Interactions among motility, fertilizing ability, and testosterone binding on spermatozoa of bonnet monkey (Macaca radiata)

Fresh ejaculates of bonnet monkeys were separated into fractions rich with highly motile and sluggishly motile spermatozoa. The motility, ability to fertilize zona-free hamster eggs, and distribution of testosterone-binding sites on spermatozoa were assessed to determine the relation between these sperm functions. Two parameters of objective assessment of motility--velocity and degree of flagellar bending--were significantly correlated with the ability to form pronuclei in zona-free hamster eggs. Only spermatozoa with good motility could form pronuclei, which might be important for assessment of the fertilizing ability. The motility was directly related to the distribution of testosterone-binding sites; the fraction having mostly motile spermatozoa was distributed over the sperm surface. The technique is simple and may be used to evaluate semen of nonhuman primates.     

Morphology and fertility of guinea pig spermatozoa aged in vivo

The morphology and fertility of spermatozoa from vasa deferentia of guinea pigs were observed following hemicastration or castration for approximately 40 days. The morphology of these aged sperm was studied from living and fixed preparations. Fertilizing ability was assayed by artificial insemination of estrous females and subsequent counting of embryos. Spermatozoa underwent morphological changes including dissociation of rouleaux, curving of tails, and loss of acrosomes; physiological changes included a decline in the number of sperm with progressive motility and increased numbers of immotile spermatozoa with time after the operations. The fertilizing capacity of spermatozoa was maintained for approximately 30 days in both groups. However, sperm from one of the hemicastrated males resulted in conception 36 days postoperation. The data suggest that the loss of motility and decline in fertilizing ability were the result of spermatozoa senescence rather than testicular androgen deficiency.     

Evaluation of the hypo-osmotic swelling test in relation with advanced methods of semen analysis

The hypo-osmotic swelling test was claimed to assess an independent functional characteristic of human spermatozoa bearing relevance to their fertilizing capacity. To test this claim, we have studied the relationship between the result of the hypo-osmotic swelling test with that of conventional semen analysis and sperm motility patterns, the semen content of adenosine triphosphate, the staining pattern to acidified aniline blue, and the zona-free hamster oocyte test. The result of the HOS test is significantly correlated with all sperm characteristics except for the aniline blue stainability and the hamster oocyte test. The capacity of spermatozoa to react in a hypo-osmotic environment expresses the same functional information as the viability test using eosine staining. It is concluded that the hypo-osmotic swelling test does not add relevant information to that obtained by routine sperm analysis with regards to the fertilizing potential of semen.     

Residual sperm function in oligozoospermia induced by testosterone enanthate administered as a potential steroid male contraceptive

To investigate the fertility of men who remain oligozoospermic despite sex steroid suppression, the in-vitro fertilizing capacity of residual spermatozoa was assessed in 30 men receiving intramuscular testosterone enanthate (TE). Spermatozoa were prepared by either Percoll or repetitive centrifugation/washing. Although the mean (+/- SEM) pretreatment zona-free hamster oocyte penetration (HOP) rates were similar (59.4 +/- 10.1 and 63.8 +/- 10.8%), following the induction of oligozoospermia the Percoll-prepared spermatozoa exhibited a penetration rate (26.9 +/- 10.2%) which was markedly greater than that obtained for sperm prepared by repetitive washing (0 +/- 0%). In addition, the partners of two men exhibiting a HOP test with Percoll-prepared spermatozoa, conceived despite a sperm concentration of 3 x 10(6) ml-1 and a negative HOP test with spermatozoa prepared by repetitive washing. These results suggest that Percoll preparation optimizes the assessment of in-vitro sperm function and that the fertility of men with TE-induced severe oligozoospermia is suppressed but not abolished.     

Characterization of the fertility of Kit haplodeficient male mice

The role of the proto-oncogene Kit expression during gonadal development, then in differentiated spermatogonia has been thoroughly established. The present study was designed to investigate the consequences of a partial defect in Kit gene expression on sperm fertilizing ability, using Kit haplodeficient mice (kitW-lacZ/+). Same inbred mice (kit+/+) were used as controls. Epididymal sperm characteristics and in vivo fertility were assessed, then in vitro-fertilization experiments were carried out for mice of both genotypes. Epididymal sperm count was drastically reduced, and sperm motility was also decreased in kitW-lacZ/+ compared with kit+/+ males. Both in vivo or in vitro fertility were greatly reduced in kitW-lacZ/+ compared with kit+/+ males. By contrast, the fertility of kitW-lacZ/+ females was apparently unaffected. Additionally, a higher number of spermatozoa with undetected acrosomal contents was revealed by fluorescein isothiocyanate-labelled Pisum sativum agglutinin acrosomal staining after epididymal sperm retrieval in kitW-lacZ/+ mice, whereas no difference was observed after induction of acrosomal reaction in mice of either genotype. Ultra-structural data confirmed the higher frequency of abnormal acrosome in spermatozoa of kitW-lacZ/+ mice. Thus, sperm production is impaired in Kit haplodeficient mice both on a quantitative and a qualitative basis. Finally, we show that one single copy of Kit gene is not sufficient to maintain genuine fertility in male mice.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Spermatozoal Fertilizing Capacity in Polyzoospermia: a Preliminary Study

