Deficiency of CDKN1A or both CDKN1A and CDKN1B affects the pubertal development of mouse Leydig cells.

Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.

Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.

An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. Time- and cost-effective biological tools to detect and screen these compounds with potential high-throughput capabilities are in ever-growing demand. We generated a knock-in zebrafish transgenic line with enhanced green fluorescent protein (EGFP) driven by the regulatory region upstream of vitellogenin 1 (vtg1), a well-studied biomarker for estrogenic exposure, using CRISPR/Cas9 technology. Exposure to 17ß-estradiol (E2: 0-625 nM) starting at 4-h post-fertilization in dechorionated embryos resulted in the significant induction of hepatic EGFP with ≥5 nM E2 as early as 3-days post-fertilization. Concentration- and time-dependent increase in the percentage of hepatic EGFP-positive larvae and extent of fluorescence expression, categorized into 3 expression levels, were observed with E2 exposure. A strong correlation between the levels of EGFP mRNA, vtg1 mRNA, and EGFP fluorescence levels were detected. Image analysis of the area and intensity of hepatic EGFP fluorescence resulted in high-fidelity quantitative measures that could be used in automated screening applications. In addition, exposure to bisphenol A (0-30 µM) resulted in quantitative responses showing promise for the use of this transgenic line to assess estrogenic activity of endocrine-disrupting chemicals. These results demonstrate that this novel knock-in zebrafish reporter allows for distinct screening of in vivo estrogenic effects, endpoints of which can be used for laboratory testing of samples for estimation of possible human and environmental risks.

Estrogen signaling is not required for prostatic bud patterning or for its disruption by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Estrogens play an important role in prostatic development, health, and disease. While estrogen signaling is essential for normal postnatal prostate development, little is known about its prenatal role in control animals. We tested the hypothesis that estrogen signaling is needed for normal male prostatic bud patterning. Budding patterns were examined by scanning electron microscopy of urogenital sinus epithelium from wild-type mice, mice lacking estrogen receptor (ER)alpha, ERbeta, or both, and wild-type mice exposed to the antiestrogen ICI 182,780. Budding phenotypes did not detectably differ among any of these groups, strongly suggesting that estrogen signaling is not needed to establish the prototypical prostatic budding pattern seen in control males. This finding contributes to our understanding of the effects of low-level estrogen exposure on early prostate development. In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can greatly alter the pattern in which prostatic buds form and reduce their number. For several reasons, including a prior observation that inhibitory effects of TCDD on prostatic budding in rats depend heavily on the sex of adjacent fetuses, we tested the hypothesis that estrogen signaling is needed for TCDD to disrupt prostatic budding. However, budding did not detectably differ among wild-type mice, or mice lacking ERalpha, ERbeta, or both, that were exposed prenatally to TCDD (5 microg/kg on embryonic day 13.5). Nor did ICI 182,780 detectably affect the response to TCDD. These results strongly suggest that estrogen signaling is not needed for TCDD to inhibit prostatic epithelial budding.

Endocrine disruption in the context of life cycles: perception and transduction of environmental cues.

Environmental and social stresses have major impacts on the life cycles of organisms. Furthermore, habitat disturbance/destruction, global climate change, and existence of endocrine disrupting chemicals (EDCs) due to human activities are increasingly likely to pose additive and synergistic stresses that could have potential deleterious effects on physiological function in vertebrates. Central to an organism's life cycle is the ability to respond to environmental cues, physical and social. Environmental signals may act directly on endocrine tissues, but most act through neural pathways, and neuroendocrine and endocrine secretions that affect changes in morphology, physiology and behavior. While most investigations focus on endocrine secretions and their effects, we know much less about perception and transduction of environmental signals. Additionally, some populations of vertebrates, from fish to mammals, temporarily resist environmental and social stresses, and breed successfully. However, many show varying degrees of failure, sometimes resulting in marked population decline. There is potential for EDCs to act at all levels of the response systems to environmental cues. Because animals live in diverse habitats, there is variation in susceptibility to disruption of response systems to environmental cues. Although this may be partly due to genetic differences at a level of receptors and/or metabolism, fundamental differences in how species perceive environmental cues and respond to them may also be major factors. Here we discuss how EDCs may interact with the perception and transduction of environmental cues that are important for all organisms in their natural world. This may introduce a new perspective on the effects of environmental endocrine disruptors.

Behavioral rhythmicity of mice lacking AhR and attenuation of light-induced phase shift by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 microg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Effects of tryptophan photoproducts in the circadian timing system: searching for a physiological role for aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

Iodide Residues in Milk Vary between Iodine-Based Teat Disinfectants.

