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R. M. HARRISON and W. R. DUKELOW

An in-vitro system for the fertilization of superovulated rabbit ova recovered from the ovarian surface and from the oviducts was used to study uterine-oviducal relationships in connection with sperm capacitation. Spermatozoa recovered from ligated uterine horns as early as 8 hr after mating were capacitated although they fertilized periovarian ova at a lower level than spermatozoa from control uteri.

In the last decade, a number of factors have been found which aid an in-vitro fertilization system. Replacement of serum by bovine serum albumin (BSA) (Brackett, 1966) and the maintenance of high humidity and prevention of evaporation from culture dishes (Brackett & Williams, 1965) have augmented the earlier work of Chang (1955, 1959) and Bedford & Chang (1962). Using the techniques first described by these workers and some new

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R. M. HARRISON and W. R. DUKELOW

Summary.

Ovulation induced in squirrel monkeys is completely blocked by daily injections of 500 μg of megestrol acetate. Levels of 50 to 250 μg either do not inhibit or only partially inhibit ovulation.

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M. T. Kane, M. Norris and R. A. P. Harrison

Summary. The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6·25 mmol inositol l−1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.

Keywords: mouse; embryo; inositol; phosphoinositides

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R. A. P. Harrison, H. M. Dott and G. C. Foster

Summary. The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA.

The response of the spermatozoa to replacement of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.

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Christine Gagliardi, John R Liukkonen, Kathrine M Phillippi-Falkenstein, Richard M Harrison and H Michael Kubisch

A retrospective analysis was performed on fertility outcomes among a colony of captive Indian rhesus monkeys. The analysis covered over 30 years and was based on 1443 females with a total of 11 453 pregnancies. Various determinants of fertility were assessed including birth rates, pregnancy loss, infant survival, interbirth intervals, and interval from last birth to death. Binary variables were analyzed with generalized linear models with random intercepts, while linear mixed models were used for analysis of continuous variables. Age of the dam was a significant factor in determining whether a pregnancy resulted in a birth and whether an infant survived the first 30 days with primiparous or older mothers being less likely to produce an infant surviving to that age. In contrast, sex proved to be the only significant factor in determining whether an infant lived to 1 year, with females being more likely to survive. The interval between births proved to be affected primarily by dam age, while the late death of an infant depressed the likelihood of an extended time interval between her last birth and her death. Overall, these results demonstrate that maternal age contributes significantly to a decline in fertility and older females can live relatively long periods following birth of their last infant.

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H. M. Dott, R. A. P. Harrison and G. C. A. Foster

Summary. Epididymal spermatozoa from bull, rabbit and ram were incubated in homologous epididymal plasma or seminal plasma in a buffered saline-based medium with or without serum albumin. The spermatozoa were either diluted directly into the medium or were washed first.

No effect of washing was observed on the subsequent reaction of the cells to the different media.

A considerable proportion of the populations of epididymal spermatozoa survived (i.e. continued to exhibit motility) for up to 22 h at 30°C in the simple saline-based medium. Initially epididymal plasma had a slight stimulatory effect on sperm motility in ram and bull but it had no effect on sperm survival in any of the 3 species. Seminal plasma stimulated motility markedly in ram initially, but in all 3 species seminal plasma was detrimental to survival: in ram even a 15-min exposure to the fluid reduced survival. Serum albumin also stimulated motility; it delayed, but did not prevent, the detrimental effect of seminal plasma, although it had no effect itself on survival.

The effects of epididymal plasma, seminal plasma and serum albumin on surface properties of epididymal spermatozoa, i.e. agglutination, sticking-to-glass and eosinophilia, were also noted. These varied between species and there was no correlation between these effects and the effects on motility and survival.

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A M Petrunkina, R A P Harrison, M Tsolova, E Jebe and E Töpfer-Petersen

The ability to maintain cellular volume is an important general physiological function, which is achieved by specific molecular mechanisms. Hypotonically induced swelling results in the opening of K+ and Cl ion channels, through which these ions exit with accompanying water loss. This process is known as regulatory volume decrease (RVD). The molecular mechanisms that control the opening of the ion channels in spermatozoa are as yet poorly understood. The present study investigated pathways of osmo-signalling using boar spermatozoa as a model. Spermatozoa were diluted into isotonic and hypotonic Hepes-buffered saline in the presence or absence of effector drugs, and at predetermined intervals volume measurements were performed electronically. Treatment with protein kinase C (PKC) inhibitors staurosporine, bismaleimide I and bismaleimide X led to dose-dependent increases of both isotonic and hypotonic volumes (P<0.05). However, as the isotonic volume was affected more than the hypotonic volume, the kinase inhibitors appeared to improve RVD, whereas activation of PKC with phorbol dibutyrate blocked RVD. The increase in isotonic cell volume induced by bismaleimide X was observed in chloride-containing medium but not in the medium in which chloride was replaced by sulphate, implying that PKC was involved in the control of chloride channel activity, e.g. by closing the channel after volume adjustment. The protein phosphatase PP1/PP2 inhibitors calyculin and okadaic acid increased the isotonic volume only slightly but they greatly increased the relative cell volume and blocked RVD. The activation of RVD processes was found to be cAMP-dependent; incubation with forskolin and papaverine improved volume regulation. Moreover, papaverine was able to overcome the negative effect of protein phosphatase inhibitors. The mechanism of sperm RVD appears to involve (a) alterations in protein phosphorylation/dephosphorylation balance brought about by PKC and PP1 and (b) a cAMP-dependent activating pathway.