Quantitative Analysis of the Human Semen Phosphorometabolome by 31P-NMR.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

Bisphenol S Reduces Pig Spermatozoa Motility through Different Intracellular Pathways and Mechanisms than Its Analog Bisphenol A.

Bisphenol A (BPA: 2,3-bis (4-hydroxyphenyl) propane) is an environmental chemical widely used in the manufacturing of epoxy polymers and many thermoplastic consumer products. Serious concerns about its safety led to the development of analogs, such as BPS (4-hydroxyphenyl sulfone). Very limited studies about BPS's impact on reproduction, specifically in spermatozoa, exist in comparison with BPA. Therefore, this work aims to study the in vitro impact of BPS in pig spermatozoa in comparison with BPA, focusing on sperm motility, intracellular signaling pathways and functional sperm parameters. We have used porcine spermatozoa as an optimal and validated in vitro cell model to investigate sperm toxicity. Pig spermatozoa were exposed to 1 and 100 µM BPS or BPA for 3 and 20 h. Both bisphenol S and A (100 µM) significantly reduce pig sperm motility in a time-dependent manner, although BPS exerts a lower and slower effect than BPA. Moreover, BPS (100 µM, 20 h) causes a significant increase in the mitochondrial reactive species, whereas it does not affect sperm viability, mitochondrial membrane potential, cell reactive oxygen species, GSK3α/ß phosphorylation or phosphorylation of PKA substrates. However, BPA (100 µM, 20 h) leads to a decrease in sperm viability, mitochondrial membrane potential, GSK3ß phosphorylation and PKA phosphorylation, also causing an increase in cell reactive oxygen species and mitochondrial reactive species. These intracellular effects and signaling pathways inhibited might contribute to explaining the BPA-triggered reduction in pig sperm motility. However, the intracellular pathways and mechanisms triggered by BPS are different, and the BPS-caused reduction in motility can be only partially attributed to an increase in mitochondrial oxidant species.

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa.

RESEARCH QUESTION: Does sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process? DESIGN: Human spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge. RESULTS: SIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca2+ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237. CONCLUSION: YK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.

The adverse impact of herbicide Roundup Ultra Plus in human spermatozoa plasma membrane is caused by its surfactant.

The scarce research about the worldwide used glyphosate-based herbicide Roundup is controversial in human reproduction, especially spermatozoa. This study investigates the in vitro effect in human spermatozoa of Roundup Ultra Plus (RUP), its active ingredient glyphosate and its non-active, surfactant. Human spermatozoa were incubated (1 h, 37 °C) in presence/absence of RUP 0.01%, glyphosate, or equivalent surfactant concentration. Motility and sperm parameters were analyzed by C.A.S.A and flow cytometry, respectively. RUP significantly increases sperm plasma membrane lipid disorganization in a concentration-dependent manner while it decreases plasma membrane integrity. RUP significantly increases the death spermatozoa population after A23187-induced acrosome reaction, without affecting sperm viability, mitochondrial membrane potential, ROS content, acrosome membrane damage, phosphatidylserine exposure, A23187-induced acrosome reaction or GSK3 phosphorylation. RUP also significantly decreases motile and the a + b sperm populations. Interestingly, all sperm effects caused by RUP 0.01% are mimicked by its surfactant POEA at equivalent concentration. However, glyphosate does not affect any sperm parameter, even using 10-times higher concentration than the RUP 0.01% equivalent. RUP disturbs lipid organization and integrity of human sperm plasma membrane and reduces motility, without affecting viability or functional parameters. Importantly, RUP adverse effects in human spermatozoa are caused by the surfactant and no by glyphosate.

Sperm Phosphoproteome: Unraveling Male Infertility.

Infertility affects approximately 15% of couples worldwide of childbearing age, and in many cases the etiology of male infertility is unknown. The current standard evaluation of semen is insufficient to establish an accurate diagnosis. Proteomics techniques, such as phosphoproteomics, applied in this field are a powerful tool to understand the mechanisms that regulate sperm functions such as motility, which is essential for successful fertilization. Among the post-translational modifications of sperm proteins, this review summarizes, from a proteomic perspective, the updated knowledge of protein phosphorylation, in human spermatozoa, as a relevant molecular mechanism involved in the regulation of sperm physiology. Specifically, the role of sperm protein phosphorylation in motility and, consequently, in sperm quality is highlighted. Additionally, through the analysis of published comparative phosphoproteomic studies, some candidate human sperm phosphoproteins associated with low sperm motility are proposed. Despite the remarkable advances in phosphoproteomics technologies, the relatively low number of studies performed in human spermatozoa suggests that phosphoproteomics has not been applied to its full potential in studying male infertility yet. Therefore, further studies will improve the application of this procedure and overcome the limitations, increasing the understanding of regulatory mechanisms underlying protein phosphorylation in sperm motility and, consequently, in male fertility.

