The other side of capacitation: role of mouse male molecules in the regulation of time and place of capacitation.

In brief: Capacitation is regulated by decapacitation factors secreted by male ducts and accessory sex glands. This revision is focused on targets and events regulated by decapacitation factors in Mus musculus and their potential use for fertility control. Abstract: Sperm capacitation is a necessary process for mammalian spermatozoa to acquire fertilization capability. This process occurs when the sperm enters the female's reproductive duct, involving a vital interplay with the uterine and oviductal environment, leading to morphological, physiological, and biochemical modifications in the male gamete. Besides, for a successful sperm capacitation, molecules are incorporated onto the sperm's surface during its passage through the male reproductive tract followed by their subsequent removal. These molecules, referred to as decapacitation factors (DFs), also regulate capacitation, preventing this process from occurring in the wrong site or at the wrong time. While decapacitation factors have been extensively studied in recent decades in species such as Mus musculus, there is no comprehensive report consolidating information on all the identified decapacitation factors and the molecular basis of their function. The aim of this review is to summarize the data related to decapacitation factors discovered and characterized in Mus musculus. Concurrently, this review aims to elucidate the implications of different decapacitation factors throughout the fertilization process (i.e. capacitation, acrosomal reaction, and fertilization), as well as the methodologies employed for their investigation. Given that mice (Mus musculus) have served as a valuable model in reproductive research due to their genetic similarity to humans, this review contributes to our understanding of the role of decapacitation factors in male fertility.

How, where and when is SPINK3 bound and removed from mouse sperm?

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.

Recombinant TrxAFNIIx4His6 improves post-thaw motility of ram sperm measured by a sperm motility tracker software.

The aim of the present study was to evaluate a freezing extender supplemented with recombinant TrxAFNIIx4His6, a reported decapacitating factor. Semen samples were diluted in tris-egg yolk medium with 0, 1.5 µM and 3.0 µM of TrxAFNIIx4His6. Computer-assisted sperm motility tracking and subpopulations evaluation showed that addition of TrxAFNIIx4His6 improved post-thaw total and progressive motility at both concentrations evaluated. TrxAFNIIx4His6 increased the sperm subpopulation with the highest progressiveness and great velocity and decreased the subpopulation of poorly motile and almost non-progressive sperm. Incorporation of TrxAFNIIx4His6 to freezing extender shows potential for the development of cryoprotection media which may lead to improved fertility after artificial insemination.

Male Decapacitation Factor SPINK3 Blocks Membrane Hyperpolarization and Calcium Entry in Mouse Sperm.

Mammalian sperm acquire ability to fertilize through a process called capacitation, occurring after ejaculation and regulated by both female molecules and male decapacitation factors. Bicarbonate and calcium present in the female reproductive tract trigger capacitation in sperm, leading to acrosomal responsiveness and hyperactivated motility. Male decapacitating factors present in the semen avert premature capacitation, until detached from the sperm surface. However, their mechanism of action remains elusive. Here we describe for the first time the molecular basis for the decapacitating action of the seminal protein SPINK3 in mouse sperm. When present in the capacitating medium, SPINK3 inhibited Src kinase, a modulator of the potassium channel responsible for plasma membrane hyperpolarization. Lack of hyperpolarization affected calcium channels activity, impairing the acquisition of acrosomal responsiveness and blocking hyperactivation. Interestingly, SPINK3 acted only on non-capacitated sperm, as it did not bind to capacitated cells. Binding selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated cells.

Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation.

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.

Recombinant SPINK3 improves ram sperm quality and in vitro fertility after cryopreservation.

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.

Extenders modify the seminal plasma ability to minimize freeze-thaw damage on ram 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.

Cryopreservation and egg yolk extender components modify the interaction between seminal plasma proteins and the sperm surface.

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.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

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 ±â€¯2.95; 40.13 ±â€¯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.

Evaluación de la eficacia de los laboratorios de embriología y anatomía patológica en el análisis de las biopsias testiculares de pacientes azoospérmicos / Evaluation of the Efficacy of the Laboratories of Embryology and Pathological Anatomy in the Analysis of Testicular Biopsies of Patients with Azoospermia

