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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.

Simple Summary In temperate regions, sheep demonstrate seasonal reproduction regulated by changes in photoperiod. This regulation is mediated by nocturnal melatonin secretion. However, in equatorial regions, with no photoperiodic changes, sheep tend to breed in all seasons of the year. Despite this, changes in seminal composition or sperm quality have been reported throughout the year. We demonstrated that melatonin concentration in seminal plasma varies between rainy and dry seasons in three Colombian breeds (Colombian Creole, Romney Marsh, and Hampshire). As melatonin can exert direct effects on ram spermatozoa, in this study we hypothesized that melatonin could modulate sperm capacitation in equatorial-located breeds as we had previously reported in seasonal breeds from temperate regions. First, we assayed two media for in vitro capacitation and found that the increment in capacitated and acrosome-reacted sperm was higher in the so-called “cocktail medium” for the three breeds and in rainy and dry seasons. The addition of melatonin to the cocktail medium partially prevented the increase in capacitated spermatozoa from all breeds and during all seasons. This study could help in understanding how melatonin affects ram reproduction in the equatorial photoperiod. Abstract In this study, we demonstrated that, in seasonal Mediterranean ovine breeds, supplementing the TALP medium with cAMP-elevating agents (the cocktail medium) is effective for achieving ram sperm capacitation, and that melatonin is able to regulate this phenomenon. We investigated the behavior under capacitating conditions using the TALP and cocktail mediums, and the response to melatonin, of spermatozoa from three sheep breeds (Colombian Creole, Romney Marsh, and Hampshire) subject to the equatorial photoperiod, during the dry and the rainy seasons. The cocktail medium was able to induce sperm capacitation, assayed by chlortetracycline staining and phosphotyrosine levels, to a greater extent than TALP, without a higher loss of viability (membrane integrity and viable spermatozoa without phosphatidylserine (PS) translocation). The addition of melatonin at 100 pM or 1 µM in the cocktail medium partially prevented the decrease in viability without PS translocation and the increase in capacitated spermatozoa from all breeds, with no significant effect on phosphotyrosine levels. Differences between breeds and seasons were evidenced. This study shows that melatonin is able to exert direct effects on spermatozoa in ovine breeds under equatorial photoperiod conditions, as it does in seasonal breeds located in temperate regions.

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2⋅–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2⋅– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.

The FecXR mutation is a variant of the ovine gene BMP15 in the Rasa Aragonesa breed. Information on the physiological importance of carrying the FecX polymorphism in rams is limited. The aim of this study was to compare semen quality, testicle characteristics, and fertility rate of rams that carry the FecXR allele. Rams (n = 15) were either FecXR allele carriers (n = 10) or non-carriers, wild type (++) (n = 5). FecXR rams exhibited higher mass motility (p &lt, 0.05), proportion of rapid sperm (p &lt, 0.05), and a lower proportion of slow sperm (p &lt, 0.0001) than did ++ rams. The presence of the FecXR allele was not associated with mean scrotal circumference or testicular length and diameter, although season had a significant (p &lt, 0.05) effect on these traits. Genotype (p &lt, 0.05) and season (p &lt, 0.01) had a significant effect on mean fertility rate, FecXR rams had a higher proportion of pregnant ewes than did ++ rams (p &lt, 0.05). In conclusion, the FecXR allele produced high-quality semen throughout the year, and corresponded with an improvement in some sperm parameters, particularly, mass motility and the proportion of rapid sperm.

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

Nitric oxide (NO&middot, ), 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&middot, 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&middot, 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&middot, 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&middot, levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO&middot, metabolism.

The steroid hormones 17-β estradiol (E2) and progesterone (P4) can regulate capacitation, hyperactive motility, and the acrosome reaction (AR) during the sperm transit through the female tract. Moreover, exogenous P4 and E2 can induce the AR in ovine spermatozoa, and progesterone receptor (PR) and estrogen receptors (ERα and ERβ) are present in these cells. Thus, to investigate whether the effects both steroid hormones in ram sperm capacitation and AR are receptor-mediated, we incubated them with receptor agonists (tanaproget 1 μM and 5 μM for PR or resveratrol 5 μM and 10 μM for ER) or antagonists (mifepristone 4 μM and 40 μM for PR or tamoxifen 5 μM and 10 μM for ER) in capacitating conditions. The addition of receptor modulators did not affect sperm viability or total motility, although changes in progressive motility were detected. The incubation with both receptor agonists increased the percentage of acrosome-reacted spermatozoa, evaluated by chlortetracycline staining, when compared with the capacitated nontreated sample (Cap-C, P0.001). Moreover, the ER agonist resveratrol 10 μM provoked a greater AR than E2 (P0.01). Furthermore, the incubation with the receptor antagonists prevented the induction of the AR by P4 or E2, as the antagonists-treated spermatozoa presented a similar CTC pattern to that of Cap-C. In conclusion, these results confirm that P4 and E2 can induce the AR in ram spermatozoa and that this effect is receptor-mediated.

