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Animal reproduction science 2010 May; 119(3-4)
Effect of season on fresh and cryopreserved stallion semen.
The objective of this study was to determine the effect of season on sperm quality variables, expression of the fertility-related protein SP22 and selected mRNA transcripts in fresh and cryopreserved stallion sperm. Four stallions were collected in e... expand abstractach of the four seasons: summer, fall, winter and spring. Ejaculates were divided and then evaluated for motility, morphology, SP22 staining and expression of selected mRNAs as either fresh semen samples or cryopreserved samples. A significant interaction between season and cryopreservation status was found for total and progressive sperm motility. RNA yield from sperm was not affected by any variable examined. There was no effect of season or cryopreservation on the relative amounts of mRNA for PGK2, TPX1, TIMP3 or ACTB. There was a tendency (P=0.1) for an effect of stallion on the relative amount of ACTB mRNA. The proportion of sperm immunostained for SP22 over the equatorial segment was affected (P<0.05) by stallion. In addition, there was an interaction (P<0.05) between season and cryopreservation status on the percentage of sperm staining for SP22 on the equatorial segment. The correlation among total motility, progressive motility and SP22 immunostaining was much greater (P<0.05) during the breeding season (March and June) than during the non-breeding season (September and December). Based on data analyzed, semen collected in the Northern Hemisphere between March and June may be best suited for cryopreservation. collapse abstract
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Animal reproduction science 2008 Mar; 105(1-2)
Saga of a sperm fertility biomarker.
A decade ago a novel sperm protein associated with the fertility of sperm was discovered by quantifying individual proteins in the sperm membrane proteome of cauda epididymal sperm from rats exposed to epididymal toxicants that compromised the fertil... expand abstractity of these sperm. Upon identification, this protein (SP22) was found to a ubiquitous, highly conserved protein never before observed in the male reproductive tract. The expression of SP22 in sperm appears driven by a testis specific mRNA transcript, and the molecule is translocated from the cytoplasmic droplet of rete testis sperm to the equatorial segment of epididymal and ejaculated sperm. The appearance of SP22 mRNA and protein coincide with the formation of pachytene spermatocytes and round spermatids, respectively, and given this testis ontogeny of SP22, we validated its use as a biomarker of fertility by extending our studies to toxicants that target spermiogenesis. Studies of both epididymal and testicular toxicants now have demonstrated that compromised SP22 gene expression is sensitive and correlated with fertility. Importantly, this applies to ejaculated sperm as well as epididymal sperm. With the goal of developing a user-friendly diagnostic assay for SP22 on epididymal and ejaculated sperm, we are attempting to identify exposed, functional domains of the protein. For this, we have generated antibodies to both full length and truncated SP22 recombinants, as well as antibodies to synthetic SP22 peptides. Each antibody has been characterized for its ability to inhibit fertilization both in utero and in vitro. Linear epitope mapping has been done for each antibody, and synthetic peptides corresponding to each epitope have been used in competition experiments designed to elucidate exposure on the sperm surface and function. Most of the linear epitopes identified appear to be exposed although there are relative differences in the degree of their exposure. Interestingly, one of the exposed epitopes does not appear to be functional, at least by itself. Many more domains of the molecule need to be studied, but based on our findings with the epitopes already identified, it seems a combinatorial targeting strategy may be beneficial. If one assumes that the protein's role in fertility resides in a single exposed epitope, or some combination of exposed epitopes, such targeting may also ultimately lead to successful modulation of the fertilizing potential of sperm. collapse abstract
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Journal of andrology 28(4)
Chronic exposure to low levels of dibromoacetic acid, a water disinfection by-product, adversely affects reproductive function in male rabbits.