Objetivos: El propósito de este estudio es evaluar la eficacia de los laboratorios de embriología y de anatomía patológica para hallar espermatozoides en las muestras de tejido testicular obtenido por biopsia testicular (testicular sperm extraction, TESE) en pacientes con azoospermia no obstructiva. Materiales y métodos: Se realizó un análisis retrospectivo y prospectivo de todos los pacientes con azoospermia no obstructiva atendidos en CRECER y en la Clínica Privada Pueyrredón, entre enero de 2006 y diciembre de 2016. En este estudio solo se incluyeron aquellos pacientes en los que la muestra obtenida con TESE fue enviada simultáneamente al anatomopatólogo y al laboratorio de embriología. Para el análisis de los resultados de las biopsias el estudio se detuvo a fines de 2016, pero el seguimiento de los pacientes continuó hasta el mes de octubre de 2017, registrándose todos aquellos casos que realizaron procedimientos de inyección intracitoplasmática de espermatozoides (intracytoplasmic sperm injection, ICSI) con muestras obtenidas de TESE y se anotó la obtención de embriones, embarazos y nacimientos. Resultados: El laboratorio de embriología halló espermatozoides en 36 de los 68 pacientes (52,9%), mientras que el laboratorio de patología solo informó presencia en 21 pacientes (30,88%). Hubo acuerdo en el hallazgo de espermatozoides entre ambos laboratorios en 20 de los 68 casos (29,41%), mientras que en 16 pacientes el laboratorio de embriología encontró espermatozoides donde el de patología no pudo hacerlo (23,53%). Al mismo tiempo, el laboratorio de patología halló espermatozoides solo en un caso en el que el de embriología informó su ausencia para la misma muestra analizada (1,47%) (p=0,0003). Conclusiones: El laboratorio de embriología es significativamente más eficaz para determinar la presencia de espermatozoides en las muestras de TESE, teniendo mejor rendimiento que el de patología, por lo que consideramos que, si las muestras fueran analizadas solo por el patólogo, se perdería la posibilidad de lograr muchos embarazos realizando ICSI más TESE.(AU)

Objectives: The purpose of this study is to evaluate the efficacy of embryology and pathological anatomy laboratories to find spermatozoa in testicular tissue samples obtained by testicular sperm extraction (TESE) in patients with non-obstructive azoospermia. Materials and methods: It was carried out a retrospective and prospective analysis of all the patients with non-obstructive azoospermia treated at CRECER and at Clínica Privada Pueyrredón, between January 2006 and December 2016. This study only includes patients in whom the sample obtained with TESE was sent at the same time to the pathology and embryology laboratory. For the analysis of the results of the biopsies, the study was stopped at the end of 2016, but the follow-up of the patients continued until October 2017, registering all those cases that performed intracytoplasmic sperm injection (ICSI) with samples obtained from TESE and wrote down the patients who´ve got embryos, pregnancies, and births. Results: The embryology laboratory found sperm in 36 of the 68 patients (52.9%), while the pathology laboratory only reported presence in 21 patients (30.88%). There was agreement in the finding of sperm between both laboratories in 20 of the 68 cases (29.41%), while in 16 patients the embryology laboratory found sperm where the pathology department could not do so (23.53%). At the same time, the pathology laboratory found sperm only in one case in which the embryology department reported its absence for the same sample analyzed (1.47%) (p=0.0003). Conclusions: The embryology laboratory is significantly more efficient to determine the presence of sperm in the samples of TESE, having better performance than the pathology one. Taking into account that, we believe that if the samples are only analyzed by the pathologist, the possibility of getting many pregnancies performing ICSI plus TESE would be lost. (AU)

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

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.

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

STUDY QUESTION: Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? SUMMARY ANSWER: Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. WHAT IS KNOWN ALREADY: In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. STUDY DESIGN, SIZE, DURATION: Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 µM), Gö6983 (PKC inhibitor, 10 µM), PD98059 (ERK-1/2 inhibitor, 30 µM), H89 and KT (PKA inhibitors, 50 µM and 100 nM, respectively), KH7 (sAC inhibitor, 10 µM), BAPTA-AM (intracellular Ca2+ chelator, 50 µM), EGTA (10 µM) and Probenecid (MRPs general inhibitor, 500 µM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 µg/ml) and bicarbonate (40 mM). PARTICIPANTS/MATERIALS, SETTING, METHODS: Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. WIDER IMPLICATIONS OF THE FINDINGS: These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests.

Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

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.

A simple technique to improve the resolution of membrane acidic proteins of the haloarchaeon Haloferax volcanii by 2D electrophoresis.

Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.

Novel inhibitory activity for serine protease inhibitor Kazal type-3 (Spink3) on human recombinant kallikreins.

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.

Regulated serine proteinase lytic system on mammalian sperm surface: there must be a role.

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.

Mouse sperm rosette: assembling during epididymal transit, in vitro disassemble, and oligosaccharide participation in the linkage material.

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.

Potato aspartic proteases (StAPs) exert cytotoxic activity on bovine and human spermatozoa.

OBJECTIVE: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) 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 StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs 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 StAP binding by an optical microscopy. RESULT(S): The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs 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 StAPs involves plasma membrane permeabilization.

Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: its role in in vitro fertilization.

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.

Partial purification and characterization of a trypsin-like serine protease from bovine sperm.

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.