Trichloroethylene exposure alters dimethylated histone three lysine four in protein kinase A signaling pathway chromatin of rat sperm†.

Histone three lysine four dimethylation (H3k4me2) in sperm is conserved across species and is linked to transgenerational epigenetic inheritance. To test whether H3K4me2 is a target for transgenerational inheritance of toxicity, a daily gavage bolus exposure of trichloroethylene (TCE) (1000 mg/kg/day) was given to rats for 14 weeks, then epididymal sperm were isolated and native chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) of H3K4me2 was performed. Differential region analysis determined there were 2608 significantly differential H3K4me2 regions after TCE exposure, 477 were significantly increased and 2131 were significantly decreased. Z-score enrichment of differential regions determined there were significantly decreased H3k4me2 in the coding and regulatory regions of genes in the PKA signaling pathway. These changes account for TCE induced spermatozoal toxicity and show H3K4me2 is a target for paternal inheritance of toxicity.

Effects of continuous bisphenol A exposure from early gestation on 90 day old rat testes function and sperm molecular profiles: A CLARITY-BPA consortium study.

Bisphenol A (BPA) is a ubiquitous industrial chemical that has been identified as an endocrine disrupting compound (EDC). There is growing concern that early life exposures to EDCs, such as BPA, can adversely affect the male reproductive tract and function. This study was conducted as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA) to further delineate the toxicities associated with continuous exposure to BPA from early gestation, and to comprehensively examine the elicited effects on testes and sperm. NCTR Sprague Dawley rat dams were gavaged from gestational day (GD) 6 until parturition, and their pups were directly gavaged daily from postnatal day (PND) 1 to 90 with BPA (2.5, 25, 250, 2500, 25,000, 250,000 µg/kg/d) or vehicle control. At PND 90, the testes and sperm were collected for evaluation. The testes were histologically evaluated for altered germ cell apoptosis, sperm production, and altered spermiation. RNA and DNA isolated from sperm were assessed for elicited changes in global mRNA transcript abundance and altered DNA methylation. Effects of BPA were observed in changes in body, testis and epididymis weights only at the highest administered dose of BPA of 250,000 µg/kg/d. Genome-wide transcriptomic and epigenomic analyses failed to detect robust alterations in sperm mRNA and DNA methylation levels. These data indicate that prolonged exposure starting in utero to BPA over a wide range of levels has little, if any, impact on the testes and sperm molecular profiles of 90 day old rats as assessed by the histopathologic, morphometric, and molecular endpoints evaluated.

Identification of sperm mRNA biomarkers associated with testis injury during preclinical testing of pharmaceutical compounds.

The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4mg/kg/day), BI665915 (0, 20, 70, 100mg/kg/d), BI665636 (0, 20, 100mg/kg/d) or BI163538 (0, 70, 150, 300mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting.

Ghrelin ameliorates adhesions in a postsurgical mouse model.

BACKGROUND: Peritoneal adhesion formation is a well-recognized consequence of abdominal and pelvic surgery, causing infertility, chronic pelvic pain, and intestinal obstruction. We hypothesized that ghrelin, a 28-amino acid peptide predominantly found in the stomach, plays an important role in preventing postoperative surgical adhesions. The purpose of this study was to develop a new surgical peritoneal adhesion model to define the role that ghrelin plays in wound healing and adhesion formation. MATERIALS AND METHODS: C57BL/6 wild-type mice (n = 40) and growth hormone secretagogue receptor-knockout (GHSR KO) mice (n = 20) underwent a midline laparotomy to establish a peritoneal adhesion model characterized by the combination of two different techniques: ischemic peritoneal buttons and cecal multiple abrasion. All mice received intraperitoneal injections with ghrelin (0.16 mg/kg) or saline twice daily for 20 d after surgery. Peritoneal ischemic buttons were harvested to determine protein expression of collagen (Masson trichrome, picrosirius red stain, and Western blot). RESULTS: The novel mouse model demonstrated consistent and easily reproducible formation of intra-abdominal adhesions. Ghrelin administration significantly reduced postoperative adhesion formation (P < 0.001) in wild-type mice. The antifibrotic effect of ghrelin in wild-type mice was confirmed by measuring collagen I protein levels via Western blot analysis. The anti-adhesion effect of ghrelin seen in wild-type mice was not detected in GHSR KO mice demonstrating that this effect is mediated by the GHSR-1a receptor. CONCLUSIONS: Ghrelin administration may improve surgical outcome by reducing peritoneal adhesion formation and fibrotic response in a mouse model.

