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W. V. Holt

Summary. The development of a tail-coiling defect in spermatozoa from a Welsh boar was studied by light and electron microscopy. The onset of tail-coiling was coincident with the passage of spermatozoa through the epididymis, since only cells isolated from regions of the duct distal to the caput epididymidis exhibited this characteristic. An intermediate form of this abnormality was detected in the proximal corpus epididymidis, where migration of the cytoplasmic droplets mainly occurred. It is suggested that defective migration of the cytoplasmic droplet might be associated with the pathogenesis of the coiled-tail defect in this animal.

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W. V. HOLT and W. H. TAM

Summary.

Using histochemical and in-vitro biochemical methods, the following steroid enzymes were found in the chin gland of the male cuis: 17 β-hydroxysteroid dehydrogenase, 5-ene-3β-hydroxysteroid dehydrogenase and 4-ene-5α-reductase. The chin gland did not metabolize [4-14C]progesterone but it did metabolize a small quantity of [7α-3H] pregnenolone to progesterone, and readily converted [4-14C]testosterone into androstenedione and 5α-androstane-3, 17-dione. These are catabolic products of testosterone. These findings, together with the possible rôle of the chin gland in sexual behaviour, may show that sebum has a communicative rôle in Galea musteloides and other mammals.

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W. V. Holt and R. D. North

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.

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R. C. JONES and W. V. HOLT

Summary.

Washing ram spermatozoa two or four times with Krebs-Henseleit-Ringer solution, to achieve dilution rates of 100- or 10,000-fold, damaged the plasma membranes, acrosomes and mitochondria of some spermatozoa. The proportion of spermatozoa with broken plasma membranes over the acrosome increased with the number of washes.

Washing once to achieve a dilution rate of 10-fold was sufficient to remove from spermatozoa all the substrate available for NADPH diaphorase and probably glucose-6-phosphate dehydrogenase, but not quite all the substrate for succinic dehydrogenase. A 10-fold dilution was also sufficient to remove glucose-6-phosphate dehydrogenase from a considerable proportion of spermatozoa, but even three washes to achieve a dilution rate of 1000-fold had little effect on succinic dehydrogenase and NADPH diaphorase activity in spermatozoa.

The effects of washing could not be attributed to repeated centrifugation of spermatozoa during the process.

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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.

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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

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L. M. Thurston, P. F. Watson, and W. V. Holt

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace–Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0–22.0 μm, 22.1–73.0 μm and 73.1–130.0 μm). The Landrace–Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1–130 μm long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean ± sd; 66.36 ± 24.70 μm) to the Landrace–Meishan introgression boar (mean ± sd; 67.09 ±21.80 μm). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.

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William V Holt and Katrien J W Van Look

Stringent selection mechanisms, in both internal and external fertilisation systems, reject all but a significant minority of the spermatozoa released at ejaculation. Sperm competition theory provides circumstantial evidence that the selection process involves mechanisms by which the quality of the fertilising spermatozoon is controlled, thereby ensuring that females and their offspring receive high quality genetic material. In this review we examine some of these selection processes to see whether they could be exploited for the improvement of laboratory tests of sperm quality. Such tests are not only required for clinical and agricultural purposes, but are increasingly needed in fields such as reproductive and environmental toxicology where the species requirement is much broader. Despite many years of research, sperm quality assessment methods continue to provide imprecise data about fertility; here we suggest that this may be a consequence of using tests that focus on the spermatozoa that would normally be unable to fertilise under natural conditions.

To achieve fertilisation a spermatozoon must be capable of responding appropriately to external signalling stimuli; those involving protein kinase-regulated flagellar function seem especially influential in governing effects ranging from non-Mendelian inheritance in mammals to sperm chemotaxis in sea urchins. Examination of the elicited responses reveals considerable heterogeneity in all species. Here we propose that this level of heterogeneity is meaningful both in terms of understanding how spermatozoa from some individuals possess fertility advantages over spermatozoa from their rivals in sperm competition, and in that the heterogeneity should be exploitable in the development of more accurate laboratory tests.

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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

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S D Johnston, N Satake, Y Zee, C López-Fernández, W V Holt, and J Gosálvez

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be ‘hot spots’ of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen–thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.