Four groups (minimum of 10/dose group) of male Dutch-belted rabbits were treated daily with dibromoacetic acid (DBA) via drinking water beginning in utero from gestation day 15 to adulthood; target dosages were 1, 5, and 50 mg DBA/kg body weight. Dev... expand abstractelopmental, prepubertal as well as postpubertal reproductive sequelae were evaluated. One (out of 22), 2 (out of 32), and 1 (out of 21) male offspring in the 1, 5, and 50 mg DBA/kg groups were unilaterally cryptorchid. There were no significant differences in serum follicle-stimulating hormone, luteinizing hormone, and testosterone (basal concentrations or in response to exogenous gonadotropin-releasing hormone) in both prepubertal and adult rabbits. Chronic exposure to DBA adversely affected the mating abilities of some rabbits. The number of sperm produced was not affected, but spermiogenesis was disrupted, resulting in unique sperm acrosomal-nuclear malformations even at the 1-mg dose level. Concentrations of SP22, a specific sperm membrane fertility protein, in detergent extracts of ejaculated sperm were significantly lower (P < .05) in all DBA-treated groups compared with controls. The conception rates following artificial insemination of a constant number of sperm for 1, 5, and 50 mg DBA/kg groups were 55% (10/18), 65% (13/20), and 55% (9/16), respectively, vs 85% (17/20) for control group. Histologic lesions in testes characterized by spermatogenic arrest predominantly at the round spermatid stage, pyknosis of differentiating germ cells, and ultimate degeneration and desquamation leaving focal vacuolation in seminiferous epithelium were evident in DBA-treated groups. Thus, male rabbits exhibit reproductive toxicity with exposure to DBA during reproductive development at dosages as low as 1 mg/kg body weight. collapse abstract
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Toxicologic pathology 34(6)
Epididymis-specific pathologic disorders in rats exposed to gossypol from weaning through puberty.
Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the ... expand abstracttreated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation. collapse abstract
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International journal of andrology 2006 Jul; 29(4)
Sexual behaviour, sperm quantity and quality after short-term streptozotocin-induced hyperglycaemia in rats.
Studies of diabetes mellitus in the streptozotocin rat model suggest that sexual dysfunctions may result from diabetes-induced alterations of the neuroendocrine-reproductive tract axis. Our investigation was performed to better define the effects of ... expand abstractshort-term hyperglycaemia on rat epididymal sperm quantity, quality and transit time, using both natural mating and artificial in utero insemination protocols. Male rats were made diabetic with streptozotocin (sc, 40 mg/kg), whereas controls received vehicle. Sexual behaviour was tested after 15 days and sperm fertilizing ability was checked 22 days after the injection through natural mating and artificial in utero insemination. Other parameters such as daily sperm production, testosterone levels, as well as sperm morphology and motility were also investigated. Fifty per cent of the diabetic animals showed no copulatory behaviour during tests and the number of animals reaching ejaculation was smaller in the diabetic group when compared with the control group (33% vs. 83%). Diabetes resulted in decreased body and reproductive organ weights, as well as diminished sperm counts in the testis and epididymis, that were associated with diminution of plasmatic testosterone levels. After natural mating, there was a decrease in the fertility in the diabetic adult male rats (25.5%) compared with control animals (81.5%). However, distal cauda epididymal sperm from diabetic rats displayed normal fertilization ability (91.5%) using in utero insemination. There were no effects of hyperglycaemia on sperm transit time in the epididymis and on spermatogenesis. Our results indicate that diabetes mellitus produces reproductive dysfunction, but does not compromise sperm fertilizing ability in the cauda epididymis in this experimental model. collapse abstract
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Neurobiology of disease 2006 Apr; 22(2)
Intersecting pathways to neurodegeneration in Parkinson's disease: effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system.
Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-s... expand abstractynuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease. collapse abstract
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Reproductive toxicology (Elmsford, N.Y.) 2004 Oct; 19(1)
Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture.