Di-n-Butyl Phthalate Induces Multinucleated Germ Cells in the Rat Fetal Testis Through a Nonproliferative Mechanism.

In utero exposure to some phthalate esters adversely affects the development of the rat seminiferous cord, causing germ cell loss and increasing the number of multinucleated germ cells (MNGs). To understand the timing of MNG formation and determine whether it requires nuclear division, timed pregnant Sprague Dawley rats were exposed to 500 mg/kg di-n-butyl phthalate (DBP) or corn oil vehicle by oral gavage on Gestational Day (GD) 17 or 18 (0 h) and euthanized after 2, 4, 6, or 24 h or given a second dose at 24 h and euthanized 48 h after the initial dose. Dams were simultaneously exposed to 0.3 M 5-bromo-2'-deoxycitidine (BrdC; converted to 5-bromo-2'-deoxyuridylate [BrdU] in vivo) through a subcutaneous micro-osmotic pump implanted at -2 h. In the testes of male fetuses, DBP induced MNGs significantly beginning at 4-6 h and dramatically by 24 h when exposure began on GD 18 but not GD 17. Seminiferous cord diameter was significantly elevated in testes of rats treated with DBP at 24 and 48 h, and cell death, measured by TUNEL assay, was significantly elevated by DBP only at 48 h, when treatment began on GD 18. TUNEL-labeled MNGs were rare. Overall BrdU labeling rate in the testis was unaffected by DBP. Only one of 606 MNGs in BrdU-labeled sections had a strongly positive nucleus, confirming a nonproliferative mechanism of MNG formation, which is a degenerative process with the potential to adversely affect testis development.

Intussusceptive-like angiogenesis in human fetal lung xenografts: Link with bronchopulmonary dysplasia-associated microvascular dysangiogenesis?

BACKGROUND: Human fetal lung xenografts display an unusual pattern of non-sprouting, plexus-forming angiogenesis that is reminiscent of the dysmorphic angioarchitecture described in bronchopulmonary dysplasia (BPD). The aim of this study was to determine the clinicopathological correlates, growth characteristics and molecular regulation of this aberrant form of graft angiogenesis. METHODS: Fetal lung xenografts, derived from 12 previable fetuses (15 to 22 weeks' gestation) and engrafted in the renal subcapsular space of SCID-beige mice, were analyzed 4 weeks posttransplantation for morphology, vascularization, proliferative activity and gene expression. RESULTS: Focal plexus-forming angiogenesis (PFA) was observed in 60/230 (26%) of xenografts. PFA was characterized by a complex network of tortuous nonsprouting vascular structures with low endothelial proliferative activity, suggestive of intussusceptive-type angiogenesis. There was no correlation between the occurrence of PFA and gestational age or time interval between delivery and engraftment. PFA was preferentially localized in the relatively hypoxic central subcapsular area. Microarray analysis suggested altered expression of 15 genes in graft regions with PFA, of which 7 are known angiogenic/lymphangiogenic regulators and 5 are known hypoxia-inducible genes. qRT-PCR analysis confirmed significant upregulation of SULF2, IGF2, and HMOX1 in graft regions with PFA. CONCLUSION: These observations in human fetal lungs ex vivo suggest that postcanalicular lungs can switch from sprouting angiogenesis to an aberrant intussusceptive-type of angiogenesis that is highly reminiscent of BPD-associated dysangiogenesis. While circumstantial evidence suggests hypoxia may be implicated, the exact triggering mechanisms, molecular regulation and clinical implications of this angiogenic switch in preterm lungs in vivo remain to be determined.

Predictors of anxiety and depression in people with colorectal cancer.

