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Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 μM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results., This research was supported by the National Agency for Scientific and Technological Promotion (ANPCyT, grant PICT-2015-3682 awarded to A.C), M.I.C was supported by Ministry of Economy and Competitiveness scholarship and the BeCAR program that facilitates the L.Z′ s stage in the UCLM, Spain.

It is known that the addition of seminal plasma (SP) or SP proteins either before freezing or post thawing show contradictory results on sperm quality and fertility due to the interference between SP and the extender. Thus, the aim of this study was to determine whether egg yolk (EY) interferes with SP ability to protect the functionality and fertility of ram sperm during freeze-thawing by modifying the interaction between seminal plasma proteins and the sperm plasma membrane. Ejaculated or epididymal ram sperm collected during the breeding season were incubated with SP in the presence or absence of EY or soybean lecithin-based extenders before cryopreservation. No significant differences were observed after thawing in sperm quality (total and progressive sperm motility, membrane integrity, plasma membrane functionality, percentage of non-capacitated sperm) between the extenders, either in presence or absence of seminal plasma (P ≥ 0.05). The amount of proteins retained by the sperm surface normalized to number of cells was diminished after freeze-thawing compared to their fresh counterparts for all the treatments (P 0.05), demonstrating that cryopreservation weakens the interaction between external proteins and the sperm surface. The electrophoretic analysis of sperm-bound proteins showed that the retention of several SP peptides onto the sperm surface (based on densitometry estimation) was affected by the presence of the diluents on both ejaculated and epididymal sperm (P 0.05). Moreover, variation was observed in the protein pattern after thawing compared to the corresponding fresh samples, suggesting that freezing affects surface protein profile. Pregnancy rate after artificial insemination at fixed time was higher (P 0.05) for samples treated with reconstituted with heterologous SP compared to those supplemented with 20% additional seminal plasma or control samples despite the presence of EY. In conclusion, both freeze-thawing and EY components affected the interaction among seminal plasma proteins and the sperm surface, although these changes were not reflected on different sperm quality parameters under our experimental conditions. In vivo fertility of sperm reconstituted with exogenous SP before freezing was improved even in the presence of EY components considering an optimal ratio SP:sperm.

Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze-thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk-based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen-thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin-based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze-thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non-capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein–lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.

The aim of the present study was to evaluate a freezing extender supplemented with recombinant TrxAFNIIx4His

The aim of this study was to investigate the dynamic changes that ram sperm experience during in vitro capacitation before and after cryopreservation. Using flow cytometry and computer assisted sperm analysis system (CASA), protein tyrosine phosphorylation and several functional parameters were evaluated in fresh and cryopreserved ram sperm incubated under capacitating and non-capacitating conditions at 0, 1, 5, 15, 30, 60, 120, 180 and 240 min. A short incubation period (5–30 min) under capacitating conditions was enough to increase mitochondrial activity and tyrosine phosphorylation in cryopreserved sperm, inducing also changes in the motility pattern, which could be related to hyperactivation. However, fresh sperm required a longer incubation (180–240 min) under capacitating conditions to undergo similar modifications. In both types of samples, tyrosine phosphorylation increased in a sequential manner in the midpiece, principal piece and tail at specific time points during in vitro capacitation. Moreover, the proportion of viable sperm with intact acrosome begun to decrease during capacitation, occurring before in cryopreserved sperm. Our findings suggest that cryopreserved ram sperm become competent for fertilization after a short exposure to capacitating conditions as a result of drastic changes inflicted by the freezing-thawing procedure, while prolonged incubations after cryopreservation severely impair sperm quality., PP-F was supported by University of Castilla-La Mancha (UCLM) fellowships. MV was supported by the Research Plan of University of Castilla- La Mancha (UCLM). MI-C was supported by the Ministry of Economy and Competitiveness fellowship.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 μM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ± 2.95; 40.13 ± 11.82 for 0.3 μM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability. Fil: Ledesma, Alba. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Buchelly Imbachí, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica; Argentina Fil: Pastore, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica; Argentina Fil: Brown, Paula. National Institute of Environmental Health Sciences; Estados Unidos Fil: Eddy, Edward Mitch. National Institute of Environmental Health Sciences; Estados Unidos Fil: Hozbor, Federico Andrés. National Institute Of Environmental Health Sciences; . Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina

[Aims]: To examine the antioxidant activity of Bacterioruberin (Bctr)‐rich extracts isolated from a hyperpigmented, genetically modified Haloferax volcanii strain (HVLON 3) and to investigate the effect on cold‐sensitive ram sperm cells., [Methods and Results]: The strain HVLON 3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg g−1 DW ). HVLON 3‐Bctr extract has higher antioxidant activity than β‐carotene (threefold) as evaluated using 2,2 diphenyl‐1‐picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC 50 4·5 × 10−5 mol l−1 vs 13·9 × 10−5 mol l−1 respectively). Different concentrations of HVLON 3‐Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 μ mol l−1 Bctr significantly improved cell viability (P < 0·0001), total and progressive motility (P < 0·0001) and sperm velocities (P = 0·0172 for curvilinear velocity VCL , P = 0·0268 for average path velocity VAP and P = 0·0181 for straight line velocity VSL ) and did not affect other parameters evaluated., [Conclusions]: HVLON 3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON 3 Bctr extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields., [Significance and Impact of the Study]: This study provides an insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited., This research was supported by the National Agency for Scientific and Technological Promotion (ANPCyT) grants PICT‐2015‐3682 and PICT‐2014‐1477 awarded to A.C. and R.D.C., respectively; UNMDP grants 15/E818, EXA819/17 and EXA823/17 awarded to M.S.C., A.C. and R.D.C, respectively.

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species. Fil: Buchelly Imbachí, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Pastore, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Greco, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Iniesta-Cuerda, M.. Universidad de Castilla-La Mancha; España Fil: Garde, J. J.. Universidad de Castilla-La Mancha; España Fil: Soler, A. J.. Universidad de Castilla-La Mancha; España Fil: Ballarin, Virginia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

During the last decades fundamental and applied aspects of mammalian ram sperm cryopreservation have been increasingly explored by scientists and biotechnologists. Many works report modifications in the composition of the freezing extenders and explore the beneficial and detrimental effects of seminal plasma or seminal plasma components in cryopreservation. Seminal plasma is known to contain stabilizing proteins, thereby this is a good start point to study the maintenance of membrane stability based on the basic knowledge of sperm physiology. However, seminal plasma composition is variable among rams and also the introduction of exogenous seminal plasma or its fractions to commercial semen can be associated with the transmission of viral diseases. Our work shows that a mouse protein, called SPINK3 (Serine Protease Inhibitor Kazal type 3) with decapacitating activity interacts with heterologous ram sperm when it is produced as a recombinant molecule. By immunocytochemistry assays we demonstrate that this protein (naturally expressed by mouse seminal vesicle under androgenic control) binds to the apical portion of both fresh and frozen ram sperm, the same localization described in mouse homologous sperm. Furthermore, it significantly improves sperm progressive motility compared to non-treated samples when it is added to freezing extenders and to dilution media after thawing. On the contrary, addition of SPINK3 does not modify sperm viability. The percentage of sperm with intact acrosome after ionophore induction was also significantly higher in sperm frozen in the presence of SPINK3 compared to control samples and the addition of SPINK3 after thawing significantly reduced both induced and non induced acrosomal loss, indicating that heterologous SPINK3 might act as a calcium inhibitor transport as described in mouse. Based on our results SPINK3 may find a place as a desirable biotechnological tool to achieve a higher proportion of competent sperm to fertilize. Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Ledesma, Alba. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentina Fil: Hozbor, Federico Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as “hyperactivated” (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables. Fil: Ledesma, Alba. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Fernández Alegre, Estela. Universidad de León; España Fil: Hozbor, Federico Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Departamento de Producción Animal; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Martínez Pastor, Felipe. Universidad de León; España

Contents This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases. Fil: Assis, Diego Magno. Universidade Federal de São Paulo; Brasil Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Juliano, María Aparecida. Universidade Federal de São Paulo; Brasil Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina

This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13 ± 2.99 and 72.27 ± 2.99 for AV and EE, respectively) and progressive motility (64.97 ± 2.64 and 63.73 ± 2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p > 0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60 ± 2.87) and sperm with stable plasma membrane (44.56 ± 2.49) comparing with the addition of SP collected by EE (35.80 ± 2.47 and 36.67 ± 1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters. Fil: Ledesma, Alba. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernández Alegre, Estela. Universidad de León; España Fil: Cano, Adriana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentina Fil: Hozbor, Federico Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Laboratorio de Biotecnología de la Reproduccion; Argentina Fil: Martínez Pastor, Felipe. Universidad de León; España Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head–head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

Summary Sperm proteolytic activities are relevant in the enzymatic mechanism of fertilization. Several authors have suggested the presence of serine proteases other than acrosin in mice and human spermatozoa. In this work we describe the characterization of a partially purified bovine sperm serine protease BSp66 and its dimmer, BSp120. Partial purification of the monomer was performed from fresh spermatozoa, while the dimer form of the protease was obtained from cryopreserved spermatozoa. The Mr of BSp120 and BSp66 estimated by zymography and gel filtration chromatography were 120 and 66 kDa, respectively. They were positively stained by Schiff-PAS reagent for glycoproteins and they both digested synthetic peptides with basic amino acids in the P1 site. Polyclonal antibodies against acrosin or proacrosin did not cross-react neither with BSp120, nor BSp66. In addition, antibodies raised in our laboratory against BSp120 and BSp66 did not recognize acrosin or proacrosin suggesting that they are not antigenically related proteins. Also, no cross- reactivity was detected with proteins in the range of 120–66 kDa when antibodies against the proteasome were used. The cellular localization of this protease by optical immunocytochemistry using specific antibodies revealed a positive signal in the apical portion of the sperm head suggesting acrosomal or membrane localization. The evidences presented here characterize BSp66 as a trypsin-like serine protease, a putative new member of this highly redundant proteolytic system of the sperm acrosome.

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post-thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.

The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.

Serine protease inhibitor Kazal-type (SPINK3)/P12/PSTI-II is a small secretory protein from mouse seminal vesicle which contains a KAZAL domain and shows calcium (Ca2+)-transport inhibitory (caltrin) activity. This molecule was obtained as a recombinant protein and its effect on capacitated sperm cells was examined. SPINK3 inhibited trypsin activityin vitrowhile the fusion protein GST-SPINK3 had no effect on this enzyme activity. The inactive GST-SPINK3 significantly reduced the percentage of spermatozoa positively stained for nitric oxide (NO) with the specific probe DAF-FM DA and NO concentration measured by Griess method in capacitated mouse sperm; the same effect was observed when sperm were capacitated under low Ca2+concentration, using either intracellular (BAPTA-AM) or extracellular Ca2+(EDTA) chelators. The percentage of sperm showing spontaneous and progesterone-induced acrosomal reaction was significantly lower in the presence of GST-SPINK3 compared to untreated capacitated spermatozoa. Interestingly, this decrease was overcome by the exogenous addition of the NO donors, sodium nitroprusside (SNP), andS-nitrosoglutathione (GSNO). Phosphorylation of sperm proteins in tyrosine residues was partially affected by GST-SPINK3, however, only GSNO was able to reverse this effect. Sperm progressive motility was not significantly diminished by GST-SPINK3 or BAPTA-AM but enhanced by the addition of SNP. This is the first report that demonstrates that SPINK3 modulates sperm physiology through a downstream reduction of endogenous NO concentration and independently of SPINK3 trypsin inhibitory activity.

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility. Fil: Bernardini, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Hozbor, Federico Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Sanchez, E.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Fornes, Miguel Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Alberio, R. H.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Serine proteases play key roles in many biological processes, regulating surface proteins that are key-points in signaling pathways. Several studies have reported the presence of members of this protease family in sperm from various species. The precise regulation of their activity is thought to be performed by specific endogenous or extrinsic inhibitors. The contribution of the sperm serine to proteases to fertilization has been demonstrated by synthetic inhibitors and several single knock out experiments, but to date, there is no evidence that links a single enzyme to a single step of fertilization. The explanation for the failure in the understanding of the "one-enzyme-one-process" hypothesis may be that sperm have multiple serine proteases as a mechanism to ensure the success of fertilization. In addition to the classical purification and expression studies, we summarized recent advances in proteomics and performed a bioinformatics search of proteases and inhibitors, providing support for the idea of redundancy. This review summarizes current knowledge about serine proteases and their inhibitors in sperm capacitation and maturation, identifies questions that need to be answered, and provides a reference for future research.