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 in vivo maternal recognition of pregnancy in sheep and the protective action that it appears to have on the in vitro procedures that are used to obtain healthy embryos are reviewed. Fil: Abecia, José Alfonso. Universidad de Zaragoza; España Fil: Forcada Miranda, Fernando. Universidad de Zaragoza; España Fil: Vazquez, Maria Isabel. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Muiño-Blanco,Teresa. Universidad de Zaragoza; España Fil: Cebrián-Pérez, José A.. Universidad de Zaragoza; España Fil: Pérez-Pe, Rosaura. Universidad de Zaragoza; España Fil: Casao, Adriana. Universidad de Zaragoza; España

The aim of this study was to determine whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and influence the out-of-season sexual activity of adult rams. In experiment 1, 24 Rasa Aragonesa ram-lambs born in September were genotyped for their RsaI and MnlI allelic variants of the MTNR1A gene, and the date of their first mounting with ejaculation after a period of semen collection training was documented. In experiment 2, the reproductive behavior, testicle size, and plasma testosterone concentrations of 18 adult rams (6 rams for each RsaI genotype) were recorded at the beginning (March) and end (May) of the seasonal anestrus. The number of days of training to achieve the first mating with ejaculation in T/T (C/C: 85.17 ± 12.08 C/T: 86.60 ± 18.87; T/T; 26.50 ± 24.50 d; P < 0.05), and G/G ram-lambs (G/G: 51.57 ± 14.99; A/G: 95.58 ± 10.95 d; P < 0.05) was significantly fewer than it was in the other genotypes. Likewise, for the RsaI genotype, 55% of the vulva-sniffing (P < 0.001), 48% of the approaches (P < 0.01), 48% of the mountings (P < 0.05) and 49% total activities (P < 0.001) were performed by T/T rams in March, and 50% of the sexual events in May (P < 0.001). For the Mnll variant, G/G rams performed a significantly (P < 0.001) larger proportion of the vulva-sniffing (41%), approaches (46%) and total activities (40%) in March, and 52% of the vulva-sniffing (P < 0.001), 43%, of the approaches (P < 0.001), 46% of the mountings (P < 0.05), and 47% of the total activities (P < 0.001) in May. Scrotal circumference, testicular volume, and plasma testosterone concentrations did not differ significantly among genotypes. Results confirmed that the polymorphisms of the MTNR1A gene sequence can influence reproductive performance in young and adult rams. Autumn-born ram-lambs that carried the T/T or G/G genotype had an advanced ability to reproduce, and T/T or G/G adult rams exhibited the most intense reproductive behavior. Genotyping might be a useful procedure for identifying the correct and rational use of rams in modern sheep farming.

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 &lt, 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 &lt, 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p &lt, 0.01) and catalase, but higher GPx activity (p &lt, 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.

Steroid hormones progesterone (P4) and 17β-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 μM in the media induced an increase in Ca2+i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H2DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification.

In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT

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.

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.

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.

Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P 0.001) in ZBA rate related to control samples in basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs. 1.31 ± 0.09 sperm bound/oocyte, respectively). A significant correlation between protein tyrosine phosphorylation and ZBA rate (P 0.05, r = 0.501) corroborated that incubation in a "high-cAMP" environment improves the fertilizing ability of ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation.

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.

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes duringin vitrocapacitation and the actions of progesterone (P4) and 17β-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40–45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERβ). ERα was located in the postacrosomal region of all the spermatozoa and ERβ on the apical region of 63.7% of the cells. The presence of ERβ was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848,P in vitrocapacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 beforein vitrocapacitation resulted in a higher (P

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.