BACKGROUND: People living with colorectal cancer are at risk of anxiety and depression. We investigated what factors were most highly associated with these. METHODS: Four hundred and ninety-six people with colorectal cancer completed the Hospital Anxiety and Depression Scale (HADS). Data on functioning, symptoms, illness perceptions and social difficulties were collected by questionnaire. Case-note-identified disease, treatment and co-morbidity data were recorded. Multiple logistic regression identified factors independently predictive of anxiety and depression caseness. RESULTS: Self-reported history of anxiety/depression predicted anxiety but not depression caseness. Depression caseness predicted anxiety caseness (p = 0.043), as did poorer self-reported cognitive functioning (p = 0.001), dyspnoea (p = 0.015) or diarrhoea (p = 0.021), reporting a high negative life and emotional impact (p < 0.001) and having difficulties with finance (p = 0.007). Having neo-adjuvant radiotherapy increased the odds of depression caseness (p = 0.007), as did poorer physical (p = 0.007), cognitive (p < 0.001) and social (p < 0.001) functioning, having constipation (p = 0.011), reporting a high negative life and emotional impact (p < 0.001), having difficulties with personal care (p = 0.022) and communicating with others (p = 0.014). CONCLUSION: Levels of anxiety caseness were similar to those of non-clinical samples, but depression caseness was higher, particularly in those who had received neo-adjuvant radiotherapy. Most factors associated with possible or probable depression may be modified with appropriate intervention.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Maturation of the developing human fetal prostate in a rodent xenograft model.

BACKGROUND: Prostate cancer is the most commonly diagnosed nonskin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. METHODS: We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate-specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. RESULTS: Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture microdissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30- and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. CONCLUSION: This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease.

Interaction between mono-(2-ethylhexyl) phthalate and retinoic acid alters Sertoli cell development during fetal mouse testis cord morphogenesis.

Phthalic acid esters (phthalates) are a class of industrial chemicals that cause developmental and reproductive toxicity, but there are significant gaps in knowledge of phthalate toxicity mechanisms. There is evidence that phthalates disrupt retinoic acid signaling in the fetal testis, potentially disrupting control of spatial and temporal patterns of testis development. Our goal was to determine how a phthalate would interact with retinoic acid signaling during fetal mouse testis development. We hypothesized that mono-(2-ethylhexyl) phthalate (MEHP) would exacerbate the adverse effect of all-trans retinoic acid (ATRA) on seminiferous cord development in the mouse fetal testis. To test this hypothesis, gestational day (GD) 14 C57BL/6 mouse testes were isolated and cultured on media containing MEHP, ATRA, or a combination of both compounds. Cultured testes were collected for global transcriptome analysis after one day in culture and for histology and immunofluorescent analysis of Sertoli cell differentiation after three days in culture. ATRA disrupted seminiferous cord morphogenesis and induced aberrant FOXL2 expression. MEHP alone had no significant effect on cord development, but combined exposure to MEHP and ATRA increased the number of FOXL2-positive cells, reduced seminiferous cord number, and increased testosterone levels, beyond the effect of ATRA alone. In RNA-seq analysis, ATRA treatment and MEHP treatment resulted in differential expression of genes 510 and 134 genes, respectively, including 70 common differentially expressed genes (DEGs) between the two treatments, including genes with known roles in fetal testis development. MEHP DEGs included RAR target genes, genes involved in angiogenesis, and developmental patterning genes, including members of the homeobox superfamily. These results support the hypothesis that MEHP modulates retinoic acid signaling in the mouse fetal testis and provide insight into potential mechanisms by which phthalates disrupt seminiferous cord morphogenesis.

Toward shared care for people with cancer: developing the model with patients and GPs.

BACKGROUND: The number of people surviving cancer for extended periods is increasing. Consequently, due to workload and quality issues, there is considerable interest in alternatives to traditional secondary care-led cancer follow-up. OBJECTIVE: To explore the views of potential recipients of shared follow-up of cancer. To conduct a modelling exercise for shared follow-up and to explore the opinions and experiences of both the patients and GPs involved. METHODS: Semi-structured audio-taped telephone or face-to-face interviews were conducted with 18 patients with a range of cancers currently attending for structured follow-up in secondary care. Six GPs and five patients (four with melanoma and one with stable metastatic colorectal cancer) took part in a shared follow-up modelling exercise. During the modelling exercise, the GPs attended 4 review meetings, which included brief training seminars, and at the conclusion 10 individuals took part in semi-structured audio-taped telephone or face-to-face interviews. RESULTS: Many rural patients, and some urban patients, would appreciate follow-up being available nearer to home with the associated benefits of time saved and easier parking and continuity of care. Patients have concerns related to the level of extra training received by the GP and loss of contact with their consultant. GPs have concerns about gaining and maintaining the clinical skills needed to conduct follow-up, especially if the numbers of patients seen are small. They also have concerns about lack of support from other GPs, and some administrative and organizational issues. CONCLUSIONS: Many patients would be willing to have GPs share their cancer follow-up with the caveat that they had received extra training and were appropriately supported by secondary care specialists. Patients attending shared care clinics appreciated a local service and longer appointment times. GPs stress the importance of maintaining their own clinical skills and reliable clinical and administrative support from secondary care.

