USP26 gene variations in fertile and infertile men.

BACKGROUND: The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. One is the ubiquitin-specific protease 26 (USP26). Five frequent mutations have been identified: 1737G>A, 1090C > T, 370-371insACA, 494T > C and 1423C>T (with the latter three usually detected in a cluster). Their role in infertility is still controversial. This study assesses the association of the most frequent USP26 mutations with male infertility and male infertility etiology factors. METHODS: The study included 300 infertile and 287 fertile men. Data were collected on ethnicity (according to maternal origin) and family history of reproduction. Clinical records from 235 infertile and 62 fertile (sperm bank donors) men were available and summarized. The five mutations were investigated by bioinformatic tools and their frequencies were assessed by restriction analysis. The results were correlated with clinical findings. Segregation of the mutations in four families was analyzed. RESULTS: The five analyzed mutations were detected in 44 men from both fertile and infertile groups. The cluster and the 1090C>T mutations showed the highest frequency among Arabs and Sephardic Jews of the infertile group, respectively. Inheritance studies showed that mutations were not always associated with the infertility trait. Mutations 1090C>T and 1737G>A were significantly associated with a history of inguinal hernia (P = 0.007 and P = 0.043, respectively). The prevalence of inguinal hernia among men with the 1090C > T mutation was 33.3% (5/15 men), higher than that reported in infertile men (6.7%). CONCLUSIONS: Mutation 1090C > T may be a new genetic risk factor for developing inguinal hernia which may be associated with impaired male fertility.

Intratubular germ cell neoplasia: associated infertility and review of the diagnostic modalities.

The incidence of testicular neoplasia has increased, and its early detection has become a pressing clinical issue. The strong association between male subfertility and risk of testicular neoplasia is consistent with the existence of common pathogenetic factors. Most forms of testicular germ tumors are believed to stem from a common precursor, intratubular germ cell neoplasia (ITGCN), also known as testicular carcinoma in situ. Identification of ITGCN cells in testicular biopsies, however, is a diagnostic challenge and markers are sorely needed to assist in the accurate identification of the lesion.

The prognostic role of the extent of Y microdeletion on spermatogenesis and maturity of Sertoli cells.

Substantial involvement of the Y chromosome in sexual development and spermatogenesis has been demonstrated. Over the last decade, varying extent of Y chromosome microdeletions have been identified among infertile patients with azoospermia or oligozoospermia. These microdeletions were clustered in three main regions named AZFa, AZFb, and AZFc. Analysis of the Y chromosome microdeletion was found to be of prognostic value in cases of infertility, both in terms of clinical management as well as for understanding the aetiology of the spermatogenesis impairment. However, the accumulated data are difficult to analyse, due to the variable extent of these deletions, the different sequence-tagged sites (STS) used to detect the microdeletions, and the non-uniformity of the histological terminology used by different investigators. This debate discusses the chances of finding testicular spermatozoa in men with a varying extent of Y chromosome microdeletions. The genotype and germ cell findings in men with AZFa microdeletions as well as those that include more than a single AZF region are reviewed, as is the effect of Y chromosome AZF microdeletions on the maturity of the Sertoli cells.

Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men.

The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.

Augmentative effects of intracellular chemotherapy penetration combined with hyperthermia in human ovarian cancer cells lines.

A significant proportion of ovarian cancer patients do not achieve a complete response to chemotherapy, due mainly to the evolution of clones resistant to cytotoxic drugs. Exploring possibilities to overcome this resistance constitutes a challenge in the study of ovarian cancer experimental therapy. In the present study, we tested the effect of hyperthermia (40 and 43 degrees C) in combination with adriamycin on three human epithelial ovarian cell lines: OC-109, OC-238, and OC-7-NU. The first was derived from the mucinous and the other two from serous cystadenocarcinomas, and all proved to be tumorigenic in nude mice. FACS analysis showed a pronounced increase in intracellular adriamycin accumulation in the presence of hyperthermia. A significant effect (P < 0.0005) observed at 40 degrees C was even more pronounced at 43 degrees C with the three cell lines. High percentages of cells (up to 70%) shifted into higher fluorescence intensities. The lines differed in their sensitivity to the hyperthermia-induced increase in adriamycin accumulation. Under mild conditions, the OC-109 line was more sensitive than the OC-238 and the OC-7-NU lines. Quantitative determination of intraneoplastic adriamycin by spectrofluorometry also showed a hyperthermia-related increase in intracellular adriamycin (P < 0.005). Our results may indicate that supranormal temperature might serve as an alternative chemosensitizer which lacks the deleterious side effects of other chemosensitizing options.

