Impairment of spermatogenesis in transgenic mice with selective overexpression of Bcl-2 in the somatic cells of the testis.

To explore the functional role of Bcl-2 in germ cell development, transgenic mice carrying 6 kilobases of the inhibin-alpha promoter were generated to express human bcl-2 gene product in the gonads. Although female transgenic mice demonstrated decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis, the adult males exhibited variable impairment of spermatogenesis. The degree of damage ranged from tubules with intraepithelial vacuoles of varying sizes to near atrophied tubules consisting of Sertoli cells and a few spermatogonia. Although there was no significant change in body weight, an approximately 34% decrease in testicular weights was noted in transgenic animals compared with wild-type mice. Gamete maturation, assessed by determining the percentage of tubules with advanced (steps 13-16) spermatids, was decreased to 44.4% of the values measured in the wild-type animals. The incidence of germ cell apoptosis increased 3.8-fold in the transgenic animals and was associated with a marked loss of germ cells. Electron microscopy of the testes further revealed large vacuoles in the Sertoli cell cytoplasm and dilations of the intracellular spaces between adjacent Sertoli cells, spermatid malformations, and increased germ cell apoptosis in the transgenic animals. There was no evidence of Sertoli cell death either by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay or electron microscopy. Leydig cell ultrastructure, cell size and numbers, and plasma levels of testosterone were not different between normal and the transgenic animals. Collectively, these results support the critical role of Bcl-2 in male germ cell development and are consistent with the gender-specific role of the Bcl-2 family members in reproduction.

XXY male mice: an experimental model for Klinefelter syndrome.

Klinefelter syndrome (47,XXY) is the most common sex chromosome aneuploidy in men. Thus, it is important to establish an experimental animal model to explore its underlying molecular mechanisms. Mice with a 41,XXY karyotype were produced by mating wild-type male mice with chimeric female mice carrying male embryonic stem cells. The objectives of the present study were to characterize the testicular phenotype of adult XXY mice and to examine the ontogeny of loss of germ cells in juvenile XXY mice. In the first experiment the testicular phenotypes of four adult XXY mice and four littermate controls (40,XY) were studied. XXY mice were identified by either Southern hybridization or karyotyping and were further confirmed by fluorescence in situ hybridization. The results showed that the testis weights of adult XXY mice (0.02 +/- 0.01 g) were dramatically decreased compared with those of the controls (0.11 +/- 0.01 g). Although no significant differences were apparent in plasma testosterone levels, the mean plasma LH and FSH levels were elevated in adult XXY mice compared with controls. The testicular histology of adult XXY mice showed small seminiferous tubules with varying degrees of intraepithelial vacuolization and a complete absence of germ cells. Hypertrophy and hyperplasia of Leydig cells were observed in the interstitium. Electron microscopic examination showed Sertoli cells containing scanty amounts of cytoplasm and irregular nuclei with prominent nucleoli. The junctional region between Sertoli cells appeared normal. In some tubules, nests of apparently degenerating Sertoli cells were found. In the second experiment the ontogeny of germ cell loss in juvenile XXY mice and their littermate controls was studied. Spermatogonia were found and appeared to be morphologically normal in juvenile XXY mice. Progressive loss of germ cells occurred within 10 days after birth. This resulted in the absence of germ cells in the adult XXY mice. We conclude that a progressive loss of germ cells occurring in early postnatal life results in the complete absence of germ cells in adult XXY mice. The XXY mouse provides an experimental model for its human XXY counterpart, Klinefelter syndrome.

Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia.