Impaired mammalian sperm function and lower phosphorylation signaling caused by the herbicide Roundup® Ultra Plus are due to its surfactant component.

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3ß phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3ß phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3ß and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress.

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.

Human sperm phosphoproteome reveals differential phosphoprotein signatures that regulate human sperm motility.

Human sperm motility is essential for fertilization and among pathologies underlying male infertility is asthenozoospermia. Nevertheless, mechanisms regulating sperm motility are not completely unraveled. This work investigates phosphoproteins underlying human sperm motility by using differential phosphoproteomic in two human sperm subpopulations: high (HM) and low (LM) motility, obtained by centrifugation in a density gradient. Phosphoproteomics (HPLC-MS/MS triple TOF), comparing human LM and HM phosphoproteomes, identified 210 phosphopeptides with different abundance that correspond with 119 sperm proteins. Analysis showed that 40% of phosphoproteins in LM spermatozoa are involved in metabolism, (catabolism, protein transport, lipid biosynthesis), 25% in spermatogenesis and sperm function, 8% in immune system and 6% in DNA repair. In HM spermatozoa, 48% of phosphoproteins are related to spermatogenesis and sperm function (motility), whereas 8% are associated to metabolism. GSK3α resulted one of the most abundant phosphoproteins in HM spermatozoa. Western blot confirmed that GSK3α phosphorylation is higher in HM spermatozoa. Summarizing, this study i) identified phosphoproteins in two human spermatozoa populations, ii) supports that human spermatozoa rely in protein phosphorylation, such as GSK3 α, to regulate sperm motility, iv) raises the challenge of using some identified human sperm phosphorylated proteins (GSK3α) as targets to develop into clinically relevant biomarkers. SIGNIFICANCE: Human sperm phosphoproteome analyzed by nano HPLC-MS/MS triple TOF identifies the differential abundance of sperm phosphoproteins in two human sperm populations exhibiting high motility (HM) and/or low motility (LM) that were isolated from normozoospermic healthy donors. Majority of human phosphoproteins found in LM spermatozoa are involved in sperm metabolism (40%), whereas those in HM spermatozoa are associated to spermatogenesis and sperm function, as motility (48%), and only 8% are associated to metabolism. One of the most abundant phosphoproteins found in HM spermatozoa is GSK3α, kinase directly involved in the regulation of sperm motility that was also validated by western blot. The biological relevance of this study is based in the fact that supports that mature human sperm cells rely in protein phosphorylation to efficiently regulate sperm motility and allows identifying those regulatory human sperm phosphoproteins. This work will clearly impacts the human reproductive field as it raises the challenge of consider identified human sperm phosphoproteins, such as GSK3α, as potential biological targets to develop into relevant biomarkers for the human clinic or assisted reproductive technology.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

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.

Boar sperm hyperactivated motility is induced by temperature via an intracellular calcium-dependent pathway.

Herein we describe a new protocol to induce boar sperm hypermotility: temperature-induced hypermotility (TIH). Briefly, spermatozoa stored at 17°C in a calcium-free Tyrode's basal medium (containing EGTA) were exposed to increased temperature by incubation at 38.5°C. Hypermotility induced by the calcium ionophore A23187 was used as a control (calcium ionophore-induced hyperactivity (CIIH)). The increase in temperature led to an increase in the percentage of hypermotile spermatozoa. When the slope of the temperature increase is near zero, sperm hyperactivity becomes a more progressive movement. Motility parameters of sperm hyperactivation induced by TIH were different from those following CIIH. Cluster analysis revealed that these two populations of hyperactivated spermatozoa are different. TIH is independent of extracellular Ca2+ but dependent on intracellular Ca2+ release. Moreover, TIH is unaffected by protein kinase A (PKA) inhibition, whereas CIIH is reduced by half in the presence of a PKA inhibitor. In conclusion, we have demonstrated that: (1) a temperature increase in boar spermatozoa is a stimulus that can induce a hyperactive population, which is differs from the hyperactive sperm population induced by calcium ionophore; (2) the temperature increase in spermatozoa triggers the release of Ca2+ from intracellular stores; (3) extracellular calcium is not required for TIH; and (4) TIH in boar spermatozoa is independent of PKA activity.

