMS: To determine the molecular basis of the intermediate extensive metaboliser (EM) CYP2D6 phenotype in healthy Gabonese subjects. METHODS: The CYP2D6 phenotype of 154 healthy Gabonese subjects was assessed by giving the subject a single dose of 30 mg dextromethorphan, and collecting their urine for the next 8 h. The CYP2D6 genotype was determined for 50 individuals of the EM phenotypic group by Southern blotting and various PCR-based procedures aimed at identifying different CYP2D6 alleles. RESULTS: We found that in the studied Gabonese population, as compared with a French population, there is significantly higher frequency of intermediate EM phenotype having lower frequency of CYP2D6 PM alleles. To clarify this discrepancy phenotype-genotype relationship was studied. We found that the CYP2D6*17 and CYP2D6*2 alleles, prevalent in this black population, are characterised by their low capacity for dextromethorphan demethylation. Our data also show that the CYP2D6*1 allele is associated with the highest in vivo activity followed by the CYP2D6*2 allele and then the CYP2D6*17 allele. CONCLUSIONS: The higher frequencies of the CYP2D6*2 and CYP2D6*17 alleles than the CYP2D6*1 allele account for the high frequency of the intermediate EM phenotype in this black population. The polymorphism of the CYP2D6 enzyme activity in African populations could have important implications for use of drugs that are substrates for CYP2D6 and have a narrow therapeutic window.

Alleles↗
PubMed1999

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare.

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.

Animals↗
PubMed1998

Maturation-promoting factor (MPF) and mitogen activated protein kinase (MAPK) expression in relation to oocyte competence for in-vitro maturation in the mare.

In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We showed that MAPK and the two subunits of MPF were present in equine oocytes whatever the nuclear stage they reached after in-vitro culture and whatever the culture medium used. In incompetent oocytes, MAPK remained in its non-phosphorylated form, supposed to be inactive. In conclusion, the incompetence of equine oocytes to resume and complete meiosis is not due to the absence of p34cdc2, cyclin B or MAPK. Our results suggest that it is more probably due to a deficiency of regulators of MPF and/or to an inability to phosphorylate MAPK.

Animals↗
PubMed1998

A high-molecular-weight preovulatory stage-related protein in equine follicular fluid and granulosa cells.

The high-molecular-weight proteins of equine follicular fluid were examined to determine whether some polypeptides are unique to certain physiological conditions. Fluids from ovarian follicles of various diameters and physiological stages during the follicular phase were recovered by ultrasound-guided follicular aspiration. Granulosa cells and cumulus-oocyte complexes (COC) were recovered by scraping the intrafollicular wall during puncture. Follicular fluids and corresponding serum, as well as granulosa cell lysates, were analyzed by one-dimensional SDS-PAGE and silver staining. COC morphology was assessed microscopically. A 200-kDa protein band was demonstrated in fluids from preovulatory follicles, in natural conditions or after induction of ovulation. This protein band was absent in fluids from follicles at earlier stages, subordinate follicles, and serum. The presence of this protein at the preovulatory (PO) stage was ascertained through recovery of the fluid from follicles twice during their growth. Its appearance was time dependent after induction of ovulation but was not induced by an intrafollicular injection of a physiological dose of progesterone. We also demonstrated the presence of this 200-kDa protein in granulosa cells lysates recovered from preovulatory follicles. The expression of this protein in the follicular fluid was related to the cumulus aspect and chromatin configuration of the enclosed COC. No relation was found between its presence in the follicular fluid at the PO stage and subsequent ovulation of the punctured follicle or embryo production. The identification of this molecule is approached and discussed. These results show a novel PO stage-related protein in equine follicular fluid, which may be involved in the differentiation and maturation mechanisms occurring in the follicle during the preovulatory period.

Animals↗
PubMed1998

Intrafollicular insulin-like growth factor-binding protein levels in equine ovarian follicles during preovulatory maturation and regression.

The profiles of insulin-like growth factor-binding proteins (IGFBPs) in follicular fluid have been characterized in a number of mammals (rats, pigs, sheep, cattle, humans) and are good indicators of follicular status. We studied the IGFBP profiles of equine serum and ovarian follicular fluid recovered at various stages of the follicular phase. The levels of IGFBPs were related to the morphology and the steroidogenic activity of the follicles. Follicular fluids were recovered by ultrasound-guided follicular aspiration. In the first experiment, the dominant follicles of 10 mares were partly punctured (aspiration of 0.5-2.2 ml of fluid) once at the early dominant stage (22-25 mm in diameter) and a second time at the preovulatory stage (PO), 34 h after induction of ovulation. Among these 10 PO follicles, 5 were classified as healthy whereas the other 5 were classified as hemorrhagic, as assessed by ultrasonic morphology and subsequent ovulation or not. In another group of mares (n = 5), the largest follicle was punctured once at the late dominant stage (33-35 mm in diameter) and then at the PO stage, 34 h after induction of ovulation. Serum was prepared at each puncture session. In the second experiment, follicular fluid was recovered from the dominant and contemporary cohort subordinate follicles (n = 5 mares). Samples were individually assayed for estradiol-17beta and progesterone content by RIA, and IGFBPs were studied by using Western ligand blotting and densitometry. Equine serum and follicular fluid displayed IGFBP at 42-44 kDa (likely corresponding to IGFBP-3), 28-32 kDa (likely corresponding to IGFBP-5), 24 kDa (likely corresponding to IGFBP-4), and 35 kDa, identified as IGFBP-2 by immunoblotting, plus one band at 120 kDa. IGFBP were clearly more abundant in serum than in fluid from healthy follicles. In the follicular fluid, 42-44-kDa IGFBP was the major binding protein, and its level was almost constant at the various physiological statuses studied. Follicular development of the dominant follicle in each mare was characterized by a decrease in intrafollicular IGFBP-2 and 28-32-kDa IGFBP levels before LH stimulation and by an increase in IGFBP-2 after LH stimulation. Follicular regression of large follicles, as well as subordinate ones, was characterized by a low level of intrafollicular estradiol-17beta and was associated with an increase in IGFBP-2, 24-kDa IGFBP, and 28-32-kDa IGFBP intrafollicular levels. Taking these results together, we have demonstrated clear correlations between the intrafollicular levels of estradiol-17beta and IGFBP-2 and 28-32-kDa IGFBP. Therefore, follicular growth and regression in the mare are associated with specific changes in IGFBP levels. These changes could be of crucial importance for follicular development in ovulation or atresia.

Animals↗
PubMed1998

Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare.

The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro maturation and measured their maturation-promoting factor activity by histone H1 kinase assay. Puncturing once at the end of the follicular phase and once during the luteal phase, or three times during the follicular phase, yielded about 11 cumulus-oocyte complexes per 22 days. The maturation rate was different between the groups, 51% in group EF, 34% in group DL (p < 0.05), and 15% in group DF (p < 0.01), and it increased with an increase in follicular diameter (p < 0.05). After in vitro culture, the H1 kinase activity was lower in oocytes that remained in germinal vesicle or dense chromatin stages than in oocytes that reached metaphase I or metaphase II (p < 0.05). The H1 kinase activity was not different between oocytes in germinal vesicle stage after in vitro maturation and immature oocytes that were not cultured in vitro, and was higher in preovulatory oocytes that reached metaphase II in vivo than in the oocytes that reached metaphase II after in vitro maturation (p < 0.001). This is the first report on kinase activity in the equine oocyte.

Animals↗