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Background: Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species-specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction., Objectives: To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process., Material and Methods: The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content., Results: The presence of glucose in the in vitro capacitating medium diminishes, in a concentration-dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non-metabolizable substrate (l-glucose) for 4 h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation-like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control., Discussion and Conclusions: Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization., (© 2024 The Author(s). Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal's sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel., (© 2023. The Author(s).)

In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14 + /PI - ), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA + /PI - ) and reactive oxygen species (ROS) production (CellRox + /PI - ), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation., (Copyright © 2022. Published by Elsevier Inc.)

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro . Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle ( n = 4) or a recent ovulation ( n = 4); the samples were kept at -80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus . Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm-oviduct interaction, fertilization , and metabolism , among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fernández-Hernández, Marinaro, Sánchez-Calabuig, García-Marín, Bragado, González-Fernández and Macías-García.)

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P <  0.05). Spermatozoa were processed and evaluated using the nano LC-MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy ( 1 H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved., Competing Interests: The authors declare no conflict of interest.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment., Competing Interests: The authors declare no conflict of interest.

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy ( 1 H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 μg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract., (© 2019 Wiley Periodicals, Inc.)

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

Frozen-thawed boar sperm have less motility and fertility capacity in comparison to fresh sperm. Glycogen Synthase Kinase 3 (GSK3) contributes to sperm motility in fresh semen. In addition, GSK3 inhibition in boar spermatozoa in fresh semen improves motility variables. The role of GSK3 on boar cryopreserved sperm, however, is still unknown. The hypothesis in the present study was that GSK3 pathway inhibition by alsterpaullone (AST) could result in enhancement of the quality of sperm afer cryopreservation. Two different strategies were evaluated: i) AST supplementation to the freezing medium (AST + Cryo); ii) AST supplementation after sperm thawing (AST + Thaw). Sperm motility was evaluated using the CASA system and different sperm quality variables were evaluated using flow cytometry, as well as amount of GSK3 phosphorylation of thawed spermatozoa after 30 and 90 min incubation at 38.5 °C. Results indicate that AST supplementation had detrimental effects on sperm viability (live spermatozoa) and mitochondrial membrane potential when it was added after thawing (P <  0.05) The AST supplementation after thawing, however, had a protective effect on plasma membrane lipid disorganization (P <  0.05). The percentage of motile spermatozoa was not modified by AST supplementation. Nonetheless, after 30 min post-thawing, STR and LIN variables (related to straightness of the movement) as well as the percentage of rapid lineal spermatozoa were increased with both AST supplementation protocols. The GSK3α phosphorylation was not modified through the incubation time in boar thawed sperm. In summary, results do not support the idea of adding AST to the cryopreservation/thawing medium to improve boar sperm quality after cryopreservation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode's basal medium or TBM) for 3, 8 and 24h or in capacitating conditions (Tyrode's complete medium or TCM) for 4h at 38.5°C or 40°C (Heat stress) in the presence or absence of 5 or 20μM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7±4.1 vs. 1.9±1.1; % mean±SEM), PM (3.1±0.9 vs. 0) and high mitochondrial membrane potential (19.5±2.2 vs. 11.8±0.8) decreased significantly in sperm incubated at 40°C for 24h in the presence of 20μM 17-AAG (control vs. 20μM 17-AAG, respectively; p<0.05). In sperm incubated at 38.5°C only a mild decrease in TM was observed (48.7±3.1 vs. 32.1±4.8; control vs. 20μM 17-AAG, respectively; p<0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study a Bayesian network (BN) has been built for the study of the objective motility of Tinca tinca spermatozoa (spz). Semen from eight 2-year-old sexually mature male tenchs was obtained and motility analyses were performed at 6-17, 23-34 and 40-51 s after activation, using computer-assisted sperm analysis (CASA) software. Motility parameters rendered by CASA were treated with a two-step cluster analysis. Three well-defined sperm subpopulations were identified, varying the proportion of spermatozoa contained in each cluster with time and male. Cluster, cinematic and time variables were used to build the BN to study the probabilistic relationships among variables and how each variable influenced the final sperm classification into one of three predefined clusters. Both network structure and conditional probabilities were calculated based on the collected data set. Results shown that almost all the variables were directly or indirectly related to each other. By doing probabilistic inference we observed that the cluster distribution corresponded to the definition provided by the cluster analysis. Also, velocity and time variables determined the cluster to which each spermatozoon belonged with a high degree of accuracy. Thus, BNs can be applied in the study of sperm motility. The construction of a BN that include fertility data opens a new way to try to clarify the roles of motility and other sperm quality indicators in fertilization., (© 2015 American Society of Andrology and European Academy of Andrology.)

During boar semen liquid preservation, extender is one of the factors that influence storage tolerance of spermatozoa. However, there are few studies about intra-breed variation in the preservation of semen quality during storage in different extenders. Similarly, boar breed is generally not considered a possible factor influencing variation in the semen storage tolerance in a particular extender. The aim of this study was to compare boar semen storage potential, in terms of the ability to maintain sperm viability and motility, of two currently used long-term extenders, MR-A and XCell. Extended semen from two breeds, Iberian and Duroc that had been stored at 17°C for up to 7 days was used. Intra- and inter-breed effect was studied. On Days 1, 4 and 7 (Day 0=day of semen collection), motility parameters and the percentage of total motile sperm and progressively motile sperm using a CASA system was evaluated. Viability (SYBR-14/PI) was evaluated by flow cytometry. Within each breed and for each storage day, there were differences between extenders, although semen tolerance to preservation was more influenced by the extender in the Iberian than in the Duroc breed. Neither breed nor extender influenced the percentage of viable spermatozoa during the storage time. Moreover, differences in motility parameters were observed between breeds, although the differences were greater when the XCell extender was used. In conclusion, both extender and breed influence motility characteristics of liquid-stored boar semen, so both aspects have to be considered in the design of comparative studies about stored boar semen quality from different breeds or with different extenders. Further studies are needed to corroborate these findings., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 μM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 μM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.

