Effects of agricultural pesticides on the reproductive system of aquatic wildlife species, with crocodilians as sentinel species.

Agricultural pesticides represent a significant class of endocrine-disrupting chemicals (EDCs) to which non-target organisms around the world are constantly exposed. Laboratory studies have found strong evidence showing the endocrine-disruptive potential of these pesticides at environmentally relevant exposure levels. Since the field of endocrine disruption continues to grow in richness and complexity, this review aims to provide an update on the effects of two agricultural pesticides that act as EDCs: atrazine and endosulfan. We will focus mainly on the effects on crocodilians due to their worldwide occurrence in tropical and sub-tropical wetland ecosystems and their ecological and physiological features, which render them vulnerable to exposure to pesticides with endocrine-disrupting action at all life stages. The results here reviewed provide important insights into the effects of hormonally active agricultural pesticides at cellular, tissue, and organ levels in the reproductive system of crocodiles. A better understanding of the effects of exposure to environmentally relevant doses of EDCs on the reproductive system of crocodilians will contribute to protect and improve the health of both wildlife species and humans.

Aging in the Syrian hamster testis: Inflammatory-oxidative status and the impact of photoperiod.

Testicular aging is linked to histological, morphological and functional alterations. In the present study, we investigated whether aging affects the inflammatory and oxidative status in the testis by comparing young adult, middle-aged adult and aged hamsters. The Syrian hamster, a thoroughly studied seasonal breeder, was chosen as the experimental model since it allows further investigations on the role of photoperiod and melatonin in testicular aging with a minimal impact of the experimental intervention on the animal well-being and the subsequent results achieved. In testes of aged hamsters, we found a decrease in melatonin concentration, a thickening of the wall of the seminiferous tubules as well as a significant increase in IL-1ß, NLRP3 and cyclooxygenase 2 expression, PGD2 production, macrophages numbers, lipid peroxidation and anti-oxidant enzyme catalase levels. Interestingly, when aged hamsters were transferred from a long day (LD) to a short day (SD) photoperiod for 16 weeks, testicular melatonin concentration increased while local inflammatory processes and oxidative stress were clearly reduced. Overall, these results indicate that melatonin might display anti-inflammatory and anti-oxidant capacities in the aged testes.

Bcl-2 correlates with tumor ploidy and nuclear morphology in early stage prostate carcinoma. A fine needle aspiration biopsy study.

We evaluated the nuclear morphology, ploidy, bcl-2 expression and in situ apoptosis in sections of fine-needle aspiration (FNA) biopsy specimens of thirty-one randomly selected Stage B prostate carcinomas. Sections of paraffin-embedded pelleted cells obtained from FNA biopsy specimens were studied. Nuclear grade was determined according to the WHO system. Nuclear morphometry and DNA ploidy were carried out using an automated image analyzer. We used immunostaining and the TUNEL method to evaluate bcl-2 expression and in situ apoptosis. The median nuclear area increased with increasing nuclear grade. Ploidy analysis showed that 54.8% of tumors were diploid, 3.2% tetraploid and 41.9% aneuploid. Bcl-2 overexpression was found in 10 of 31 tumors. There was a significant positive correlation between bcl-2 expression and nuclear area (r(s): 0.45 p < 0.01). Nine of ten bcl-2-positive tumors had a nuclear area larger than the median of the series, and 70% of bcl-2-positive tumors were of the aneuploid type. The apoptotic index had a negative correlation with nuclear area, and the lowest indexes were found in aneuploid tumors. Bcl-2 expression showed a highly significant association with both parameters of high aggressiveness: nuclear size and aneuploidy. The combined evaluation of nuclear morphology, ploidy and cell survival parameters might better identify patients with poor prognosis among early stage prostate carcinomas diagnosed by FNA biopsies.

Characterization of fibroblastic cell plasticity in the lamina propria of the rat uterine cervix at term.

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never alpha-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.

Differential distribution of the fibres of the collagenous and elastic systems and of glycosaminoglycans in the rat pubic joint.