Small RNAs in Rat Sperm Are a Predictive and Sensitive Biomarker of Exposure to the Testicular Toxicant Ethylene Glycol Monomethyl Ether.

Testicular histology and semen parameters are considered the gold standards when determining male reproductive toxicity. Ethylene glycol monomethyl ether (EGME) is a testicular toxicant with well-described effects on histopathology and sperm parameters. To compare the predictivity and sensitivity of molecular biomarkers of testicular toxicity to the traditional endpoints, small RNAs in the sperm were analyzed by next generation RNA-sequencing (RNA-seq). Adult rats were exposed to 0, 50, 60, or 75 mg/kg EGME by oral gavage for 5 consecutive days. Testis histology, epididymal sperm motility, and sperm small RNAs, including microRNAs (miRNAs), mRNA fragments, piwi-interacting RNAs (piRNAs), and tRNA fragments (tRFs), were analyzed 5 weeks after cessation of exposure. Testicular histology showed a significant dose-dependent increase in retained spermatid heads (RSH), while sperm motility declined with increasing dose. RNA-sequencing of sperm small RNAs was used to identify significant dose-dependent changes in percent mRNA fragments (of total reads), percent miRNAs (of total reads), average tRF length, average piRNA length, and piRNA and tRF length-distributions. Discriminant analysis showed relatively low predictivity of exposure based on RSH or motility compared to the average read length of all assigned RNAs. Benchmark dose (BMD) modeling resulted in a BMD of 62 mg/kg using RSH, whereas average read length of all assigned RNAs resulted in a BMD of 47 mg/kg. These results showed that sperm small RNAs are sensitive and predictive biomarkers of EGME-induced male reproductive toxicity.

All-trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. II: Modulation of Mono-(2-ethylhexyl) Phthalate Toxicity.

Humans are universally exposed to low levels of phthalate esters (phthalates), which are used to plasticize polyvinyl chloride. Phthalates exert adverse effects on the development of seminiferous cords in the fetal testis through unknown toxicity pathways. To investigate the hypothesis that phthalates alter seminiferous cord development by disrupting retinoic acid (RA) signaling in the fetal testis, gestational day 15 fetal rat testes were exposed for 1-3 days to 10-6 M all-trans retinoic acid (ATRA) alone or in combination with 10-6-10-4 M mono-(2-ethylhexyl) phthalate (MEHP) in ex vivo culture. As previously reported, exogenous ATRA reduced seminiferous cord number. This effect was attenuated in a concentration-dependent fashion by MEHP co-exposure. ATRA and MEHP-exposed testes were depleted of DDX4-positive germ cells but not Sertoli cells. MEHP alone enhanced the expression of the RA receptor target Rbp1 and the ovary development-associated genes Wnt4 and Nr0b1, and suppressed expression of the Leydig cell marker, Star, and the germ cell markers, Ddx4 and Pou5f1. In co-exposures, MEHP predominantly enhanced the gene expression effects of ATRA, but the Wnt4 and Nr0b1 concentration-responses were nonlinear. Similarly, ATRA increased the number of cells expressing the granulosa cell marker FOXL2 in testis cultures, but this induction was attenuated by addition of MEHP. These results indicate that MEHP can both enhance and inhibit actions of ATRA during fetal testis development and provide evidence that RA signaling is a target for phthalate toxicity in the fetal testis.

Spermatozoal large RNA content is associated with semen characteristics, sociodemographic and lifestyle factors.

