Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane.

The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization (e.g., lipid rafts). Herein, we determined the effects of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the sperm plasma membrane, using Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit ß), liquid chromatography - mass spectroscopy (LC-MS) analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence were used to describe and compare sperm fluidity, membrane composition, as well as lipid raft composition and distribution among sperm types. Stripped sperm became more fluid following motility activation (40% increase in highly fluid cells characterized by a 2 × increase in fluorescence) and contained lipid rafts restricted to the midpiece. Testicular harvest yielded sperm with characteristics similar to stripped sperm. By contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process. Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decrease in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm. Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to substantially affect the sperm plasma membrane, but not always detrimentally. These results provide multiple potential biomarkers of sperm quality as well as insight into sources of sub-fertility resulting from use of ARTs.

Equine Oviductal Organoid Generation and Cryopreservation.

Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids.

Multifocal Necrotizing Myopathy in Northern Elephant Seal ( Mirounga angustirostris) Pups, San Miguel Island, California.

A field study addressing causes of mortality in freshly dead northern elephant seals ( Mirounga angustirostris, Gill, 1866) was conducted on San Miguel Island, California, in February 2015. Necropsies were performed on 18 pups ranging in age from stillbirths to approximately 7 to 8 weeks. The primary gross diagnoses in these pups included trauma, myopathy, starvation/emaciation, infections, congenital anomalies, and perinatal mortality. However, 6 (33%) had a previously unrecognized myopathy characterized by multiple white streaks that were most obvious within the inner layer of the abdominal wall and the small innermost ventral intercostal muscles. Following histological examination, 2 more pups from San Miguel Island and 6 pups from The Marine Mammal Center (Sausalito, California) were found to have similar lesions. Histologically, the lesions within the skeletal muscles were characterized by a multifocal polyphasic, mild to severe, acute to subacute necrotizing myopathy with mineralization. Acute necrosis and degeneration characterized by pyknotic nuclei, eosinophilic cytoplasm and cytoplasmic vacuolization were found in smooth muscle myocytes within the urinary bladder and digestive system. Degeneration of myocytes was present in the tunica media of a few small- to medium-sized vessels and was characterized by a vacuolar degeneration and occasionally necrosis. This condition has been termed multifocal necrotizing myopathy. A cause of this myopathy was not identified.

Perinatal Bisphenol A Exposure Induces Chronic Inflammation in Rabbit Offspring via Modulation of Gut Bacteria and Their Metabolites.

Bisphenol A (BPA) accumulates in the maturing gut and liver in utero and is known to alter gut bacterial profiles in offspring. Gut bacterial dysbiosis may contribute to chronic colonic and systemic inflammation. We hypothesized that perinatal BPA exposure-induced intestinal (and liver) inflammation in offspring is due to alterations in the microbiome and colonic metabolome. The 16S rRNA amplicon sequencing analysis revealed differences in beta diversity with a significant reduction in the relative abundances of short-chain fatty acid (SCFA) producers such as Oscillospira and Ruminococcaceae due to BPA exposure. Furthermore, BPA exposure reduced fecal SCFA levels and increased systemic lipopolysaccharide (LPS) levels. BPA exposure-increased intestinal permeability was ameliorated by the addition of SCFA in vitro. Metabolic fingerprints revealed alterations in global metabolism and amino acid metabolism. Thus, our findings indicate that perinatal BPA exposure may cause gut bacterial dysbiosis and altered metabolite profiles, particularly SCFA profiles, leading to chronic colon and liver inflammation. IMPORTANCE Emerging evidence suggests that environmental toxicants may influence inflammation-promoted chronic disease susceptibility during early life. BPA, an environmental endocrine disruptor, can transfer across the placenta and accumulate in fetal gut and liver. However, underlying mechanisms for BPA-induced colonic and liver inflammation are not fully elucidated. In this report, we show how perinatal BPA exposure in rabbits alters gut microbiota and their metabolite profiles, which leads to colonic and liver inflammation as well as to increased gut permeability as measured by elevated serum lipopolysaccharide (LPS) levels in the offspring. Also, perinatal BPA exposure leads to reduced levels of gut bacterial diversity and bacterial metabolites (short-chain fatty acids [SCFA]) and elevated gut permeability-three common early biomarkers of inflammation-promoted chronic diseases. In addition, we showed that SCFA ameliorated BPA-induced intestinal permeability in vitro. Thus, our study results suggest that correcting environmental toxicant-induced bacterial dysbiosis early in life may reduce the risk of chronic diseases later in life.

