Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase

In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.     

Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.     

Artificial insemination with seminal plasma improves the reproductive performance of frozen-thawed boar epididymal spermatozoa

Frozen-thawed epididymal spermatozoa have good fertilization capability in vitro; however, their artificial insemination conception rate is less than half of that of frozen-thawed ejaculated spermatozoa. Because the addition of seminal plasma to the thawing solution enhances the in vivo fertilizing ability of frozen-thawed ejaculated spermatozoa, we hypothesized that the reproductive performance of frozen-thawed epididymal spermatozoa could also be improved by the inclusion of seminal plasma. When frozenthawed epididymal spermatozoa were incubated for up to 6 hours, the motility of the sperm significantly decreased in a time-dependent manner. The acrosomal membrane was damaged in the majority of frozen-thawed epididymal spermatozoa. The addition of seminal plasma to the thawing solution significantly decreased the percentage of sperm with abnormal acrosomes and increased their total motility in a dose-dependent manner. Furthermore, the addition of seminal plasma reduced the abundance of a 15-kDa tyrosinephosphorylated protein in frozen-thawed sperm, and the maximum effect was observed at 15% (vol/vol) seminal plasma. When cryopreserved epididymal spermatozoa from 3 different boars were thawed with a 15% (vol/vol) seminal plasma-containing solution, the conception rate and mean litter size obtained by artificial insemination were significantly increased as compared with those in the control without seminal plasma. From these results, we concluded that the addition of seminal plasma to the thawing solution is a key step in obtaining an optimal number of piglets by artificial insemination using frozen-thawed boar epididymal spermatozoa.     

Lack of effect of exogenous prolactin on the fertilizing capacity of human spermatozoa in vitro

The effect of exogenous prolactin (PRL) on the fertilizing capacity of human spermatozoa in vitro was evaluated by the zona-free hamster ova penetration assay. Bovine and ovine PRL, in both physiological and pharmacological concentrations, failed to increase the penetration after 5 hr of incubation in vitro. The percent motility of spermatozoa was not influenced by PRL during the incubation period. Exogenous PRL does not affect the fertilizing capacity of human spermatozoa in vitro. The role of prolactin, normally present in seminal plasma and female reproductive tract fluid, in the human spermatozoal capacitation process merits further investigation.     

Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation

In this study, we evaluated the effects of glutathione (L-g-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intra-cellular reactive oxygen species generation, sperm chromatin condensation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins, a reduction of the reactive oxygen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of decondensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.     

Evaluation of acrosome on Papanicolaou-stained sperm and its relationship to the in vitro fertilizing capacity of human spermatozoa

Papanicolaou-stained sperm smears from 1150 ejaculates evaluated from infertility clinic patients and 166 ejaculates in an in vitro fertilization program were studied for the presence of acrosomal abnormalities. Morphologically, a normal acrosome was defined as a sperm having a ratio of 1:1 to 2:1 between the acrosome and postacrosomal part in an oval or round, uniformly shaped head. Acrosomal abnormality was identified in at least a few of the spermatozoa in all the ejaculates (range 8-98%) and appeared to be closely related to normal sperm morphology (r = 0.85). A significantly higher (p less than 0.01) correlation (r = 0.31) was noted between the presence of normal acrosome and in vitro fertilizing capacity of spermatozoa as compared to other standard semen characteristics. Acrosome can be identified on Papanicolaou-stained smears, and since it appears to be related to fertility it may be beneficial to evaluate the acrosome morphologically during routine semen analysis.     

Binding of seminal vesicle proteins to the plasma membrane of rat spermatozoa in vivo and in vitro

The association of seminal vesicle (SV) proteins with rat spermatozoa has been studied in vivo and in vitro. SV proteins bind to the sperm plasma membrane after ejaculation but are removed progressively from the sperm plasma membrane in the female genital tract. Although some of these remain bound to spermatozoa when they reach the oviducts, they do not seem to be present at the time of fertilization. This could indicate a putative role for these SV proteins in pre-fertilization events. In addition, the binding of SV antigens was studied in vitro. It was observed that the ability to bind SV proteins is gained by the spermatozoa during epididymal maturation, and is first detectable in spermatozoa collected from the cauda epididymis. On the other hand, the binding is regulated by other proteins present in the ejaculate which are secreted by the coagulating glands. Experiments also showed that mouse spermatozoa are able to bind rat SV proteins, indicating that the binding is not a highly species-specific phenomenon.     