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

The calcium/CaMKKalpha/beta and the cAMP/PKA pathways are essential upstream regulators of AMPK activity in boar spermatozoa.

Spermatozoa successfully fertilize oocytes depending on cell energy-sensitive processes. We recently showed that the cell energy sensor, the AMP-activated protein kinase (AMPK), plays a relevant role in spermatozoa by regulating motility as well as plasma membrane organization and acrosomal integrity, and contributes to the maintenance of mitochondrial membrane potential. As the signaling pathways that control AMPK activity have been studied exclusively in somatic cells, our aim is to investigate the intracellular pathways that regulate AMPK phosphorylation at Thr(172) (activity) in male germ cells. Boar spermatozoa were incubated under different conditions in the presence or absence of Ca(2+), 8Br-cAMP, IBMX, PMA, the AMPK activator A769662, or inhibitors of PKA, PKC, or CaMKKalpha/beta. AMPK phosphorylation was evaluated by Western blot using anti-phospho-Thr(172)-AMPK antibody. Data show that AMPK phosphorylation in spermatozoa is potently stimulated by an elevation of cAMP levels through the activation of PKA, as the PKA inhibitor H89 blocks phospho-Thr(172)-AMPK. Another mechanism to potently activate AMPK is Ca(2+) that acts through two pathways, PKA (blocked by H89) and CaMKKalpha/beta (blocked by STO-609). Moreover, phospho-Thr(172)-AMPK levels greatly increased upon PKC activation induced by PMA, and the PKC inhibitor Ro-32-0432 inhibits TCM-induced AMPK activation. Different stimuli considered as cell stresses (rotenone, cyanide, sorbitol, and complete absence of intracellular Ca(2+) by BAPTA-AM) also cause AMPK phosphorylation in spermatozoa. In summary, AMPK activity in boar spermatozoa is regulated upstream by different kinases, such as PKA, CaMKKalpha/beta, and PKC, as well as by the essential intracellular messengers for spermatozoan function, Ca(2+) and cAMP levels.

Adenosine monophosphate-activated kinase, AMPK, is involved in the maintenance of the quality of extended boar semen during long-term storage.

Boar semen preservation for later use in artificial insemination is performed by diluting semen in an appropriate medium and then lowering the temperature to decrease spermatozoa metabolism. The adenosine monophosphate-activated kinase, AMPK, is a key cell energy sensor that controls cell metabolism and recently has been identified in boar spermatozoa. Our aim was to investigate the role of AMPK in spermatozoa functional parameters including motility, mitochondrial membrane potential, plasma membrane integrity, acrosome integrity, and cell viability during long-term boar semen storage at 17 °C in Beltsville thawing solution. Boar seminal doses were diluted in Beltsville thawing solution in the presence or absence of different concentrations of AMPK inhibitor, compound C (1, 10, and 30 µM) and evaluations were performed at 1, 2, 4, 7, or 10 days. Data demonstrate that AMPK becomes phosphorylated at threonine(172) (active) during storage of boar semen reaching maximum levels at Day 7. Moreover, AMPK inhibition during boar semen storage causes: (1) a potent inhibition of spermatozoa motility; (2) a reduction in the percentage of spermatozoa showing high mitochondria membrane potential; (3) a rise in the percentage of spermatozoa displaying high plasma membrane scrambling; and (4) a loss of acrosomal membrane integrity. Our study suggests that AMPK activity plays an important role in the maintenance of the spermatozoa quality during long-term storage of boar semen.

AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa.

Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein kinase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (∆Ψm), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30µM), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPKα protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr(172)-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa. Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high ∆Ψm. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa. This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function.

AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility.

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca(2+) and bicarbonate TCM, time from 1-24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca(2+) and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.

Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa.