Programmed cell death occurs spontaneously during spermatogenesis and can be induced in a cell- and stage-specific manner by mild testicular hyperthermia. Studies using transgenic mice suggest the involvement of Bcl-2 proteins in regulating germ cell apoptosis. To delineate further the pathways involved, we examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in rat testes after transient exposure to heat (43 degrees C for 15 min). Germ cell apoptosis, involving exclusively early (I-IV) and late (XII-XIV) stages, was activated within 6 h. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization within 0.5 h of heating as assessed by immunocytochemical methods. In contrast, Bcl-2 is distributed both in the cytoplasm and nucleus in those cell types susceptible to heat-induced apoptosis. Despite the striking redistribution, Bax levels remained unchanged as determined by Western analysis; Bcl-2 levels increased significantly by 6 h after heat exposure. Reverse transcription-polymerase chain reaction analysis indicated no change in either Bax or Bcl-2 mRNA levels in response to heat, suggesting the involvement of post-transcriptional rather than transcriptional mechanisms mediating their activity. The marked subcellular redistribution of Bax prior to activation of apoptosis and the increase in Bcl-2 suggest an involvement of Bcl-2 family members in heat-induced apoptotic death of germ cells.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in normal and pathological adult human epididymis.

The pathogenesis of the aberrant development of the male genital tract (epididymis, vas deferens and seminal vesicles) seen in patients with congenital bilateral absence of the vas deferens (CBAVD) is still unclear. Since men with CBAVD carry mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), it is likely that CFTR mRNA of the translated protein plays a major role in the pathogenesis of CBAVD. The aim of this study was to compare the pattern of expression of CFTR mRNA in epididymides of men with CBAVD and other types of obstruction (post-vasectomy and post-inflammatory) with that of normal non-obstructed adult epididymis. Epididymal biopsies were obtained at the time of microsurgical epididymal sperm aspiration procedures or during vaso-epididymostomy reanastomosis. A normal epididymis was obtained from an orchiectomy specimen. After standard processing for in situ hybridization, tissue sections were hybridized with CFTR gene-probe labelled by incorporation of digoxigenin-dUTP. After hybridization the signal was detected by an alkaline phosphatase-tagged antidigoxigenin antibody. CFTR mRNA was clearly identified in the columnar epithelium of the normal adult epididymis and vas deferens and the signal intensity was greatest in the most proximal regions of the caput epididymis. In contrast, men with genital tract obstructions due to CBAVD or post-vasectomy or post-inflammatory obstructions, had sloughing of the epithelial cells lining the lumen and as a consequence CFTR mRNA expression was lacking. In one subject (post-vasectomy obstruction), some residual caput epididymal epithelium was preserved and CFTR mRNA was detected. The abundant CFTR mRNA expression in the proximal caput of the epididymis and vas deferens under normal conditions strongly favours the hypothesis of an early obstructive process in the pathogenesis of CBAVD. The absent or severely reduced activity of CFTR protein affects the ionic exchange and fluid content within the epididymal lumen and this, in turn, can lead to excessive viscosity of the epididymal fluid, sloughing of epithelial cells expressing CFTR and further reduction in the amount of CFTR activity. As a consequence, variable segments of the epididymis and the vas deferens may be blocked and progressively obliterated. The epididymal lumen obstruction could also sustain the anatomical defects by not allowing testosterone to exert a local action on the mesonephric duct.

Prolactin disorders.

OBJECTIVE: To review the pathophysiology, clinical manifestations, current diagnostic procedures, and treatment options for disorders involving PRL production. Common clinical dilemmas are discussed in a pragmatic fashion to guide the practitioner. DESIGN: A world literature search of basic sciences and medical articles from the last three decades was performed using computerized MEDLINE. Recent endocrine and reproductive endocrine textbooks also were reviewed. Studies were selected for their degree of contribution to the basic sciences and clinical understanding of the disorder and for the quality of their study design and content. The information was summarized and grouped according to its relevance and application to specific sections of the manuscript. Studies were evaluated and critically used to support the views of the authors and to suggest specific clinical management strategies. RESULT(S): Disorders derived from abnormal PRL production are relatively common in clinical practice. Infertility, menstrual disorders, and galactorrhea are the most frequent manifestations encountered in women. Although frequently benign, the disorder occasionally may have severe consequences. CONCLUSION(S): An understanding of the underlying physiology and pathophysiology coupled with the awareness of the heterogeneous presentation of this disorder should help the clinician to approach it successfully.