Computer-assisted sperm morphometry analysis was used to determine the effects of cryopreservation on boar sperm head and midpiece morphometry. Sperm-rich fractions were collected from five mature boars. Three microscope slides were prepared from single extended sperm samples prior freezing and post-thawing. All slides were stained with Hemacolor, and 250 sperm images were obtained from each slide. The sperm head dimensions for length, width, area, perimeter and four shape factors and sperm-midpiece dimensions for area, width, angle and distance were determined in each spermatozoa. The effects of sperm freezing on sperm dimensions within and among boars were determined. A previous discriminant analysis of the results was able to correctly classify a 78.3 and 82% of fresh and frozen-thawed spermatozoa respectively. Sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for length, width, area and perimeter. Sperm midpieces were also significantly smaller in cryopreserved spermatozoa for width and area. The highest changes in morphometric dimensions after the freeze-thawing process were found in the midpiece of spermatozoa. The variability of morphometric measurements only was significantly different between fresh and thawed samples for head rugosity and midpiece area. The effects of cryopreservation on morphometric parameters were similar in the boars, which allow us to conclude that cryopreservation process does not have a different effect in each individual boar. In summary, morphometric changes associated with the cryopreservation process on boar spermatozoa do not apparently depends on an effect at individual level.

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.

We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by mevalonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apoptosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinositide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovastatin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.

We have previously shown that lovastatin induces apoptosis in spontaneously immortalized rat brain neuroblasts. Focal adhesion proteins and protein kinase Cdelta (PKCdelta) have been implicated in the regulation of apoptosis. We found that lovastatin exposure induced focal adhesion kinase, Crk-associated substrate (p130(Cas)), PKCdelta cleavage and caspase-3 activation in a concentration-dependent manner. Lovastatin effects were fully prevented by mevalonate. The cleavage of p130(Cas) was almost completely inhibited by z-DEVD-fmk, a specific caspase-3 inhibitor, and z-VAD-fmk, a broad spectrum caspase inhibitor, indicating that cleavage is mediated by caspase-3. In contrast, the lovastatin-induced cleavage of PKCdelta was only blocked by z-VAD-fmk suggesting that PKCdelta cleavage is caspase-dependent but caspase-3-independent. Additionally, z-VAD-fmk partially prevented lovastatin-induced neuroblast apoptosis. The present data show that lovastatin may induce neuroblast apoptosis by both caspase-dependent and independent pathways. These findings may suggest that the caspase-dependent component leading to the neuroblast cell death is likely to involve the cleavage of focal adhesion proteins and PKCdelta, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin.

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.

PKC-delta is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-delta TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-delta TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-delta TYR-P by activation of the low affinity CCK(A) receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-delta TYR-P but instead, increased it. A23187 did not stimulate PKC-delta TYR-P. Wortmannin and LY 294002 did not alter CCK-stimulated PKC-delta TYR-P. GF 109203X, at low concentrations, increased PKC-delta TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, Gö 6976 and safingol, caused a similar increase in TPA- and CCK-stimulated PKC-delta TYR-P. These results demonstrate that CCK(A) receptor activation causes PKC-delta TYR-P through activation of only one of its two receptor affinity states. This PKC-delta TYR-P is not directly influenced by changes in [Ca(2+)](i); however, the resultant activation of PKC-alpha has an inhibitory effect. Therefore, CCK activates both stimulatory and inhibitory PKC cascades regulating PKC-delta TYR-P and, hence, likely plays an important role in regulating PKC-delta degradation and cellular abundance.

Some of the effects of several oncogenes, integrins, growth factors, and neuropeptides are mediated by tyrosine phosphorylation of the non-receptor tyrosine kinase p125(FAK) and the cytoskeletal protein paxillin. We have demonstrated that different stimuli cause tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The aim of the present study was to determine whether exogenous NO activates this pathway. We demonstrate that in isolated rat pancreatic acini, a NO donor, sodium nitroprusside (SNP) stimulates, in a dose- and time-dependent way, tyrosine phosphorylation of p125(FAK) and paxillin. The same effects could be observed after incubating acini with 8-Br-cGMP. Moreover, the stimulation caused by SNP was completely abolished by two different guanylyl cyclase inhibitors, methylene blue, and LY-83583. These inhibitors also diminished unstimulated phosphorylation of p125(FAK) and paxillin. We conclude that in rat pancreatic acini exogenous NO causes p125(FAK) and paxillin tyrosine phosphorylation that is mediated by a guanylyl cyclase-dependent pathway., (Copyright 2000 Academic Press.)

Increased levels of plasmatic calcitonin (PC) have been found in patients afflicted with liver cirrhosis (LC). Different circulating polypeptides of calcitonin with contrasting molecular weight (MW) have been observed by way of several methods of identification. The aim of our study is identify these types of PC in patients afflicted with LC, using a gel chromatography technique, comparing them with those obtained from patients with thyroid medullar carcinoma (TMC). A first PC peak with MW of 30,000 daltons (D) corresponding to macromolecular types of PC of LC was observed. Furthermore, a second peak of PC, corresponding to a synthetic human calcitonin and to monomers of PC in patients with TMC was noted. This indicates is a slight increase of monomeric PC in patients with LC, although its biological function is unknown.