Coexpression of human chorionic gonadotropin beta subunit and its receptor in nontrophoblastic gynecological cancer.

A considerable number of biochemical and physiologic studies evaluate the roles of gonadotropins in carcinogenesis. Latest reports show that human chorionic gonadotropin (hCG), and especially its beta subunit, are secreted by a variety of malignant tumors of different origin. However, the mechanism of hCG action and its role in tumor development is not known yet. This study, with the help of reverse transcription-polymerase chain reaction and immunohistochemistry, is an attempt to document the molecular presence of the hCGbeta and luteinizing hormone/hCG receptor (LH/hCGR) in the ovarian, endometrial, and uterine cervix cancer tissues. The LH/hCGR, coexpressed with hCGbeta, may act as a potential mediator of hCG action in nontrophoblastic gynecological cancers.

Distribution of the DAZ gene transcripts in human testis.

Involvement of variety of genes, especially located on Y chromosome, is critical for the regulation of spermatogenesis. In particular, fertility candidate genes such as deleted in azoospermia (DAZ) are believed to have important function in sperm production, since DAZ is frequently deleted in azoospermic and severy oligozoospermic men. The role of the DAZ gene is supported by its exclusive expression in the testis and by its deletion in about 10% of azoospermic and severely oligozoospermic patients. The distribution of DAZ transcripts in seminiferous epithelium of human testis is reported in the present study. The use of Adobe Photoshop and Scion Image softwares allowed for semi-quantitative analysis of in situ RT-PCR (ISRT-PCR) results. The intensity of ISRT-PCR product's fluorescence was different within individual seminiferous tubules. It was clearly shown by using the pseudocolour scale and transforming the intensity of the fluorescence into levels of greyscale images. The more intense fluorescence characterised single spermatogonia and those organized in small groups inside separate tubules. The most intense accumulation of DAZ mRNA was observed in spermatogonia.

Influence of cycloheximide on apoptosis in CHO cells, induced by ethane 1,2-dimethanesulphonate (EDS).

Ethane 1,2-dimethanesulphonate (EDS) causes apoptotic death of Leydig cells. Additionally, EDS causes damage of Chinese Hamster Ovary (CHO) cells but the occurrence of apoptosis is not such plentiful. The present study tested whether the inhibition of protein synthesis by cycloheximide (CHX) would influence apoptosis of CHO cells, induced by EDS. The study compounds induced morphological changes in CHO cell typical for apoptosis. An active form of caspase-9 and an alternation of mitochondrial transmembrane potential were also observed. In our study, a more cumulative effect of the CHX and EDS on apoptosis was observed, when both compounds were simultaneously employed. The obtained results indicated that synthesis of antiapoptotic proteins plays a very important role in the inhibition of apoptosis.

The influence of photodynamic reaction on R2C cells.

Photodynamic therapy (PDT) represents a therapeutic approach in which photosensitised neoplastic cells undergo destruction under effect of light. In this study we have attempted to define effects of photochemotherapy on R2C cells, sensitised with protoporphyrin IX (PpIX) and to find out whether inhibition of gene expression by cycloheximide affects development of lesions in the cells. The photosensitised cells were exposed to visible light and development of apoptotic and necrotic lesions was followed in the cells, using the fluorescent staining with propidium iodide and Hoechst 33342. The experiments demonstrated that PpIX and light, acting in parallel, induce development of apoptotic and necrotic lesions in R2C cells. Intensity of the lesions correlated with concentration of the applied photosensitiser and with duration of light exposure. Using cycloheximide, we also inhibited protein expression in cells photosensitised with protoporphyrin before they were exposed to light. In the latter case, development of apoptosis was clearly intensified which might be explained by inhibition of anti-apoptotic protein synthesis in the cells.

Plasma membrane translocation of phosphatidylserine in human spermatozoa.

The purpose of the study was to examine the phosphatidylserine translocation in human spermatozoa membrane during capacitation. Material consisted of human semen from normozoospermic men. Spermatozoa were stained with fluorescein-labelled annexin V. The presence and distribution of annexin V binding sites were analysed using the fluorescence microscope. Within first 60 min afterejaculation, 5-39% viable annexin V-positive spermatozoa were detected. The annexin V binding sites were found mainly in the midpiece. After 4 to 8 h of incubation of spermatozoa in capacitation medium (BMI), the number of cells positively stained with annexin V increased. After capacitation, the localisations of phosphatidylserine was changed and the annexin V binding sites were found also in the acrosomal region but never in the equatorial area. The process of the phosphatidylserine translocation observed during our experiments may reflect changes of the plasma membrane occurring during capacitation or, less likely, apoptosis of spermatozoa.

Chlamydia trachomatis infection in women with CIN and invasive uterine cervix cancer. Significance of hormonal status.

