Summary. The preparation of a purified fraction of ram sperm plasma membranes is described and validated in this paper. Lipid analyses were performed on both the membrane preparation and whole spermatozoa; the main differences were that plasma membranes showed a significantly higher cholesterol:phospholipid molar ratio and a higher sphingomyelin content than did whole spermatozoa, but a lower proportional content of phosphatidylethanolamine. Enzymic assays revealed the presence of two distinct adenosine triphosphatases (ATPases) in the membrane fraction, activated independently by calcium and sodium ions. Arrhenius plots of the calcium-stimulated ATPase activity demonstrated that a change in energy of activation occurred in the region of 23°C; it is believed that this is evidence for the occurrence of a thermal phase transition in the lipid environment of the enzyme molecules.
W. V. Holt and R. D. North
Summary. A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36°C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24°C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24°C to 30°C, but the reasons for this are unclear.
It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.
W. V. Holt and R. D. North
Summary. The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form.
Spermatozoa fixed at 39°C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4·4% (± 1·6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by > 0·025% Triton X-100 before immunofluorescence, actin was localized in the post-acrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15°C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10°C markedly increased the proportion of immunoreactive cells (mean ± s.e.m.; 12 ± 4·5% at 15°C; 27 ± 4·5% at 10°C; n = 4 ejaculates). Further cooling to 5°C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfils a stabilizing function in spermatozoa.
Keywords: actin; spermatozoa; membranes; cryopreservation; cytoskeleton; ram
W. V. Holt, H. D. M. Moore, R. D. North, T. D. Hartman, and J. K. Hodges
Summary. Hormonal detection of urinary pregnanediol-3α-glucuronide proved an effective method of monitoring the progress of oestrous cycles in the blackbuck; observation of sexual behaviour in a vasectomized male was, however, a more practical procedure. Good correlation was observed between the occurrence of minimal pregnanediol concentrations in females and the maximal behavioural response by the male. On the basis of intervals between periods of behaviourally detected oestrus, a mean cycle length of 16·9 days (± 0·62, s.e.m.) was derived from 12 cycles (4 animals).
Eleven females were inseminated in this study, 8 with freshly collected semen and 3 with frozen semen; 6 calves were obtained, 1 after the use of frozen semen. Pregnancy was monitored by measurements of pregnanediol-3α-glucuronide excretion and by ultrasound scanning. The mean interval between insemination and parturition was 183·3 days inclusive, ranging from 182 to 186 days.
Keywords: blackbuck; artificial insemination; oestrous cycles; oestrus detection; pregnancy