The pubic joint of male and female rats was studied at the light- and electron microscopical levels using methods that selectively disclose the extracellular matrix fibres and glycosaminoglycans. The interpubic tissue showed no difference between sexes (including pregnant and intrapartum females). The medial ends of the pubic bones were covered by articular caps of hyaline cartilage that blended in the midline. The whole articular cartilage was covered dorsally and ventrally (as well as craneally and caudally) by a typical perichondrium. The differential distribution of the fibres of the collagenous and elastic systems in the pubic joint agreed with the results reported in the literature for other rat cartilages. Collagen fibres, composed mainly of type-I collagen, were localised to the fibrous perichondrium and bone. Type-II collagen was localised to the central nucleus of hyaline cartilage, whereas reticulin fibres (rich in type-III collagen) were found in the adventitial loose connective tissue adherent to the most superficial layer of the perichondrium. The central nucleus of hyaline cartilage possessed the two types of elastic-related fibres: elaunin fibres were localised mainly to the chondrogenic layer of the perichondrium, whereas oxytalan fibres were found in the matrix that surrounded the chondrocytes. The bulk of the glycosaminoglycans present in the pubic joint cartilage corresponded to hyaluronic acid and chondroitin sulphate. The propriety of classification of the rat pubic joint as a true synchondrosis (instead of symphysis), and the fact that the unaltered pelvis of the rat seems to be adequate for normal parturition, are discussed.

Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissue at term.

Ripening and dilation of the rat cervix at term involves a widespread reduction in the density and organization of collagen fibers following a polymorphonuclear eosinophilic invasion. These are hormonally regulated events; however, the correlation between hormonal milieu and these morphological changes is not well understood. To investigate the role of preparturient relaxin and estradiol-17ss (E2) in cervical collagen remodeling and eosinophilic infiltration, pregnant rats were either sham-ovariectomized (group C) or ovariectomized in the morning of Day 22. Ovariectomized rats were treated with E2 (group OE), relaxin (group OR), E2 plus relaxin (group OER), or hormone vehicles (group O). There were 4 or 5 animals per group. Cervices were taken from animals killed approximately 1 h before expected parturition. Five-micrometer serial sections of paraffin-embedded cervices were stained with either Sirius Red in alkaline solution to measure eosinophil infiltration or in Picrosirius to measure collagen birefringence. Ovariectomized rats treated with E2 (group OE or OER) showed high levels of eosinophilic infiltration that did not differ from those in group C intact controls. However, the values of eosinophilic infiltration in ovariectomized rats treated with relaxin or hormone vehicles (groups OR and O) were low and far below (p < 0.01) those of groups OE and C. In ovariectomized rats treated with E2 alone (group OE), the widespread reduction in collagen organization that occurred in group C controls was not observed. It was only in ovariectomized rats treated with relaxin or E2 + relaxin (groups OR and OER) that the values of birefringence were low, and they were as low as in control group C. In conclusion, this study indicates that eosinophilic infiltration and collagen remodeling in the rat cervix at term are under the control of different hormones: E2 stimulates eosinophilic invasion, and relaxin promotes a widespread reorganization of collagen fibers.

Proliferative activity and steroid hormone receptor status in male breast carcinoma.

Hormonal factors have been implicated in the development of both female and male breast cancers (MBC). However, MBCs are rare and seem to have different biological behavior than those of females. The aim of this study was to evaluate proliferative activity and to establish an association with steroid hormone receptor concentration and clinicopathological parameters in MBC. Proliferative activity was assessed in 18 MBC by mitotic figure counts and immunohistochemical evaluation of MIB-1 and proliferating cell nuclear antigen (PCNA). Estrogen (ER), progesterone (PR) and androgen (AR) receptors were evaluated in serial section from the same tumor by immunohistochemistry. PCNA (range 17-73%; mean, 51.6%) and MIB-1 (range 18.5-58%; mean 38.4%) were positive correlated with the mitotic rate. High proliferative activity assessed either by mitotic index or MIB-1 expression was associated with more poorly differentiated tumors. Sixty one percent (11/18) of the tumors were ER+, 72% (13/18) PR+ and 38.5% (5/13) AR+. Proliferative activity in tumors displaying ER+/PR+ phenotype showed a tendency to be higher than in ER-/PR- tumors. This difference was statistically significant when MIB-1 expression was used as proliferation marker. An association between AR concentration and age at diagnosis was found; in the AR negative group (8/13) mean age at diagnosis was 54.4 +/- 7.3 which was significantly lower than the age of patients with AR+ tumors, 63.2 +/- 11.1 (5/13). Results presented here show that decreased androgen action (AR-) within the breast might contribute to an earlier development of MBC. Besides that, the presence of ER and PR in carcinoma cells is considered to provide a growth advantage as shown by the positive association between the phenotype (ER+/PR+) and high proliferative activity. These results add information for a better understanding of hormonal control of MBC growth and development.

