Presence of melatonin-catabolizing non-specific enzymes myeloperoxidase and indoleamine 2,3-dioxygenase in the ram reproductive tract.

The melatonin catabolism is very complex and not completely understood. Melatonin can be metabolized by free radical interaction, but also pseudo-enzymatically or by enzymatic pathways. We have previously detected the existence of melatonin-synthesizing enzymes and melatonin receptors MT1 and MT2 in the ram reproductive tract; thus, in order to start to elucidate melatonin catabolism in these organs, we have investigated the presence of the melatonin-catabolizing enzymes indoleamine 2,3-dioxygenase (IDO, both IDO1 and IDO2 isoforms) and myeloperoxidase (MPO) in testis, epididymis and accessory glands. Gene expression analyses by real-time PCR showed the presence of MPO, IDO1 and IDO2 in all the organs of the ram reproductive tract and revealed that MPO is the main melatonin-catabolizing enzyme, which is mainly expressed in the testis and the bulbourethral glands (p < .05). These results were further corroborated by immunohistochemical staining, and by Western blot. Likewise, MPO was also evidenced in epididymal and ejaculated spermatozoa by indirect immunofluorescence and Western blot. In conclusion, melatonin-catabolizing enzymes MPO, IDO1 and IDO2 are expressed in the ram reproductive tract, and MPO is the most expressed one, mainly in the testis and the bulbourethral glands. The presented results warrant further studies on the function of these enzymes and their melatonin-metabolizing activity.

Melatonin reduces cAMP-stimulated capacitation of ram spermatozoa.

The presence of melatonin receptors on the surface of ram spermatozoa has led to speculation about melatonin having a role in sperm functionality. The aim of this study was to elucidate the mechanism through which melatonin regulates ram sperm capacitation induced by a cocktail containing cAMP-elevating agents. Cocktail samples capacitated in the presence of 1µM melatonin showed lower percentages of capacitated spermatozoa (chlortetracycline staining; P<0.001) together with a decrease in protein tyrosine phosphorylation (P<0.01) and lower levels of reactive oxygen species (ROS) and cAMP (P<0.05) compared with cocktail samples without the hormone. Determination of kinematic parameters, together with principal component and cluster analyses, allowed us to define four sperm subpopulations (SP). After 3h of incubation with cAMP-elevating agents, the percentages of spermatozoa belonging to SP1 (high straightness) and SP4 (less-vigorous spermatozoa with non-linear motility) increased while SP2 and SP3 (rapid spermatozoa starting hyperactivation or already hyperactivated) decreased compared with the control sample. The presence of melatonin at 100 pM and 10nM restored these subpopulations to values closer to those found in the control sample. These results indicate that melatonin at micromolar concentrations modulates ram sperm capacitation induced by cAMP-elevating agents, reducing ROS and cAMP levels, whereas at lower concentrations melatonin modifies motile sperm subpopulations. These findings warrant further studies on the potential use of melatonin for controlling capacitation in artificial insemination procedures.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels.

This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone-induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation-associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5 µm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4 h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on in vitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1 µm melatonin and used for in vitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of in vitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix.

Role of melatonin on embryo viability in sheep.

Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day-night perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes (fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function, reduced antiluteolytic mechanisms, or improved embryo quality. This review focuses on the effects of melatonin on embryo viability and summarises the processes by which this hormone affects the ovary, follicle, oocyte, corpus luteum and embryo. Moreover, the effects of melatonin on the mechanisms of invivo maternal recognition of pregnancy in sheep and the protective action that it appears to have on the invitro procedures that are used to obtain healthy embryos are reviewed.

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes†.

The mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38) signaling cascades are involved in triggering apoptosis in somatic cells. Given that spermatozoa are able to undergo apoptosis, we tested the hypothesis that these pathways might be functional in ram spermatozoa as two signal transduction mechanisms that contribute to the modulation of capacitation and apoptosis. Indirect immunofluorescence and western blot analysis evidenced the presence of JNK and p38 in ram spermatozoa. To verify the involvement of these enzymes in sperm physiology, we determined the effect of specific inhibitors of JNK or p38 on in vitro capacitation induced with either cAMP-elevating agents or epidermal growth factor (EGF). Both inhibitions reduced the EGF-induced capacitation with a decrease in the chlortetracycline capacitated-sperm pattern, protein tyrosine phosphorylation, phosphatidylserine externalization, caspase-3 and -7 activation, and the proportion of DNA-damaged spermatozoa. No significant changes were found in the high-cAMP capacitated samples. The addition of 3.4 mg/ml seminal plasma proteins (SPPs) to the EGF-containing samples, either alone or together with each inhibitor, resulted in a decreased proportion of capacitated sperm pattern, protein tyrosine phosphorylation, loss of plasma membrane integrity, and apoptotic alterations. Furthermore, SPPs significantly reduced the phosphorylation level of JNK and p38 MAPK (active forms). These findings show a relationship between capacitation and apoptosis, and represent a step forward in the knowledge of the SPP protective mechanism in spermatozoa.

