Semen-coagulating protein, SVS2, in mouse seminal plasma controls sperm fertility

Mammalian seminal plasma is known to contain a decapacitation factor(s) that prevents capacitation and thus, the fertility of sperm. This phenomenon has been observed in experiments conducted in vitro that assessed the inhibition of epididymal sperm fertility by seminal plasma or by the purified decapacitation factor. However, the phenomenon of decapacitation has not yet been characterized in vivo. In the present study, we demonstrate that seminal vesicle protein secretion 2 (SVS2), which is a 40-kDa basic protein and a major component of the copulatory plug, enters the uterus and interacts with ejaculated sperm heads after copulation. The SVS2-binding region of sperm changed from the postacrosomal region to the equatorial segment, while the sperm migrated through the uterus and finally disappeared in the oviduct. Furthermore, SVS2 reduced the fertility of epididymal sperm. The sperm treated with SVS2 decreased the percentage of fertilized oocytes from 60% to 10%. The capacitation state was assessed by protein tyrosine phosphorylation and the comprehensiveness of the acrosome reaction. SVS2 functioned to maintain sperm in the uncapacitated state and to reverse capacitated sperm to the uncapacitated state. We found that the fertility of ejaculated sperm is associated with SVS2 distribution in the female reproductive tract. These results indicate that SVS2 functions as a decapacitation factor for mouse sperm.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa

The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion.     

Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.     

Application of seminal plasma in sex-sorting and sperm cryopreservation

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Components in seminal plasma regulating sperm transport and elimination

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.     

Effect of heterologous and homologous seminal plasma on stallion sperm quality

Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to investigate the effect of adding back SP to colloid centrifuged spermatozoa to determine its effect on spermatozoa; and (2) to investigate whether the stallion's own SP had a greater or lesser effect than heterologous SP. Conventional semen doses were sent from a stud overnight to the laboratory using standard transport conditions. Once at the laboratory, the semen samples were used for single layer centrifugation with Androcoll-E, and the resulting sperm preparations were treated with heterologous SP. Adding SP had a small but significant effect on sperm motility but no effect on the proportion of spermatozoa that had acrosome reacted. There were significant increases in hydrogen peroxide production and chromatin damage (P < 0.001). When homologous and heterologous SP were compared, considerable variation was observed between stallions, so that it was not possible to predict whether homologous or heterologous SP, or no SP, will produce the best motility for spermatozoa from any given stallion. Therefore, it is necessary to test different combinations of spermatozoa and SP to find the optimal effect on motility. The SP from most stallions increased reactive oxygen species and chromatin damage. In conclusion, the interaction between SP and spermatozoa depends on the origin of both SP and spermatozoa. If it is desirable to add SP to stallion sperm samples, it should be done directly before insemination rather than before storage, because of increased hydrogen peroxide production and sperm chromatin damage.     

Effects of seminal vesicle removal on fertility and uterine sperm motility in the house mouse

The relative significance of the accessory glands of the male reproductive tract in fertility is unclear. To clarify the role of the seminal vesicles, fertility and uterine sperm motility were determined before and after removal of seminal vesicles in the house mouse. After removal of seminal vesicles, the pregnancy rate (number of females pregnant/number of females X 100) was reduced and the time to birth was increased, while the average litter size was not changed. Fertilization, determined by examining the oocytes 30 h after mating, was highly variable after matings with males whose seminal vesicles were removed; in some cases none of the oocytes were fertilized. The motility of sperm recovered from the uterus 1 h after matings with males before and after seminal vesicle removal and sham operations was analyzed using a videomicrographic system. The motility of uterine sperm was less progressive with more lateral displacement of the head about the trajectory and a less linear trajectory after removal of the seminal vesicles. Sham-operated animals showed no consistent changes in motility of uterine sperm. The changes in sperm motility could contribute to the reduction in fertilization since sperm motility is necessary for transport in the female reproductive tract and interaction with the oocytes.     

Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa

The effects of different dilutions of seminal plasma (SP) on the qualitative characteristics of rabbit spermatozoa and on their fertilising ability were analysed. Ejaculated semen was centrifuged twice and the sperm resuspended in media with decreasing ratios of SP/Tris: (1/2; 1/5; 1/10; 1/20; 1/30; 1/100) until the complete substitution was with SP. The control constituted sperm in undiluted SP. Samples were maintained at 37 degrees C and kinetic analysis done at fixed intervals (0-6h). Also the thiobarbituric reactive substances (TBA-RS) values were determined. Rabbit sperm suspended in Tris, or with extremely low content of SP, lost motility and viability within 1-3h, while sperm suspended in SP either undiluted or diluted up to 10-fold, showed similar motility during the 6h period (from 39 to 49%). Further dilutions of SP (1/20-1/30) had no effect during the initial 2h of storage but thereafter the decline of motility was more marked (after 6h: from 0 to 17%). Kinetic parameters followed the same trend and differences were particularly marked after storage: the highest values were in samples diluted up to 1/10; a sharp decline in motility characteristics was observed at higher dilutions. The addition of SP (1/2 v/v) to immotile sperm reactivated 35.5% of cells. However, SP did not significantly affect fertility rate or litter size possible involving an interaction with the female reproductive tract. SP reduced lipid oxidation (TBA-RS) of semen only after storage. A positive correlation between final TBA-RS and cell viability indicated that peroxidation was one of the cause of rabbit sperm deterioration during conservation.     

Inclusion of seminal plasma in sperm cryopreservation of Iberian pig

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process.     

Identification of sperm forward motility-related proteins in human seminal plasma

Seminal plasma, an amorphous material that exists in semen, contains proteins related to sperm forward motility. Employing affinity chromatography with ConA beads and protein ultrafiltration, we isolated and concentrated proteins from heated human seminal plasma. Results of computer-assisted semen analyses (CASA) demonstrated that the forward motility index of bovine spermatozoa from the epididymal caput, incubated with proteins and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone (P < 0.01). The electrophoreses revealed that the protein bands with high molecular weights in the gel of PAGE changed into low molecular weights in the gel of SDS-PAGE. Furthermore, proteins from a separated portion of the PAGE gel were still able to stimulate spermatozoa from the epididymal caput to gain forward motility. Two-dimensional (2D)-gel electrophoresis and mass spectrometry indicated that spots focused on the portion seemed, according to their amino acid sequences, to be like human alpha-1-antitrypsin and zinc-alpha-2-glycoprotein (ZAG) precursors. Western blot analysis showed the presence of these two proteins in seminal plasma. These proteins, related to the forward motility of spermatozoa in human seminal plasma, may play important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract.     

Comparison of sperm viability with seminal plasma metal levels

This study compares the semen levels of lead (Pb), cadmium (Cd), and aluminum (Al) in relation to live sperm in semen samples from 64 apparently healthy men. The measured levels were separated into live sperm count tertiles (<25% [18 subjects], 25–50% [26 subjects], and >50% [20 subjects]). The mean ± SD for each group was calculated, and the difference between the means of the high and low tertiles were compared by ANOVA. Significant differences were observed between the high and low live sperm groups for Pb (p < 0.01) and Al (p < 0.05), but not Cd. Spearman’s rank correlation between sperm viability and the semen plasma metal levels showed a direct relation to Mg (p < 0.05). However, there was an inverse relation to lead (p < 0.001), cadmium (p < 0.01), and aluminum (p < 0.01). There was no significant correlation between Ca and Zn. Linear regression between the live sperm counts and semen level of the three metals show that metal levels were inversely correlated with the percentage of live sperm (p < 0.001, <0.01). Apparently, the presence of these metals in the environment and in seminal plasma exerts a toxic effect on sperm.     

Characterization of radiolabeled components of human sperm surface and seminal plasma

Externally oriented components on the human sperm cell surface and components in human seminal plasma were labeled by enzymatic iodination with lactoperoxidase and [¹²⁵I] NaI. SDS-7.5% PAGE of labeled sperm surface resolved one minor and four major components with approximate molecular weights of 92, 72, 46, 30, and 20K daltons, respectively. SDS-7.5% PAGE of labeled seminal plasma resolved five components with approximate molecular weights of 74, 51, 43, 28, and 20K daltons. Three of the five moieties seen on the sperm surface and in seminal plasma were similar in molecular weight. This suggested that these surface components were adsorbed from seminal secretions. Because the iodination procedure used labels both proteins and lipids, labeled sperm surface and labeled seminal plasma were subjected to isopycnic density gradient centrifugation to identify the chemical composition of the radioiodinated components. With human sperm surface, two areas of radioactivity were resolved in CsCl gradients, one corresponding to protein and the other to lipid. With human seminal plasma, only one area of radioactivity, corresponding to protein, was identified. Electrophoretic analysis of each peak of radioactivity obtained from the gradients demonstrated that all of the sperm surface and four of five seminal plasma components were in the protein fractions. All three of the seminal plasma components which correspond to sperm surface components were recovered in the protein fraction. This observation supports our hypothesis that some of the proteins labeled on the human sperm cell surface are adsorbed from seminal secretions.