Majority of iodine found in dairy milk comes from the diet and teat disinfection products used during milking process. The objective of this study was to evaluate the effects of 4 iodine-based teat dips on milk iodide concentrations varying in iodine level (0.25% vs. 0.5%, w/w), normal low viscosity dip versus barrier dip, and application method (dip vs. spray) to ensure safe iodine levels in dairy milk when these products are used. The iodine exposure study was performed during a 2-wk period. The trial farm was purged of all iodine-based disinfection products for 21 d during a prestudy "washout period," which resulted in baseline milk iodide range of 145 to 182 ppb. During the experiment, iodine-based teat dips were used as post-milking teat disinfectants and compared to a non-iodine control disinfectant. Milk iodide residue levels for each treatment was evaluated from composited group samples. Introduction of different iodine-based teat disinfectants increased iodide residue content in milk relative to the control by between 8 and 29 µg/L when averaged across the full trial period. However, residues levels for any treatment remained well below the consumable limit of 500 µg/L. The 0.5% iodine disinfectant increased milk iodide levels by 20 µg/L more compared to the 0.25% iodine. Compared to dip-cup application, spray application significantly increased milk iodide residue by 21 µg/L and utilized approximately 23% more teat dip. This carefully controlled study demonstrated an increase in milk iodide concentrations from iodine disinfectants, but increases were small and within acceptable limits.

4-Vinylcyclohexene diepoxide reduces fertility in female Siberian hamsters when treated during their reproductively active and quiescent states.

The industrial compound 4-vinylcyclohexene diepoxide (VCD) destroys ovarian follicles and reduces fertility in rodents, but to date VCD has not been tested in species that experience seasonal anestrus. To determine if VCD destroys follicles when administered during reproductive quiescence, Siberian hamsters were treated with VCD (240mg/kg i.p. daily for 10 days) during short days, and outcomes were compared with reproductively active females that were maintained and treated in long days. Primordial follicle numbers were significantly reduced by VCD under both day lengths, and reproductive quiescence in short days did not appear to render the ovaries less susceptible to VCD-induced follicle depletion. Independent of day length and reproductive state, VCD-treated hamsters weaned substantially fewer offspring than controls. These results suggest that time of year may not be an important consideration for optimizing use of VCD in the field when the target pest species is a seasonally breeding rodent.

Brain transcriptome sequencing and assembly of three songbird model systems for the study of social behavior.

Emberizid sparrows (emberizidae) have played a prominent role in the study of avian vocal communication and social behavior. We present here brain transcriptomes for three emberizid model systems, song sparrow Melospiza melodia, white-throated sparrow Zonotrichia albicollis, and Gambel's white-crowned sparrow Zonotrichia leucophrys gambelii. Each of the assemblies covered fully or in part, over 89% of the previously annotated protein coding genes in the zebra finch Taeniopygia guttata, with 16,846, 15,805, and 16,646 unique BLAST hits in song, white-throated and white-crowned sparrows, respectively. As in previous studies, we find tissue of origin (auditory forebrain versus hypothalamus and whole brain) as an important determinant of overall expression profile. We also demonstrate the successful isolation of RNA and RNA-sequencing from post-mortem samples from building strikes and suggest that such an approach could be useful when traditional sampling opportunities are limited. These transcriptomes will be an important resource for the study of social behavior in birds and for data driven annotation of forthcoming whole genome sequences for these and other bird species.

Detection of diisocyanates in nesting material associated with mortality in pigeon chicks.

Diisocyanates, commonly used in the production of polyurethane foams, paints, elastomers, varnishes, and coatings, are considered among the most hazardous inhalation toxicants. The present report describes 2 unusual cases of mortality in pigeon chicks associated with nesting material contaminated by diisocyanates. Case 1 was submitted by a racing pigeon breeder who had lost all the hatchlings (n = 125) following replacement of the nesting material with a different lot. All adult birds appeared healthy, and hatchability was not significantly affected, but hatchlings became lethargic and dyspneic after a day of hatch. At necropsy, dark wet lungs were found in the hatchlings. Case 2 was submitted by a show-roller pigeon breeder. In this case, the owner reported lower hatchability, and all hatchlings (approximately 100) died within 2 days of hatching with clinical signs similar to the first case. Necropsy did not reveal any significant findings. For both cases, nesting materials were screened for toxic compounds using gas chromatography-mass spectrometry. Toluene-2,4-diisocyanate (approximately 190-290 ppm) and 4,4'-methylene diphenyl diisocyanate (unquantified) were detected in the nesting pads. While there is very limited information on toxicosis in birds, there are reports of inhalant exposure of diisocyanates causing pulmonary edema and death in various mammalian species. Although cause-effect relationship of mortality and the nesting material was not established in the present cases, the presence of toxic compounds in the nesting materials is a cause for concern. Further investigation is needed to determine the prevalence and toxicity of diisocyanates-contaminated nesting material in avian species.

RNA interference of gonadotropin-inhibitory hormone gene induces arousal in songbirds.