Background: One of the main drawbacks of refrigerated and frozen-thawed semen is lipid peroxidation and the subsequent generation of reactive oxygen species from cellular metabolism. In this study, we tested the hypothesis that the use of antioxidant compounds improves the frozen-thawed sperm quality. We analysed the effect of different antioxidants, or antioxidant-like additives, upon the processes involved in cooling and freeze-thawing ram semen. Methods: Sperm motility, plasma membrane integrity and stability, and mitochondrial membrane potential (MMP) were tested immediately after thawing and following incubation for 3 h and 6 h at 37°C. Results: The addition of oleic/linoleic acid did not improve sperm viability, although it resulted in increased motility after refrigeration and rewarming, similar to that found with pyruvic acid. In frozen-thawed samples, the effect of 75 mM ascorbic acid was beneficial and improved viability, membrane stability, MMP and motility. Pyruvic acid, melatonin, pinoline and N-acetyl cysteine, or the combination of certain antioxidants such as oleic/linoleic acids with tocopherol, lipoic and ascorbic acids, melatonin and pinoline, N-acetyl cysteine and GSH did not significantly improve the frozenthawed sperm quality. Thawed samples supplemented with lecithin scored higher (p

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., Author summary In the field of the biology of reproduction, it is very important to develop new methods that allow us to predict the breeding capacity of males of certain species. In recent years, the use of Computer Assisted Sperm Analysis systems (that allow the user to analyze certain parameters of sperm that have been shown to be related to fertility), has been fundamental in advancing this knowledge. In addition, new parameters are being investigated that are believed to help characterize seminal quality. However, most of the software used to analyze these parameters is commercial and private, and the user is not allowed to add new features. Although there were already open source alternatives to analyze certain parameters of seminal quality, in this work we have integrated several tools into one, with an easy-to-use interface for the user, which we hope will help the scientific community to develop new functionalities and methods. The tool is free and open-source.

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

Melatonin increases in vivo embryo viability in sheep and improves the blastocyst rate on in vitro sheep embryo culture. To determine whether this melatonin effect is receptor-mediated, we evaluated the presence of melatonin receptors MT1 and MT2 on in vitro-obtained sheep embryos by means of RT-PCR and indirect immunofluorescence (IIF). For in vitro embryo production, oocytes were collected from ovaries of adult ewes and matured with TCM-199; 10% oestrus sheep serum; FSH and LH, 10μg mL−1 each; cysteamine, 100 μM; sodium pyruvate, 0.3 mM; penicillin G, 100IU mL−1; and streptomycin sulfate, 100μg mL−1 for 24h at 39°C and 5% CO2. Matured oocytes were transferred to a fertilization medium (SOF without glucose, with 2% oestrus sheep serum; heparin, 10μg mL−1; and hypotaurine, 1μg mL−1) and fertilized with swim-up selected spermatozoa at a final concentration of 106 cells mL−1. After 36h, the cleaved embryos were incubated in culture medium (SOF with amino acids, 0.4% BSA; l-glutamine, 1 mM; penicillin G, 100IU mL−1; and streptomycin sulfate, 100μg mL−1) for 8 days at 39°C with 5% CO2, 5% O2, and 90% N2. In vitro fertilization was repeated 3 times. Day 8 hatched blastocysts were selected for RT-PCR or IIF. For RT-PCR assays, RNA from 5 to 10 blastocysts was extracted and reverse transcribed with SuperScript™ III CellsDirect™ cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PCR amplification was carried out in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR mix contained 2μL of embryo cDNA, iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc., Waltham, MA, USA) and 200nM of the MT1 (forward: 5′-CTCCATCCTCATCTTCACCATC-3′, reverse: 5′-GGCTCACCACAAACACATTC-3′, 113bp) or MT2 (forward: 5′-GCTGAGAGAATGGAGCGATATG-3′, reverse: 5′-GTCCACAGTGAGAAGCCATC-3′, 81bp) primers. The RT-PCR products were visualised under ultraviolet light on 2% agarose gel in a TBE buffer (Tris, 0.9 M; boric acid, 0.9 M; and EDTA, 20mM) with 0.5μL mL−1 ethidium bromide. Ovine testis was used as positive control. For IIF, 5 blastocysts were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 5% BSA in PBS for 2h at RT. Embryos were incubated overnight at 4°C with MTNR1A mouse polyclonal antibody (Abnova, Taipei City, Taiwan) for MT1, and rabbit melatonin receptor 1B antibody (Acris Antibodies, Atlanta, GA, USA) for MT2, both diluted 1/50 in PBS with 1% BSA. Secondary antibodies were Alexa Fluor 594 chicken anti-mouse (Invitrogen, Carlsbad, CA, USA) for MT1 and Alexa Fluor 488 chicken anti-rabbit for MT2, diluted 1/800 in PBS containing 1% BSA. Embryos were mounted in slides and visualised on a Nikon Eclipse E-400 microscope (Nikon, Tokyo, Japan). The RT-PCR revealed a single band of 113bp for MT1 and another one of 81bp for MT2 in both the embryo samples and the positive control, which confirmed the gene expression of melatonin receptors in the ovine embryo. The IIF located the MT1 receptor around the nucleus of the trophoblastic cells, whereas MT2 was over the nucleus in the same cells. These results indicate the presence of melatonin receptors in sheep blastocyst, which could mediate the positive effects of this hormone on embryo viability.

