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D. S. Gonzales, J. C. Pinheiro and B. D. Bavister

Time-lapse videomicrography was used to determine the timing of early developmental events in hamster embryos in vitro. The time intervals from pronuclear envelope breakdown to the completion of the first cleavage (Dt 2), second cleavage (Dt 4 = 2–4 cells), third cleavage (Dt 8 = 4–8 cells), blastocyst formation, and zona escape were precisely measured to determine whether the variable 'time' (t) can be used to predict the developmental potential of preimplantation embryos. The range of the developmental time interval (Dt) from the second to the third cleavage divisions (Dt 8) provided the best indicator for predicting the probabilities of blastocyst formation and zona escape (P= 0.015 and 0.041, respectively). Dt 8 was subdivided into consecutive time cutoff points of ≤750, ≤800, ≤850 and ≤900 min. Of the embryos that took ≤750 min to complete the third cleavage division, 92% developed into blastocysts and 69% escaped from their zonae pellucidae. When the completion of Dt 8 extended to ≤900 min, the percentages decreased to 75% and 49% for blastocyst formation and zona escape, respectively. This study identifies a specific developmental time interval and a model whereby time can be used as a noninvasive parameter to predict embryo developmental potential in vitro.

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B. D. BAVISTER, R. G. EDWARDS and P. C. STEPTOE

Positive identification of the sperm midpiece and tail in the vitellus establishes beyond reasonable doubt that pronucleate eggs are undergoing fertilization. Previously Edwards, Bavister & Steptoe (1969) tentatively identified sperm midpieces in pronucleate human eggs fertilized in vitro. Unequivocal evidence of midpieces and tails in eggs undergoing fertilization is now presented.

Oocytes recovered from two ovaries excised 3 hr previously were cultured in a mixture containing three parts follicular fluid : one part of Bavister's medium (Bavister, 1969). Four samples of follicular fluid were used, three being straw-coloured and a fourth, which was slightly pink, probably came from an atretic follicle. After 36 to 37 hr in culture, the eggs were washed through two or three changes of Bavister's medium, the dilution of the follicular fluid being approximately 1:100. Ejaculated spermatozoa were washed twice in this medium and then added to some of the eggs at a concentration of

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C. R. Brown, N. Clarke, M. Aiken and B. D. Bavister

Summary. Hamster zonae pellucidae were obtained from follicular oocytes, superovulated eggs, and eggs fertilized in vivo or in vitro. Zonae were labelled with N-succinimidyl-3(4-hydroxy,5-[125I]iodophenyl)propionate, and compared on single-and two-dimensional SDS-PAGE. Single-dimensional electrophoresis showed considerable differences between zona categories in the amount of label that they incorporated; follicular zonae incorporated the least label and zonae from eggs fertilized in vivo the most. On two-dimensional electrophoresis, polypeptides from 3 of the 4 zona categories migrated into 4 major groups: two of these groups each with M r 150 000–250 000 were within the M r range of ZP1, and two others, at M r 90 000 and 55 000, appeared to be analogous to ZP2 and ZP3, respectively. The fourth zona category (zonae from eggs fertilized in vivo) showed a changed polypeptide profile as well as incorporating the most label; one of the polypeptides, M r 150 000–250 000, was undetectable, but a train of M r 70 000–90 000 polypeptides and a discrete polypeptide at M r 20 000 were new. Since this changed profile did not occur in zonae from superovulated eggs, or in zonae from eggs fertilized in vitro, a synergism between oviducal factors and factors from the spermatozoon or egg, or both, towards the zona in vivo is indicated.

Keywords: fertilization; zona pellucida; electrophoresis; hamster

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B. D. Bavister, A. F. Chen and P. C. Fu

Summary. Homogenates of hamster and bovine glands contain a "sperm motility factor" (SMF) that stimulates the motility of hamster epididymal spermatozoa in vitro. The potency of these adrenal preparations was severely attenuated after gel filtration on a Sephadex G-10 column. This loss of activity was ascribed to the retardation and separation of co-factors for SMF which appeared to be catecholamines. The sperm motility-stimulating activity of the SMF-containing fractions was fully restored by addition of either the 'retarded' fractions or catecholamines (epinephrine or norepinephrine). Neither the catecholamines nor the 'retarded' fractions were able to sustain vigorous sperm motility in the absence of the SMF-containing fractions. The potentiating action of catecholamines on SMF was mimicked by the adrenergic agonists isoproterenol and phenylephrine and inhibited by the α-adrenergic antagonist phentolamine, but not by the β-adrenergic antagonist propranolol. Our results indicate that one or more catecholamines are essential cofactors of SMF and demonstrate that hamster spermatozoa require catecholamines for their motility in vitro.

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C. L. Chatot, C. A. Ziomek, B. D. Bavister, J. L. Lewis and I. Torres

Summary. One-cell CF-1 × B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mm-glutamine and 0·1 mm-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3–4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33·7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.

Keywords: mouse; embryo; 2-cell block; glutamine; glucose