Gonadotropin-inhibitory hormone (GnIH) was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi) of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts.

Activation of aryl hydrocarbon receptor signaling by cotton balls used for environmental enrichment.

Dioxins are nearly ubiquitous environmental contaminants that are produced as byproducts during industrial processes, including the bleaching of paper and textiles. Contamination of animal bedding material with dioxins has been a concern for both laboratory and farm animals. The objective of this study was to determine whether the presence of cotton balls, provided to mice for enrichment, caused induction of the cytochrome P450 1A1 gene (Cyp1A1), which typically is stimulated through activation of the aryl hydrocarbon receptor (AhR) by dioxins and dioxin-like compounds. Cyp1A1 transcripts and protein in the liver were increased significantly by either exposure to cotton balls or treatment with a single dose of 2,3,7,8-tetrachlorodibenzo-para-dioxin. Unexposed controls displayed low levels of Cyp1A1 transcript and undetectable levels of CYP1A1 protein. These results suggest that cotton balls are potentially contaminated with dioxins and/or dioxin-like compounds that act as potent inducers of Cyp1a1 in laboratory animals if used as nesting material. This study underscores the necessity of considering dioxin content in products used for enrichment in animal facilities.

Seasonal differences of gene expression profiles in song sparrow (Melospiza melodia) hypothalamus in relation to territorial aggression.

BACKGROUND: Male song sparrows (Melospiza melodia) are territorial year-round; however, neuroendocrine responses to simulated territorial intrusion (STI) differ between breeding (spring) and non-breeding seasons (autumn). In spring, exposure to STI leads to increases in luteinizing hormone and testosterone, but not in autumn. These observations suggest that there are fundamental differences in the mechanisms driving neuroendocrine responses to STI between seasons. Microarrays, spotted with EST cDNA clones of zebra finch, were used to explore gene expression profiles in the hypothalamus after territorial aggression in two different seasons. METHODOLOGY/PRINCIPAL FINDINGS: Free-living territorial male song sparrows were exposed to either conspecific or heterospecific (control) males in an STI in spring and autumn. Behavioral data were recorded, whole hypothalami were collected, and microarray hybridizations were performed. Quantitative PCR was performed for validation. Our results show 262 cDNAs were differentially expressed between spring and autumn in the control birds. There were 173 cDNAs significantly affected by STI in autumn; however, only 67 were significantly affected by STI in spring. There were 88 cDNAs that showed significant interactions in both season and STI. CONCLUSIONS/SIGNIFICANCE: Results suggest that STI drives differential genomic responses in the hypothalamus in the spring vs. autumn. The number of cDNAs differentially expressed in relation to season was greater than in relation to social interactions, suggesting major underlying seasonal effects in the hypothalamus which may determine the differential response upon social interaction. Functional pathway analyses implicated genes that regulate thyroid hormone action and neuroplasticity as targets of this neuroendocrine regulation.

Increased proliferation but decreased steroidogenic capacity in Leydig cells from mice lacking cyclin-dependent kinase inhibitor 1B.

Proliferating cells express cyclins, cell cycle regulatory proteins that regulate the activity of cyclin-dependent kinases (CDKs). The actions of CDKs are regulated by specific inhibitors, the CDK inhibitors (CDKIs), which are comprised of the Cip/Kip and INK4 families. Expression of the Cip/Kip CDKI 1B (Cdkn1b, encoding protein CDKN1B, also called p27(kip1)) in developing Leydig cells (LCs) has been reported, but the function of CDKN1B in LCs is unclear. The goal of the present study was to determine the effects of CDKN1B on LC proliferation and steroidogenesis by examining these parameters in Cdkn1b knockout (Cdkn1b(-/-)) mice. LC proliferation was measured by bromodeoxyuridine incorporation. Testicular testosterone levels, mRNA levels, and enzyme activities of steroidogenic enzymes were compared in Cdkn1b(-/-) and Cdkn1b(+/+) mice. The labeling index of LCs in Cdkn1b(-/-) mice was 1.5% +/- 0.2%, almost 7-fold higher than 0.2% +/- 0.08% (P < 0.001) in the Cdkn1b(+/+) control mice. LC number per testis in Cdkn1b(-/-) mice was 2-fold that seen in the Cdkn1b(+/+) control mice. However, testicular testosterone levels, mRNA levels of steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), and 3beta-hydroxtsteroid dehydrogenase 6 (Hsd3b6), and their respective proteins, were significantly lower in Cdkn1b(-/-) mice. We conclude that deficiency of CDKN1B increased LC proliferation, but decreased steroidogenesis. Thus, CDKN1B is an important regulator of LC development and function.

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl on gene expression in the ovaries of immature Sprague-Dawley rats.