In women with CIN at fertile age and those over 50 years of age, EGFR expression is lower in the presence of Chlamydia trachomatis (Cht) infection. In all Cht infected women over 50 years of age expression of Ki 67 is higher; the increase is significant among women with invasive carcinoma. In these groups of women with CIN and invasive carcinoma TGF-alpha expression is insignificantly augmented. Chronic Cht infection is associated with cervical hypertrophy.

Detection of Chlamydia trachomatis infection in women with CIN and invasive carcinoma. Controversial results of different methods.

Chlamydia (Ch.) trachomatis infection as a sexually transmitted disease is highly important, but reliable methods of diagnosing it remain to be worked out. We used three methods of detection: an immunoenzymatic technique for detection of Ch. trachomatis antigen in endocervical material, in situ PCR, and enzyme-immuno assay for detection of IgG class anti-Ch. trachomatis antibodies in serum. We have compared the IS-PCR technique and method of detection of the endocervical antigen. We have not confirmed compatibility of the results obtained in these two methods. Parallel positive results obtained in patient serum and detection of chlamydial DNA by IS-PCR have been accepted to be indicative of persistent infection of Ch. Trachomatis.

Detection of DAZ mRNA distribution in human testis using reverse transcription in situ PCR technique (RT-ISPCR).

Normal course of spermatogenesis reflects appropriate expression of specific genes in spermatogenic cells. In the present study, the localization of DAZ mRNA in human testis obtained from two fertile organ donors has been determined. It was established that DAZ mRNA is located in the germinal epithelium of seminiferous tubules. The most intensive accumulation of DAZ transcript was observed in primary spermatocytes. The results indicate that expression of DAZ gene begins in spermatogonia and that distribution of DAZ mRNA most likely correlates with the stage of the human seminiferous cycle.

Production of human papillomavirus type 16 early proteins in Bac-To-Bac Expression System (GibcoBRL).

Human papillomavirus type 16 (HPV16) is a major agent in cervical cancer etiology. Its early proteins are responsible for virus persistence, replication and initiation of neoplastic disease. In the present study we describe a use of baculovirus-insect cell expression system for production and study of HPV16 E2 and E4 proteins. The E2 protein binds specifically to viral DNA and E4 protein shows characteristic cytopathic effects on cells.

Expression of progesterone membrane receptor in spermatozoa from normozoospermic and oligozoospermic men.

The aim of the study was to assess the expression of progesterone membrane receptors in human spermatozoa before and after capacitation. The sperm of 16 men with normozoospermia and 48 men with oligozoospermia was examined. Progesterone-BSA complex labelled with fluorescein isothiocyanate (P-BSA-FITC) was applied to visualise the progesterone receptors. The spermatozoa were capacitated with BM1 medium. In freshly ejaculated sperm from normozoospermic men, P-BSA-FITC staining (bright fluorescence of acrosome region) was observed in 50.1+/-5.1% of spermatozoa. Following capacitation in BM1 medium, the percentage of P-BSA-FITC stained spermatozoa increased to 69.9+/-3.3%. In sperm from slight oligozoospermia the percentage of P-BSA-FITC stained cells before and after capacitation was 48.2+/-8.5% and 67.9+/-4.2%, respectively. In men with severe oligozoospermia the percentage of P-BSA-FITC stained cells was 11.9+/-1.7% and 11.9+/-1.9%, respectively. It is supposed that progesterone membrane receptors in human spermatozoa are gradually exposed during capacitation. Disturbances in the expression of progesterone membrane receptors might be involved in male infertility.

Studies on the effect of photodynamic therapy on in vitro cultured neoplastic cells.

Photodynamic therapy (PDT) represents a therapeutic approach in which photosensitised neoplastic cells undergo destruction under the effect of light. In this study we have attempted to define effects of PDT on CHO cells, sensitised with protoporphyrin IX (PpIX). The photosensitised CHO cells were exposed to a visible light and development of apoptotic and necrotic lesions was followed in the cells, using the fluorescent staining with propidium iodide and Hoechst 33342. The experiments demonstrated that PpIX and light, acting in parallel, induce development of apoptotic and necrotic lesions in the cells. Intensity of the lesions was correlated with the concentration of the applied photosensibiliser and with the duration of exposure to light. The control experiments suggest that development of apoptosis in the applied model probably reflect mitochondrial damage, while processes developing close to the cell membrane are responsible for necrosis. In order to corroborate the obtained results, ultrastructural studies were performed on experimental groups in which evident apoptotic lesions were observed in the cells.

Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture.