During the capacitation process, spermatozoa acquire the ability to fertilize an oocyte, and upregulation of cAMP-dependent protein tyrosine phosphorylation occurs. Recently, Src family tyrosine kinase (SFK) has been involved in spermatozoa capacitation as a key PKA-dependent tyrosine kinase in several species. This work investigates the expression and role of SFK in porcine spermatozoa. SFK members Lyn and Yes are identified in porcine spermatozoa by western blotting as well as two proteins named SFK1 and SFK2 were also detected by their tyrosine 416 phosphorylation, a key residue for SFK activation. Spermatozoa with SFK1 and SFK2 increase their Y416 phosphorylation time-dependently under capacitating conditions compared with noncapacitating conditions. The specific SFK inhibitor SU6656 unaffected porcine spermatozoa motility or viability. Moreover, SFK inhibition in spermatozoa under capacitating conditions leads to a twofold increase in both nonstimulated and calcium-induced acrosome reaction. Our data show that capacitating conditions lead to a time-dependent increase in actin polymerization in boar spermatozoa and that long-term incubation with SFK inhibitor causes a reduction in the F-actin content. In summary, this work shows that the SFK members Lyn and Yes are expressed in porcine spermatozoa and that SFK1 and SFK2 are phosphorylated (activated) during capacitation. Our results point out the important role exerted by SFK in the acrosome reaction, likely mediated in part by its involvement in the actin polymerization process that accompanies capacitation, and rule out its involvement in porcine spermatozoa motility.

Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase-3A serine 21 phosphorylation in boar spermatozoa.

The cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase-3alpha (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase-resistant cell permeable cAMP analogue 8Br-cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro-32-0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM- or 8Br-cAMP-stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility.

CCK1 and 2 receptors are expressed in immortalized rat brain neuroblasts: intracellular signals after cholecystokinin stimulation.

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system (CNS) where it promotes important functions by activation of receptors CCK1 and CCK2. Our aim was to investigate CCK receptors expression and their downstream intracellular signaling in immortalized rat brain neuroblasts. Results show that CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein are expressed in neuroblasts. CCK incubation of neuroblasts leads to stimulation in a time-dependent manner of several signaling pathways, such as tyrosine phosphorylation of adaptor proteins paxillin and p130(Cas), phosphorylation of p44/p42 ERKs as well as PKB (Ser473). Moreover, CCK-8 stimulates the DNA-binding activity of the transcription factor AP-1. The CCK2 receptor agonist gastrin stimulates ERK1/2 phosphorylation in a comparable degree as CCK does. ERK1/2 phosphorylation activated by CCK-8 was markedly inhibited by the CCK2 receptor antagonist CR2945. Incubation for 48 h with CCK-8 increases neuroblasts viability in a similar degree as EGF. In summary, our data clearly identify CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein in brain neuroblasts and show that incubation with CCK promotes cell proliferation and activates the phosphorylation of survival transduction pathways. Stimulation of ERK1/2 phosphorylation by CCK is mainly mediated by the CCK2 receptor. Moreover, this work might provide a novel model of proliferating neuronal cells to further study the biochemical mechanisms by which the neuropeptide CCK exerts its actions in the CNS.

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules.

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca(2+)-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca(2+)](i) decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK(A) receptor states. CCK stimulated an association of Lyn with PKC-delta, Shc, p125(FAK) and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130(CAS) or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125(FAK), PYK2, PKC-delta and Shc, which play central roles in CCK's effects on acinar cell function.

Adapter protein CRKII signaling is involved in the rat pancreatic acini response to reactive oxygen species.

Recent studies demonstrate that reactive oxygen species (ROS) are important mediators of acute pancreatitis, whether induced experimentally or in necrotizing pancreatitis in humans; however, the cellular processes involved remain unclear. Adapter protein CrkII, plays a central role for convergence of cellular signals from different stimuli. Cholecystokinin (CCK), which induces pancreatitis, stimulates CrkII tyrosine phosphorylation and CrkII protein complexes, raising the possibility it can be important in the acinar cell responses to ROS. Therefore, our aim was to investigate whether CrkII signaling is involved in the biological response of rat pancreatic acini to H2O2 and the intracellular mediators implicated. Treatment of isolated rat pancreatic acini with H2O2 rapidly stimulates CrkII phosphorylation, measured as electrophoretic mobility shift and by using a phosphospecific antibody (pTyr221). Tyrosine kinase blocker B44 inhibits the higher phosphorylation state, demonstrating that it occurs mainly in tyrosine residues. H2O2-induced CrkII phosphorylation is time- and concentration-dependent, showing maximal effect with 3 mM H2O2 at 5 min. The intracellular pathways induced by H2O2 leading to CrkII tyrosine phosphorylation do not involve PKC, intracellular calcium, PI3-K or the actin cytoskeleton integrity. ROS generation clearly promotes the formation of protein complex CrkII-PYK2. In conclusion, ROS clearly affect the key adapter protein CrkII signaling by two ways: stimulation of CkII phosphorylation and a functional consequence: formation of CrkII-protein complexes. Because of its central role in activating more distal pathways, CrkII might likely play an important role in the ability of ROS to induce pancreatic cellular injury and pancreatitis.