Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a "Sertoli cell only" phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.

The human steroid sulfatase gene (STS) is located on the distal X chromosome short arm close to the pseudoautosomal region but in a segment ofDNA that is unique to the X chromosome. In contrast to most X chromosomeencoded genes, STS expression is not extinguished during the process of X chromosome inactivation. Deficiency of STS (steryl-sulfatase; steryl-sulfate sulfohydrolase, EC 3.1.6.2) activity produces the syndrome of X chromosome-linked ichthyosis, which is one of the most common inborn errors of metabolism in man. Approximately 90% of STS-individuals have large deletions at the STS locus. We and others have found that the end points of such deletions are heterogeneous in their location. One recently ascertained subject was observed to have a 40-kilobase deletion that is entirely intragenic, permitting the cloning and sequencing of the deletion junction. Studies of this patient and of other X chromosome sequences in other subjects permit some insight into the mechanism(s) responsible for generating frequent deletions on the short arm of the X chromosome.

We hypothesized that macrophage activation results in nitric oxide (NO) production and that this NO acts directly on Leydig cells (LC) to alter androgen synthesis. Both peritoneal macrophages and a murine macrophage cell line (RAW 264.7) were activated in vitro by sequential exposure to interferon-gamma (50 U/ml) and then bacterial lipopolysaccharide (LPS; 100 ng/ml) for 24 h each. At various times after initiation of activation, selected wells were harvested for identification of messenger RNA for inducible NO synthase by RT-PCR. Amplicons of the predicted 651-bp product were isolated, cloned, and sequenced to validate the PCR procedure. Such amplicons first appeared between 2-4 h after exposure to LPS, and staining increased in intensity for the rest of the study. Nitrite accumulation followed a similar time course. Similarly treated wells were washed after 24-h activation and cocultured with purified LC for a final 24-h incubation in the absence of interferon-gamma and LPS. Basal and LH-stimulated production of androgen was estimated by RIA. In some experiments the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) was added during activation and coculture. Coculture of LC with quiescent macrophages altered neither basal nor LH-stimulated androgen production. Coculture with either type of activated macrophage did not alter basal, but significantly reduced (by 50%) LH-stimulated, androgen production. N(omega)-Nitro-L-arginine methyl ester and C-PTIO blocked the inhibitory effect. The NO donor S-nitroso-N-acetyl penicillamine at concentrations greater than 10(-5) M significantly inhibited LH-stimulated androgen production by purified LC (P < 0.01). The inhibitory effect of S-nitroso-N-acetyl penicillamine was evident when exposure exceeded 4 h. Intermediates of steroidogenesis were added to elucidate the site of NO inhibition. The enzymatic inhibition occurred at least in part at 17alpha-hydroxylase/C(17/20) lyase (P450c17). Enzyme inhibition was reversed by C-PTIO. Northern blot analysis indicated that accumulation of messenger RNA for P450c17 was not significantly altered. Therefore, activation of macrophages results in decreased androgen production by cocultured LC. The inhibition is mediated in part by macrophage-derived NO acting directly on the LC via inhibition of at least one of the P450 steroidogenic enzymes.

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Purpose: Circulating tumor cells (CTC) have been recently accepted by the Food and Drug Administration of the United States as a prognostic tool in advanced prostate cancer. However, a number of questions remain about the use of the test. The optimal clinical cut-off has never been determined. Also, the predictive value of CTCs in the setting of low-burden advanced prostate cancer has not been evaluated. Herein we describe our experience with the CellSearch method of CTC enumeration. Experimental Design: CTCs enumerated from 100 patients with castration-resistant prostate cancer were correlated with clinicopathologic characteristics and conventional biomarkers, such as prostatespecific antigen and lactate dehydrogenase. Patients received ongoing medical oncologic follow-up for up to 26 months, and overall survival status was documented.Results: Forty-nine of the patients (49%) were alive at the end of the study. CTC counts correlate well with overall survival (P < 0.001) but are also tightly interrelated to other biomarkers. Threshold analysis identified 4 CTC/7.5 cc (compared with the approved value of 5) as an optimal cut-off value with respect to correlation with survival outcomes as well as predictive of metastatic disease. Univariate analysis confirmed a tight interrelationship between cut-off CTC values and biomarkers. Multivariate analysis with bootstrap sampling validation identified lactate dehydrogenase (P = 0.002) and CTCs (P = 0.001) as independently prognostically significant. Conclusions: Baseline CTC values provide important prognostic information and specific prediction of metastatic disease. Their presence correlates with classic biomarkers. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1904 -13)