Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 +/- 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertility in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water.     

Study on the fertility of epididymal sperms

One approach to the treatment of obstructive azoospermic cases in whom either vasovasostomy cannot be performed or the vas deferens is absent is to construct an artificial spermatocele in the epididymis. By using this method to recover sperms for use in AIH, we have succeeded in achieving pregnancy in only 2 of 35 cases, leading us to investigate the fertility of the recovered sperms. Sperm fertility was investigated by the hypoosmotic swelling (HOS) test, observation by confocal laser-scanning microscope (CLSM), flow cytometry and hamster egg sperm penetration test. The percentage of swollen sperm determined by the HOS test was 15.0-83.3%, with a mean of 47.6 +/- 16.1%, significantly lower than the values of the normal group and the infertile male group, excluding cases of obstructed azoospermia. Examination by confocal laser-scanning microscope of samples stained with PI and FITC-PSA or PI and FITC-Con A stain revealed viable and dead spermatozoa as well as viable and dead acrosome-reacted spermatozoa. In addition, fertility was evaluated from the distribution of spermatozoa in each area from the flow cytometry percentage. This method was shown previously to be useful for the evaluation of fertility as it demonstrated the presence of numerous spermatozoa in the fertile area of cases who did not succeeded in pregnancy. The hamster egg sperm penetration test yielded a fertility rate of 0-25% with a mean of 8.2 +/- 10.0%, which was significantly lower than the value of the normal group, but was not significantly different from the infertile group. The findings of this study indicated that the fertility of epididymal sperms is low, thereby pointing to the need for studies to improve the materials used in the artificial spermatocele as well as the method of sperm recovery. Furthermore, our findings suggest that flow cytometry may be used to select the epididymal sperms with the highest fertility for sperm recovery.     

Factors affecting preservation and fertility of bull sperm: a brief review

This paper is a brief review of the factors that determine the number of sperm required for insemination to obtain high fertility and ways that sperm viability might be prolonged. Damage to sperm during freezing results in a requirement, after thawing, of about 6 x 10(6) motile sperm (> 10 x 10(6) total) per insemination to achieve near-maximal fertility, whereas 2.5 x 10(6) motile fresh sperm result in high nonreturn rates. Multiple inseminations to bracket the time of ovulation are usually not economical except in superovulated cows. Earlier unpublished work on sperm packaging for slow release in the cow and methods for stabilizing membranes to increase sperm survival time in the cow are discussed. Current studies are directed towards reducing catabolic metabolism of sperm and studying membrane changes during freezing and thawing and during incubation with bovine oviduct epithelial cells. Studies with bull sperm indicate that the choline and ethanolamine phosphoglyceride components of their membranes represent an unstable configuration. Exposure of sperm to liposomes with the sterol cholesterol can alter the phospholipid bilayer and increase capacitation time. Similar approaches may produce sperm with a longer fertilizing life following insemination. New procedures in vitro permit low cost modelling of fertilization, which will facilitate research by reducing the cost of studies in vivo.     

Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners

BACKGROUND: Semen analysis is part of the routine assessment of infertile couples. WHO defines a sperm concentration above 20x10(6) per mL seminal fluid as normal. We studied the association between semen quality and the probability of conception in a single menstrual cycle in Danish couples with no previous reproductive experience. METHODS: In 1992-94, we invited 52,255 trades-union members aged 20-35 years, who lived with a partner and had no children to take part in the study; 430 couples agreed. The couples discontinued use of contraception, and were followed up for six menstrual cycles or until a pregnancy was verified within this period. Each man was asked to provide a semen sample at enrolment (which was analysed without freezing). Women kept a daily record of vaginal bleeding and sexual activity. The association between semen quality and likelihood of pregnancy was assessed by logistic regression, adjusted for sexual activity and female factors associated with low fertility. RESULTS: There were 256 (59.5%) pregnancies among the 430 couples: 165 (65.0%) among those with a sperm concentration of 40x10(6)/mL or more and 84 (51.2%) among those with lower sperm concentrations. The probability of conception increased with increasing sperm concentration up to 40x10(6)/mL, but any higher sperm density was not associated with additional likelihood of pregnancy. The proportion of sperm with normal morphology was strongly related to likelihood of pregnancy independently of sperm concentration. Semen volume and motility were of limited value in pregnancy prediction. INTERPRETATION: Our study suggests that the current WHO guidelines for normal semen quality should be used with caution. Some men with sperm counts above the lower limit of the normal range defined by WHO may in fact be subfertile.     