We have previously shown that 3,3',4,4',5-pentachlorobiphenyl (PCB-126) vertically transferred from dams potentially exerts a direct effect on the ovaries of offspring and adversely affects female puberty. To investigate its toxicological targets in ovarian tissues, mRNAs encoding representative peptides that regulate follicular development in granulosa cells, theca cells, and oocytes were quantified using ovaries collected on postnatal days (PND) 5, 15, and 24 from the offspring of dams administered oral doses of 0, 1 or 3 microg/kg PCB-126 starting 2 weeks prior to mating and continuing until 20 days after delivery. Quantification using the real-time RT-PCR method revealed that PCB-126 lowered the amounts of mRNAs that encoded the inhibin alpha- and inhibin/activin beta A-subunits from PND 15 onwards; the amounts of mRNAs for inhibin/activin beta B-subunit, follicle-stimulating hormone (FSH) receptor, and aromatase on PND 15; and the amounts of luteinizing hormone receptor mRNA on PND 24 compared with those of the age-matched controls. In contrast, no differences were noted for mRNAs encoding c-kit, growth differentiation factor-9, bone morphogenetic protein-15, or kit ligand for any of the age groups examined. The serum FSH level on PND 24 was higher than that in the control. Since the earliest effects on the mRNAs in the rat ovaries were observed in those expressed in the granulosa cells of the growing follicles after the antral follicles had developed, molecules in granulosa cells but not in oocytes during the early stages of the antral follicles might be the primary targets of vertically transferred PCB-126.

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl (PCB-126) on the reproductive development of female rats.

In order to study the effects of vertically transferred coplanar polychlorinated biphenyls on female reproductive development, female rat offspring from dams of Sprague-Dawley strain, which received daily oral administration of vehicle (corn oil) or 1 or 3 microg/kg of 3,3',4,4',5-pentachlorobiphenyl (PCB-126) from 2 weeks prior to mating with intact males until 20 days after delivery were examined from birth until puberty. Hepatic expression of the aryl hydrocarbon receptor (AhR)-inducible enzyme cytochrome P450 1A1 (CYP1A1) was detected in all offspring from PCB-126-exposed dams, indicating vertical transfer of PCB-126. Furthermore, quantification of ovarian mRNAs encoding CYP1A1, AhR and ARNT demonstrated that the ovary equipped the AhR-signaling system through which transcription of the CYP1A1 gene was enhanced in a dose-dependent manner. Exposure to PCB-126 retarded the growth of offspring in both exposed groups, while the viability of the neonates of the exposed groups was comparable to that of the oil-exposed controls. The exposure to 3 mug/kg/day reduced the ovarian weight on postnatal day (PND) 24, with atresia of most of the antral follicles and delayed vaginal opening. Exposure to 1 microg/kg/day did not produce such effects; however, both doses of PCB-126 induced external urogenital anomalies, such as vaginal thread and hypospadias, in all of the PCB-126-exposed female offspring. These results indicate that vertically transferred PCB-126 is potent enough to exert a direct effect on the ovary and adversely affect female puberty by altering the morphological and functional development of the female reproductive system.

Altered prostatic epithelial proliferation and apoptosis, prostatic development, and serum testosterone in mice lacking cyclin-dependent kinase inhibitors.

Normal prostatic development and some prostatic diseases involve altered expression of the cell-cycle regulators p27 and p21 (also known as CDKN1B and CDKN1A, respectively). To determine the role of these proteins in the prostate, we examined prostatic phenotype and development in mice lacking p27 and/or p21. In p27-knockout (p27KO) mice, epithelial proliferation was increased 2- and 3.8-fold in the ventral and dorsolateral prostate, respectively, versus wild-type (WT) mice, although prostatic weights were not different. Epithelial apoptosis was increased in p27KO mice and may account for the lack of a concurrent increase in weight. Testosterone deficiency observed in this group was not the cause of this increase, because vehicle- and testosterone-treated p27KO mice had similar percentages of apoptotic cells. Also observed was a trend toward a decreased functional epithelial cytodifferentiation, indicating a potential role of p27 in this process. Conversely, dorsolateral prostate and seminal vesicle (SV) of p21-knockout (p21KO) mice, and all prostatic lobes and SV of p21/p27 double-knockout mice, weighed significantly less compared to the WT mice, and their epithelial proliferation was normal. Decreased testosterone concentrations may contribute to the decreased prostatic weights. However, other factors may be involved, because testosterone replacement only partially restored prostatic weights. We conclude that loss of p27 increases prostatic epithelial proliferation and alters differentiation but does not result in prostatic hyperplasia because of increased epithelial cell loss. The p21KO mice showed phenotypes distinctly different from those of p27KO mice, suggesting nonredundant roles of p21 and p27 in prostatic development. Loss of p27 or of both p21 and p27 results in serum testosterone deficiency, complicating analysis of the prostatic effects of these cell-cycle regulators.

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice.

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.