Men with the spermatological symptom of polyzoospermia (greater than 250 X 10(6) sperm/ml) have been reported to seldom impregnate their wives. It was the aim of this study to investigate the spermatozoal fertilizing capacity in polyzoospermia by the human sperm and zona-free hamster ova penetration bioassay. General semen characteristics and in vitro spermatozoal fertilizing capacity were studied in 12 polyzoospermic male partners of couples of infertile marriages. The results were compared with those from a control group of normospermic fertile men (n = 22). No significant differences in sperm motility, normal morphology and in vitro spermatozoal fertilizing capacity were found between the two groups. The polyzoospermic men we studied did not appear to have any defect with the spermatozoal fertilizing capacity, as assessed by the heterologous sperm--ova penetration bioassay. The apparent impairment of fertility and higher abortion rate in couples with polyzoospermic male partners, as described in the literature, may be related to chromosomal aberrations and/or other unknown functional defect of the spermatozoa.     

Fumarase Activity in Human Ejaculate Relationship to Sperm-Motility or -Penetration of Zona-Free Hamster Eggs

Fumarase activity, sperm count, sperm motility and in vitro fertilizing ability of spermatozoa were determined in semen samples of 69 men attending an infertility clinic. There existed a significant correlation between sperm count and fumarase activity (r = 0.52). No significant relationship was observed between sperm motility or hamster egg fertilizing ability of the spermatozoa and fumarase activity. From these results it is concluded that fumarase activity in human ejaculates can probably not be used to define the fertilizing ability of spermatozoa.
Fumarase-Aktivität im menschlichen Ejakulat Beziehungen zwischen Spermatozoen-Motilität oder -Penetration in Zona-freie-Hamster-Eier

Zusammenfassung

An den Spermaproben von 69 Männern wurde die Fumarase-Aktivität, die Spermatozoendichte, die Spermatozoenmotilität und die in-vitro-Fertilisierungs-Fähigkeit von Spermatozoen untersucht. Dabei konnte eine signifikante Beziehung zwischen der Spermatozoendichte und der Fumarase-Aktivität (r = 0,52) festgestellt werden. Zwischen der Spermatozoenmotilität oder der Fertilisierungs-Fähigkeit menschlicher Spermatozoen für Hamster-Eier ließ sich dagegen keine Signifikanz zur Fumarase-Aktivität herstellen. Aus den Ergebnissen wird die Schlußfolgerung gezogen, daß die Fumarase-Aktivität im menschlichen Ejakulat im allgemeinen nicht für die Beurteilung der Fertilisierungs-Fähigkeit der Spermatozoen herangezogen werden kann.     

Exposure to ethanol during capacitation impairs the fertilizing ability of human spermatozoa in vitro

To validate earlier findings, mainly in laboratory animals, the effect of ethanol on the fertilizing ability of human spermatozoa has been investigated. Ethanol added to the capacitation medium reduced the penetration of zona-free hamster eggs by human spermatozoa in a dose-dependent manner at concentrations from 50 to 500 mg % (0.05-0.5%). Fertilizing capacity was at least partially restored by washing in ethanol-free medium. Ethanol exposure before capacitation had a slight stimulatory effect on the penetration of spermatozoa into zona-free hamster ova. The motility of spermatozoa was not altered significantly, either quantitatively or qualitatively, by the presence of ethanol in the capacitation medium. These results suggest that the decrease in fertilizing ability of spermatozoa induced by ethanol during capacitation is due to a specific action on the capacitation process.     

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa

The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes afier stimulation with 2μmol A23187 per litre was increased by the hrther addition of 0.6 or 3.6 mmol pentoxifjrlline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm headd/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 μmol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that CAMP may act synergistically with Ca²⁺ to stimulate the acrosome reaction. Pentoxifjrlline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca²⁺ signal produced by binding to the zona pellucida.     