Lack of HLA-molecules on human spermatozoa and in seminal plasma

Summary. The expression of human leukocyte antigens (HLA) on ejaculated spermatozoa and on lymphocytes was compared by flow cytometry using monoclonal antibodies towards HLA class I (pan-HLA-A, -B, -C) and class II (DR) antigens. Soluble antigens of HLA class I (s HLA-A, -B, -C) in seminal plasma and in blood plasma were monitored with an elisa technique. Lymphocytes showed specific fluorescence after incubation with the antibodies against HLA class I and class II (DR), whereas, on spermatozoa no positive immunofluorescence could be detected. No antibodies were bound to any significant extent either after modifications of sperm preparation (density gradient centrifugation, swim up-technique, addition of azide, foetal calf serum or benzamidine chloride) or after treatment of spermatozoa with detergens. Furthermore, different concentrations of soluble HLA-A, -B, -C in seminal plasma and in blood plasma were detected. The latter one showed soluble HLA about four-fold more concentrated than the seminal plasma (x ± SD: 262.5 ± 144.4 nmol 1-¹vs. 62.5 ± 27.1 nmol 1-¹). These results suggest, that the HLA-expression differs between human spermatozoa and somatic cells.     

Involvement of the calcium-specific protease, calpain, in the fertilizing capacity of human spermatozoa

We recently reported the novel finding that human spermatozoa contain the calcium (Ca2+)-dependent protease, calpain. In somatic cells this protease mediates several cellular activities regulated by Ca2+ including membrane fusibility during cell-to-cell interactions. In this paper we examined the participation of sperm calpain in sperm-oocyte penetration, a process that is dependent on Ca2+ and involves membrane fusion between the two cells. Oocyte penetration was assessed using ejaculated spermatozoa from fertile men and zona-free hamster oocytes. Penetration rate was impaired by the presence of the active-site calpain inhibitor, calpain inhibitor-I, in a dose-dependent manner. At 1 mM, penetration scores were reduced by 65% (p < 0.01; n=5). The effects did not involve the oocyte, nor did the inhibitor alter sperm motility. Similar inhibitory effects on sperm penetration capacity were observed with specific antibodies directed either against calpain-I or calpain-II, the two forms of calpains described in somatic cells. At 1:1000 antibody dilution, penetration was inhibited 50 and 60% with anti-calpain-I and anti-calpain-II antibodies, respectively (p < 0.01; n=6). Furthermore, a combination of these two antibodies reduced the penetration rates by 75% (p < 0.01; n=6). We conclude that calpain inhibitor and anti-calpain antibodies impair human sperm capacity to fuse and penetrate the oocyte. These findings suggest that sperm calpain is a novel component of the biochemical processes that regulate the fertilizing capacity of human spermatozoa.     

Determination of fatty acid profile in ram spermatozoa and seminal plasma

Fatty acids are important in male reproductive function because they are associated with membrane fluidity, acrosome reaction, sperm motility and viability, but limited information exists about the fatty acid profile of ram semen. Our aim was to determine the fatty acid composition in ram spermatozoa and seminal plasma. Sixty ejaculates were obtained from three ram (20 ejaculates/ram) using artificial vagina. Ram spermatozoa (RS) and seminal plasma (SP) were separated using centrifugation, and the fatty acids were analysed by gas chromatography. Total lipids obtained in ram spermatozoa were 1.8% and 1.6% in seminal plasma. Saturated fatty acid (SFA) was proportionally major in SP (66.6%) that RS (49.9%). The highest proportions of SFA corresponded to C4:0 (RS = 16.3% and SP = 28.8%) and C16:0 (RS = 16.3% and PS = 20%). The most important unsaturated fatty acid (UFA) was docosahexaenoic acid (DHA), 44.9% in RS and 31.5% in SP. The profile of fatty acid and their proportions showed differences between spermatozoa and seminal plasma.     

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.     