In many mammals, sperm associations had been observed, but not in the mouse. In this work, mouse sperm rosettes are morphologically described inside the epididymis and during its dissolution in a culture medium. Also characterized are the saccharides present in the linking material. Sperm association and other epididymal actions are supported by sperm during epididymal transit and are verified at the caudal region, suggesting a relation between epididymal transit and sperm maturation. In drops of epididymal content obtained from distal (cauda), but not from proximal (caput and corpus) regions; dissolved in culture medium, rosettes appear to be 10 to 15 motile sperm joined by their heads. After 3 min, sperm progressively detach, disassembling the rosette. These structures are studied by several techniques, including optic, electronic (scanning electron microscopy and transmission electron microscopy), and video microscopy. At the ultrastructural level, a dense network of electron-dense material was observed between sperm heads, joining them. Based on previous works in rat, several lectins were used to characterize the type of saccharides present in this linking material. To avoid the contact between sperm and epididymal fluid from distal region—that probably exerts an influence on sperm association—a ligature was placed between caput and corpus. This epididymal content isolated from caput did not display any rosettes after 28 days. Anat Rec, 2007. © 2007 Wiley-Liss, Inc.

Objective To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases ( St APs) on bovine and human sperm. Design Controlled laboratory study. Setting Three research laboratories at a university of biologic science. Animal(s) and Donor(s) Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. Intervention(s) The effect of St APs on sperm motility was studied in vitro by incubation of different concentrations of St APs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different St AP concentrations. The effect of St APs was evaluated by human erythrocyte lysis, as a control in somatic cells. The St APs binding was monitored by fluorescence. Main Outcome Measure(s) Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled St AP binding by an optical microscopy. Result(s) The St APs reduced sperm motility in a dose-dependent manner, and 25 μM of St AP1 and 35 μM of St AP3 completely abolished the progressive motility. The St APs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, St APs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. Conclusion(s) The spermicidal effect of St APs involves plasma membrane permeabilization.

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.

Fertilization in mammals comprises a sequence of events leading to the fusion of sperm and oocyte membranes. Although proteases are known to be involved in this process, their role in fertilization is controversial. There is extensive work on the characterization of proteolytic systems, including serine proteases, which demonstrates that acrosomal proteases can be distinguished among the sperm of different mammalian species on the basis of the gelatin-hydrolyzing activity on SDS–PAGE by the quantity and variety of the enzymes. In this report, we investigated the occurrence and activity of the serine protease BSp66, previously characterized in bovine spermatozoa, in various mammalian sperm. A protein with a molecular mass of 66 kDa cross-reacted with heterologous antibodies against bovine BSp66 when sperm extracts of several mammalian species were analyzed by Western blot. In agreement, proteolytic activity corresponding to the molecular mass of BSp66 was detected by gelatin zymography in all the species analyzed. This protein was located on the acrosomal region of sperm cells by immunofluorescence methods. We concluded that BSp66 is widespread in mammalian sperm, with a conserved location in the acrosomal region.

BSp120 and BSp66 are trypsin-like serine proteases from bovine spermatozoa. The former is active in cryopreserved sperm samples while the latter shows proteolytic activity in recently obtained fresh sperm. Both proteases are immunologically related and co-localize in the apical portion of the sperm head. In Western blots with specific antibodies, sperm samples incubated with reducing agents showed a decrease in the amount of BSp120, while BSp66 was detected with both anti-BSp120 and anti-BSp66 antibodies. BSp120 was evident in frozen intact spermatozoa after 60 days of semen cryopreservation and the kinetic of appearance of this protein was coincident with the decrease in the amount of BSp66. Identical results were obtained by freezing sperm extracts from fresh semen at -20 degrees C. Our results suggest that BSp120 results from disulfide bond-dimerization of BSp66 and that this process may be induced by temperatures below zero in both intact spermatozoa and in sperm extracts.