Melatonin MT1 and MT2 Receptors in the Ram Reproductive Tract.

Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates.

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state.

Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species.

Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding. For this purpose, the presence of melatonin receptors and the levels of melatonin in seminal plasma have been examined in several species: donkey and stallion as long-day breeders; red deer as a wild, short-day, highly seasonal breeder (epididymal spermatozoa); bull as a conventional nonseasonal breeder; boar as a seasonal breeder under management techniques; and dog as possible a seasonal breeder not regulated by melatonin. We have detected measurable levels of melatonin in the seminal plasma of all ejaculated semen samples (from donkey, stallion, boar, bull, and dog). Also, and for the first time, we have demonstrated the presence of MT1 and MT2 melatonin receptors in the spermatozoa of all these species, regardless their type of reproduction or sperm source (ejaculated or epididymal), using indirect immunofluorescence techniques and Western blotting. Our findings suggest that melatonin and melatonin receptors may be universally distributed in the reproductive system of mammals and that the sperm melatonin receptors cells may not be necessarily related with seasonal reproduction. Furthermore, the presence of MT1 at the cytoplasmic droplet in immature ejaculated stallion spermatozoa found in one sample and epididymal red deer spermatozoa suggests that melatonin may be involved in specific functions during spermatogenesis and sperm maturation, like protecting spermatozoa from oxidative damage, this activity being mediated through these receptors.

New evidence of melatonin receptor contribution to ram sperm functionality.

The present study analysed the involvement of melatonin, acting via its receptors (MT1 and MT2), in ram sperm functionality. Indirect immunofluorescence assays revealed no changes in the distribution or intensity of MT1 receptors, whereas different subpopulations were established for MT2 receptors in control, in vitro capacitated and acrosome-reacted ram spermatozoa. Chlortetracycline staining revealed the following correlations between the pattern of staining for MT2 receptors in: (1) non-capacitated (NC) sperm rate and the proportion of spermatozoa with equal immunostaining intensity in the acrosome and post-acrosome (r=0.59, P<0.001); (2) in capacitated (C) sperm rate and the proportion of spermatozoa with stronger reactivity in the acrosome (r=0.60, P<0.001); and (3) in acrosome-reacted (AR) sperm rate and the proportion of spermatozoa with more intense staining on the post-acrosome (r=0.67, P<0.001). Incubation of swim-up-selected samples with either 1µM melatonin or MT1 and MT2 receptor agonists (2-phenylmelatonin 1µM and 8-Methoxy-2-propionamidotetralin (8M-PDOT) 1µM and 10nM) at 39°C and 5% CO2 for 3h resulted in a higher proportion of the NC pattern compared with the control group (P<0.05), whereas treatment with MT1 and MT2 receptor antagonists (luzindole 1µM and 4-phenyl-2-propionamidotetralin (4P-PDOT) 1µM and 10nM) decreased the proportion of spermatozoa exhibiting the NC pattern (P<0.001) concomitant with an increase in those exhibiting the C pattern (P<0.01). In conclusion, melatonin exerts a modulating effect on ram sperm functionality, primarily via activation of the MT2 receptor.

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs).

Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b).

Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

New insights into the mechanisms of ram sperm protection by seminal plasma proteins.

To provide new insights into the mechanisms through which seminal plasma proteins (SPP) are able to protect spermatozoa, we tested the hypothesis that apoptosis can contribute to the negative effect of refrigeration on ram spermatozoa, and that SPP prevent this damage. Having proved the presence of key constituents of apoptosis-related pathways in ram sperm protein extracts, we carried out a comparative analysis of the effects of the addition of SPP before refrigeration (15 °C, 30 min) and induced-apoptosis with betulinic acid or fibroblast-associated receptor ligand, assessing sperm quality parameters and apoptotic markers. The protective effect of SPP on plasma membrane integrity and potential, motility and mitochondrial inner membrane potential, and surface (cardiolipin content) was evidenced in refrigerated and induced-apoptosis samples. The addition of SPP resulted in lower values of phosphatidylserine externalization, DNA damage, and caspase activity. Therefore, apoptosis in fresh or refrigerated ram spermatozoa can occur due to activation of both the extrinsic and the intrinsic mediated pathway, and SPP might interfere with both pathways. The addition of SPP also resulted in higher proportions of viable, noncapacitated sperm and fertilizing ability (ZBA rate). This report demonstrates that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, and proposes the possibility that SPP might interfere with the extrinsic and the intrinsic apoptotic pathways. This opens new, interesting perspectives for the study of cellular regulatory mechanisms in spermatozoa that could be crucial for the improvement of ram semen preservation protocols.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14.

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76-85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

A novel epidermal growth factor-dependent extracellular signal-regulated MAP kinase cascade involved in sperm functionality in sheep.