Enhancing effect of ATP on intracellular adriamycin penetration in human ovarian cancer cell lines.

Ovarian cancer has the highest mortality rate of all gynecological malignancies probably due to the evolution of clones resistant to cytotoxic drugs. Exploring possibilities to overcome such resistance constitutes a challenge in this study. We present the effect of adenosine triphosphate (ATP), serving as a chemosensitizer, in combination with adriamycin on three human ovarian cancer cell lines of epithelial origin, OC-109, OC-238 and OC-7-NU, obtained from malignant ascites of different patients, and were proven to be tumorigenic in nude mice. The three lines differ in their sensitivity to the ATP-induced increase in adriamycin accumulation. FACS analysis showed a pronounced increase in intracellular adriamycin accumulation after treatment with various concentrations of ATP. In the OC-238 line, a 50.1% increase was observed at a low ATP concentration (200 microM), whereas higher concentrations (400 microM and 500 microM) were needed to obtain an increase in ADR accumulation of 30% with the other two lines. Our study demonstrates that ATP improves the penetration of adriamycin at the neoplastic cellular level. Furthermore, our results may indicate that intratumoral ATP may serve as an alternative chemosensitizer which lacks the deleterious side effects of other chemosensitizing options.

Drug resistance and its counteraction by cyclosporin A in function of metastatic potential in the Lewis lung carcinoma system.

Tumor progression (TP) is often accompanied by evolution of drug resistant clones. Decreased intracellular accumulation of cytotoxic agents is probably the major mechanism of drug resistance. In the present study, we tried to examine the possibility to overcome the resistance to adriamycin (ADR) treatment, by cyclosporin A (CS) in two models of TP in the Lewis lung carcinoma (3LL) system. The first model consisted in the comparison of primary tumor cells (3LL-PT) to metastatic cells (3LL-MT) and the second consisted in comparison of lung metastases of the highly malignant variant D122 to those of the parental 3LL tumor. Cyclosporin had a weak augmenting effect on ADR uptake, in the two more malignant cell variants and no influence on the 3LL-PT cells, according to FACS analysis. Cytofluorometry also showed practically no effect of CS on cell size, unlike the effect of other chemosensitizers, such as membrane active agents. In order to find out whether CS counteracts resistance to ADR despite the fact that it does not increase cytotoxic agent uptake, we examined its effect on in vitro proliferative capacity of the 3LL-PT cells. CS in combination with ADR had a more pronounced effect, as compared to single treatments on cell proliferation. The low effect of CS on ADR uptake according to FACS analysis, and by contrast, its efficiency to overcome resistance to ADR according to the in vitro growth results, suggest that the mechanism of the CS action as a chemosensitizer is not related to the p-glycoprotein (P-G-P), known to be overexpressed in the typical multidrug-resistance (MDR) phenotype. A better understanding of the complexity of MDR mechanisms may contribute to the design of new modalities to overcome this phenomenon, which still limits effectiveness of cancer cure, to the early stages of the disease.

Effect of a membrane-active agent on uptake of adriamycin in Lewis lung carcinoma cells derived from 'primary' and 'metastatic' growths.

The treatment of metastatic growth still constitutes a challenge for cancer research. Tumor progression is often accompanied by a loss in sensitivity to previously efficient drugs. Decreased intracellular accumulation of cytotoxic agents is probably the major reason for drug resistance, although other mechanisms have also been described. Properties of the cell membrane have been shown to determine the metastatic phenotype. This cellular organelle is also responsible for the multiple drug resistance phenomenon. Membrane-active agents may increase cell permeability to drugs, counteracting thereby drug resistance. In the present study the effect of the nonionic detergent Tween 80 on sensitivity to adriamycin (ADR) of cells derived from Lewis lung carcinoma 'primary tumors' (PT)-the local growth following subcutaneous inoculation - and 'metastatic tumors' (MT) - lung tumors which develop following intravenous injection - was compared. Flow cytometry analysis demonstrated several differences between cells derived from the PT and the MT: (1) single ADR treatment showed that MT cells possessed a lower percentage of a high ADR permeability subpopulation than PT cells; (2) a dose-dependent shift to a higher ADR accumulating population was seen in the presence of Tween 80 for both cell types. However, the increase in percentage of high ADR permeability cells was more pronounced in MT (up to x4.7) than in PT (up to x1.3) cells. This differential effect of the membrane-acting agent was evident at various ADR (10-50 micrograms) and Tween 80 (0.1-0.4%) concentrations. The present results corroborate previous data obtained in our group in another tumor progression model, AKR lymphoma.

