Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine.

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (>52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers-the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa-which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates.

Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Melatonin membrane receptors MT1 and MT2 are expressed in ram spermatozoa from non-seasonal breeds.

In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT1 and MT2. We have previously reported their presence in ram spermatozoa from males located in temperate climates, but there is no information on their presence in spermatozoa from rams in areas with an equatorial photoperiod (12L:12D). Thus, we have investigated the existence and cellular distribution of melatonin receptors in spermatozoa from three sheep breeds in Colombia (Colombian Creole, Hampshire, and Romney Marsh) during dry and rainy seasons, using indirect immunofluorescence and western blot. Our results indicated the presence of melatonin receptors in spermatozoa from these rams, and that their distribution differs from that previously found in spermatozoa from rams in temperate climates. Moreover, two new immunotypes of MT2 were identified: type N, with staining only in the neck, and type E with a band of immunofluorescence in the upper part of the post-acrosome and the apical edge. Likewise, differences between breeds and climate seasons were detected for both receptors. However, densitometry analysis of western blot bands only revealed differences between seasons in the Creole rams for MT1 and the Romney Marsh rams for MT2, whereas differences between breeds were only detected for MT2. It could be inferred that melatonin receptors in rams subjected to an equatorial photoperiod might be more closely related to sperm quality than seasonal control. Therefore, the presence of these receptors suggests that melatonin could be a useful tool to increase the fertility of rams located in tropical or equatorial climates.

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

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.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson's correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

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.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod.

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

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.

Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm.

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

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.