Age and seasonal variation in testis and baculum morphology in East Greenland polar bears (Ursus maritimus) in relation to high concentrations of persistent organic pollutants.

Persistent organic pollutants (POPs) are found in high concentrations in the Artic. Polar bears (Ursus maritimus) are one of the most exposed mammals in the Arctic and are thereby vulnerable to reproductive disruption. The aim of this study was to investigate male polar bear reproduction based on a detailed evaluation of testis histology and to assess possible effects of environmental chemicals on male polar bear reproduction. Reproductive groups that were identified based on histology were as follows: actively reproductive (REP), non-reproductive either with degenerated testes (DEG), undeveloped seminiferous tubules (UND), or morphology in-transition (INT). Categorization into these groups was supported by significant differences in testis and baculum measurements among REP, DEG, and UND, as well as differences in the area and diameter of seminiferous tubules among REP, DEG, and UND. These results show that it is possible to identify the reproductive stage in polar bears even if capture date and or age is lacking. Based on testis morphology we suggest that adult male polar bears from East Greenland have active spermatogenesis in February to June, and inactive degenerated testes in August to January. January to February was the main period of reproductive transition, characterised by a shift between inactive and active spermatogenesis. Baculum and testis size measurements decreased significantly with increasing concentrations of the chlordane metabolite oxychlordane, suggesting a potential impact on male reproductive success. Half of the investigated polar bears in REP group displayed signs of disorganization of the spermatogenesis which might be a sign of disrupted reproduction. However, no correlations with levels of the investigated POPs were detected. Reproductive organ measurements in polar bears differed significantly between REP and DEG groups, which cannot be explained by age, and therefore should be considered when investigating the effect of POPs on male reproduction.

Low-dose exposure to Bisphenol A during development has limited effects on male reproduction in midpubertal and aging Fischer 344 rats.

Low doses of Bisphenol A (BPA) during development may affect reproduction. In this study, Fischer 344 rats were exposed to 0.5 or 50 µg BPA/kg bw/day via drinking water from gestational day 3.5 to postnatal day 22. Anogenital distance, organ weight, histopathology of reproductive organs, hormone analysis and sperm morphology were evaluated in male offspring. In this study no major effects of BPA on male reproduction in midpubertal (postnatal day 35) or adult (12-month-old) rats were revealed, apart from a higher prevalence of mild inflammatory cell infiltrate in cauda epididymis in adult rats exposed to 50 µg BPA/kg bw/day. No BPA-related effects on sexual development were seen but care should be taken when evaluating histopathology in midpuberty testis due to large morphological variation. Results from the present study show no major signs of altered male reproduction in rats exposed to low doses of BPA during gestation and lactation.

New computerized staging method to analyze mink testicular tissue in environmental research.

Histopathology of testicular tissue is considered to be the most sensitive tool to detect adverse effects on male reproduction. When assessing tissue damage, seminiferous epithelium needs to be classified into different stages to detect certain cell damages; but stage identification is a demanding task. The authors present a method to identify the 12 stages in mink testicular tissue. The staging system uses Gata-4 immunohistochemistry to visualize acrosome development and proved to be both intraobserver-reproducible and interobserver-reproducible with a substantial agreement of 83.6% (kappa = 0.81) and 70.5% (kappa = 0.67), respectively. To further advance and objectify this method, they present a computerized staging system that identifies these 12 stages. This program has an agreement of 52.8% (kappa 0.47) with the consensus staging by 2 investigators. The authors propose a pooling of the stages into 5 groups based on morphology, stage transition, and toxicologically important endpoints. The computerized program then reached a substantial agreement of 76.7% (kappa = 0.69). The computerized staging tool uses local ternary patterns to describe the texture of the tubules and a support vector machine classifier to learn which textures correspond to which stages. The results have the potential to modernize the tedious staging process required in toxicological evaluation of testicular tissue, especially if combined with whole-slide imaging and automated tubular segmentation. Environ Toxicol Chem 2017;36:156-164. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Effect of pre-fixation delay and freezing on mink testicular endpoints for environmental research.