Circulating miRNAs as Potential Alternative Cell Signaling Associated with Maternal Recognition of Pregnancy in the Mare.

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy.

Embryo mortality in Isg15-/- mice is exacerbated by environmental stress.

The interferon-stimulated gene 15 (Isg15) encodes a ubiquitin-like protein that is induced in the endometrium by pregnancy in mice, humans, and ruminants. Because ISG15 is a component of the innate immune system, we hypothesized that development of the embryo, fetus, and postnatal pup may be impaired in mice lacking Isg15 (Isg15(-/-)) and that this development would be further impaired in response to environmental insults such as hypoxia. The number of implantation sites, resorption sites, dead embryos, and the changes in overall gross morphology of the uterus were evaluated in Isg15(-/-) mice on Days 7.5 and 12.5 postcoitum (dpc). Postnatal development also was monitored from birth to 12 wk of age. On 7.5 dpc, the number of implantation sites and serum progesterone concentrations were similar. However, embryo mortality increased (P < 0.05) in Isg15(-/-) dams by 12.5 dpc, resulting in smaller litter sizes (4.26 ± 0.21 embryos; n = 83 litters) compared to Isg15(+/+) females (7.78 ± 0.29 pups; n = 47 litters). Embryo mortality in Isg15(-/-) mice was further exacerbated to 70% when dams were stressed through housing under hypoxic conditions (PB = 445 mmHg; 6.5-12.5 dpc). Transmission electron microscopy revealed lesions in antimesometrial decidua as well as trophoblast cells adjacent to decidual cells on 7.5 dpc. ISG15 was localized to mesometrial decidua on 7.5 dpc. By 12.5 dpc, ISG15 was intensely localized to the labyrinth of the placenta. By 7.5 dpc, uterine natural killer cell migration into the mesometrial pole was diminished by 65% and was less prevalent in Isg15(-/-) compared to Isg15(+/+) deciduum. Postnatal growth rate of offspring that survived to birth from Isg15(-/-) and Isg15(+/+) dams was not different. Embryo mortality occurs in pregnant Isg15(-/-) mice, is exacerbated by environmental insults like maternal hypoxia, and might result from impaired early decidualization, vascular development, and formation of the labyrinth.

Colon carcinogenesis: influence of Western diet-induced obesity and targeting stem cells using dietary bioactive compounds.

Colon cancer strikes more than 1 million people annually and is responsible for more than 500,000 cancer deaths worldwide. Recent evidence suggests that the majority of malignancies, including colon cancer are driven by cancer stem cells (CSCs) that are resistant to current chemotherapeutic approaches leading to cancer relapse. Wnt signaling plays a critical role in colon stem cell renewal and carcinogenesis. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a Wnt target gene, and aldehyde dehydrogenase 1 B1 (ALDH1B1) are good markers for normal and malignant human colon stem cells. Diet contributes to 20% to 42% of all human cancers and 50% to 90% of colon cancer. Recent evidence shows that the Western diet has a causative link to colon cancer; however, mechanisms of action are not fully elucidated. Western diet-induced obesity elevates systemic insulin-like growth factor-1 and insulin levels, which could lead to elevated proliferation and suppressed apoptosis of CSCs through PI3K/AKT/Wnt pathway. Although conventional chemotherapy targets the PI3K/AKT pathways and can significantly reduce tumor size, it fails to eliminate CSCs and has serious side effects. Dietary bioactive compounds such as grape seed extract, curcumin, lycopene, and resveratrol have promising chemopreventive effects, without serious side effects on various types of cancers due to their direct and indirect actions on CSC self-renewal pathways such as the Wnt pathway. Understanding the role of CSCs in diet-induced colon cancer will aid in development of evidence-based dietary chemopreventive strategies and/or therapeutic agents targeting CSCs.

Phthalate-induced pathology in the foetal testis involves more than decreased testosterone production.

Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have been noted; e.g., following foetal exposure, abnormal clustering of Leydig cells or decreased production of testosterone that is manifested in rats does not occur in mice or humans. Nonetheless, other facets of testicular dysgenesis occur in both rats and mice as well as in some other species tested. We recently published a comprehensive evaluation of the foetal rat testis proteome, following in utero exposure to diethylhexyl phthalate (DEHP), which revealed changes in individual proteins that are known to be factors in cellular differentiation and migration or related to the capacity of the foetal Leydig cell to produce testosterone and fit a pathway network in which each is regulated directly or indirectly by oestradiol. Plasma oestradiol indeed was found to be elevated approximately twofold in 19-day-old DEHP-exposed foetal male rats. In this brief review, we discuss our new findings vis-à-vis 'oestrogen hypothesis' as a cause for testicular dysgenesis syndrome.

Echocardiographic evidence of left ventricular hypertrophy in obese dogs.

BACKGROUND: Cardiomyopathy of obesity occurs in humans, but the gross and cellular myocardial response to obesity in dogs is not well defined. OBJECTIVES: To characterize in vivo myocardial morphology and function in normotensive obese dogs, and quantitate collagen, triglyceride and myocyte cross-sectional area (CSA) in postmortem tissues from obese dogs. ANIMALS: Echocardiographic-Doppler measurements of normotensive obese dogs (n = 19) without historical or physical examination evidence of disease, and lean healthy dogs (n = 19) matched for age and ideal weight. Postmortem data were obtained from a separate population of 4 obese and 12 lean dogs without evidence of cardiac disease. METHODS: A prospective, observational study of myocardial morphology and function was conducted by echocardiographic-Doppler measurement. Left ventricular (LV) tissue was collected for quantitation of triglyceride, collagen, and myocyte CSA. RESULTS: Compared with lean control dogs, obese dogs had increased systolic blood pressure (obese 153 ± 19 mm Hg; lean 133 ± 20 mm Hg; P = .003), and increased LV free wall thickness at end-diastole (obese 9.9 ± 1.8 mm, lean 8.7 ± 1.5 mm; P = .03) and end-systole (obese 15.2 ± 2.3 mm, lean 12.9 ± 2.3 mm; P = .004). Isovolumic relaxation time was prolonged in 7/19 (37%) of obese dogs, compared with normal ranges. Myocardial triglyceride and collagen content and myocyte CSA were similar between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: As in humans, LV hypertrophy and diastolic dysfunction can be an early myocardial change in some obese dogs.

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol.

Significant research has been focused on phthalate-induced alterations in male reproductive development. Studies on rodents have prompted the notion that a syndrome exists in the human male which includes phenotypic alterations such as hypospadias, cryptorchidism, poor semen quality, and even testicular cancer. Each phenotype in this 'testicular dysgenesis syndrome' is predicated on reduction in testosterone production by the fetal Leydig cell. We sought to examine the relationship between dysgenesis and steroidogenic capacity in the fetal rat testis more stringently by incorporating lower exposures than those typically used, conducting a comprehensive, non-targeted quantitative evaluation of the fetal testis proteome, and relating alterations in individual proteins to the capacity of the fetal Leydig cell to produce testosterone, and histopathology of the fetal testis. Pregnant dams were dosed orally from gestation day (GD) 13-19 with 0, 10, or 100 mg diethylhexyl phthalate (DEHP)/kg body weight per day. Each endpoint was represented by 16l. Clustering of Leydig cells occurred before any significant decrease in the capacity of the GD19 Leydig cell to produce testosterone. At 100 mg DEHP/kg, testosterone production was reduced significantly, Leydig cell clusters became quite large, and additional dysgenetic changes were observed in the fetal testis. Of 23 proteins whose expression was altered significantly at both DEHP exposure levels, seven were found to be correlated with and predictive of the quantified endpoints. None of these proteins have been previously implicated with DEHP exposure. Notably, pathway analysis revealed that these seven proteins fit a pathway network in which each is regulated directly or indirectly by estradiol.

Overexpression of follistatin in the mouse epididymis disrupts fluid resorption and sperm transit in testicular excurrent ducts.

Activin is a well-established modulator of male and female reproduction that stimulates the synthesis and secretion of follicle-stimulating hormone. Nonpituitary effects of activin have also been reported, although the paracrine actions of this growth factor in several reproductive tissues are not well understood. To identify the paracrine functions of activin during mammary gland morphogenesis and tumor progression, we produced transgenic mice that overexpress follistatin (FST), an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-Fst mice were constructed to assess the role of activin in females, expression of the transgene was also observed in the testes and epididymides of males. While all 17 transgenic founder males exhibited copulatory behavior and produced vaginal plugs in females, only one produced live offspring. In contrast, transgenic females were fertile, permitting expansion of transgenic mouse lines. Light and transmission electron microscopic examination of the transgenic testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Sperm collected from the caudae epididymidis of MMTV-Fst males had detached heads and were immotile. Together, these data reveal that activin signaling is essential for normal testicular excurrent duct function and that its blockade impairs fertility. These results also suggest that selective inhibitors of activin signaling may provide a useful approach for the development of male contraceptives without compromising androgen synthesis and actions.

