Serum albumin and HCO3- regulate separate pools of ATP in human spermatozoa.

STUDY QUESTION: Do the known capacitating agents HCO(3)(-) and serum albumin regulate the generation of ATP required for sperm motility and capacitation? SUMMARY ANSWER: Serum albumin and HCO(3)(-) seem to regulate two separate pools of ATP by different mechanisms in human spermatozoa. WHAT IS KNOWN ALREADY: Sperm capacitation is a maturation process that naturally occurs in the female reproductive tract preparing the sperm cell for fertilization. It is a highly energy-depending process as it involves hyperactive motility and substantial levels of protein phosphorylation. STUDY DESIGN, SIZE, DURATION: Human sperm cells from four (motility experiments) and three (all other experiments) healthy donors were used. Untreated cells were compared with cells treated with HCO(3)(-) and serum albumin for up to 4 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in glycolysis and mitochondrial respiration rates upon treatment with serum albumin and HCO(3)(-) were analysed by metabolic tracing of (13)C-labelled substrates and respirometry studies, respectively. Levels of hyperactive spermatozoa and ATP content were measured during 4 h of incubation under capacitating conditions. MAIN RESULTS AND THE ROLE OF CHANCE: We found that HCO(3)(-) significantly (P < 0.05) increased glycolytic flux by >3-folds via a cAMP/PKA sensitive pathway. This was accompanied by an increase in hyperactive motility. In contrast, serum albumin significantly increased endogenous ATP levels by 50% without stimulating hyperactive motility or glycolysis, indicating that this pool of ATP is separately located from the HCO(3)(-)-induced ATP. The increase in ATP induced by albumin could be mimicked by treatment with the cholesterol acceptors 2-hydroxypropyl- and methyl-ß-cyclodextrin and counteracted by co-incubation with cholesterol sulphate to the level of the non-treated control (P < 0.05), pointing to cholesterol extraction from the sperm cell membrane as the main mechanism. However, the concentration of cyclodextrins needed to directly detect cholesterol extraction from the sperm cells was not compatible with maintenance of sperm viability. The increase in ATP seemed not to be dependent on the sperm-specific Ca(2+) channel CatSper. Finally, we demonstrated that neither HCO(3)(-) nor serum albumin stimulated mitochondrial respiration rates. However, serum albumin increased the respiratory capacity of mitochondria by >50%, an effect that was counteracted by HCO(3)(-). LIMITATIONS, REASONS FOR CAUTION: Great variation in motility and capacitation is observed between sperm cells from different species. Hence, caution should be taken when extrapolating the findings in this work on human spermatozoa to sperm from other species. WIDER IMPLICATIONS OF THE FINDINGS: It is already established that an efficient energy-generation is required to support sperm motility and capacitation. However, the mechanisms explaining how ATP production is regulated in spermatozoa are not fully understood. Our findings indicate that HCO(3)(-) stimulates hyperactive motility by increasing glycolytic flux and ATP production in a cAMP/PKA sensitive fashion. On the other hand, serum albumin seems to increase ATP concentration at a different location and by a mechanism different from glycolysis that involves extraction of cholesterol from the sperm cell membrane. These new insights into sperm metabolism may pave the way for both the development of new and improved male contraceptives and optimized assisted reproduction techniques. STUDY FUNDING: The work was funded by Spermatech AS, The University of Oslo and the Research Council of Norway. COMPETING INTEREST(S): T.H.H. and K.R.R. are employees at Spermatech. B.S.S is a shareholder in Spermatech.

Ghrelin is related to lower brain reward activation during touch.

The gut hormone ghrelin drives food motivation and increases food intake, but it is also involved in the anticipation of and response to rewards other than food. This pre-registered study investigated how naturally varying ghrelin concentrations affect the processing of touch as a social reward in humans. Sixty-seven volunteers received slow caressing touch (so-called CT-targeted touch) as a social reward and control touch on their shins during 3T functional imaging on two test days. On one occasion, participants were fasted, and on another, they received a meal. On each occasion, plasma ghrelin was measured at three time points. All touch was rated as more pleasant after the meal, but there was no association between ghrelin concentrations and pleasantness. CT-targeted touch was rated as the most pleasant and activated somatosensory and reward networks (whole brain). A region-of-interest in the right medial orbitofrontal cortex (mOFC) showed lower activation during all touches, the higher the ghrelin concentrations were. During CT-targeted touch, a larger satiety response (ghrelin decrease after the meal) was associated with higher mOFC activation, and this mOFC activation was associated with higher experienced pleasantness. Overall, higher ghrelin concentrations appear to be related to a lower reward value for touch. Ghrelin may reduce the value of social stimuli, such as touch, to promote food search and intake in a state of low energy. This suggests that the role of ghrelin goes beyond assigning value to food reward.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex.

