Carmen J. Serrano

T-type Ca2+ channels and α1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction

There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage‐dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the α 1A, α 1B, α 1C, α 1D and α 1E genes in mouse testis and in purified spermatogenic cell populations with RT‐PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only α 1E, and to a minor extent α 1A, are expressed in spermatogenic cells. In agreement with these findings, only T‐type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch‐clamp recordings of pachytene spermatocytes. The results suggest that low‐threshold Ca2+ channels are the dihydropyridine‐sensitive channels involved in the sperm acrosome reaction.

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca2+ has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca2+ in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1–7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca2+ channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca2+‐dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co‐immunolocalized with caveolin‐1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca2+ flux. Furthermore, by using fluorescein‐coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin‐ and Trp‐independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.

Transient receptor potential (TRPC) channels in human sperm: expression, cellular localization and involvement in the regulation of flagellar motility.

Capacitative Ca2+ entry is a process whereby the activation of Ca2+ influx through the plasma membrane is triggered by depletion of intracellular Ca2+ stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca2+ channels. Recent evidence indicates that capacitative Ca2+ entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription‐polymerase chain reaction (RT‐PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer‐assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.

Voltage-dependent Ca2+ channel subunit expression and immunolocalization in mouse spermatogenic cells and sperm

Though voltage‐dependent Ca2+ channels contribute to the orchestratation of sperm differentiation and function, many questions remain concerning their molecular architecture. This study shows that α1A and α1C Ca2+ channel pore‐forming subunits are expressed in spermatogenic cells. In addition, it provides what is to our knowledge the first evidence for the presence of the Ca2+ channel β auxiliary subunits in spermatogenic cells and sperm. Using RT‐PCR we demonstrated the expression of all four known genes encoding the β subunits in spermatogenic cells. Specific antibodies detected three of these proteins in spermatogenic cells and sperm. In spermatogenic cells both α1 and β subunits are diffusely distributed throughout the cytoplasm while in sperm they appear to be regionally localized.

TRPM8, a Versatile Channel in Human Sperm

Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

Susceptibility to Infectious Diseases Based on Antimicrobial Peptide Production

ABSTRACT In the last few years, the great impact of antimicrobial peptides on infectious disease susceptibility and natural resistance has been reported. In some cases, susceptibility to diseases is related to antimicrobial peptide polymorphisms and gene copy numbers, but for the vast majority of infectious diseases, these phenomena need to be elucidated. This review is focused on the current knowledge about susceptibility and resistance conferred by genetic variations in antimicrobial peptide expression in infectious diseases.

Identification of distinct K+ channels in mouse spermatogenic cells and sperm.

Potassium (K+) channels are believed to regulate mammalian sperm acquisition of fertilising capacity. However, the molecular identity of these proteins in sperm has not been elucidated. In this report, using immunoconfocal and electron microscopy we show that a minimum of four different classes of K+ channels (Kv1.1, Kv1.2, Kv3.1 and GIRK1) are present and regionally distributed over the surface of mouse epididymal sperm. In addition, the use of reverse transcription polymerase chain reaction on RNA from mouse spermatogenic cells allowed the amplification of multiple transcripts corresponding to the channels identified by immunocytochemistry. Consistent with this, whole-cell patch-clamp recordings showed the expression of at least two different outwardly rectifying K+ currents in spermatogenic cells.

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti‐microbial peptides in tuberculosis. The cathelin‐related anti‐microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well‐characterized models of latent or chronic infection and progressive disease. The lung of non‐infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post‐infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelecidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis.

Expression and vitamin D-mediated regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy skin and in diabetic foot ulcers

Diabetic foot ulcers (DFUs) are chronic wounds with high matrix metalloproteinase (MMP) activity, and are a frequent complication on diabetics. This work studied the expression of selected MMP and tissue inhibitor of metalloproteinases (TIMP) gene family members in DFU and normal skin biopsies, and in vitamin D-treated keratinocytes cultured from those biopsies. We report for the first time the expression of some of these genes in healthy skin. Our results suggest that vitamin D may modulate the expression of some MMP gene family members in keratinocytes. Gene expression in DFU and in non-diabetic healthy skin (control) biopsies was evaluated by RT-qPCR for MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-19, TIMP-1 and TIMP-2, and also by immunohistochemistry for MMP-1 and MMP-9. Primary keratinocytes cultured from DFU and healthy skin biopsies were used for gene expression analyses of selected MMPs and TIMPs by RT-qPCR, both in the presence and absence of calcitriol. The expression of MMP-1, MMP-8, MMP-9, MMP-10, and TIMP-2 in healthy skin is reported here for the first time. DFUs showed increased MMP-1, MMP-9 and TIMP-1 expression, compared to healthy skin. Calcitriol down-regulated MMP-1 and MMP-10 expression in DFU-derived keratinocytes but not in those derived from healthy skin. Our data demonstrate the expression of certain MMPs that had not been previously described in healthy skin, and further support previous reports of MMP and TIMP up-regulation in DFUs. Our results point to calcitriol as a potential modulator for the expression of certain MMP members in DFUs.

Trifluoroacetylated adducts in spermatozoa, testes, liver and plasma and CYP2E1 induction in rats after subchronic inhalatory exposure to halothane

The induction of cytochrome P450 (CYP) 2E1 in testes and liver and the presence of trifluoroacetylated (TFA) adducts in spermatozoa, testes, liver and plasma were investigated in rats subchronically exposed by inhalation to halothane (15 ppm/4 h/day/5 days/week/9 weeks). After halothane exposure, p-nitrophenol hydroxylase (p-NPH) activity increased 3.2-fold and CYP2E1 apo-protein content 7-fold in testes, whereas in liver, p-NPH increased 2.3-fold and CYP2E1 apoprotein content 1.4-fold. These results suggest a differential inductive effect of halothane on CYP2E1 in these tissues. Moreover, TFA adducts were present in microsomes of testis and liver and in plasma of halothane-treated rats. The immunoblot analysis of testicular microsomes showed two intense TFA protein bands of 63 and 59 kDa, whereas in liver three intense bands of 100, 76 and 63 kDa were observed. Bands of similar molecular weights to those observed in liver were detected in the plasma of halothane-treated animals. In addition, TFA adducts were detected by immunofluorescence in spermatozoa, probably in the acrosome and/or perinuclear theca region, and in the distal tail of spermatozoa. The increase in CYP2E1 apoprotein and p-NPH activity observed in testis and liver microsomes suggests that halothane induces its own biotransformation both hepatically and extrahepatically and in addition, that the nature of the TFA adducts will depend on the proteins present in each tissue. Also, the presence of TFA adducts in spermatozoa may result from the activation of halothane in the reproductive tract. The detailed mechanism of TFA adduct formation and its consequences on the spermatozoa function remain to be fully clarified.