Chlamydia trachomatis Can Persist in Joint Tissue After Antibiotic Treatment in Chronic Reiter's Syndrome / Reactive Arthritis.

Considerable evidence suggests that viable Chlamydia trachomatis are present in joint tissues of patients with Reiter's syndrome/reactive arthritis (RS/ReA), but the use of antibiotics to treat such patients remains controversial. We investigated the continued presence of chlamydia in synovial tissues of patients with RS/ReA; these patients had been treated with antibiotics for relatively extended periods, had shown clinical improvement, but had persistent active disease. Knee synovial tissue was obtained from two patients with RS/ReA and two controls with osteoarthritis (OA). Each sample was screened for chlamydia by culture, direct fluorescent antibody assay (DFA), in situ hybridization (ISH), and polymerase chain reaction (PCR).Synovial tissues from antibiotic-treated RS/ReA patients were negative for chlamydia when analyzed by culture and DFA, but positive when analyzed by ISH for chlamydial RNA and by PCR for chlamydial DNA. Samples from OA patients were negative by all screening methods. Thus, antibiotic treatment does not appear to easily eradicate chlamydia from the joints of RS/ReA patients. Rather, the organism can persist in synovial tissue in a form not detectable by routine laboratory screening methods. Further studies are needed to determine whether antibiotic regimens other than those used here can eradicate synovial chlamydia and to determine how this relates to disease activity. Optimal therapy for patients with RS/ ReA is therefore not yet clear.

Identification of a cAMP-dependent protein-DNA interaction at a sequence near the yeast mitochondrial rRNA genes.

We have shown that expression of yeast mitochondrial (mt) rRNA genes (S. cerevisiae) is controlled in a cAMP-dependent manner via PKA, suggesting a trans-activation process involving phosphorylation-dependent protein-mt DNA interaction. We used filter-binding assays, mt protein extracts, and mt DNA from a rho-mutant strain retaining the 21S rRNA gene to demonstrate such an interaction. Competition assays with the cloned 21S-related mt DNA fragment undergoing interaction showed that a sequence in that fragment is present in mt DNA from a rho-strain retaining the 16S mt rRNA gene, but not in a VAR1-retaining rho-strain that lacks cAMP-mediated mt transcription. The sequence of the 21S-related mt DNA fragment undergoing protein interaction includes a GC cluster; that GC cluster sequence is also present near the 16S gene but not near VAR1. These and other data are consistent with a role for the GC cluster in cAMP-mediated expression of mt rRNA genes.

Case report: in situ hybridization for detection of inapparent infection with Chlamydia trachomatis in synovial tissue of a patient with Reiter's syndrome.

The authors have shown that protein antigens, RNA, and DNA from Chlamydia trachomatis are present in synovial tissues of patients with Reiter's syndrome (RS). However, those studies gave no insight into the host cell type involved or the precise tissue location of the bacteria. To address such issues, the authors developed an in situ hybridization system to detect chlamydia, and they used that system to examine synovial biopsies from a patient with RS and a patient without RS. The in situ system uses a previously described digoxigenin-labeled DNA probe that hybridizes with chlamydial 16S rRNA sequences in paraformaldehyde-fixed samples. Control studies with chlamydia-infected and uninfected HeLa cells confirmed that the in situ system is as sensitive as is direct fluorescence cytology for detection of the organism. Morphology of host and chlamydia cells is preserved after hybridization. Studies using synovial tissue from an osteoarthritis patient produced no in situ hybridization signal, but similar hybridization to tissue from a culture-/direct fluorescence cytology- negative RS patient had a strong intracellular signal for chlamydia within a subsynovial cell layer. These in situ hybridization results confirm the extensive presence of chlamydia in synovia and extend the authors' earlier observation that chlamydia RNA is present in the synovia of patients with RS. The data also confirm their electron microscopy studies, indicating that chlamydia are intracellular in synovial tissue, and they further show that infected host cells are located beneath the synovial lining.