The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA... expand abstract) and bromochloro (BCA) acetic acid are developmentally toxic in mouse whole embryo culture. Human exposure to these contaminants in drinking water would involve simultaneous exposure to all three HAs. This study explores the question of developmental toxicity interactions between these compounds. Gestational day (GD) 9.5 rat embryos were exposed to various concentrations of the three HAs (singly or in combination) for 48 h and then evaluated for dysmorphology. The embryonic effects from exposure to the single compounds and mixtures were evaluated using developmental score (DEVSC) as the parameter of comparison. Concentrations of individual compounds and mixtures were chosen (based on a dose-additivity model) which were predicted to produce scores 10 or 20% lower than control levels. Evaluations were performed on all possible combinations of the three HAs. The HAs were dysmorphogenic and resulted in primarily rotation and heart defects and to a lesser extent prosencephalic, visceral arch, and eye defects. The percent anomalies in the rat were comparable to those previously published for the mouse at comparable toxicant concentration. There was a low incidence of neural tube defects in the rat following exposure to the HAs. The rat neural tube appeared less sensitive to the HAs than did the mouse resulting in a higher rate of neural tube dysmorphology in the mouse. Following exposures to BCA and DBA, alone and in combination, there was a significant incidence of delayed embryonic caudal development with apparent normal development anterior to the second visceral arch. The developmental scores for embryos exposed to combinations of the three compounds, when compared to scores for embryos exposed to the single compounds, indicated that the dose-additivity model adequately predicted the observed toxicity and that the developmental toxicity of these water disinfection by-products appears to be additive in whole embryo culture (WEC). collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2004 Sep; 81(2)
Continuous exposure to dibromoacetic acid delays pubertal development and compromises sperm quality in the rat.
Previously our work on the haloacid by-products of drinking water disinfection focused on adult exposures. Herein we evaluate the consequence of continuous exposure to dibromoacetic acid (DBA) via drinking water through reproductive development into ... expand abstractadulthood. An initial study in which offspring were exposed from gestation day (GD) 15 through adulthood revealed significant delays in preputial separation and vaginal opening, dose-related decreases in the fertility of cauda epididymal sperm, and dose-related diminutions in the sperm membrane protein SP22. Subsequent studies consisted of groups in which exposure ceased on postnatal day 21 (PND 21) versus adulthood. For each exposure, animals were evaluated after puberty (PND 56) as well as at adulthood (PND 120). Exposure to 4, 40, or 400 ppm DBA from GD 15 through PND 21 failed to result in any significant reproductive alterations. By contrast, continuous exposure until adulthood resulted in dose-related alterations consistent with those observed in the dose-finding study. Preputial separation and vaginal opening were delayed 4 and 3 days in males and females exposed to 400 ppm (76.3 mg/kg) DBA. This was associated with increased responsiveness of both the testis and ovary to hCG ex vivo; hCG-stimulated testosterone production by testicular parenchyma on PND 56 was increased at 4 ppm (0.6 mg/kg) DBA and higher. Finally, the quality of proximal cauda epididymal sperm was compromised by continuous exposure to DBA. The sperm membrane proteome was altered in a dose-related manner with SP22, and one of its charged variants, diminished at 40 ppm (3.6 mg/kg) DBA and higher. As more sensitive endpoints are evaluated, lower effect levels can be attributed to haloacid exposure. We are now extending our evaluations to epidemiology studies designed to evaluate sperm quality in men exposed to varying levels of disinfection by-products. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2004 Sep; 81(2)
Haloacid induced alterations in fertility and the sperm biomarker SP22 in the rat are additive: validation of an ELISA.
Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of c... expand abstractauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether fertility and levels of SP22 on sperm were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22. In a dose finding study, animals were exposed by oral gavage daily for 14 days to: BCA alone at 1.6, 4, and 8 mg/kg; DBA at equimolar levels of 2, 5, and 10 mg/kg; and two binary mixtures of 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. The ED(50)s for the decrease in SP22 quantified by two-dimensional SDS-PAGE were 7.2 and 4.6 mg/kg for DBA and BCA. The ED(50)s for the decrease in SP22 quantified by ELISA were 8.1 and 5.9 mg/kg for DBA and BCA. The definitive study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. The ED(50)s for decreases in fertility assessed by intrauterine insemination were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA. Immunolocalization of SP22 in spermatocytes and spermatids, as well as on the cytoplasmic droplet and the equatorial segment of luminal sperm, was decreased by the DBA + BCA mixture. The decrease in SP22 in testicular parenchyma was comparable to that observed for sperm extracts. Based on 2D SDS-PAGE, ELISA, or fertility the haloacid-induced decreases in SP22 or fertility were additive or synergistic. The correlation between SP22 levels by ELISA and fertility was r(2) = 0.72 compared to 0.82 for SP22 levels by 2D SDS-PAGE and fertility, validating SP22 quantitation by ELISA. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2004 Jun; 80(1)
Chronic exposure to dibromoacetic acid, a water disinfection byproduct, diminishes primordial follicle populations in the rabbit.
