Inhibition of mTOR pathway decreases the expression of pre-meiotic and meiotic markers throughout postnatal development and in adult testes in mice.

Rapamycin (mTOR inhibitor) has been reported to have negative effect on human male gonadal function. Previously, we showed that mTOR signalling molecules are expressed during early spermatogenesis in mice. The objective of this study was to investigate the role of mTOR signalling in meiosis both during the first wave of spermatogenesis and also during adult spermatogenesis. Day 5 post-partum mice were administered rapamycin and retinoic acid (RA; a Stra8 activator), and expression of p-p70S6K and Stra8 proteins was evaluated. p-p70S6K and Stra8 protein expressions decreased in post-natal testes after rapamycin treatment. Stra8 protein expression increased after RA and rapamycin+RA administrations in post-natal testes. In adult mice, rapamycin was administrated for 1 or 4 weeks. Morphological analysis for testicular damage and TUNEL assay was performed. After rapamycin administration, germ cell loss increased in adult testes. Ultrastructural analysis revealed disorganised testicular morphology and vacuolisation. The number of apoptotic germ cells increased after 4 weeks rapamycin administration. Stra8 and Dmc1 expressions decreased in 4 weeks rapamycin group, whereas Sycp3 and VASA expression did not change. Our findings suggest that mTOR pathway has an important role in meiotic progress of male germ cells both during first wave of spermatogenesis and in adult mice.

Inhibition of poly(ADP-ribose) polymerase may have preventive potential for varicocoele-associated testicular damage in rats.

Varicocele is ordinarily accompanied by testicular damage and male infertility. Several theories have been proposed to explain the detrimental effect of varicocele on testis tissue, including the possible effects of oxidative stress. The poly(ADP-ribose) polymerase (PARP) pathway has been established as a major downstream intracellular pathway of oxidative stress. Recently we have reported that PARP pathway has been activated in varicocele-induced rat testicular damage model. The aim of the present study was to investigate the possible protective effect of PARP inhibition in varicocele-associated testicular damage. Fifty male Wistar rats were divided into five groups: control, sham, varicocele-induced, varicocele-induced 1,5-isoquinolinediol (ISO, a PARP inhibitor)-treated, and ISO treated groups. The ISO-treated rats received intraperitoneal injections of 3 mg/kg ISO daily for 13 weeks. After 13 weeks of varicocele induction, body and testes weights were investigated in all groups. Histopathology of testes were evaluated by light microscopy. Expressions of PAR, p53 and cytochrome c were detected by immunohistochemistry and cleaved PARP-1, PAR, p53 and cytochrome c by western blot. The degree of apoptosis was determined by TUNEL. Light microscopy revealed testicular damage comprising various degrees of seminiferous tubule degeneration in varicocele-induced rats and their testes weights decreased significantly, whereas ISO administration prevented it. Expressions of cleaved PARP-1, PAR, cytochrome c, and p53 increased significantly in varicocele-induced rats, whereas the level of these molecules were similar to controls in varicocele-induced rats treated with ISO. In conclusion, increased PARP activation in testes seems to be related with testicular damage and apoptosis associated with varicocele and pharmacological inhibition of this pathway might be an effective intervention to prevent varicocele-induced testicular injury.

Effect of abamectin exposure on semen parameters indicative of reduced sperm maturity: a study on farmworkers in Antalya (Turkey).

Environmental exposure to pesticides may cause serious health risks including fertility and reproductive function. The aim of this study was to highlight whether there is a relationship between exposure to abamectin and male fertility parameters of farmworkers. Twenty male farmworkers who were using abamectin and 20 men not exposed to pesticides were recruited as experimental and control groups, respectively. Semen analysis, molecular markers of sperm maturity and serum reproductive hormone levels were evaluated. In experimental group, high plasma abamectin levels were detected. These men have decreased sperm motility. Moreover, diminished molecular markers of sperm maturity, such as decreased hyaluronic acid (HA) binding of sperm, increased numbers of aniline blue positive sperm and increased percentage of creatine kinase (CK) positive sperm, were observed in abamectin-exposed men. Their serum testosterone, LH and FSH levels did not change significantly. We conclude that exposure to abamectin may impair male fertility by effecting semen quality.

Mechanical stimulation of nitric oxide synthesizing mechanisms in erythrocytes.