Sperm capacitation is characterized by a series of significant biochemical and biophysical modifications. Unlike the case with most other mammalian species, ram spermatozoa are difficult to capacitate in vitro. We have already suggested that unusually high levels of intracellular phosphodiesterases would account for cAMP levels that are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of capacitation. In this study, we have 1) investigated the presence of the epidermal growth factor receptor (EGFR) and ERK1/2, a specific subset of the mammalian mitogen-activated protein kinase (MAPK) family, in ram spermatozoa and their involvement in capacitation; 2) searched for possible cross talk between the EGF effect and PKA pathway; and 3) explored a possible relationship between the EGF effect and the MAPK family that may underlie modulation of ram sperm capacitation. Indirect immunofluorescence evidenced the presence of EGFR and ERK in fresh ram spermatozoa. Western blot analysis confirmed both that EGFR is in the active form and that phosphorylation of Tyr845 increased after incubation with EGF. The proportion of CTC capacitated-sperm pattern and protein tyrosine phosphorylation significantly increased in the presence of EGF as well as the phosphorylation state (activation) of ERK. The specific inhibition of EGFR, PKA, or MEK reduced capacitation and protein tyrosine phosphorylation induced by EGF. We propose a working model for the molecular mechanism of the signaling cascade involved in ram sperm capacitation. These findings should improve our understanding of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells.

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.

Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes.

BACKGROUND: Some breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and non-breeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity. METHODS: Seminal plasma from nine Rasa Aragonesa rams were collected for one year, and their levels of melatonin, testosterone, superoxide dismutase (SOD), glutathione reductase (GRD), glutathione peroxidase (GPX) and catalase (CAT) were measured. RESULTS: All samples presented measurable quantities of hormones and antioxidant enzymes. Both hormones showed monthly variations, with a decrease after the winter solstice and a rise after the summer solstice that reached the maximum levels in October-November, and a marked seasonal variation (P < 0.01) with higher levels in the breeding season. The yearly pattern of GRD and catalase was close to that of melatonin, and GRD showed a significant seasonal variation (P < 0.01) with a higher activity during the breeding season. Linear regression analysis between the studied hormones and antioxidant enzymes showed a significant correlation between melatonin and testosterone, GRD, SOD and catalase. CONCLUSIONS: These results show the presence of melatonin and testosterone in ram seminal plasma, and that both hormones have seasonal variations, and support the idea that seasonal variations of fertility in the ram involve interplay between melatonin and the antioxidant defence system.

Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate.

We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.

Ultrastructural study of the ability of seminal plasma proteins to protect ram spermatozoa against cold-shock.

The process of sperm cryopreservation, involving cooling, freezing, and thawing, induces serious detrimental changes in sperm function. The plasma and acrosomal membranes of spermatozoa are considered to be the primary site of these modifications due to thermal, mechanical, chemical, and osmotic stress. In previous studies, we demonstrated the ability of seminal plasma (SP) proteins to protect ram spermatozoa against cold-shock by using biochemical markers and scanning electron microscopy. In this study, we have attempted to examine the potential protective effect of SP proteins in membrane ultrastructure of ram spermatozoa subjected to cold-shock, by means of transmission electron microscopy (TEM). All the experiments were carried out with fresh spermatozoa freed from SP by a dextran/swim-up procedure. The high proportion of viable spermatozoa found in the swim-up obtained sample decreased drastically after the cold-shock treatment, and a considerable blebbing and vesiculation of the plasma and acrosomal membranes was found. The addition of SP proteins increased the sperm resistance to damage due to cold-shock (48% membrane-intact spermatozoa versus 15% in the control sample), and TEM analysis revealed that membrane alteration was prevented. This protective effect seems to be specific for SP proteins, as the addition of BSA did not provide any protection.

Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation.

Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca(2+) are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-beta-cyclodextrin (M-beta-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-beta-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars.

The study evaluated the protective effect of seminal plasma (SP) added to freezing extender against cryopreservation injuries to boar spermatozoa. Pooled sperm-rich fractions collected from 9 fertile boars were frozen in 0.5-mL straws after being extended in a conventional freezing extender either alone or supplemented with 5% of SPs (SP1-SP4) collected from the sperm-rich fractions (diluted 1:1, vol/vol, in Beltsville Thawing Solution extender) from 4 boars (1-4) with known sperm cryosurvival (poor, moderate, and good sperm freezers). Cryopreservation injuries were assessed in terms of postthaw sperm motility (assessed by computer-assisted sperm analysis), viability (plasma membrane and acrosome integrity assessed simultaneously by flow cytometry), membrane lipid peroxidation (malondialdehyde [MDA] production), and the ability of thawed spermatozoa to fertilize in vitro-matured homologous oocytes. The addition of SP from good sperm freezers (SP3 and SP4) improved (P < .01) the motility and viability of thawed spermatozoa without any influence on MDA production. Moreover, SP from good sperm freezers also increased (P < .05) the percentage of penetrated (SP3) and polyspermic oocytes (SP4) with respect to the control. Neither the total amount of SP proteins, protein profiles, nor antioxidant capacity of the different SPs were related to the various cryosurvival/fertilizing capacities of the processed spermatozoa.