Lack of relationship between the expression of Hsp27 heat shock estrogen receptor-associated protein and estrogen receptor or progesterone receptor status in male breast carcinoma.

Estrogen, through estrogen receptors (ERs), may regulate the synthesis of progesterone receptors (PRs) and of a heat shock estrogen receptor-associated protein (hsp27). In female breast carcinoma (FBC) both proteins serve as surrogate indicators for the presence of functional ERs. In addition, the expression of these proteins was related to other prognostic indicators of value in female breast tumours. Endocrine disorders, hormone therapy and altered estrogen metabolism have been associated with the development of male breast cancer (MBC), suggesting that evaluation of the expression of ER, PR and hsp27 might improve our understanding of the biology of this tumour. ER and PR status and hsp27 expression were evaluated by immunohistochemistry in 16 primary MBC patients. The interrelationships between these parameters were established and compared with the clinicopathological data on the tumours. Ten (56%) MBC patients were ER-positive, 69% were PR-positive and all samples were hsp27-positive. Our series of MBC patients showed a positive correlation between ERs and PRs, however there was a lack of correlation between hsp27 and ERs or PRs. MBCs did not exhibit any correlation between the biomarkers studied and known prognostic indicators for females (e.g. Scarff-Bloom-Richardson (SBR) or modified SBR (MSBR) grade, T stage, lymph node status). This is the first published series reporting the incidence of hsp27 in MBC. The lack of association between the expression of ERs and hsp27 found in MBC differs from the results reported for FBC, moreover the expression of ERs, PRs or hsp27 did not correlate with the clinicopathological parameters that have prognostic value in females. Although the data were obtained from a relatively small sample population, our findings suggest that MBC and FBC are biologically different tumours with respect to the expression of the studied proteins.

Dissociation in the control of cervical eosinophilic infiltration and collagenolysis at the end of pregnancy or after pseudopregnancy in ovariectomized steroid-treated rats.

Previous studies have demonstrated that during parturition the physiological ripening that permits dilation of the cervical canal is due to a widespread collagenolysis that follows a heavy polymorphonuclear leukocyte invasion of the uterine cervix. The aim of this study was to investigate whether there is any association between the ovarian steroid hormones involved in rat parturition and this phenomenon. Pregnant or pseudopregnant rats were ovariectomized (OVX) at Day 9 and then given a hormonal treatment (sufficient to maintain fetal viability) until Day 23. Cervical biopsies, taken from animals killed intrapartum or 1 h before expected parturition, were studied for eosinophilic infiltration and collagen birefringence. Intrapartum or sham-OVX pregnant rats showed a massive eosinophilic infiltration and a widespread collagenolysis as indicated by a loss of collagen birefringence. Ovariectomized pregnant rats treated with estrogen plus progesterone or with progesterone alone showed neither infiltration nor collagenolysis. In rats OVX during pseudopregnancy, estrogen given alone induced a significant infiltration of eosinophils in the cervical stroma; however, treatment with the combination of estrogen and progesterone was not able to promote eosinophilic infiltration. Collagenolysis was absent in all pseudopregnant animals. These results show that estrogen induced a cervical eosinophilic infiltration in rats but that when progesterone was added to the estrogen treatment the infiltration was not present; in addition, none of the steroid hormones assessed were responsible for the collagenolysis found in the cervical tissue at term.