Extender components and surfactants affect boar sperm function and membrane behavior during cryopreservation

To determine how the individual components of extenders affected boar sperm function and membrane structure and to test a new surfactant's cryoprotective ability, boar sperm were cryopreserved in straws in BF5 extender plus or minus egg yolk plus or minus glycerol plus or minus a surfactant (Orvus ES Paste [OEP] or various concentrations of Pluronic F-127). After thawing, sperm function and fluidity of the isolated head plasma membrane (HPM) were determined. Total motility and adenosine triphosphate content (a measure of viability) were superior postthaw in sperm extended in egg yolk plus glycerol (P < 0.05); neither surfactant improved function. Egg yolk plus any other ingredients improved normal acrosome morphology, whereas a combined measure of motility and normal acrosome morphology was better in the presence of 0.33% OEP or 0.1% Pluronic F-127 (P < 0.05 vs. controls). Head plasma membrane was isolated from freshly collected spermatozoa and spermatozoa cryopreserved in the various extenders. Membrane fluidity was monitored with the probes cis-parinaric acid (cPNA), transparinaric acid (tPNA), and 1,6-diphenyl-1 ,3,5-hexatriene (DPH). The cPNA and the DPH monitor the fluidity of gel and liquid-crystalline areas of the membrane, whereas the tPNA preferentially monitors the gel-phase domains of the membrane. Additionally, DPH monitors the hydrophobic core of the bilayer. In the HPM from fresh sperm, the fluidity of each domain changed over time in a manner unique to that domain, and the behavior of the DPH domain varied among boars. The fluidity dynamics of each domain responded uniquely to cryopreservation. The cPNA domain was unaffected, the tPNA domain was altered by four of the eight extenders, and all extenders affected the fluidity of the DPH domain. Membrane structure was significantly correlated with cell function for sperm cryopreserved in extenders that preserved viability and motility. Sperm cryopreserved in egg yolk plus glycerol plus either OEP or 0.1% Pluronic F-127 functioned best when the bulk domains were less fluid initially and the gel domain solidified more slowly. Therefore, the behavior of domains in the HPM of boar spermatozoa is affected by cryopreservation and is related to the postthaw function of boar sperm cryopreserved in different extenders.     

Antagonism of SR 90107A/Org 31540-induced bleeding by protamine sulfate in rats and mice

The neutralization by protamine sulfate of bleeding enhancement induced by the potent anti-factor Xa pentasaccharide SR 90107A/Org 31540 and by heparin has been studied in rats and mice. Bleeding, as measured by transection of the tail of anaesthetised rats or mice, was increased following the administration of heparin and very high doses of SR 90107A/Org 31540. In rats, i.v. doses of 0.6 mg/kg heparin or 15 mg/kg SR 90107A/Org 31540 were required to enhance bleeding time to approximately the same extent (5- or 7-fold increase), whereas in mice a 13-fold increase in blood loss was observed with i.v. doses of 3 mg/kg heparin or 10 mg/kg SR 90107A/Org 31540. Protamine sulfate (10 mg/kg i.v.) reduced bleeding in rats and mice induced by both compounds. It also neutralized the anti-factor Xa activity as well as the antithrombotic activity of heparin as observed in venous thrombosis models in both species. However, protamine sulfate neither affected the anti-factor Xa activity nor the antithrombotic activity of SR 90107A/Org 31540 in rats and mice. The present results suggest that protamine sulfate may be regarded as a potential antidote to neutralize bleeding side-effects in cases of SR 90107A/Org 31540 overdosing.     

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.     