Effect of hyperthermia and thermochemotherapy on primary and metastatic tumour cells of AKR lymphoma.

The cure of metastatic disease constitutes a serious problem. Recent findings showed cell membrane differences between slightly and highly metastasizing tumour cells, suggesting that since this cell organelle determines the metastatic phenotype, it might serve as a target for future drugs programmed against advanced cancer. The cell membrane has also been shown to be involved in the evolution of drug resistance which often accompanies tumour progression. In the present study, the effect of hyperthermia--an antitumoral treatment modality partly exerting its effect on the cell membrane--on primary and metastatic AKR lymphoma cells was compared. The effect of hyperthermia in conjunction with adriamycin (ADR) on the two cell types was also tested. Hyperthermic treatment, alone and in combination with ADR, was more effective in reducing the tumorigenicity of cells derived from metastatic tumours than of the primary tumour cells. Fluorescent microscopy and cytofluorometry showed that the increased effect of ADR by hyperthermia was due to an increased drug uptake at the supranormal temperature.

Long-term physical exercise retards trabecular bone loss in lumbar vertebrae of aging female mice.

The present study examined the effect of long-term, moderate physical exercise on trabecular bone volume (TBV), calcium content, 3H-proline uptake, and the activities of alkaline and acid phosphatases in lumbar vertebrae of aging and senescent mice. It became apparent that if physical activity starts at an early stage of life, i.e., prior to middle age and is extended until old age, it exerts beneficial effects on trabecular bone mass and mineralization. Such a positive effect is not obtained if the training program is initiated after middle age. The training-induced reduction in bone loss was accompanied by a significant decrease in acid phosphatase activity whereas no changes took place with regard to the activity of alkaline phosphatase. Long-term physical exercise also enhanced the uptake of 3H-proline by lining cells along the bone trabecules. In spite of its moderate nature, the endured training program served as a stress factor for the involved animals, a fact that was manifested by an increase in the serum levels of corticosterone. Thus, it seems that whereas young animals respond favorably to such a stimulatory stress, older animals lose this ability of adaptation.

Correlation between alkaline and acid phosphatase activities and age-related osteopenia in murine vertebrae.

Lumbar vertebrae (L4) from CW-1 female mice were examined for age-related changes in alkaline and acid phosphatase activities from young to old age. Histochemically, both enzymes were encountered along the bony surfaces of both trabecular and cortical bones with no significant age-related changes in their distribution. Biochemical determinations of bone alkaline phosphatase (Alk'ase) activity revealed that for a given unit level of bone or the bone as a whole no significant changes took place, whereas acid phosphatase (Acid'ase) activity was found to have increased significantly with age. A high positive correlative relationship was noted between the calcium content and the trabecular bone volume of the same vertebrae. It may, therefore, be proposed that age-related bone loss in mice could be attributed to an enhanced resorption rather than to a substantial reduction in the formative potential of bone cells.

Age-related bone loss in lumbar vertebrae of CW-1 female mice: a histomorphometric study.

Age-related changes in vertebral cortical and trabecular bone were quantitated in female CW-1 mice. Histomorphometric measurements involved the use of two different systems of image analysis: Olympus Cue 2 and Zeiss Morphomat 10. The peak of bone mass, both cortical and trabecular, was found in mice aged 13.5 months. Thereafter, there was a progressive decline in the area occupied by bone tissue and this became highly significant in elderly female and male mice. A loss of about 60% of bone tissue was encountered in both the trabecular and cortical bone, as determined by automated image analysis system. Hence, aging CW-1 mice reveal structural features consistent with vertebral osteopenia. Despite the fact that to date we lack the precise etiology for the above-mentioned phenomenon, it is suggested that the laboratory mouse might serve as an appropriate experimental model for investigations related to age-related bone loss.