Successful lowering of epididymal carnitine by administration of pivalate to rats

This study was designed to lower the epididymal content of carnitine in male rats and to examine subsequent effects on fertility and sperm motility. As carnitine is transported from serum into the epididymal lumen a method to lower serum carnitine was sought. Administration of 20 mmol/L sodium pivalate in the drinking water for up to 5 weeks substantially lowered serum carnitine (to 20% of control levels within 1 week) and reduced epididymal carnitine content (to 25% of control levels in the proximal and 52% of control in distal regions) within 2 weeks. Carnitine in distal cauda epididymal fluid was also reduced (to 30% of control levels) but no changes were observed in the sperm carnitine content. The percentage motility and kinematic parameters of spermatozoa released from four epididymal regions and diluted into artificial medium were unaltered by the treatment, and all males retained their fertility in mating tests performed at weekly intervals. Increasing the dose of sodium pivalate administered to 60 mmol/L for 2 weeks lowered serum carnitine concentration more but did not further decrease epididymal carnitine content and altered neither sperm motility nor male fertility. The rat epididymis secretes an excessive amount of carnitine into its lumen so that substantially lowering the tissue content does not reduce sperm carnitine or affect their motility or fertilizing ability.     

Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility

A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia.     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Effects of cotinine on sperm motility, membrane function, and fertilizing capacity in vitro

We evaluated the effect of cotinine on sperm fertilizing capacity in vitro. Human spermatozoa were washed and re-suspended in medium containing albumin and various concentrations of cotinine (0, 100, 200, 400, or 800 ng/ml). After an 8-h incubation period, sperm motility, hypoosmotic swelling test (HOST) outcome, and the percentage of hyperactivated spermatozoa were assayed. Aliquots of spermatozoa were then processed for the zona-free hamster oocyte sperm penetration assay (SPA) or hamster ooplasmic injections. Spermatozoa exposed to concentrations of cotinine equal to 400 or 800 ng/ml demonstrated significantly smaller outcomes for all of the above with the exception of after hamster ooplasmic injections, where high cotinine concentrations did not affect sperm viability or sperm capacity to undergo decondensation and activate hamster oocytes. It appears that cotinine concentrations of 400 or 800 ng/ml exert a detrimental effect on sperm motility, membrane function, and the ability to undergo capacitation. In addition, the current findings suggest that smokers with a high seminal plasma cotinine concentration who participate in assisted reproduction programs may be treated with intracytoplasmic sperm injections (ICSI) rather than conventional in vitro fertilization (IVF) trials. Received: 25 November 1998 / Accepted: 24 September 1999     

Analysis of spermatozoa from the proximal vas deferens of vasectomized men

This study assessed the condition of spermatozoa from the proximal vas deferens of men after vasectomy. The fluids of both proximal vas deferens were collected from 67 vasectomized men by cannulating the vas deferens at the time of vasectomy reversal. Selected sperm parameters were analysed after incubation of the spermatozoa for 30 min at 37°C. Spera concentration in the proximal vas from vasectomized men (16 312 ± 21 496 million per ml, geometric mean: 7948 ± 398 million per ml) was significantly higher than that of fertile men and was maintained at a constant level independent of the duration of vas obstruction. The means of sperm motility (36.2 ± 26.2%), spermatozoa with normal morphology (50.7 ± 21.7%), sperm viability (53.0 ± 25.3%) and hypo-osmotic swelling test (HOS-test, 53.9 ± 21.7%) were statistically lower than the respective values for normal fertile men. There was no significant correlation between the duration of vas obstruction and the above semen parameters. In 46.4% of vas fluids all spermatozoa were immotile and this condition was more common after 3 years of vasectomy. Immotile spermatozoa in the proximal vas fluids at the time of vasectomy reversal may be an important factor for predicting semen quality and fertilizing ability after vasovasostomy. There were no significant differences in the results of sperm-cervical mucus penetration test (CMPT) between spermatozoa fiom vasectomized and fertile men. Antisperm antibodies on the surface of spermatozoa from the vas of vasectomized men were determined by the immunobead test (IBT; 78.6% for IgG, 32.1% for IgA) and sperm cervical mucus contact test (SCMC, 36.4%). The presence of antisperm antibodies on the spermatozoa from the vas of vasectomized men may explain, in part, the lower pregnancy rate after vasovasostomy. These parameters of spermatozoa from the proximal vas of vasectomized men may closely reflect those in the cauda epididymis after vasectomy.     

Effect of human sperm exposure to progesterone on sperm-oocyte fusion and sperm-zona pellucida binding under various experimental conditions

In this study, the effect of human sperm exposure to progesterone on sperm/oocyte fusion, using the hamster egg penetration test, and on sperm/zona pellucida (ZP) binding, using the hemizona assay, was investigated under various experimental conditions. A brief exposure of human spermatozoa to progesterone exerted a stimulatory effect on sperm/oocyte fusion which was dose-dependent, capacitation-dependent, influenced by the source of serum albumin in capacitating medium, and was higher than that produced by the exposure to progesterone from the onset of capacitation. The exposure of capacitated spermatozoa to progesterone during 20 min-spermatozoa/ZP-coincubation produced an enhancement of ZP-binding, which was not significantly influenced by the source of serum albumin in capacitating medium. A significantly lower ZP-binding was exhibited by spermatozoa exposed to progesterone from the beginning of capacitation. These results indicate that progesterone exerts a stimulatory effect on human sperm's fertilizing ability, which occurs mainly in post-capacitation events directly involved in sperm/oocyte fusion and in ZP-binding. Conditions optimizing these effects are provided. They should be taken into account in the standardization of experimental and clinical studies designed to evaluate the response of human spermatozoa to progesterone.     

