Citation Rodríguez‐Martínez H, Kvist U, Ernerudh J, Sanz L, Calvete JJ. Seminal Plasma Proteins: What Role Do They Play? Am J Reprod Immunol 2011; 66 (Suppl. 1): 11–22Problem  Semen is a heterogenous and complex cell suspension in a protein‐rich fluid with different functions, some of them well known, others still obscure.Method of study  This paper reviews, comparatively, our current knowledge on the growing field of proteomics of the SP and its relevance in relation to the in vivo situation, for the sake of reproductive biology, diagnostics and treatment.Results  Ejaculated spermatozoa, primarily bathing in cauda epididymal fluid, are (in vitro) bulky, exposed to most, if not all, secretions from the accessory sexual glands. In vivo, however, not all spermatozoa are necessarily exposed to all secretions from these glands, because sperm cohorts are delivered in differential order and bathe in seminal plasma (SP) with different concentrations of constituents, including peptides and proteins. Proteins are relevant for sperm function and relate to sperm interactions with the various environments along the female genital tract towards the oocyte vestments. Specific peptides and proteins act as signals for the female immune system to modulate sperm rejection or tolerance, perhaps even influencing the relative intrinsic fertility of the male and/or couple by attaining a status of maternal tolerance towards embryo and placental development.Conclusions  Proteins of the seminal plasma have an ample panorama of action, and some appear responsible for establishing fertility.

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Finding a laboratory test reliable enough to predict the potential fertility of a given semen sample or a given sire for artificial insemination (AI) is still considered utopian, as indicated by the modest correlations seen between results obtained in vitro and field fertility. Male fertility is complex, and depends upon a heterogeneous population of spermatozoa interacting at various levels of the female genital tract, the vestments of the oocyte, and the oocyte itself. For this reason, laboratory assessment of semen must include the testing of most sperm attributes relevant for fertilization and embryo development, not only in individual spermatozoa but within a large sperm population as well. Strategies for the discovery of in vitro predictors of semen fertility require evaluations of low sperm doses for AI, so that differences in innate in vivo fertility can be accurately detected.

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The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (rZK0.789, P!0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Djm) post-thaw (rZK0.689, P!0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was rZ0.772, P!0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering 'apoptotic-like changes' that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, against cryopreservation injuries to boar spermatozoa. In experiment 1, the lowest BHT concentrations able to reduce lipid peroxidation in boar spermatozoa were determined. Nine BHT concentrations (ranging from 0.025 to 3.2 mM) were evaluated, and the lowest (P <.05) production of malondialdehyde (MDA), as an indicator of lipid peroxidation, was obtained when BHT ranged from 0.2 to 1.6 mM. In experiment 2, sperm survivability was evaluated when BHT was added to a postthaw freezing extender by measuring the degree of sperm lipid peroxidation (using MDA production) and by measuring parameter such as motility, plasma membrane and acrosome integrity, and cell apoptosis. The ability of thawed spermatozoa to fertilize in vitro-matured oocytes and of embryos to develop to the blastocyst stage in vitro was also assessed. Pooled sperm-rich fractions collected from 3 mature Pietrain boars were frozen in 0.5-mL straws after dilution with lactose-egg yolk-glycerol-Orvus ES Paste extender supplemented with 0, 0.2, 0.4, 0.8, and 1.6 mM BHT. Postthaw sperm survival, evaluated 30 and 150 minutes after thawing, was higher in BHT-treated spermatozoa, being significant (P <.05) when the freezing extender was supplemented with 0.2, 0.4, and 0.8 mM BHT. The addition of BHT to the freezing extender resulted in a significant (P <.05) decrease in the MDA concentration in thawed spermatozoa, irrespective of the level of BHT used. BHT had no effect on oocyte cleavage rates, but the development to blastocyst was improved for embryos derived from spermatozoa frozen in extender supplemented with 0.4 mM BHT (16% vs 29% of blastocysts per total oocytes; P <.05). In conclusion, under the conditions tested in the present study, the addition of BHT to the freezing extender improved the overall efficiency of thawed boar spermatozoa.

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.