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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. British Journal of Pharmacology (2003) 138, 351–358. doi:10.1038/sj.bjp.0705039

Objective To investigate the frequency and spectrum of lipophilic Malassezia yeasts within the area of the glans penis and preputial space in healthy men, most of whom were uncircumcised. Subjects and methods As the glans penis and the prepuce are abundant in sebaceous glands, 130 clinically healthy men were examined for the presence of Malassezia spp. Two impression preparations each were made on modified Dixon (mDixon) and Leeming-Notman agar. The media were used with no added cycloheximide to allow the identification of the full spectrum of Candida yeasts. After 10 days of incubation at 32 °C Malassezia and Candida yeasts were differentiated by micromorphological and biochemical criteria. ResultsMalassezia spp. were found as part of the microflora of the glans penis in 49% of the men. The two media yielded comparable results for isolation frequency and spectrum: On mDixon agar, 47.6% of the isolates were identified as M. sympodialis, 42.8% as M. globosa, 4.8% as M. pachydermatis and 2.4% as M. furfur. Candida spp. (especially C. albicans) were found in 20.8%. There were no significant differences in the distribution of different Malassezia spp with age. ConclusionsMalassezia yeasts may belong to the resident microflora of the male genital region, which is supported by the finding that free sebaceous glands (‘Tyson's glands') normally occur at this site. They may also be involved in pathological processes of the glans penis, e.g. balanitis, seborrheic eczema or psoriasis.

A 63-year-old patient, known to have suffered from lymphogranulomatosis X for 4 years is reported, in whom a cutaneous cryptococcosis appeared as first sign of a disseminated cryptococcosis. Despite systemic therapy with amphotericin B, the patient died after a period of 2 months. Differential diagnosis of skin tumours of immunosuppressed patients includes rare skin mycoses, and both histological and mycological examinations should be performed.

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein alpha-subunits, and A 86, which detects all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein alpha-subunits failed to detect any specific antigens in enriched tail membranes. AS 36, recognizing the beta 2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive alpha-subunits. However, membrane preparations of nonpurified human spermatozoa contained alpha i2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein alpha-subunits; the putative beta-subunit was identified as a beta 2-subunit.

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4°C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.