Gunther Wennemuth

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation.

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO3- and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25°C. Local perfusion with HCO3- produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO3- begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8±0.2 seconds at 15 mM HCO3- and 17.5±0.4 seconds at 1 mM HCO3-. Removal of external HCO3- allows a slow return to basal beat frequency over ∼10 minutes. Increases in beat symmetry accompany the accelerating action of HCO3-. As in our past work, HCO3- also facilitates opening of voltagegated Ca2+ channels, increasing the depolarization-evoked rate of rise of intracellular Ca2+ concentration by more than fivefold. This action also is detectable at 1 mM HCO3- and occurs with an apparent halftime of ∼60 seconds at 15 mM HCO3-. The dual actions of HCO3- respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO3- on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 μM cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca2+ concentration from 17±4 to 41±6 nM second-1. However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO3-, perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO3- produces during sperm activation.

Calcium Clearance Mechanisms of Mouse Sperm

The spermatozoon is specialized for a single vital role in fertilization. Past studies show that Ca2+ signals produced by the opening of plasma membrane entry channels initiate several events required for the sperm to reach and enter the egg but reveal little about how resting [Ca2+]i is maintained or restored after elevation. We examined these homeostatic mechanisms by monitoring the kinetics of recovery from depolarizing stimuli under conditions intended to inhibit candidate mechanisms for sequestration or extrusion of Ca2+ from the cytosol. We found that the Ca2+-ATPase pump of the plasma membrane performs the major task of Ca2+ clearance. It is essential in the final stages of recovery to achieve a low resting [Ca2+]i. With immunomethods we found a ∼130-kD plasma membrane Ca2+-ATPase protein on Western blots of whole sperm extracts and showed immunolocalization to the proximal principal piece of the flagellum. The plasma membrane Na+-Ca2+ exchanger also exports Ca2+ when [Ca2+]i is elevated. Simultaneous inhibition of both mechanisms of extrusion revealed an additional contribution to clearance from a CCCP-sensitive component, presumably sequestration by the mitochondria. Involvement of SERCA pumps was not clearly detected. Many aspects of the kinetics of Ca2+ clearance observed in the presence and absence of inhibitors were reproduced in a mathematical model based on known and assumed kinetic parameters. The model predicts that when cytosolic [Ca2+] is at 1 μM, the rates of removal by the Ca2+-ATPase, Na+-Ca2+-exchanger, mitochondrial uniporter, and SERCA pump are ∼1.0, 0.35, 0.33, and 0 μmole l−1 s−1, rates substantially slower than those reported for other cells studied by similar methods. According to the model, the Na+-Ca2+ exchanger is poised so that it may run in reverse at resting [Ca2+]i levels. We conclude that the essential functions of sperm do not require the ability to recover rapidly from globally elevated cytosolic [Ca2+].

Release of Macrophage Migration Inhibitory Factor and CXCL8/Interleukin-8 from Lung Epithelial Cells Rendered Necrotic by Influenza A Virus Infection

ABSTRACT Bronchiolar epithelial cells are the prime targets for influenza A virus infection. It still remains to be clarified which signals are generated from these cells to initiate an immune response. Among chemokines, viral infection of primary lung epithelial cells triggered exclusively the release of CXCL8/interleukin-8 (IL-8), which contrasts with our previous observation that influenza A virus induced in monocytes the expression of mononuclear-leukocyte-attracting chemokines and even suppressed the production of neutrophil-attracting chemokines. Therefore, we speculated that it may be advantageous for respiratory epithelial cells to release primarily neutrophil-attracting CXCL8/IL-8 since neutrophils rapidly remove necrotic debris and are the first line of defense against bacterial superinfections. This concept has also been supported by our finding that influenza A virus infection led to necrosis of lung epithelial cells. This is in striking contrast to previous studies where influenza A virus infection induced apoptosis in monocytes and epithelial cells from origins other than the lung. Thus, the cell type instead of the virus determines which death pathway will be followed. In addition to the release of CXCL8/IL-8, we obtained a massive release of macrophage migration inhibitory factor (MIF) from virus-infected lung cells. However, whereas the CXCL8/IL-8 secretion was accompanied by induced gene activation, the transcription rate of MIF remained unchanged during the infection course and the virus-induced MIF release was predominantly a discharge from intracellular stores, suggesting that MIF is passively released upon cell death. Despite virus induced necrosis, the passively liberated MIF remained bioactive. Considering the well-established immunostimulatory effects of MIF on different leukocyte subsets, is its very likely that enhanced levels of MIF may contribute to the host immune response during the acute phase of influenza A virus infection in humans.

Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread

Abstract Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

Angiotensin II-mediated calcium signals and mitogenesis in human prostate stromal cell line hPCPs

1 Western blots and immunocytochemistry were used to detect angiotensin 1 (AT1) and angiotensin 2 (AT2) receptors in human primary cultures of the prostate stromal compartment (hPCPs). Immunohistochemistry was performed on human prostate tissue‐embedded paraffin. In addition, pharmacological tools were applied in combination with photometry experiments to characterize the physiological activity of AT1 and AT2 receptors in hPCPs cell culture. A proliferation assay was used to describe the mitogenic activity of angiotensin II (Ang II) on hPCPs cells. 2 Only the AT1 receptor was detected in Western blot analysis. Immunocytochemistry of hPCPs cells showed that the AT1 receptor is present in both the smooth muscle type and the fibroblastic type. In the stromal compartment of human prostate tissue, immunoreaction with antibodies against the AT1 receptor was detectable. 3 Fura‐2‐loaded hPCPs cells showed an instantaneous and linear rise in free intracellular calcium ion concentration ([Ca2+]i) after local perfusion with Ang II in concentrations of 10u2003nM. Removing of external calcium or emptying intracellular calcium stores before Ang II application diminished or abolished this [Ca2+]i response. 4 The response to Ang II was also diminished when hPCPs cells were perfused with the AT1 receptor inhibitor losartan prior to Ang II application. No inhibition of the [Ca2+]i increase was detectable after perfusion with PD 123319, a specific inhibitor of the AT2 receptor. 5 hPCPs cells were stimulated with Ang II in various concentrations over a period of 2 days. The subsequently performed proliferation assay revealed a mitogenic effect of Ang II on hPCPs in concentrations starting at 10u2003nM. This effect could be inhibited by losartan.

Macrophage Migration Inhibitory Factor-Induced Ca2+ Response in Rat Testicular Peritubular Cells

Abstract Macrophage migration inhibitory factor (MIF), originally described as a T-cell product, has recently been identified in several endocrine organs. In the rat testis, MIF is secreted by the Leydig cells into testicular interstitial fluid that directly contacts Sertoli and peritubular cells. To investigate whether MIF is involved in calcium-dependent signal transduction, we have isolated rat Sertoli and peritubular cells. Despite progress in understanding functional properties of MIF, the molecular mechanism of MIF action in target cells is almost completely unknown. Here we find that recombinant MIF evokes a transient increase in calcium levels in peritubular cells but not in Sertoli cells from dissociated rat testis. Concentrations in the range between 12.5 ng/ml and 120 ng/ml of recombinant MIF were found to be effective, with 50 ng/ml yielding the largest increase in intracellular calcium. Preincubation of MIF with a neutralizing monoclonal antibody specifically blocked the response. Incubation of the peritubular cells in calcium-free buffer clearly decreased the evoked response in intracellular calcium concentration. However, the calcium response was greatly decreased by thapsigargin, an inhibitor of the Ca2+ ATPase of the endoplasmic reticulum. The results strongly indicate that calcium is mobilized from reticulum stores during MIF-mediated signal transduction in the testis. In conclusion, our results provide the first characterization of MIF signal transduction in the testis and suggest that signaling from Leydig cells to peritubular cells through MIF is mediated by receptors coupled to release of intracellular calcium.

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

Characterization of the Olfactory Receptors Expressed in Human Spermatozoa.

The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa.

Distribution of endogenous and exogenous 5'-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5′-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5′-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.

Bradykinin increases intracellular calcium levels in rat testis peritubular cells via the B2 receptor subtype

RT–PCR and Western blots were used to detect bradykinin B2 receptors in testis and isolated peritubular cells of pre‐pubertal rats. RT–PCR demonstrated expression of a single transcript, whereas Western blots showed up to three specific bands that were in accordance with the described native, glycosylated and dimeric form of B2 receptor proteins, respectively. Fura‐2‐loaded peritubular cells responded with an instantaneous, linear and transient rise in [Ca2+]i after adding bradykinin. Stimulation of cells with bradykinin concentrations between 1 μM and 1 pM showed a dose dependent increase of [Ca2+]i. The calcium response to bradykinin was diminished after stimulation of peritubular cells in calcium‐free buffer. After blocking the SERCA‐pumps by thapsigargin and subsequent stimulation with bradykinin, no rise of [Ca2+]i was appreciated. Multiple stimulation of a single peritubular cell by local perfusion with a brief addition of BK (10 nM) resulted in a fast and immediate response. However, the second and third stimuli had slower rise rates and diminished [Ca2+]i peaks, showing desensitization of the kinin receptor. Addition of the bradykinin B1 receptor agonist [des‐Arg9]‐bradykinin (100 nM) to Fura‐2‐loaded peritubular cells did not change the [Ca2+]i. However, the B2 receptor antagonist HOE 140 (100 nM) strongly inhibited the bradykinin‐induced calcium response. We conclude that the bradykinin‐induced increase in [Ca2+]i, in testicular peritubular cells is mediated by the stimulation of kinin receptors of the B2 subtype.