Ultrastructural evaluation of semen to assess effects of exposure to toxicants.

Conventional light microscopic evaluation of a semen ejaculate does not fully utilize potential indicators of functional impairment in sperm organelles. The technique described here facilitates critical evaluation of morphological features of spermatozoal organelles at an ultrastructural level and helps identify vulnerable targets. Compared with a battery of sperm function assays employed in andrology clinics, this relatively less expensive technique efficiently uses semen as biopsy material and thus serves as a comprehensive means to assess the impact of toxicants on the male reproductive system.

Docosahexaenoic acid supplementation does not improve Western diet-induced cardiomyopathy in rats.

Obesity increases risk for cardiomyopathy in the absence of hypertension, diabetes or ischemia. The fatty acid milieu, modulated by diet, may modify myocardial structure and function, lending partial explanation for the array of cardiomyopathic phenotypy. We sought to identify gross, cellular and ultrastructural myocardial changes associated with Western diet intake, and subsequent modification with docosahexaenoic acid (DHA) supplementation. Wistar and Sprague-Dawley (SD) rats received 1 of 3 diets: control (CON); Western (WES); Western + DHA (WES+DHA). After 12 weeks of treatment, echocardiography was performed and myocardial adiponectin, fatty acids, collagen, area occupied by lipid and myocytes, and ultrastructure were determined. Strain effects included higher serum adiponectin in Wistar rats, and differences in myocardial fatty acid composition. Diet effects were evident in that both WES and WES+DHA feeding were associated with similarly increased left ventricular (LV) diastolic cranial wall thickness (LVW(cr/d)) and decreased diastolic internal diameter (LVID(d)), compared to CON. Unexpectedly, WES+DHA feeding was associated additionally with increased thickness of the LV cranial wall during systole (LVW(cr/s)) and the caudal wall during diastole (LVW(ca/d)) compared to CON; this was observed concomitantly with increased serum and myocardial adiponectin. Diastolic dysfunction was present in WES+DHA rats compared to both WES and CON. Myocyte cross sectional area (CSA) was greater in WES compared to CON rats. In both fat-fed groups, transmission electron microscopy (TEM) revealed myofibril degeneration, disorganized mitochondrial cristae, lipid inclusions and vacuolation. In the absence of hypertension and whole body insulin resistance, WES+DHA intake was associated with more global LV thickening and with diastolic dysfunction, compared to WES feeding alone. Myocyte hypertrophy, possibly related to subcellular injury, is an early change that may contribute to gross hypertrophy. Strain differences in adipokines and myocardial fatty acid accretion may underlie heterogeneous data from rodent studies.

Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle.

Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.

Testicular germ cell tumours in dogs are predominantly of spermatocytic seminoma type and are frequently associated with somatic cell tumours.

Unlike seminomas in humans, seminomas in animals are not typically sub-classified as classical or spermatocytic types. To compare testicular germ cell tumours (TGCT) in dogs with those of men, archived tissues from 347 cases of canine testicular tumours were morphologically evaluated and characterized using human classification criteria. Histopathological and immunohistological analysis of PLAP, KIT, DAZ and DMRT1 expression revealed that canine seminomas closely resemble human spermatocytic seminomas. In addition, a relatively frequent concomitant presence of somatic cell tumours was noted in canine TGCT. None of the canine TGCT evaluated demonstrated the presence of carcinoma in situ cells, a standard feature of human classical seminomas, suggesting that classical seminomas either do not occur in dogs or are rare in occurrence. Canine spermatocytic seminomas may provide a useful model for this rare human neoplasm.