Lectin from Chelidonium majus L. (CML) significantly stimulates the proliferation of human lymphocytes and has hemagglutination activity towards group B human erythrocytes and potent antimicrobial properties against multiresistant enterococci and staphylococci. In the present work we describe the effect of lectin from Chelidonium majus L on normal and cancercells in culture in vitro. The studies were performed on three types of cells: CHO, R2C and on normal mouse fibroblasts. Effects on the cultures were examined 24 h after addition of CML. Exposure to CML resulted in growth inhibition of CHO and R2C cells but not of fibroblasts. Moreover, evident apoptotic lesions were observed in CHO cells and less well marked apoptotic lesions in R2C cells. In contrast, only insignificant numbers of fibroblasts reacted to the applied lectin.

Studies on the influence of aneuploidy of spermatozoa obtained from patients with oligozoospermia on their structure.

The presence of aneuploidy in spermatozoa influences their biological characteristics, especially their ability to fertilise the ovum. The aim of the present study was to investigate if aneuploidy is accompanied by any changes in the morphology of spermatozoa in oligozoospermic patients. For this purpose, the percentage of aneuploid cells in sperm and the correlation between the specific morphological forms of spermatozoa and aneuploidy were evaluated. The study proved a negative correlation between DNA content of aneuploid and normal spermatozoa. A weak positive correlation was demonstrated between the presence of aneuploid spermatozoa and DNA content of spermatozoa with large heads. No such correlations could be detected for DNA content of the remaining morphological forms of spermatozoa. Thus, men with a lowered number of spermatozoa and/or with abnormal spermatozoal morphology should have their spermatozoal DNA content tested in order to evaluate the degree of aneuploidy, especially in cases where in vitro fertilisation is intended.

Studies on the involvement of endogenous neuropeptides in the control of thymocyte proliferation in the rat.

The possible involvement of endogenous vasoactive intestinal peptide (VIP), cholecystokinin (CCK) and neurotensin (NT) in the control of thymocyte proliferation has been investigated in vivo in the immature rat. For this task, we have studied the effects of the administration of selective antagonists of the receptors of the three neuropeptides on the mitotic index (% of metaphase-arrested cells after vincristin injection) of thymocytes. Both CCK- and TN-receptor antagonists were ineffective. In contrast, two VIP receptor antagonists (VIP-As) enhanced the mitotic index of thymocytes. VIP reversed the effect of VIP-As, but when administered alone it did not alter the mitotic activity of thymocytes. In light of these findings, we conclude that endogenous VIP exerts a maximal tonic inhibitory influence on the basal proliferative activity of rat thymocytes, while endogenous CCK and NT do not play a relevant modulatory role in this process.

Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?

When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language.

Significance of DNA ploidy measurements in Spitz nevi.

In 28 Spitz nevi DNA content was determined by video-imaging cytometry. The nevi were selected for this study because of difficulties in differentiation from melanoma and heterogeneity of this type of nevus. Morphological features of Spitz nevi and differences helpful for differentiation between Spitz nevi and malignant melanoma were identified. DNA ploidy was measured in paraffin embedded and fresh tissue material from each patient and the results were comparable. The sample preparation process and video-imaging method are presented in this study. Twenty two (78.6%) lesions contained diploid cell populations, 5 (17.9%) aneuploid and 1 (3.6%) tetraploid cell population. A significant correlation was observed between DNA ploidy measured in fresh tissue and retrospective material. The results indicate the presence of abnormal DNA content in some of the lesions. This observation does not indicate that DNA ploidy cytometry is useful for the differentiation of Spitz nevi from malignant melanoma.

[Ultrastructure of human aneuploid spermatozoa]. / Badania ultrastrukturalne plemników wykazujacych aneuploidie DNA.

The goal of the study was to evaluate ultrastructural changes in aneuploid human spermatozoa. The sperm was collected from seven males with teratozoospermia. In all patients ultrastructural changes in spermatozoa were noted: inappropriate nuclear chromatin condensation, changes in the formation of the acrosome and pathology of the mobile apparatus. These changes suggest a defect in spermatogenesis at the level of spermatide or spermatocyte. Ultrastructural spermatozoa studies may reveal new elements related to factors responsible for decreased sperm fertilizing ability.

[Investigation of occurrence of deletion of the DAZ gene among men with azoospermia and severe oligozoospermia]. / Badania nad wystepowaniem delecji w genie DAZ u mezczyzn z azoospermia i cieza oligozooospermia.

A genetic etiology has been proposed for severe forms of idiopathic male infertility and involvement of variety of genes is critical for the regulation of spermatogenesis. The role of DAZ gene is supported by its exclusive expression in the testis and deletions of DNA sequence within this gene are associated with azoospermia or severe oligozoospermia. The objective of our study was to validate the PCR strategy, using primers based on cDNA sequence, for the detection of genomic aberration among males with azoospermia or severe oligozoospermia who carry mutations in the DAZ gene. Using the PCR technology, deletion of the DAZ gene in one infertile male with azoospermia was detected. No deletion was detected in any of the remaining 15 patients.