Semen analysis is one of the standard diagnostic tools currently used to assess male infertility and reproductive toxicity. However, semen analysis has a limited ability to separate fertile from infertile populations. Additional methods to detect impaired fertility are needed. The purpose of the present study was to evaluate how spermatozoal RNA content varies with sociodemographic and behavior/lifestyle factors, and to determine if spermatozoal large and small RNAs discriminate normal from abnormal spermatozoa. Semen specimens were collected from 133 men aged between 18 to 55 years undergoing semen analysis as part of couple infertility evaluation while 10 proven fertile donors were recruited as control group. Semen samples were classified as normal or abnormal according to World Health Organization (WHO) 2010 criteria. Sperm RNAs were extracted after somatic cells were lysed, and the association of large or small RNA content with semen quality and sociodemographic and behavioral/lifestyle factors was evaluated using a generalized additive model and one-way ANOVA. Inverse relationship was observed between large RNA content and sperm parameters such as sperm count, density and motility. Large RNA content per sperm was significantly increased in semen samples showing abnormal number of round cells. Furthermore, sperm motility was inversely associated with spermatozoal small RNA contents. Grouping donors by the number of semen abnormalities, we observed significant increased spermatozoal large and small RNA content in men with more than two semen abnormalities. Alcohol consumption was strongly associated with increased large RNA per sperm concentration after adjustment for age and BMI. Our study demonstrates a strong relationship between spermatozoal large RNA content and poor semen characteristics that may lead to a role in the assessment of male fertility, and may be used as an endpoint for reproductive toxicology risk assessment.

All-Trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. I: Altered Seminiferous Cord Maturation and Testicular Cell Fate.

Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10-8 to 10-6 M) or vehicle for 3 days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1 day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3 days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.

Differential toxicity of water versus gavage exposure to trichloroethylene in rats.

Trichloroethylene (TCE) is a persistent environmental contaminant that causes male reproductive toxicity. We investigated whether transient increases in TCE exposure modulated male reproductive toxicity by exposing rats via daily oral to repeated gavage exposures (1000 mg/kg/day) and through drinking water (0.6% TCE) for 14 weeks. The gavage route resulted in reversible reduction of epididymis weight, and reduced body weight that persisted for up to 12-weeks after cessation of exposure. Physiologically-based pharmacokinetic modeling predicted that the gavage route results in higher Cmax and AUC exposure of TCE compared to drinking water exposure, explaining the observed differences in toxicity between dosing regimens.

Fetal mouse phthalate exposure shows that Gonocyte multinucleation is not associated with decreased testicular testosterone.

The rat has been explored in detail for its in utero susceptibility to male reproductive tract malformation following phthalate exposure. Few other species have been studied in detail, and it is important for both mechanistic and risk assessment purposes to understand the species specificity of this response. We investigated the response of the fetal mouse testis to phthalate exposure and compared these results with those previously obtained from the rat. Initial experiments using a variety of phthalate congeners (monobutyl phthalate, di-(n-butyl) phthalate, or mono (2-ethylhexyl) phthalate) and exposure paradigms did not reduce fetal mouse testis testosterone levels. Pharmacokinetic data after a single 500 mg/kg di-(n-butyl)-phthalate (DBP) exposure on mouse gestation day (gd) 18 demonstrated that the concentrations and kinetics of the active metabolite monobutyl phthalate (MBP) in fetal and maternal plasma were similar to the rat. After a single 500 mg/kg or multiple day 250 mg/kg fetal mouse DBP exposure, rapid and dynamic changes in testis gene expression were observed, including induction of immediate early genes. Unlike the rat, expression of genes involved in cholesterol homeostasis and steroidogenesis were not decreased and were increased in a few cases. Similar to the rat, however, a 250- or 500-mg DBP/kg/day mouse exposure from gd 16 through 18 significantly increased seminiferous cord diameter, the number of multinucleated gonocytes per cord, and the number of nuclei per multinucleated gonocyte. Together, these results demonstrate that fetal mouse and rat phthalate exposure both induce immediate early gene expression and disrupt seminiferous cord and gonocyte development. This response in the mouse occurs without a measurable decrease in testicular testosterone, suggesting that altered seminiferous cord formation and gonocyte multinucleation may not be mechanistically linked to lowered testosterone.

Ghrelin modulates testicular damage in a cryptorchid mouse model.