The fertilization potential provides a fast block to polyspermy in lamprey eggs

At fertilization, the membrane potential of the egg of the lamprey, Lampetra japonica, shifted rapidly from its resting value of -12 to +36 mV and gradually returned to about the same resting level (fertilization potential). The amplitude of depolarization was influenced by the external Cl- concentration and by an anion channel blocker, DIDS, indicating that the positive shift of membrane potential resulted from Cl- efflux. A similar change in membrane potential (activation potential) was observed when the unfertilized egg was pricked with a fine needle or treated with A23187 to induce parthenogenetic activation. Pricking at the animal pole region (predetermined site for sperm entry) resulted in the occurrence of an immediate activation potential and the initiation of cortical granule exocytosis. A time lag between the pricking and the occurrence of the activation potential was observed when the egg was pricked at a distance from the animal pole. In this instance, the activation potential was produced immediately before the propagating cortical granule exocytosis initiated at the pricked site reached the animal pole region. Sperm-egg fusion was blocked in eggs voltage-clamped at +20 to +40 mV and inseminated, whereas it took place in eggs clamped at -60 to 0 mV. However, most eggs clamped at +20 to +40 mV did activate, indicating that the voltage dependence of egg activation differs from that of sperm-egg fusion. Although eggs voltage-clamped at negative membrane potentials permitted multiple sperm to fuse with the egg plasma membrane, the nucleus of the fused sperm did not necessarily enter the ooplasm. We conclude that: (1) A fast electrical block against polyspermy operates in this species and is effective for about 160 sec of the onset of the positive shift; (2) the opening of Cl- channels is responsible for the potential change; (3) the channels are largely localized in the animal pole region; (4) during voltage clamp at positive potentials, eggs can be activated without sperm-egg fusion; and (5) during voltage clamp at negative potentials, sperm-egg fusion occurs, but sperm entry into the egg cytoplasm does not always proceed.     

Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with 14C-FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis.     

The correlation of sperm chromatin decondensation following in vitro exposure to heparin and sperm penetration rates

PURPOSE: The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes. METHODS: Twenty-two donors of known fertility and 105 patients undergoing evaluation at an andrology laboratory were evaluated by standard World Health Organization semen analysis techniques and a modified sperm penetration assay (SPA). An aliquot was also incubated for 60 min and Ham's F10 medium containing 50 USP/ml heparin. The percentage of sperm undergoing chromatin decondensation was evaluated and correlated to SPA rates and semen quality parameters. RESULTS: No significant correlation was observed between semen parameters and decondensation rates. A nonsignificant (P = 0.11) inverse correlation (P = -0.21) was observed between SPA rates and chromatin decondensation. Significant (P < 0.001) differences were observed in the decondensation rate of donors (3.7 +/- 0.6), patients with normal SPA rates (7.8 +/- 1.5), and patients with decreased SPA rates (21.7 +/- 1.8). The decondensation rates were significantly different (P < 0.01) between patients with a normal SPA rate and patients with a decreased SPA rate. CONCLUSIONS: These data indicate a significant inverse relationship between the SPA rate, which has previously been shown to correlate highly with fertilization ability and heparin-induced sperm chromatin decondensation.     

Human apolipoprotein E allele-specific brain expressing transgenic mice

OBJECTIVES: Several reports indicate a secular decline of human sperm counts. It is still not known if these findings are artifacts related to shortcomings in the data and applied methodologies. Even less is known about possible mechanisms, but it has been proposed that potential changes may be related to disruption of the hormonal regulation of testicular development in prenatal life. The objective of this study was to examine whether sperm count was related to year of birth. METHODS: An analysis was made of the sperm count of 1196 men participating in 10 cross-sectional occupational sperm studies in 3 regions of Denmark from 1986 through 1995. RESULTS: The median sperm concentration was 63 million per milliliter for men born in 1937-1949 and 52 million per milliliter for men born in 1970 or later, and the median total sperm was 206 million and 117 million, respectively. The inverse relationship between sperm concentration and year of birth was statistically significant even after adjustment for duration of sexual abstinence, season of the year, and study population. However, bias because of differential participation related to age and fertility or lack of comparability across the populations cannot be ruled out. CONCLUSIONS: The apparent decline of sperm count with increasing year of birth is compatible with the hypothesis of a common risk factor for male reproductive health operating in prenatal life or early childhood, but the evidence is circumstantial. Age-related selection bias is an alternative and perhaps not a less likely explanation.     

Liquid storage of ram semen: a review

Research from 1930 to 1992 is reviewed with regard to storage of semen at reduced (0-5 degrees C or 10-15 degrees C) and at ambient temperatures. Diluents investigated have included synthetic buffers combined with sugars and egg yolk or its fractions, milk from various sources, glycine, and other substances. Irrespective of the diluent, dilution rate, temperature or conditions of storage, the spermatozoa deteriorate with time of storage. Changes include reduction in motility and morphological integrity of spermatozoa, accompanied by a decline in their survival in the female reproductive tract, reduction in fertility and increased embryonic loss. In critical studies, fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination, but after intrauterine insemination some spermatozoa maintained their fertilizing capacity up to 10 days. Laparoscopic intrauterine or transcervical inseminations could be the means of improvement of fertility. These methods may eliminate the problem of sperm transport through the cervix and ageing of spermatozoa in the reproductive tract, thereby improving fertilization of ova and reducing embryonic loss.     

