SEXUAL COMPOSITION and ANALYSIS OF REPRODUCTIVE FEMALES IN THE NORTH ATLANTIC RIGHT WHALE, EUBALAENA GLACIALIS, POPULATION

To test hypotheses involving reproduction and demographics, the sex of individuals must be established, but many species of Cetacea are not obviously dimorphic. In the North Atlantic right whale, Eubalaena glacialis , population, the sex of 61 males and 55 females had been determined previously by observation of the urogenital region, and the sex of 43 more females had been inferred from repeated sightings with a calf. To confirm the sex of some of these animals and to identify the sex of mote animals, genomic DNA was isolated from skin samples of 95 individual right whales (54 from among those described above and 41 additional recognizable individuals). The DNA was surveyed using the human Y-chromosome probe pDP1007. With Eco RI-digested DNA, a clear, sex-discriminating banding pattern was apparent. This method verified the sex of all 54 animals whose sex was previously known or inferred and identified the sex of an additional 41 recognizable individuals. A total of 89 male and 111 female right whales was identified in the population. The most unbiased estimate of sex ratio available is the 36 male and 34 female calves identified by genital morphology and DNA techniques. The sex ratio of this sample does not differ significantly from unity (P = 0.811). Only 38% (58/152) of the females in the North Atlantic population are known to have been reproductively successful compared with 54% in the population of right whales in the western South Atlantic. The population growth rate reported for the North Atlantic population is only 33% of that reported for right whales in the South Atlantic. Thirteen adult North Atlantic females have been identified that have not been known to calve during the past 11 yr. These data suggest that the absence of measurable recovery may be due to a combination of fewer actively reproducing females and lower reproductive rates of some females.     

NEW ZEALAND HERD STRANDING SITES DO NOT RELATE TO GEOMAGNETIC TOPOGRAPHY

Recent theories relating cetacean stranding sites to geomagnetic topography were tested for the New Zealand coastline. New Zealand herd strandings showed no relationship to either perpendicular geomagnetic contours or magnetic minima, nor did whales appear to be avoiding magnetic gradients. Single-live stranding sites also showed no geomagnetic relationship.     

Possible involvement of a sialylated component of the sperm plasma membrane in sperm-zona interaction in the mouse

We have studied the molecular mechanisms of gamete interaction in vitro in the laboratory mouse, Mus musculus. In particular, we were interested in whether this interaction is similar to a lectin-hapten-mediated process. Inhibition of sperm-zona binding was examined using various concentrations (.25 mM to 50 mM) of different sugars (sialic acid α-methylmannose, glucose, fucose, galactose, and N-acetyl-glucosamine). Sperm-zona binding was significantly decreased when eggs were pretreated with 10 mM of sialic acid or α-methylmannose but not by other sugars tested. Furthermore, treatment of capacitated sperm with neuraminidase destroyed their ability to bind and fertilize eggs. We have also used a specific lectin for sialic acid from the horseshoe crab (Limulus polyphemus) to agglutinate mouse sperm. The lectin (.120 ng/ml) mediated agglutination of mouse sperm (10⁵ sperm/ml) was routinely observed to increase from a 10% agglutination immediately following their isolation from the epididymis to 100% agglutination 90 minutes later. Collectively, these results suggest the appearance of specific sugar moieties on the surface of the sperm plasma membrane which, in this particular species of mouse, are sialylated glycoproteins acting as ligands for specific receptors on the surface of the egg. These are the first data to indicate that sperm-egg recognition and attachment is a lectin-hapten-mediated process in the mouse.     

Procedures for obtaining high percentages of viable in vitro capacitated hamster sperm

Suspensions of nearly 100% viable golden hamster sperm were prepared by passing washed cauda epididymal sperm through a column of 0.25–0.3 mm glass beads. Incubations of these viable sperm under in vitro capacitation conditions in volumes of 0.1 or 1 ml (2–2.5 × 10⁶/ml) resulted in 85–92% viable sperm after four hours and 45 minutes of incubation. More than 70% of these sperm were judged to have been capacitated after four hours and 45 minutes of incubation on the basis of their having undergone acrosome reactions and the presence of high numbers of sperm exhibiting the activated motility characteristic of capacitated hamster sperm. Thus, for the first time, procedures are available that will yield large numbers of viable capacitated sperm for biochemical analysis and that will also allow other studies of hamster sperm capacitation with minimum interference from molecules released from dead sperm.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

The movement characteristics of rabbit spermatozoa before and after activation

Spermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five- to 10-minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high-speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules

The keratinous capsules surrounding rat sperm mitochondria were isolated 24 days after intratesticular injections of [⁷⁵Se] selenite or [³⁵S] cysteine. Dodecyl sulfate-polyacrylamide gel electrophoresis of purified, doubly labeled mitochondrial capsules revealed only a single ⁷⁵Se-labeled component, whose molecular weight was 17,000, in agreement with previously reported observations obtained with cruder sperm fractions. Most of the ³⁵S label and the major zone of stained protein on the gels coincided with the position of ⁷⁵Se, suggesting that selenium is associated with a cysteine-rich structural protein. The level of selenium in rat sperm, 195 ± 3.2 ng/10⁸ sperm (approximately 30 ppm), determined by hydride generation and atomic absorption spectrophotometry, is consistent with a structural function for this trace element in the sperm.     

Effects on spermiogenesis in the mouse of a male sterile neurological mutation,Purkinje cell degeneration

Purkinje cell degeneration (pcd) is a neurological mutation in the mouse that causes male sterility, but not female sterility. In order to assess the effects of this mutation on spermiogenesis, the structure of the testis and of epididymal spermatozoa was examined by transmission and scanning electron microscopy. In the mutant males, the sperm count was reduced, sperm were nonmotile, and 93% of the sperm were characterized by structural abnormalities of the head, the tail, or both. In the testes of mutant mice, Sertoli cell structure was normal, as were also the early stages of spermiogenesis. However, the elongating and maturing spermatids were characterized by abnormally shaped heads and tails with extraneous and ectopic outer dense fibers. These defects were common in the testes of the mutant mice and rare in the testes of the littermate control mice. It was concluded that the structural abnormalities of the pcd sperm occurred during spermiogenesis and were not due to degeneration of the sperm in the epididymis. These structural abnormalities are similar to those found in all other reported male sterile mutants of the mouse; therefore, although they are caused by the expression of the pcd gene, they are not unique to the expression of this gene.     

Rapid,simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins

A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.