It has been previously demonstrated that red blood cells (RBC) possess functional nitric oxide (NO) synthesizing mechanisms. RBC are also equipped with variety of intracellular control mechanisms, and respond to mechanical forces and to various biological stimuli by increased release of ATP. Nitric oxide has also been demonstrated to be released from RBC under certain circumstances, and it has been hypothesized that NO synthase (NOS), which is located in both the RBC membrane and cytoplasm, might be activated by mechanical factors. The present study aimed at investigating NOS activation and NO export induced by mechanical stress applied to RBC in suspension. Heparinized venous blood samples were obtained from healthy, adult volunteers and their hematocrit adjusted to 0.4 l/l. The RBC suspensions were equilibrated at room temperature (22+/-2 degrees C) with either room air or made hypoxic (36 mmHg, approximately 70% saturation) using moisturized 100% nitrogen. The samples were then continuously pumped through a glass tube (diameter = 0.06 cm; length = 33 cm) for 30 min using a dual syringe pump to maintain a wall shear stress of 0.5-2 Pa with NO concentrations in the RBC suspensions measured electrochemically. NO concentration significantly increased under the influence of 2 Pa in hypoxic RBC suspensions: 105.0+/-14.2 nM to 127.1+/-12.0 nM as the peak value at 20 min of perfusion. No increase was observed at lower levels of shear stress. Plasma nitrite/nitrate concentrations were measured in samples obtained at five minute intervals. Application of fluid shear stress to hypoxic RBC suspensions resulted in a significant, time-dependent increase of plasma nitrite/nitrate levels, reaching to 14.7+/-1.5 microM from a control value of 11.2+/-1.3 microM. The presence of the non-specific NOS inhibitor L-NAME (10(-3) M) prevented this increment. Additionally, both eNOS and serine 1177 phosphorylated eNOS immuno-fluorescence staining in RBC cytoplasm were shown to increase in response to applied shear stress. Our results support the hypothesis that RBC NO synthase is activated and that export of NO from RBC is enhanced by mechanical stress.

The distribution of angiopoietin-1, angiopoietin-2 and their receptors tie-1 and tie-2 in the very early human placenta.

Angiopoietins are integral to vasculogenesis and angiogenesis, which play crucial roles in the growth and development of the placenta. The current study assessed expression of angiopoietins (Ang-1 and Ang-2) and their receptors (Tie-1 and Tie-2) during development of the early human placenta. First-trimester placental tissues were obtained from women undergoing curettage during normal pregnancies. The use of immunohistochemistry (IHC) showed that Ang-1 was primarily localized to syncytiotrophoblasts where it displayed moderate immunoreactivity, whereas weak immunoreactivity for Ang-1 was observed in endothelial cells and angiogenic cell cords (ACC). Strong immunoreactivity for Ang-2 was also found predominantly in syncytiotrophoblasts with lower immunostaining levels evident in cytotrophoblasts. Moderate immunoreactivity for Ang-2 was observed in endothelial cells, ACC and Hofbauer cells. By contrast, the trophoblastic shell, as well as endothelial cells and ACC exhibited strong staining intensity for Tie-1 with the strongest immunoreactivity for Tie-2 observed in cytotrophoblasts, ACC and endothelial cells. Western blotting of tissue extracts confirmed the IHC results. Previous studies focused on VEGF and its receptors in controlling vasculogenesis and angiogenesis in human placenta. However, the specific localization patterns of angiopoietins and their receptors revealed by the current study emphasize the importance of these molecules in placental vascular development. Functional studies aimed at identifying the molecular mechanisms of actions of these factors and receptors may prove essential in elucidating the pathophysiology of placental disorders such as intrauterine growth restriction and pre-eclampsia.

Immunolocalizations of VEGF, its receptors flt-1, KDR and TGF-beta's in epithelial ovarian tumors.