Disturbances of nuclear condensation in human spermatozoa: search for mutations in the genes for protamine 1, protamine 2 and transition protein 1

During spermiogenesis, the successive replacement of the somatic histones by basic proteins, the transition proteins and protamines, allows normal sperm nuclear condensation. It was suggested that disturbances in nuclear condensation may result in male infertility. Here we report the first molecular analysis of the structure of three genes which code for germ cell-specific nuclear proteins, namely protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 1 (TNP1) in infertile men with disturbed sperm chromatin condensation. In 36 infertile men whose spermatozoa showed a positive reaction with aniline blue, which is an indication for the presence of histones in the nuclei, the complete nucleotide sequences of the coding regions and 5' and 3' untranslated regions of the three genes were evaluated. In addition, 10 infertile patients with oligoasthenoteratozoospermia were studied in the same way, as well as nine infertile patients whose spermatozoa showed a reduction of the protamine 2 content. We did not detect any mutation in the three genes in any of the patients. We assume that the disturbances in the sperm chromatin condensation of our patients, and those described in the literature, are not primarily due to mutations in the genes for PRM1, PRM2 and TNP1.     

Determining the cost of care through clinical pathways

To clarify the role of urokinase plasminogen activator (uPA) in the mechanisms of regulating sperm motility and the ability of fertilizing, we investigated the quantities and activities of uPA in human seminal plasma and on the membrane of spermatozoa. Semens were harvested from 22 infertile patients with asthenospermia and 20 healthy fertile men according to WHO standards. To quantify the membrane-bound uPA in the samples, polyclonal antibodies against human urokinase were employed by means of a sandwich ELISA. The uPA activities in seminal plasma and on the surface of spermatozoa were determined using A-garose-Fibrine-Plate method and the experiment of immunological identification with polyclonal antibodies against urokinase. In lysates of spermatozoa, significantly lower levels of uPA (23.1 +/- 7.35 mu/10(6) cells) and uPA activity (5.13 +/- 3.85 mu/10(6) cells) were found in patient group as compared to healthy fertile men exhibiting normospermia (29.89 +/- 9.40 mu/10(6) cells and 10.17 +/- 6.18 mu/10(6) cells). In seminal plasma, uPA activity in patient group (2134 +/- 1581.3 IU/L) was also found significantly lower than that of normal group (3365 +/- 1859.5 IU/L). Positive correlations were observed between sperm motility and uPA quantities (r = 0.48, P < 0.005), as well as with uPA activities (r = 0.45, P < 0.005). Thus, it is inferred that membrane associated uPA on human spermatozoa may be related directly to sperm motility and fertility.     

Effect of lipid peroxidation on cryopreserved semen quality in patients with testicular or nontesticular cancer

OBJECTIVES: Although pre-freeze and post-thaw semen quality in patients with cancer is poor, it is not clear whether lipid peroxidation affects semen quality. This study assessed (1) whether poor semen quality in patients with cancer is caused by lipid peroxidation, and (2) whether patient age or sperm motility is associated with lipid peroxidation. METHODS: Lipid peroxidation was measured by determining malonaldehyde levels using the thiobarbituric acid method. Malonaldehyde levels were measured in cryopreserved semen specimens from patients with testicular (n = 15) or nontesticular cancer (n = 16) and normal men (control subjects, n = 20). A computer-assisted semen analyzer was used to determine the sperm concentration and motility before and after cryopreservation. RESULTS: Malonaldehyde levels did not differ in frozen-thawed semen specimens among patients with testicular (25.90 +/- 1.00 nM/10(8) sperm/hr) or nontesticular cancer (24.48 +/- 1.66 nM/10(8) sperm/hr), and control subjects (24.86 +/- 1.43 nM/10(8) sperm/hr). Malonaldehyde levels did not correlate with post-thaw sperm motility or patient age in patients with testicular or nontesticular cancer. Post-thaw sperm motility from patients with testicular or nontesticular cancer was significantly lower than that in normal subjects. CONCLUSIONS: The poor post-thaw semen quality in patients with testicular or nontesticular cancer is not related to lipid peroxidation but may be caused by other factors such as sperm membrane stress induced during the freeze-thaw process.     

Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review

When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsaturated:saturated fatty acids in the phospholipids and a low cholesterol content. The high unsaturated fatty acid content of sperm also makes them susceptible to damage from peroxidation which adversely affects motility, metabolism, ultrastructure and fertility. Hydroxynonenal, a product of fatty acid peroxidation, depresses the motility and oxygen uptake of ram sperm in vitro and may react with the -SH groups of the axonemal microtubules. High calcium concentrations in the external medium may decrease the motility and metabolism of sperm and 'calcium intoxication' may be a factor in cold shock. Lowering the environmental temperature increases calcium uptake by sperm and the effect is aggravated if the rate of cooling is rapid. Phospholipids, particularly those in egg yolk, protect sperm to some extent from cold shock and also prevent increased calcium flux into the sperm. Suggestions are made for increasing the life span of sperm during preservation and microencapsulation by adding agents that may stabilize membranes, counter peroxidation and decrease calcium uptake.     

Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg

The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with fertilin in sperm-egg fusion.     

Surface localization of the sea urchin egg receptor for sperm

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.     

Development of an outcome measure to document pain relief for home hospice patients: a collaboration between nursing education and practice

Epididymal glycosidases play a role in sperm maturation by modifying sperm surface glycoproteins. To study the effects of ethanol on epididymal sperm maturation, ethanol (3 g/kg body weight as 25%, v/v) was administered to a group of rats by gastric-intubation twice daily for 30 days. In another group, rats were also treated with alcohol for 30 days but were then withdrawn from treatment for 30 days to assess the reversibility of ethanol-induced effects. Ethanol-induced changes in epididymal tissue and sperm glycosidases, cauda epididymal sperm motility and the fertility of rats were assessed. Ethanol treatment caused a marked decrease in the specific activities of glycosidases in both tissues and spermatozoa from epididymal segments. Cauda epididymal sperm motility and the fertility of ethanol-treated rats were significantly impaired compared to control rats fed an isocaloric diet. These changes are likely to be the consequence of direct and indirect effects of ethanol mediated through subnormal testosterone and dihydrotestosterone. Most of these changes were found to be reversible. The present study suggests that impaired activity of sperm glycosidases may be one of the factors responsible for defective sperm motility and fertilizing potential in ethanol-treated rats.     

In vitro maturation and fertilization techniques for assessment of semen quality and boar fertility

The reliability of using different in vitro-derived measures of sperm quality to predict boar fertility was examined. On three occasions during a 20-wk period of breeding, special collections of the first sperm-rich fraction of the ejaculate from six boars were carried out. After in vitro capacitation procedures, three dilutions (5 x 10(5), 1.25 x 10(5), and 3.125 x 10(4) sperm/mL) of these semen samples were used in a standardized in vitro fertilization (IVF) test with oocytes recovered from prepubertal slaughterhouse ovaries and matured in vitro. Routine assessments of sperm motility, concentration, and morphology were also carried out for all collections used for AI during the 20-wk period. Semen from the same ejaculate, processed according to normal commercial practice using the AndroHEP extender, was used to inseminate equal numbers of recently weaned sows with either 3 x 10(9) or 2 x 10(9) total sperm, three times during the estrous period. Data from a total of 444 sows were used to determine boar fertility; between 12 and 54 sows were bred with each semen dose across the six boars. All measures of sperm fertilizing ability in vitro were different among boars (all P < .05) and use of different semen dilutions for IVF allowed further discrimination of apparent sperm quality among boars. The laboratory evaluation of semen collected during the period of breeding indicated effects of boar on ejaculate volume, total number of sperm per ejaculate, motility, and the percentage of sperm with normal morphology (all P < .01). Sperm dose used in AI had no effect on farrowing rate (80.7 vs 81.5%), but the lower AI dose resulted in a reduction (P < .05) in total numbers born (10.8 vs 10.0). For all three semen dilutions, estimated potential embryo production rate accounted for up to 70% of the variation in litter size obtained with 3 x 10(9) sperm per AI dose, and the number of sperm attached per oocyte was a major factor accounting for variation in litter size obtained with 2 x 10(9) sperm per AI dose. These IVF variables may, therefore, be effective indicators of boar sperm quality for use in AI. With 2 x 10(9) sperm per AI dose, the percentage of sperm with normal morphology also explained a large part of the variance in litter size born (R2 = .59), indicating that morphological characteristics are a useful measure of semen quality.