To determine if dibromoacetic acid (DBA) affects ovarian folliculogenesis, four groups of female Dutch-belted rabbits were exposed daily to 0, 1, 5, or 50 mg DBA/kg body weight in drinking water beginning in utero from gestation day 15 throughout lif... expand abstracte. Functionality of the endocrine axis was assessed by measuring serum concentrations of gonadotropins following an im injection of 10 microg GnRH at 12 (prepubertal; n = 6/dose group) and 24 (postpubertal; n = 10/dose group) weeks of age. A day after GnRH challenge, number of ovulation sites and ovarian weights were determined at necropsy. Left ovaries were processed for histopathology, serially sectioned at 6 microm, and every twelfth section stained with hematoxylin and eosin was evaluated. All healthy follicles were categorized as primordial, primary, small preantral, large preantral, or small antral follicles. The area of each section evaluated was measured and the number of follicles in each category expressed per mm2 unit area. In prepubertal animals, DBA caused a reduction in number of primordial follicles (p < 0.05) and total healthy follicles (p < 0.05) at 50 mg/kg dose level. In adult animals, there were fewer primordial follicles in both the 5 (p < 0.01) and 50 (p = 0.1) mg/kg dose groups. No profound changes in gonadotropin profiles were observed. Although chronic exposure to DBA did not appear to have an effect on late follicular development or ovulation, DBA did reduce the population of primordial follicles. The long-term health consequences of diminished primordial follicles are unknown, but it is very likely that reproductive senescence would occur earlier. collapse abstract
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Proceedings of the National Academy of Sciences of the United States of America 2004 Jan; 101(3)
Phthalate-induced Leydig cell hyperplasia is associated with multiple endocrine disturbances.
The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction i... expand abstractndustry, and di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment. Daily human exposure to DEHP in the U.S. is significant, and occupational and clinical exposures from DEHP-plasticized medical devices, e.g., blood bags, hemodialysis tubing, and nasogastric feeding tubes, increase body burden levels. We investigated the effects of chronic exposures to low environmentally relevant DEHP levels on testicular function. Our data show that prolonged exposures to this agent induced high levels of the gonadotropin luteinizing hormone and increased the serum concentrations of sex hormones [testosterone and 17beta-estradiol (E2)] by >50%. Increased proliferative activity in Leydig cells was evidenced by enhanced expression of cell cycle proteins, as determined by RT-PCR. The numbers of Leydig cells in the testis of DEHP-treated rats were 40-60% higher than in control rats, indicating induction of Leydig cell hyperplasia. DEHP-induced elevations in serum testosterone and E2 levels suggest the possibility of multiple crosstalks between androgen, estrogen, and steroid hormone receptors, whereas the presence of estrogen receptors in nonreproductive tissues, e.g., cardiovascular system and bones, implies that the increases in serum E2 levels have implications beyond reproduction, including systemic physiology. Analysis of the effects of phthalate exposures on gonadotropin and steroid hormone levels should form part of overall risk assessment in human populations. collapse abstract
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Endocrinology 2004 Jan; 145(2)
Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells.
Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 microg/kg.d (a dose that approximates BPA levels in ... expand abstractthe environment) from postnatal d 21-35 suppressed serum LH (0.21 +/- 0.05 ng/ml; vs. control, 0.52 +/- 0.04; P < 0.01) and testosterone (T) levels (1.62 +/- 0.16 ng/ml; vs. control, 2.52 +/- 0.21; P < 0.05), in association with decreased LHbeta and increased estrogen receptor beta pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nm BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17alpha-hydroxylase/17-20 lyase. BPA decreased serum 17beta-estradiol levels from 0.31 +/- 0.02 ng/ml (control) to 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.23 +/- 0.03 ng/ml in rats exposed to 2.4 microg, 10 microg, or 100 mg/kg.d BPA, respectively, from 21-35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 +/- 34 (control) to 261 +/- 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility. collapse abstract
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Birth defects research. Part B, Developmental and reproductive toxicology 2003 Jul; 68(4)
Effects of gestational exposure to ethane dimethanesulfonate in CD-1 mice: microtia and preliminary hearing tests.
