Luis J. Garcia-Marin

Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen.

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.

Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3α

Sperm functions are critically controlled through the phosphorylation state of specific proteins. Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (alpha and beta), the enzyme activity of which is inhibited by serine phosphorylation. Recent studies suggest that GSK3 is involved in the control of bovine sperm motility. Our aim was to investigate whether GSK3 is present in porcine spermatozoa and its role in the function of these cells. This work shows that both isoforms of GSK3 are present in whole cell lysates of porcine sperm and are phosphorylated on serine in spermatozoa stimulated with the cAMP analog, 8Br-cAMP. A parallel increase in serine phosphorylation of the isoform GSK3alpha, but not in the isoform GSK3beta, is observed after treatments that also induce a significant increase in porcine sperm velocity parameters. Therefore, a significant positive correlation among straight-line velocity, circular velocity, average velocity, rapid-speed spermatozoa, and GSK3alpha serine phosphorylation levels exists. Inhibition of GSK3 activity by alsterpaullone leads to a significant increase in the percentage of rapid- and medium-speed spermatozoa as well as in all sperm velocity parameters and coefficients. Moreover, pretreatment of porcine spermatozoa with alsterpaullone significantly increased the percentage of capacitated porcine spermatozoa and presents no effect in the number of acrosome-reacted porcine spermatozoa. Our work suggests that the isoform GSK3alpha plays a negative role in the regulation of porcine sperm motility and points out the possibility that sperm motile quality might be modulated according the activity state of GSK3alpha.

The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C

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.

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

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.

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 Ca2+ 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 Ca2+ 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.

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.

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‐3α (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. J. Cell. Biochem. 109: 65–73, 2010.

Cholecystokinin-stimulated tyrosine phosphorylation of PKC-δ in pancreatic acinar cells is regulated bidirectionally by PKC activation

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.

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.

Lovastatin inhibits the growth and survival pathway of phosphoinositide 3-kinase/protein kinase B in immortalized rat brain neuroblasts.

We previously showed that lovastatin, an HMG‐CoA reductase inhibitor, suppresses cell growth by inducing apoptosis in rat brain neuroblasts. Our aim was to study intracellular signalling induced by lovastatin in neuroblasts. Lovastatin significantly decreases the phosphoinositide 3‐kinase (PI3‐K) activity in a concentration‐dependent manner. Expression of p85 subunit and its association with phosphotyrosine‐containing proteins are unaffected by lovastatin. Lovastatin decreases protein kinase B (PKB)/Akt phosphorylation, and its downstream effectors, p70S6K and the eukaryotic initiation factor 4E (eIF4E) regulatory protein 1, 4E‐BP1, in a concentration‐dependent manner, and reduces p70S6K expression. Lovastatin effects are fully prevented with mevalonate. Only the highest dose of PI3‐K inhibitors that significantly reduce PI3‐K kinase activity induces apoptosis in neuroblasts but to a lower degree than lovastatin. In summary, this work shows that treatment of brain neuroblasts with lovastatin leads to an inhibition of the main pathway that controls cell growth and survival, PI3‐K/PKB and the subsequent blockade of downstream proteins implicated in the regulation of protein synthesis. This work suggests that inactivation of the antiapoptotic PI3‐K appears insufficient to induce the degree of neuroblasts apoptosis provoked by lovastatin, which must necessarily involve other intracellular pathways. These findings might contribute to elucidate the molecular mechanisms of some statins effects in the central nervous system.