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Chicken spermatozoa may remain in the female oviduct for a prolonged period before induction of the acrosome reaction on contact with the inner perivitelline layer (IPVL). By contrast, the acrosome reaction may be induced very rapidly in vitro in the presence of IPVL and Ca2 +. In the present study, we examined the extent to which the chicken acrosome reaction can be induced in media of various compositions in the presence or absence of IPVL and/or Ca2 + and other factors known to be efficient in mammals. We also compared the efficacy of perivitelline layer (PL) taken at various states of oocyte maturation in initiating the reaction. The acrosome reaction was induced in less than 5 min in the presence of Ca2 + and IPVL. Incubation of spermatozoa in different saline media (Beltsville poultry semen extender (BPSE); Dulbecco's modified eagle medium; NaCl-TES buffer) without IPVL showed a significant induction of acrosome reaction in BPSE supplemented with 5 mM Ca2 + and in the three media after supplementation with Ca2 + and Ca2 + ionophore A23187. By contrast, the acrosome reaction was never induced without Ca2 +. BSA, NaHCO3, and progesterone did not stimulate the acrosome reaction. Ca2 + plus PL taken at various physiological states (follicle IPVL, ovulated IPVL, oviposited IPVL, and/or outer perivitelline layer) strongly stimulated the acrosome reaction, the latest states being the most efficient. Although PL induced the acrosome reaction in the presence of extracellular Ca2 +, it was not possible to induce hyperactivation in chicken spermatozoa. Taken together, these results emphasize the central role of Ca2 + in the in vitro initiation of the acrosome reaction in chickens and show specific features of this induction in birds.
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A series of experiments was conducted in domestic fowl to investigate the consequences of five generations of divergent selection for increased (L+) or decreased (L−) numbers of hatched chicks. After artificial insemination with pooled ejaculates within the same line (L+ males × L+ hens or L− males × L− hens), significant differences were observed between L+ and L− hens for mean fertility rates (L+ 94.8%, L− 70.2%, P < 0.0001) and for effective and maximum duration of fertility (P < 0.00001). A comparison of the overall laying performance and shell quality between the two selected lines showed that L− hens laid fewer eggs than L+ hens (P < 0.00001) and L− eggs had poorer shell quality (shell breaking strength) than L+ eggs (P < 0.00001). These observations were associated with significantly higher percentages of early embryo death in eggs from L− hens compared with L+ hens. Another series of experiments revealed the presence of larger initial populations of spermatozoa in the sperm storage tubules as well as in the perivitelline layer of eggs from L+ hens. The populations of spermatozoa in the sperm storage tubules of commercial laying hens inseminated with pooled semen samples from L+ males was compared with those in hens inseminated with samples from L− males to determine whether the variations in oviductal sperm storage between the two lines were male dependent. No significant differences between the populations of spermatozoa present in the sperm storage tubules of either group of hens could be detected at any of the intervals examined after insemination (days 1, 3 and 10). Finally, an experiment conducted on hens originating from the two selected lines indicated that the utero–vaginal junction of L+ hens contained significantly more sperm storage tubules compared with L− hens (P < 0.01). It is concluded that selection based on overall reproductive performance modifies the number of eggs capable of developing viable embryos and also influences the efficacy of initial sperm storage by increasing or altering the population of sperm storage tubules located in the utero–vaginal junction. Such changes have major consequences on the duration of the fertile period, which in avian species is directly dependent on both the actual population of spermatozoa stored in the oviduct and on their rate of release from the storage sites.
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The differences in the hydrolytic activity of chicken spermatozoa towards the inner perivitelline layer in situ and when isolated from the ovum were investigated. Points of hydrolysis produced by chicken spermatozoa on the inner perivitelline layer of laid and freshly ovulated eggs were viewed as dark holes after staining with fluorescein-conjugated wheat-germ agglutinin. In laid eggs that had been fertilized in vivo, these holes appeared more frequently on the perivitelline layer overlying the blastodisc at the animal pole than on that overlying the vegetal pole. However, in an assay in which fragments of the inner perivitelline layer from freshly ovulated eggs were incubated with spermatozoa in vitro, points of hydrolysis appeared with similar frequency on the perivitelline layer from over the germinal disc at the animal pole or from that over the vegetal pole. No difference could be demonstrated in the electrophoretic profile of peptides extracted from the inner perivitelline layer at the animal or vegetal pole. The factor(s) responsible for either increased attraction of spermatozoa to, or for increased activity at, the perivitelline layer of the animal pole does not appear to be associated with the perivitelline layer itself.
CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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INRA, USC1361, Bron, France
Université de Lyon, Lyon 1, UMR846, Lyon, France
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CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
Université François Rabelais de Tours, F-37041 Tours, France
IFCE, F-37380 Nouzilly, France
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Abstract
Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of ‘protamine’, a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.
Reproduction (2016) 151 527–538