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.

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P0.01) of membrane integrity, lower PS translocation (P0.05) and DNA damage (P0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.

Despite considerable cryobiology research there is no industry standard for the concentration to which ram spermatozoa should be diluted before freezing. Ram semen is highly concentrated and often frozen at a high sperm concentration, necessitating the use of small laparoscopic insemination doses. The aim of this paper was to ascertain the effect of dilution on the integrity of frozen-thawed ram spermatozoa. In the first experiment, spermatozoa were extended with a Tris-buffered diluent before freezing or after thawing to yield a final sperm concentration of 20 × 10 6 /ml, or were not diluted. Motility characteristics, viability and acrosome integrity of spermatozoa were analysed over a 6 h incubation period at 37 ◦ C. In the second experiment, spermatozoa were either diluted before freezing, subjected to sex-sorting or not diluted before freezing. Thawed spermatozoa were separated into sub-populations using centrifugal counter-current distribution (CCCD) and the profile of partition and functional integrity (viability, chlortetracycline status and Annexin-V binding) in the sub-populations assessed. Dilution before freezing significantly improved post-thaw viability, acrosome integrity and total motility whereas dilution post-thaw decreased viability and motility of spermatozoa. Sperm heterogeneity, as assessed by CCCD profile, was not different for control, diluted and sex-sorted spermatozoa. Analysis of CCCD subpopulations showed the proportion of functional cells (displaying the F-Pattern or no PS translocation) was similar for all sperm types. The results show that ram spermatozoa retain normal function at higher pre-freeze dilution rates than are commonly used in the sheep industry. The application of these findings would result in more practicable and functional artificial insemination doses. © 2009 Elsevier B.V. All rights reserved.

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.

Contents The objectives of this study were two. First, to compare three base media with different sugar composition as an initial step to achieve a good chemically-defined extender for ram sperm refrigeration. The second one, to determine which sperm quality parameters may be more useful for revealing differences between sperm samples. One medium contained 200 mm sucrose and 2.8 mm glucose (SM), another only disaccharides (D) such as sucrose, trehalose, maltose and lactose (75 mm each); and the third one (D+M) included a mix of monosaccharides (50 mm glucose, 20 mm fructose and 20 mm galactose,) and the same disaccharides as in D (50 mm each). Ram semen samples diluted in the mentioned media were refrigerated at 5°C for 1 h, and rewarmed upto 37°C in order to mimic the temperature in the female reproductive tract. Addition of monosaccharides to the extender did not produce a better preservation of motility or viability after cooling. The supplementation with other disaccharides apart from sucrose did not enhance the viability either. Thus, after cooling and rewarming, there were no significant differences in sperm viability (membrane integrity evaluated by CFDA/PI staining) or the percentage of progressive motile and rapid sperm (evaluated by CASA) between the three media. However, the percentage of viable non-capacitated sperm evaluated by the chlortetracycline (CTC) assay was higher and sperm oxygen consumption was lower in SM than in D and in D+M. Although the apoptosis-like markers [phosphatidylserine exposure assessed by Annexin V/CFDA staining and DNA-damage evaluated by TUNEL assay] showed a continuous increment throughout the process with all diluents, the percentage of sperm with damaged DNA at the end of the process was significantly lower in SM than in the other two media (p

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.

The purpose of this study was to determine the presence of actin in ejaculated ram spermatozoa and the changes of localization that actin undergoes as a consequence of certain in vitro-induced physiological states. Using indirect immunofluorescence (IIF), three different patterns of staining (defined immunotypes) were established in ejaculated sperm. The three sperm immunotypes showed actin labelling in flagellum, neck and post-acrosomal area, differing on the labelling in the acrosomal region that was complete in immunotype 1, partial (frequently concentrated in the apical area, punctuate form) in immunotype 2, and totally absent in immunotype 3. The main subpopulation in ejaculate was immunotype 1 that represented 68% of total sperm, while 21% corresponded to immunotype 2 and only 10% corresponded to immunotype 3. Selection of high-quality sperm using a dextran/swim-up procedure hardly influenced the proportion of each immunotype resulting in a slight increase in type 1 sperm. Cold-shock treatment and in vitro capacitation induced a partial loss of actin labelling in the acrosomal area, whereas the ionophore-induced acrosomal exocytosis provoked a total loss of the acrosomal actin labelling, a phenomenon partially inhibited by phalloidin.