Selective activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase type I (cAKI), but not type II, is sufficient to mediate inhibition of T cell replication induced through the antigen-specific T cell receptor-CD3 (TCR-CD3) complex. Immunocytochemistry and immunoprecipitation studies of the molecular mechanism by which cAKI inhibits TCR-CD3-dependent T cell replication demonstrated that regulatory subunit I alpha, along with its associated kinase activity, translocated to and interacted with the TCR-CD3 complex during T cell activation and capping. Regulatory subunit II alpha did not. When stimulated by cAMP, the cAKI localized to the TCR-CD3 complex may release kinase activity that, through phosphorylation, might uncouple the TCR-CD3 complex from intracellular signaling systems.

Kinetic properties of the C-terminal Src kinase, p50csk.

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu, Tyr) as substrate, phosphotransferase activity was linear for 7-8 min with Mg2+ and 5 min with Mn2+. With Mg2+ and Mn2+, respectively, K(m) (ATP) was 56.9 +/- 6.2 and 5.4 +/- 0.6 microM and Vmax was 293 +/- 52 and 217 +/- 38 pmol phosphate transferred (microgram Csk)-1 min-1. Optimal concentrations of Mg2+ and Mn2+ were 4-10 mM and 2-3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na+, K+) and divalent (Ca2+) cations, the sensitivity being 2-5-fold higher with Mg2+ than Mn2+. Physiological concentrations of Ca2+ (less than 10 microM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 microM strongly inhibited Csk catalytic activity towards poly(Glu, Tyr) in the presence of Mn2+, but not in the presence of Mg2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

Specificity in the cAMP/PKA signaling pathway. Differential expression,regulation, and subcellular localization of subunits of PKA.

A large number of hormones, neurotransmitters and other signal substances utilize adenosine 3',5' cyclic monophosphate (cAMP) as an intracellular second messenger. Cyclic AMP regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the cAMP-dependent protein kinase (PKA). The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP-engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we will describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

Specificity in the cAMP/PKA signaling pathway. differential expression, regulation, and subcellular localization of subunits of PKA.

A large number of hormones, neurotransmitters and other signal substances utilize cyclic adenosine 3 5 cyclic monophosphate (cAMP) as an intracellular second messenger. Cyclic AMP regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription The principle intracellular target for cAMP in mammalian cells is the cAMP-dependent protein kinase (PKA). The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here, we will describe features of PKA signaling pathway that may contribute to explain how differential effects of cAMP may be maintained in this pathway.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may occur.

Cyclic-AMP-dependent protein kinase (PKA) in testicular cells. Cell specific expression, differential regulation and targeting of subunits of PKA.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

PKAI as a potential target for therapeutic intervention.

We have mapped a molecular mechanism for the impaired T-cell function in HIV infection and common variable immunodeficiency (CVI). Protein kinase A type I (PKAI) has a key role as an inhibitor of immune function in T lymphocytes and is activated following antigen receptor triggering. T cells from patients with HIV infection and CVI have increased activation of PKAI. This inhibits immune function and proliferation of T cells. Selective antagonists that block cAMP action through PKAI improve the immune function of T cells from HIV-infected patients up to 300%. Furthermore, combination of cAMP antagonists with interleukin-2 normalized immune responses of T cells from all patients examined and stimulated immune function of T cells from HIV-infected patients up to 600%. In addition, in vitro experiments indicate that approximately 50% of patients with CVI have a T-cell dysfunction that might benefit from a treatment reversing PKAI hyperactivation. This outlines PKAI as a potentially attractive drug target for immunomodulating therapy in HIV infection, as well as for the treatment of other immunodeficiency disorders such as CVI.

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle.

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.

[Genetic modification of the mouse]. / Genetisk skreddersøm av mus.

Genetic modification of mice by homologous recombination has rapidly become a central tool within molecular medicine and molecular biology. The use of such techniques allows precise mutation of single genes and study of the direct consequences in intact animals. A decade has passed since the a series of landmark studies demonstrated the feasibility of genetically modifying embryonic stem cells in mice by homologous recombination. This initiated the whole field of gene targeting, or "knockout" technology in mice. This article reviews briefly the historical and technical development of knockout technology, and the application to selected mouse models within cancer biology, immunology and neurobiology. Refinements of this type of genetic modification, with tissue- or time-specific genetic ablation or mutations, represent the next step in technology development. The combined use of Cre/loxP and homologous recombination has opened for a wide spectrum of possibilities, reaching far beyond null mutations. The rapid evolvement of the whole field has opened for genetic engineering--in the presise sense of the word--in whole animals.

Two novel brain-specific splice variants of the murine Cbeta gene of cAMP-dependent protein kinase.