OBJECTIVE: Angiogenesis is an essential factor for growth, differentiation, invasion and metastasis of tumors. In this study, we aimed to evaluate the immunolocalizations of vascular endothelial growth factor (VEGF), its receptors flt-1, KDR/flk-1, and transforming growth factor-beta's (TGF-beta) in epithelial ovarian tumors, utilizing indirect immunohistochemistry to understand the role of the angiogenic events in ovarian neoplasia. METHODS: Tissue blocks from 40 patients who had ovarian pathology (borderline serous-mucinous tumor and malignant serous-mucinous adenocarcinoma of the ovary) were included in this study. All formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies against VEGF, flt-1, KDR/flk-1, TGF-beta1, TGF-beta2 and TGF-beta3 using the avidin-biotin-peroxidase method. H-SCORE, a semi-quantitative grading system, was used to compare immunohistochemical staining intensities. RESULTS: Positive VEGF immunoreactivity was concentrated in the epithelial and stromal parts of all the ovarian samples and the endothelial cells in the stroma were also stained. Increased immunoreactivity of VEGF was observed in malignant ovarian adenocarcinomas compared to the borderline tumors of the ovary. VEGF receptors, flt-1 and KDR/flk-1 immunoreactivities were detected not only in vascular endothelial cells, but also in tumor cells at malignant sites. Immunoreactivities of VEGF and its receptors were coexpressed in tumor cells of the ovarian carcinoma. While immunoreactivities of TGF-beta1 and TGF-beta2 were both overexpressed in malignant ovarian carcinomas, immunoreactivity of TGF-beta3 was still mild. CONCLUSION: Our results suggest that overexpression of VEGF, its receptors flt-1, KDR/flk-1 and TGF-beta interaction may play an important role in the ovarian cancer biology, with potential effects on tumor growth and angiogenesis. New therapeutic strategies using VEGF and TGF-beta antagonists could obtain an additional approach to the treatment ovarian carcinoma by inhibiting angiogenesis.

Sequential expression of VEGF and its receptors in human placental villi during very early pregnancy: differences between placental vasculogenesis and angiogenesis.

Vascularization within the human placenta is the result of the de novo formation of vessels derived from pluripotent precursor cells in the mesenchymal core of the villi. Vascularization of placental villi starts at around day 21 post conception (p.c.) with a four somite embryo. At this stage progenitors of haemangiogenic cells differentiate to form first vessels. These progenitor cells are thought to be directly derived from mesenchymal cells rather than originating from fetal blood cells. We investigated the relation between differentiation of stromal cells towards endothelial cells and vascular structures and the expression pattern of the respective growth factors. Using transmission electron microscopy and immunohistochemistry (for VEGF, Flt-1, Flk-1, CD14, CD34, and CD68) the development of placental vasculogenesis during very early stages of pregnancy (days 22-48 p.c.) was studied. We found that VEGF is strongly expressed in villous cytotrophoblast cells and subsequently in Hofbauer cells while its receptors Flt-1 and Flk-1 are found on vasculogenic and angiogenic precursor cells. The developmental expression and secretion of VEGF suggests its involvement in recruitment, maintenance and formation of first angiogenic cells and vessels. Interactions between VEGF and Flk-1 and Flt-1 may regulate placental vasculogenesis and angiogenesis in a paracrine and autocrine manner. The sequential expression of growth factors in different cell types may point to the fact that placental vasculogenesis and angiogenesis are clearly distinct events.

Biochemical markers of sperm function: male fertility and sperm selection for ICSI.

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.

Structural differentiation of human uterine luminal and glandular epithelium during early pregnancy: an ultrastructural and immunohistochemical study.

The differentiation of human endometrial epithelium is a dynamic event that occurs throughout the menstrual cycle and early pregnancy. The structural transformation and differentiation of human uterine luminal and glandular epithelium of early human pregnancy (n=14) was investigated ultrastructurally and immunohistochemically using antibodies against cytokeratin (CT), endothelial marker CD31, Fas, and proliferating cell nuclear antigen (PCNA). Ultrastructurally, luminal epithelial cells showed distinctive euchromatic nuclei with prominent nucleoli and relatively loose cell membranes in all poles (apical to basal). Subcellular components were easily recognized in luminal epithelium except in degenerating cells. Mainly two cell types, dark and clear cells, formed the glandular epithelium. In the early gestation period, microvilli were abundant on the apical and apico-lateral poles of these cells. Only a few cytoplasmic projections were observed in dark cells. Numerous cilia were observed on the apical pole of some clear cells, located at the adluminal segment. In contrast, dark cells lacked cilia, nuclear channels, or giant mitochondrial profiles. Glycogen synthesis and apocrine secretion were recognizable for several days during early gestation. The apocrine secretory activity differed among dark cells of the glandular epithelium. The immunoreactivity of PCNA and Fas, and ultrastructural observations in the glandular epithelium suggest that, even in different segments of the same gland, epithelial cells do not regress during early gestation, but proliferate, perhaps representing a resistance against trophoblastic invasion. These morphological and molecular changes suggest that both luminal and glandular epithelium may play an important role in cellular defense and limitation for trophoblastic invasion during early pregnancy since plasma membrane alterations of the surface epithelium take place at the apical, basal and lateral poles compared to early secretory phase endometrial cells. Besides glandular epithelium may be consequently responsible for uterine secretions, which may be critical for early embryo development.