Acrosomal status of human spermatozoa after follicular fluid or calcium ionophore challenge in relation to semen parameters and fertilizing capacity in vitro

Summary.  The proportion of spermatozoa that undergo spontaneous acrosome reaction in vitro is relatively low. The proportion can be enhanced by incubation with either biological inducers such as follicular fluid or chemicals like calcium ionophore. It has been suggested that improper acro-somal reaction may be a cause of fertilization failure in vitro. The objectives of the present study were to assess the acrosomal status of human sperm following follicular fluid or calcium ionophore treatment and to analyse the relationship between spontaneous and induced acrosome reaction and fertilization rates in vitro by standard in vitro fertilization (IVF) technology. In all, 53 semen samples (22 normal and 31 subnormal) were studied. The effect of calcium ionophore A 23187 and follicular fluid was assessed using the fluorescence activated cell sorter. IVF results were evaluated in relation to the acrosome status of the sperm samples. Our results demonstrate that the effect of follicular fluid on the acrosomal status correlated positively with the effect obtained by the calcium ionophore (Pearson's correlation r = 0.45). A significantly higher percentage of maximal acrosome change ( P <0.02) was found in cases where fertilization occurred (19/27), than in sperm samples that did not achieve fertilization in vitro (8/27). The present finding that follicular fluid induced acrosome reaction can serve as a predictive tool which is as good as the ionophore treatment for assessing IVF outcome, supports the use of this method for clinical purposes.     

Effect of seminal plasma fractions from entire and vasectomized rams on the motility characteristics, membrane status, and in vitro fertility of ram spermatozoa

Whole seminal plasma from ram semen collected before and after vasectomy was separated into 2 fractions, supernatant and pellet of vesicles, and their protein profiles characterized by one-dimensional (1D) gel electrophoresis. The effects of autologous whole seminal plasma and these fractions on motility characteristics (assessed subjectively and by computer-assisted sperm analysis), membrane status (assessed by chlortetracycline staining patterns), and in vitro fertility (assessed by fertilization success and timing of fertilization events) of washed frozen-thawed ram spermatozoa were studied. Regardless of vasectomy, whole seminal plasma and supernatant displayed similar protein patterns. These fractions, when included in the postthaw buffer, improved the motility characteristics (59.6% +/- 6.21% and 39.6% +/- 6.21% vs 31.7% +/- 6.46% and 15.5% +/- 6.46% total motility) and membrane integrity (36.6% +/- 8.52% and 31.2% +/- 8.19% vs 30.3% +/- 11.49% and 21.6% +/- 10.28% B staining pattern [characteristic of capacitated acrosome-intact cells] for whole seminal plasma and supernatant vs control at 3 and 6 hours of postthaw incubation, respectively) of frozen-thawed spermatozoa and improved their ability to fertilize in vitro-matured oocytes compared with control buffer without seminal plasma fractions (25.3%, 47.4%, and 37.4% vs 12.3%, 20.2%, and 20.5% oocytes fertilized for spermatozoa incubated with supernatant vs control at 2, 6, and 18 hours after insemination, respectively). Vesicles were absent from semen collected after vasectomy. Pellets of vesicles collected before vasectomy had no effect on spermatozoa at their normal protein concentration but marginally improved both motility characteristics and in vitro fertility, possibly due to contamination from supernatant proteins, when their concentration in the postthaw medium was increased by threefold. It was concluded that the vesicle-free supernatant fraction of seminal plasma, but not the seminal plasma membrane vesicles, improved the function and fertility of frozen-thawed ram spermatozoa when added to the postthaw medium.     

Enzyme comparative study of spermatozoa and seminal plasma in normal and subfertile man

A semiquantitative colorimetric micromethod (APIZYM) was used to study the enzyme profiles of seminal plasma and of spermatozoa. Reactions with 65 different substrates are simultaneously tested in a single specimen. These substrates (principally naphtolic) allow the detection of hydrolytic enzymes (esterases, phosphatases, and peptidases) and of dehydrogenases potentially involved in sperm metabolism and in the process of fertilization. The usual sperm enzymes were regularly observed: C3-C4 esterases, amino acid arylamidases, acrosine, phosphatases, glutamyl transpeptidase, and various osidases. Among the dehydrogenases we observed a striking predominance of the enzymes of the hexose monophosphate shunt and of LDH. Seminal plasma has an enzyme pattern very similar to that of spermatozoa except for the absence of acrosine and of some dehydrogenases. This unexpected similarity is discussed. The gametes from subfertile donors do not at first sight differ in their enzyme pattern from those from fertile donors. Moreover, we found no marked differences between zymograms of seminal plasma from normal, subfertile, or even azoospermic patients. Deep freezing does not modify the hydrolytic enzymes of human sperm either quantitatively of qualitatively, but the dehydrogenases of the hexose monophosphate shunt are adversely affected (60% loss of activity for G 6 PDH, and 30% for 6 PGDH); LDH is not affected. The consequences on fertilizing capacity of frozen semen are discussed.     