Cryptorchidism or undescended testis (UDT) is a common congenital abnormality associated with increased risk for developing male infertility and testicular cancer. This study elucidated the effects of endogenous ghrelin or growth hormone secretagogue receptor (GHSR) deletion on mouse reproductive performance and evaluated the ability of ghrelin to prevent testicular damage in a surgical cryptorchid mouse model. Reciprocal matings with heterozygous/homozygous ghrelin and GHSR knockout mice were performed. Litter size and germ cell apoptosis were recorded and testicular histological evaluations were performed. Wild type and GHSR knockout adult mice were subjected to creation of unilateral surgical cryptorchidism that is a model of heat-induced germ cell death. All mice were randomly separated into two groups: treatment with ghrelin or with saline. To assess testicular damage, the following endpoints were evaluated: testis weight, seminiferous tubule diameter, percentage of seminiferous tubules with spermatids and with multinucleated giant cells. Our findings indicated that endogenous ghrelin deletion altered male fertility. Moreover, ghrelin treatment ameliorated the testicular weight changes caused by surgically induced cryptorchidism. Testicular histopathology revealed a significant preservation of spermatogenesis and seminiferous tubule diameter in the ghrelin-treated cryptorchid testes of GHSR KO mice, suggesting that this protective effect of ghrelin was mediated by an unknown mechanism. In conclusion, ghrelin therapy could be useful to suppress testicular damage induced by hyperthermia, and future investigations will focus on the underlying mechanisms by which ghrelin mitigates testicular damage.

Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat.

The tumor suppressor protein p53 (TP53) has many functions in cell cycle regulation, apoptosis, and DNA damage repair and is also involved in spermatogenesis in the mouse. To evaluate the role of p53 in spermatogenesis in the rat, we characterized testis biology in adult males of a novel p53 knockout rat (SD-Tp53tm1sage ). p53 knockout rats exhibited variable levels of testicular atrophy, including significantly decreased testis weights, atrophic seminiferous tubules, decreased seminiferous tubule diameter, and elevated spermatocyte TUNEL labeling rates, indicating a dysfunction in spermatogenesis. Phosphorylated histone H2AX protein levels and distribution were similar in the non-atrophic seminiferous tubules of both genotypes, showing evidence of pre-synaptic DNA double-strand breaks in leptotene and zygotene spermatocytes, preceding cell death in p53 knockout rat testes. Quantification of the spermatogonial stem cell (SSC) proliferation rate with bromodeoxyuridine (BrdU) labeling, in addition to staining with the undifferentiated type A spermatogonial marker GDNF family receptor alpha-1 (GFRA1), indicated that the undifferentiated spermatogonial population was normal in p53 knockout rats. Following exposure to 0.5 or 5 Gy X-ray, p53 knockout rats exhibited no germ cell apoptotic response beyond their unirradiated phenotype, while germ cell death in wild-type rat testes was elevated to a level similar to the unexposed p53 knockout rats. This study indicates that seminiferous tubule atrophy occurs following spontaneous, elevated levels of spermatocyte death in the p53 knockout rat. This phenomenon is variable across individual rats. These results indicate a critical role for p53 in rat germ cell survival and spermatogenesis.

Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-ß Signaling Pathway.

Post-operative adhesions are a critical problem in pelvic and abdominal surgery despite a multitude of studies dedicated to finding modalities to prevent their occurrence. Ghrelin administration promotes an anti-fibrotic response in a surgical mouse model of adhesion-induction, but the mechanisms mediating this effect have not been established. In the current study, the molecular mechanisms that underlie the anti-adhesion effect of ghrelin were investigated. Post-surgical adhesions were experimentally created in C57BL/6 wild-type mice via a combination of ischemic peritoneal buttons and cecal multiple abrasions. Ghrelin or saline intraperitoneal injections were given twice daily from two days before surgery to selected time points post-surgically to assess the phenotypic and molecular effects of treatment (1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) after surgery). Endpoints included the scoring of adhesions and gene and protein expression analysis of pro-fibrogenic factors conducted on peritoneal ischemic tissue by quantitative PCR and Western blot. Ghrelin administration significantly reduced post-surgical adhesions and down-regulated pro-inflammatory gene and protein expression, including Tgfb3 and Tgfbr2. The up-regulation of inhibitory proteins Smad6 and Smad7 confirmed the ghrelin-induced blockage of TGF-ß signaling. Ghrelin is a candidate therapeutic drug for post-operative adhesion prevention, inhibiting inflammatory responses via blockage of the TGF-ß signaling pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.