The effect of melatonin implants administered during non-breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer-assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46-75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona-pellucida binding assays, using spermatozoa from experiment 1, obtained 60-70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen-thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non-breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46-60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non-breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.

The role of seminal plasma (SP) in mammalian sperm function remains largely a matter of speculation as both inhibitory and stimulating effects have been found. Specific components of SP, particularly proteins, are adsorbed onto the surface of ejaculated sperm as they pass through the male and female reproductive tracts. These sperm coating components seem to have the important function of maintaining the stability of the membrane up to the process of capacitation (decapacitation factors). Therefore, they must be removed, modified or masked before the spermatozoa undergo the acrosome reaction, an essential process for successful fertilization. It is well known that low temperatures alter the function of spermatozoa. Cold shock results in the destabilization of sperm membranes and impairment of sperm function, and it is also well known that ram spermatozoa are more sensitive to cold-shock stress than those of other species. The addition of SP proteins to spermatozoa before and/or after cooling is able to minimize cryoinjury effects. The major proteins in ram SP which are able to protect and repair the cold-shock damage to sperm contain fibronectin-II domains. The significance of this domain and the role of these proteins in sperm capacitation and gamete interaction are discussed.

Certain features of capacitated or frozen-thawed spermatozoa have been considered to be an apoptosis-like phenomenon, and, it has been suggested that the presence of apoptotic sperm in seminal doses could be one of the reasons for poor fertility. The objective of this study was to determine whether phosphatidylserine (PS) translocation, caspase activity and DNA fragmentation, which are considered to be apoptotic markers in somatic cells, occur in ram sperm. Fresh ejaculates and sperm samples in different physiological state (cold-shocked, in vitro capacitated and acrosome-reacted (AR)) were compared. Simultaneous staining with 6-carboxifluorescein diacetate (6-CFDA) and Annexin V-Cy3.18 (AnnV) revealed four different sperm subpopulations in ejaculates. The main subpopulation was composed of viable cells without PS exposure (CFDA+/AnnV-). A total of 40.8% of sperm showed inverted PS, with two levels of alteration: CFDA+/AnnV+ in midpiece ("type I AnnV+"), and in acrosome and midpiece ("type II AnnV+"). The fewest subpopulation contained non-viable cells showing Annexin labelling in the entire cell (CFDA-/AnnV+). Labeling of caspases-3 and -7 by immunocytochemistry revealing different sperm subtypes depending on their localization in apical, equatorial, post-acrosomal regions and tail. The results obtained by western-blot showed, for the first time to our knowledge, that caspase-like proteins are present in fresh ram semen as both inactive and active forms. The proportion of sperm with fragmented DNA [terminal transferase-mediated dUDP nick end-labeling (TUNEL)-positive] were found rarely (2.7+/-0.5%) in all fresh ejaculates involved in this study. The analysis of total activity of both caspases by a fluorometric method showed a decrease in vitro capacitated and acrosome-reacted samples as well as in cryoinjured samples. However, the percentage of TUNEL-positive sperm demonstrating DNA fragmentation was significantly increased after in vitro induced capacitation and acrosome reaction, as well as after cold-shock although this augment was not significant. PS exposure is not totally dependent on caspases in ram spermatozoa as the addition of a caspase inhibitor prevented the increase in PS inversion due to incubation in capacitating conditions but not to the ionophore-induced acrosome reaction or cold-shock.

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed.

Melatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine-N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT) was analyzed by RT-PCR, q-PCR and Western-blot in ram testes, epididymis, and accessory glands. The RT-PCR showed the presence of the m-RNA codifying both AANAT and ASTM in all the tissues under study, but the q-PCR and Western-blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

Spermatozoa require substantially more ATP than other cells, not only for sustaining sperm motility but also for regulating protein phosphorylation during capacitation. In this study, we have reported for the first time the presence of the two key enzymes of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in ovine spermatozoa by indirect immunofluorescence, Western blotting, in-gel activity, and reverse transcription polymerase chain reaction analysis. We found that the activity of both enzymes significantly increased after in vitro capacitation in the presence of high-cAMP levels, with a concomitant increase in protein tyrosine phosphorylation and in the proportion of sperm-capacitated pattern assessed by the chlortetracycline staining. These results suggest that PPP is related with the progress of capacitation and that a relationship between calcium compartmentalization, protein tyrosine phosphorylation and PPP seems to exist. This is the first report that shows a connection between the PPP, cAMP/PKA signaling pathways and sperm capacitation. These findings can be of high-biological importance to improve our knowledge of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.

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.

The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this study, we investigate the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides the evaluation of viability and the capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the phosphatidylserine translocation and caspase-3 and -7 activities. This is the first report, to our knowledge, that evidences the presence of active caspases in ram sperm. The results confirm the better ability of the dextran/swim-up procedure to select nonapoptotic spermatozoa, in addition to viable and noncapacitated sperm, compared with other sperm preparation methods. This should be considered to enhance results of artificial insemination techniques in ovine reproduction.

Efficient animal production involves accurate estimations of fertilizing ability. One key factor is the plasma membrane of the sperm cell, which is actively involved in the cascade of events before oocyte fusion. Many methods are used to analyze the characteristics of this membrane, including partition in aqueous two-phase systems which is an efficient method to analyze sperm surface changes accounting for loss of viability and different functional states. Centrifugal countercurrent distribution (CCCD) analysis can also be used in an aqueous two-phase system to determine the relationship between sperm parameters and in vivo fertility in ewes. In a previous work, we found a significant correlation between two post-CCCD parameters (heterogeneity and recovered viability) and field fertility when the same sample was used after cervical AI. The present study was intended to find out whether the control of several external factors that affect reproductive efficiency is able to increase the correlation coefficient between post-CCCD parameters and fertility. Thus, 90 Rasa aragonesa ewes were controlled on the same farm and received intrauterine inseminations using the same technical equipment. The fertilizing ability of the raw semen and sperm samples selected by a dextran/swim-up process was compared using a low number of spermatozoa per insemination (7 x 10(7)) to enhance possible fertility differences. A new post-CCCD parameter was considered; the loss of viability (LV) occurred during the CCCD process. This variable denotes the sperm surviving ability and corresponds to the difference between the total number of viable cells loaded and recovered after the CCCD run. The mean fertility of eight sperm control samples was 60% (range: 25-76%), and there was no significant correlation between standard parameters and in vivo fertility. LV ranged from 2 to 69% (average 27%) and was negatively correlated with fertility (r = -0.914, P < 0.01). Ejaculate heterogeneity (H) ranged from 20 to 47% and was positively, but not significantly, correlated with fertility (r = 0.391). A predictive equation for fertility was deduced by multiple analysis with a very high correlation coefficient (r = 0.967), and level of significance (P < 0.005): predictive fertility PF = 52.546 - 0.594 LV + 0.665 H. The mean fertility of 13 swim-up selected samples was 63% (range: 25-86%). Again, only parameters derived from the CCCD analysis were highly correlated with fertility, especially LV and H (P < 0.05).

This study reveals that the chick embryo has active the machinery for the production and degradation of the amyloid beta peptide characteristic of Alzheimer's disease. We cloned the principal beta-amyloid precursor protein isoforms in the chick embryo and observed that they are highly homologous to the human sequences and identical at the C-terminal sequence, including the amyloid beta domain. Mammals such as rat or mouse, more commonly used as animal models of human diseases, have a distinct amyloid beta sequence. The distribution of beta-amyloid precursor protein isoforms in the chick embryo revealed that, as in humans, their expression is ubiquitous and the prototype beta-amyloid precursor protein-695 predominated in the nervous system. We also found that the chick embryo expresses the genes for the main proteolytic proteases implicated in the production of amyloid beta, including BACE-1, BACE-2, presenilin-1, presenilin-2 and nicastrin, as well as the amyloid beta-degrading enzyme neprilysin, or ADAM-17, a protease implicated in the non-amyloidogenic processing of beta-amyloid precursor protein. We have also found that between amyloid beta40 and amyloid beta42, this latter seems to be the major amyloid beta peptide produced during chick embryogenesis. The chick embryo appears as a suitable natural model to study cell biology and developmental function of beta-amyloid precursor protein and a potential assay system for drugs that regulate beta-amyloid precursor protein processing.