Morphology of seminal and swim-up spermatozoa and the outcome of in vitro fertilization and embryo transfer

Tubal infertility was treated by in vitro fertilization-embryo transfer (IVF-ET) in 112 couples. Twenty-eight pregnancies were obtained in 140 treatment cycles. Couples are accepted for treatment in our IVF-ET programme if previous semen samples fulfil the inclusion criteria: ejaculate volume greater than 1.5 ml, concentration of spermatozoa greater than 15 x 10(6) ml-1, greater than 40% motile spermatozoa, and greater than 25% spermatozoa with normal morphology. In order to determine to which extent IVF-ET treatment results are influenced by sperm morphology, within this selected group of patients, we have retrospectively analysed the data from both original semen samples and swim-up preparations. The sperm morphology was not related to the outcome of treatment in terms of fertilization (ovum cleavage rate), early embryo development, or pregnancy. Nor was any relationship detected between early embryo development or pregnancy and the degree of improvement in morphology resulting from the swim-up procedure. However, if improvement in morphology by swim-up was high, ovum cleavage rate was low. Sperm morphology within the limits set by our inclusion criteria could not predict the outcome of IVF-ET treatment. It is further concluded that the presence of abnormal spermatozoa at the site of fertilization may be without harm if only the number of normal sperms is high enough.     

Distribution of alpha-, gamma (+beta)- and delta-tocopherol in the seminal plasma, spermatozoa and seminal vesicles of rabbit

Dietary vitamin E supplementation plays a key role in animal reproduction by protecting germ cells from oxidative damage. Recently, alpha-tocopherol homologues (namely, beta-, gamma- and delta-tocopherol) have been the object of increasing research because of their peculiar nonantioxidant properties. We found that these tocol-derived compounds are not homogeneously distributed among semen components. Alpha-T was the major vitamin E homologue found in all semen fractions. Half of the total gamma (+beta)-T was found in germ cells, while more than 50% of total delta-T was preferentially accumulated in seminal plasma. The concentration of various tocol-derived compounds depended on their relative amounts in diet and the competition for saturable enzymes implicated in their metabolism. A higher concentration of delta-T in seminal plasma may be related to its more polar nature. However, the biological function of this compound in semen remains to be cleared. To our knowledge, this is the first study aimed at identifying alpha-tocopherol homologues in rabbit semen fractions.     

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.     

Reduced activities of triple adenosine triphosphatase in seminal plasma and spermatozoa of patients with oligoasthenospermia

Na+- and K+-activated and Mg2+-dependent adenosine triphosphatase, Mg2+-activated adenosine triphosphatase and Ca2+- and Mg2+-activated adenosine triphosphatase activities were determined in washed spermatozoa and in two fractions (pellet and supernatant) of seminal plasma of oligoasthenospermic patients and men with normal spermiograms. The activities of triple adenosine triphosphatase oligoasthenospermics were significantly lower than those of normals. The morphologic features of spermatozoa of oligoasthenospermics were of normal standards. Possible explanations for the significantly lowered triple adenosine triphosphatase activities from patients with oligoasthenospermia are given with special reference to the ion transport functions of the triple adenosine triphosphatase enzyme system.     

Tissue inhibitors of metalloproteinases 1 and 2 in human seminal plasma and their association with spermatozoa

A 24-kDa heparin binding protein recently identified as tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in bovine seminal fluid was suggested to play an important role in bull fertility. As no data are present for men, the concentrations of tissue inhibitors of metalloproteinases 1 (TIMP-1) and TIMP-2 were quantified in human seminal plasma of normozoospermic and azoospermic men using enzyme-linked immunosorbent assay methods. TIMP-1 and 2 were not significantly different in both groups and there were no relationships between the concentrations of both TIMPs and other sperm characteristics. It is assumed that TIMPs are released from accessory sex glands.