We have previously characterized two murine cAMP-dependent protein kinase catalytic subunit genes, Calpha and Cbeta1. Targeted disruption of the Cbeta1 promoter revealed two splice variants of the Cbeta catalytic subunit gene (designated Cbeta2 and Cbeta3) that continue to be expressed. These variants arise from unique promoters and are brain-specific. Cbeta2 is expressed in several discrete areas in the limbic system. These include the lateral septum, the bed nucleus of the stria terminalis, the ventral medial hypothalamus, and the amygdala. In the neocortex, expression is highest in cortical areas such as the prefrontal and insular cortex that are associated limbic structures. By contrast, Cbeta1 is most highly expressed in the cortex and hippocampus and is also present in all non-neuronal tissues examined. Cbeta3 is expressed at very low levels with wide distribution throughout the brain. Both the Cbeta2 and Cbeta3 variants are enzymatically active and induce gene expression in transient transfections with a cAMP response element-reporter construct. This activity is inhibited by protein kinase A regulatory subunits, the protein kinase inhibitor, and the chemical inhibitor H-89. We also demonstrate that Cbeta1 is myristoylated at the amino terminus like the Calpha isoform, but neither Cbeta2 nor Cbeta3 is myristoylated. The discrete expression of Cbeta variants in the brain suggests specific functional roles in neuronal signaling.

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Cyclic AMP sensitive signalling by the CD28 marker requires concomitant stimulation by the T-cell antigen receptor (TCR/CD3) complex.

We have previously demonstrated that activation of cAMP-dependent protein kinase (cAK) type I (cAKI, RI alpha 2-C beta 2) mediates the inhibitory effects of cAMP on T-cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T-cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3- and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti-CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T-cell replication. Combined stimulation with anti-CD3 and anti-CD28, which gives a synergistic response in T-cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP-dependent inhibition. When PMA was added in addition to anti-CD3 and anti-CD28, the inhibitory effect of cAMP on both T-cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3-, CD28- and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T-cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T-cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP).

Cellular location and age-dependent changes of the regulatory subunits of cAMP-dependent protein kinase in rat testis.

This study was undertaken to examine the expression and cellular location of the various cAMP-dependent protein kinase (PKA) subunits in different testicular cell types, using cDNA probes, isoenzyme-specific antibodies and activity measurements. Amounts of mRNA and protein were examined in cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), Leydig cell tumours as well as whole testes from rats of various ages. In Sertoli cells, there was a good correlation between the amount of mRNA and the respective immunoreactive proteins. In other types of cell, such as germ cells and Leydig tumour cells, this was not always the case. Large amounts of RII beta mRNA were found in Leydig tumour cells, whereas the amount of immunoreactive protein was low. Furthermore, large amounts of small-sized, germ cell-specific mRNAs for RI alpha (1.7 kb) and RII alpha (2.2 kb) were also found in the developing rat testis after 30 to 40 days of age, but the large amounts of mRNA were only partially reflected at the protein level. Pachytene spermatocytes and round spermatids were practically devoid of both RII alpha and RII beta protein. During spermatid differentiation, there was a decrease in RI alpha and an increase in RII alpha protein. Cell specific distribution of the various PKA subunits in testicular cell types is described. In some types of cell, discrepancies between mRNA and protein were demonstrated, which clearly suggest cell specific differences in translational efficiencies for some of these mRNAs, particularly the small-sized mRNAs for RI alpha and RII alpha in meiotic and post-meiotic germ cells.

Fractionation and identification of metaphase-specific phosphorylated forms of high-mobility-group proteins.

In the present work chromatography on phosphocellulose and blue Sepharose have been used to fractionate the different phosphorylated forms of the low-molecular-mass high-mobility-group (HMG) proteins from metaphase arrested HeLa cells. The proteins in the different fractions from the blue Sepharose column were analysed by acetic acid/urea gel electrophoresis. Aliquots from the same fractions were also treated with alkaline phosphatase and the dephosphorylated and phosphorylated proteins were then compared by electrophoresis to identify the phosphorylated proteins. It was found that HMG 14 consisted of a mixture of an unphosphorylated and two phosphorylated forms, while HMG Y existed as one homogeneous superphosphorylated form. These findings remove previous uncertainty about phosphorylation of HMG Y and HMG 14. The presence of HMG M and phosphorylated forms of HMG 17 was confirmed. Peptide mapping of HMG I and HMG M gave further evidence that HMG M is a superphosphorylated form of HMG I, and it is suggested that the term HMG Im be used instead of HMG M. The results suggested that HMG I and Y from HeLa cells contained at least three and two metaphase-specific phosphate groups respectively, while HMG 14 and 17 both consisted of an unphosphorylated form and two phosphorylated forms. A protein corresponding to HMG Im from HeLa cells was also found to be present in metaphase-arrested human lymphocytes, while HMG I and from two different rodent species seemed to be less phosphorylated then their counterparts from HeLa metaphase cells.