Boar Proacrosin Correlation between Total Proacrosin Content and Sperm Fertilizing Capacity

A method of acidic extraction at pH-1.3 was used for quantitative estimation of the proacrosin and acrosin content in boar spermatozoa after various times of storage in four different sperm dilutors. The proacrosin and acrosin contents were correlated with the results of biological tests for sperm penetration in zona-free hamster eggs and sperm survival. The results have shown that proacrosin quantitation should be a convenient biochemical test of sperm fertilizing capacity.     

Contribution of the male factor to unexplained infertility: a review

The more exhaustive the evaluation of couples with unexplained infertility, the more likely is the opportunity for detecting the aetiological factor responsible for infertility. Transport of spermatozoa through the upper genital tract and their ability to fertilize the oocyte are two obscure areas for the conventional evaluation of infertility. Although research in the former area is limited, there is indirect evidence that impaired sperm transport could be one of the causes of infertility in some couples with otherwise unexplained infertility. On the other hand, the availability of sperm function tests and the correlation of their results with in-vitro fertilization rates have allowed the detection of a previously hidden male factor in couples with unexplained infertility. It has been demonstrated that couples suffering unexplained infertility have significantly lower in-vitro fertilization rates in comparison with patients with tubal problems. These results can be explained because several case control studies in patients with unexplained infertility have reported defects in capacitation and sperm motion characteristics, binding of the spermatozoa to the zona pellucida, acrosome reaction, acrosin activity of the spermatozoa, and the ability of the spermatozoa to penetrate zona-free hamster cocytes. These observations suggest that methods for assessing the fertilizing capacity of the spermatozoa have to be incorporated in the evaluation of couples with unexplained infertility in order to amplify the scope of the workup and to better decide the appropiate treatment for these couples.     

Penetration of sperm from teratospermic men into zona-free hamster eggs

The ability of sperm samples from male partners of infertile couples, with isolated teratospermia (ITS), to penetrate zona-free hamster eggs was examined. The ITS patients had a significantly lower proportion of normal spermatozoa than did a control group of men of proven fertility (23.6% and 47.8%, respectively; P less than 0.001), while the mean total sperm count and the % motility did not differ between the two groups. The mean hamster egg penetration rate of sperm from ITS patients was 2.64% compared to 31.1% in the control group. A significant negative correlation was found in the ITS group between the proportion of a) abnormal forms and the ability to penetrate zona-free hamster eggs, and b) pyriform-shaped sperm and the penetration rate. Of the sperm parameters which were examined, only the morphology could be correlated with the rate of penetration.     

Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei.

The relationship between thiol-disulfide status and acridine orange fluorescence of testicular, epididymal, and ejaculated spermatozoa in several mammalian species was investigated. Spermatozoa were fixed with acetic alcohol, stained with acridine orange, and examined with a fluorescence microscope. The majority of the nuclei of testicular spermatozoa of the hamster, mouse, and rabbit exhibited red acridine orange fluorescence. The proportion of sperm nuclei with red acridine orange fluorescence decreased as the spermatozoa descended the epididymis. Red acridine orange fluorescence was replaced by green acridine orange fluorescence. The site in the epididymis where 100% of the nuclei exhibited green fluorescence was the distal caput in the mouse, the corpus in the rabbit, and the proximal cauda in the hamster. In semen samples from men with proven fertility, normal semen parameters, or both, about 60% to 90% of the nuclei exhibited green acridine orange fluorescence. The proportion of sperm nuclei exhibiting green acridine orange fluorescence was higher in the spermatozoa pellet (containing highly motile spermatozoa) obtained by centrifugation through a Percoll gradient. From experiments using disulfide-reducing, thiol-oxidizing and thiol-detecting agents, we concluded that sperm nuclei fluoresce red when they are treated with acid while their DNA-associated protamines are poor in disulfides. Under such conditions, DNA is vulnerable to denaturation. Acridine orange binds to denatured (single-stranded) DNA as aggregates and emits red fluorescence. In contrast, when sperm nuclei are treated with acid while their DNA-associated protamines are rich in disulfides, DNA is resistant to denaturation. Acridine orange binds to native (double-stranded) DNA as a monomer and emits green fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)