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

BACKGROUND: Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model. METHODS: We determined the expression of 128 miRNAs by real time PCR in early-gestational (gestational day (GD) 42) and mid-gestational (GD75) sheep ovaries and testes. Expression data were further examined and validated by bioinformatic analysis. RESULTS: Expression analysis revealed significant differences between ovaries and testes among 24 miRNAs at GD42, and 43 miRNAs at GD75. Bioinformatic analysis revealed that a number of differentially expressed miRNAs are predicted to target genes known to be important in mammalian gonadal development, including ESR1, CYP19A1, and SOX9. In situ hybridization revealed miR-22 localization within fetal testicular cords. As estrogen signaling is important in human and sheep ovarian development, these data indicate that miR-22 is involved in repressing estrogen signaling within fetal testes. CONCLUSIONS: Based on our results we postulate that gene expression networks underlying fetal gonadal development are regulated by miRNAs.

Cottontail rabbit papillomavirus in Langerhans cells in Sylvilagus spp.

A wildlife sanctuary presented an adult female cottontail rabbit (Sylvilagus spp.), age unknown, to the Colorado State University Pathology service for postmortem examination. Gross examination revealed numerous pigmented wartlike lesions arising from the skin of the head surrounding the ears, eyes, nares, mouth, and dorsum. Masses were firm, friable, and easily detached from the underlying skin. Differential diagnoses included Cottontail rabbit papillomavirus, Rabbit fibroma virus, and Myxoma virus. Histological examination revealed multiple papillary masses lined by stratified squamous epithelial cells with central cores of fibrovascular connective tissue and parakeratotic hyperkeratosis. Cells of the Stratum spinosum were frequently swollen with abundant perinuclear, cytoplasmic, clearing, and occasional intranuclear basophilic, glassy, spherical inclusions up to 3 microm in diameter. The lesions were consistent with Cottontail rabbit papillomavirus infection. Papilloma virus antigens were identified by immunohistochemistry. In addition, papillomavirus particles were identified by transmission electron microscopy within Langerhans cells of the epidermis, suggesting a unique mechanism for systemic dissemination of the virus. The present case report highlights the finding of viral particles within the Langerhans cells and suggests a novel mechanism of pathogenesis.

Testicular microlithiasis and neoplastic lesions in wild eland (Tragelaphus oryx): possible effects of exposure to environmental pollutants?

The purpose of the study was to compare wildlife in the proximity and away from the sources of known industrial pollution. Macroscopic, focal, gritty areas that appeared white were observed in the testes of all 24 South African eland (Tragelaphus oryx) culled in the Rietvlei Nature Reserve (RNR; n=17) between 2001 and 2003 and Suikerbosrand Nature Reserve (SNR; n=7) in 2004. Histopathological evaluation of testes showed multiple intratubular dystrophic calcifications, focal areas of sperm stasis and interstitial chronic cell infiltrates with fibrosis. Spermatogenesis was generally impaired; a few atypical germ cells were also encountered. Sertoli cell vacuolization and sloughing of the seminiferous epithelium were evident. Adenomatous changes of the rete testis, reflective of possible chronic estrogenic exposure, were found. In testes collected from three reference eland in 2007 from the Molopo Nature Reserve (MNR) in the Kalahari/Kgalagadi Desert, except for one focal area of sperm stasis and another with microcalcification, the seminiferous epithelium as well as collecting/rete tubules were normal. Analyses of fat tissue for environmental pollutants showed that 11 out of 17 RNR eland contained a detectable estrogenic chemical p-nonylphenol (mean+/-SD: 184.8+/-24.6 microg/kg fat); no organochlorine chemicals or polychlorinated biphenyls were detected. Of the 7 SNR eland, 5 had detectable octylphenol residues (50.2+/-30.9 microg/kg fat), 3 had detectable p-nonylphenol (137.8+/-77.9 microg/kg fat), 3 had o-p'-DDT (114.9+/-31.1 microg/kg fat), 3 had p-p'-DDT (127.3+/-49.9 microg/kg(79.5+/-30.4 microg/kg fat) and 5 contained o-p'-DDE (27.7+/-9.9 microg/kg fat). One eland from the MNR contained one 70.6 microg o-p'-DDT/kg fat and another p-p'-DDE 61.3 microg/kg fat. Therefore, in eland with testicular abnormalities, significant amounts of various estrogenic chemicals were bioaccumulated in fat samples. It therefore seems likely that the lesions found in RNR and SNR were associated with the relatively high body-burden of environmental pollutants (phenols), although the possibility of systemic infections cannot be ruled out. No testicular abnormalities were found in reference eland. These findings are the first indication of mammalian wildlife being affected by environmental pollution of endocrine disrupting chemicals in South Africa.