There is growing interest in using wild animals to monitor the real-life cocktail effect of environmental chemicals on male reproduction. However, practical difficulties, such as long distances to the laboratory, generally prolong the time between euthanisation and specimen handling. For instance, tissue fixation is often performed on frozen material or on material where deterioration has started, which may affect tissue morphology. This study examined the effect of pre-fixation delay and freezing on mink testicular endpoints in order to determine robust endpoints in suboptimally handled specimens. Sexually mature farmed mink (n=30) selected at culling were divided into six groups and subjected to different time intervals between euthanisation and fixation or freezing: 0 hours (fixed immediately post mortem), 6 hours, 18 hours, 30 hours, 42 hours, or frozen 6 hours post mortem and thawed overnight. Unaffected endpoints when pre-fixation storage was extended to 30 hours included: area and diameter of the seminiferous tubules, length and weight of the testes, and acrosomes marked with Gata-4. Epithelial height, Sertoli cells marked with Gata-4 and cell morphology were affected endpoints after 6 hours of storage. Freezing the tissue prior to fixation severely altered cell morphology and reduced testicular weight, tubular diameter and area. Morphological changes seen after 6 hours included shredded germ cells and excess cytoplasm in seminiferous tubular lumen, chromatin rearrangements and increased germ cell death. Extended delay before fixation and freezing affected many endpoints in the mink testicular tissue. Some of these endpoints may mimic chemically induced effects, which is important to consider when evaluating specimens from wild animals for environmental toxicity.

Effects of genistein on oestrogen and progesterone receptor, proliferative marker Ki-67 and carbonic anhydrase localisation in the uterus and cervix of gilts after insemination.

Soya products are routinely fed to domestic animals as an important source of protein. The aim of this work was to study how the phytooestrogen genistein, supplemented at a feed relevant level, affects the morphology and distribution of reproductive hormone receptors, proliferative activities and carbonic anhydrase (CA) in the uterus and cervix of gilts. Eleven gilts were fed a soya-free diet. Six were given genistein (1 mg/kg bw) twice daily for eight days starting three days before expected oestrus. Five gilts were used as controls. All gilts were inseminated (AI) one day after signs of standing oestrus and euthanized three days after AI. Samples from the uterus and cervix were processed for morphometric evaluation, immunohistochemical localisation of oestrogen receptors α and ß (ERα and ERß), progesterone receptor (PR), proliferative marker Ki-67 and histochemical localisation of CA. Nuclear staining for ERß was detected in surface epithelial, glandular and some stromal cells in the uterus and in the cervix surface epithelial cells. ERα and PR were observed in surface epithelium, subepithelial stromal cells and smooth muscle cells of uterus and cervix, and glandular cells of the uterus. Ki-67 positive cells were recorded in uterine and cervical surface epithelium and subepithelial stromal layer. CA was mainly confined to glandular cells of the uterus. Immunohistochemical results were evaluated using semi-quantitative image analysis. Statistic comparison between groups revealed no differences. However, intra-treatment evaluation and correlations indicate that the supplementation of genistein modulates the expression pattern of all receptors and Ki-67, which may induce cellular activities in both the uterus and cervix of early pregnant gilts.

Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids.

BACKGROUND: Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids. METHODS: Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3-4 mg/kg/day) for approximately 3 months. Plasma testosterone and total and free triiodothyronine (T3) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out. RESULTS: No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6). A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant. CONCLUSION: Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T3; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.

Carbonic anhydrase in mouse testis and epididymis; transfer of isozyme IV to spermatozoa during passage.

Spermatozoa are subjected to major changes as they pass through the epididymal duct. The aim of the present study was to describe the distribution of carbonic anhydrase (CA) in the mouse testis and epididymis using a histochemical technique showing total catalytic activity, in combination with immunohistochemistry for the two important isoforms CAs II and IV. By comparing normal mice with CA II-deficient mice, we were able to study membrane-bound CA without influence from the ubiquitous cytoplasmic CA II. Spermatozoa, when studied in both the scanning electron and light microscope, were found to pickup membrane-bound CA IV during their passage through the epididymal duct. The transfer appeared to take place in the proximal part of the corpus, where the apical membrane and vesicles of principal cells were richly supplied with CA IV. In addition to CA IV, another membrane-bound isozyme was located in basolateral membranes of principal cells. Cytoplasmic CA II was found in varying amounts in apical/narrow cells and principal cells of the corpus in control animals. The significance of CA for pH-regulating processes vital for sperm storage and motility is discussed. A function in HCO3- transport during sperm capacitation at fertilization is suggested for the CA IV found in spermatozoa.