BACKGROUND: Microtia is a reduction in pinna size, usually seen in humans in conjunction with other medical conditions. We report microtia in CD-1 mice after gestational exposure to ethane dimethanesulfonate (EDS), an alkylating agent and adult rat L... expand abstracteydig cell toxicant. METHODS: Time-pregnant CD-1 mice were administered 0, 80, or 160 mg EDS/kg on gestation days (GD) 11-17, or 0 or 160 mg EDS/kg on GD 11-13, GD 13-15 or GD 15-17. Pinnae were measured on postnatal days (PND) 4, 8, 18, and 28; and were observed for detachment from birth through PND 8. Branchial-arch derived skeletal structures and histology of the pinna was examined on PND 4 and 24. Brainstem auditory evoked response (BAER) tests were carried out at approximately PND 160 to determine possible effects on hearing. RESULTS: All offspring of EDS-treated dams exhibited bilateral, dose-related decreases in pinna size. Gestational exposure during GD 11-13 produced smaller ears than during GD 13-15 or 15-17, but not as small as the GD 11-17 regimen. Ossification of other pharyngeal arch derivatives was delayed whereas histology was unremarkable. BAER analysis showed a decrease in the proportion of adult offspring producing a quantifiable response to varied auditory stimuli among EDS-treated litters. CONCLUSIONS: Gestational exposure to EDS affects pinna development in the mouse, with a broad period of sensitivity during the second half of gestation. Microtia induced by EDS may be associated with hearing deficits, suggesting functional importance of pinna size or additional effects of EDS on ear development not detected by morphological examination. collapse abstract
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The Journal of biological chemistry 2003 Sep; 278(38)
L166P mutant DJ-1, causative for recessive Parkinson's disease, is degraded through the ubiquitin-proteasome system.
Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second ... expand abstractis a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell. collapse abstract
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Biology of reproduction 2003 Aug; 69(3)
Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse.
Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant ... expand abstractCD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development. collapse abstract
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Journal of andrology 23(2)
Postweaning exposure to gossypol results in epididymis-specific effects throughout puberty and adulthood in rats.
Gossypol, a yellow pigment found in cottonseeds, well known for its antifertility properties in animals, has been used as a contraceptive by men. The aims of this work were to evaluate the effects of gossypol throughout sexual development of male rat... expand abstracts and to provide additional data to clarify the target site or sites of this compound in the male reproductive system. Gossypol (15 mg/kg per day) was given to animals from weaning through prepuberty (41 days), early puberty (51 days), puberty (61 days), and sexual maturity (91 days). Ventral prostate weight and fructose levels were similar in control and treated rats, suggesting that androgen levels were normal. No histological effects on the testis were detected, but there was a significant decrease in the sperm concentration in the cauda epididymidis of gossypol-treated animals killed at 61 and 91 days, as well as a significant increase in abnormal sperm in the vas deferens of treated animals. Moreover, the histology of the cauda epididymidis of the rats treated throughout puberty (ie, until days 51 and 61) showed a great number of round bodies in the lumen of the epididymis. These structures stained for the epididymis-specific protein E. Collectively, the data demonstrate that the epididymis is a target of gossypol when postweaning exposure extends throughout pubertal development, and that whereas more subtle histological effects commence around puberty, indicators reproductive competence are compromised in adulthood. collapse abstract
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Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 2002 Jul; 132(4)
Identification and molecular cloning of Xenopus laevis SP22, a protein associated with fertilization in mammals.
SP22 is a novel sperm protein that has been shown to be highly correlated with fertility in rats. SP22 homologues have been studied in mouse and man but a definitive role for the protein has not yet been established. Using a polyclonal IgG to recombi... expand abstractnant rat SP22, we detected the presence of this protein in Xenopus laevis tissues. Moreover, a Xenopus EST was found that shares a high degree of similarity with rat SP22 and the derived sequence codes for an 189 aa protein that is very similar to rat SP22. Finally, using Western blotting and RT-PCR analyses, we investigated the expression of Xenopus SP22 both in adult tissues and during embyronic development. SP22 protein expression predominated in the adult testis, and both mRNA and protein levels diminished subsequent to the initial day following fertilization. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2002 Jun; 68(1)
Bromochloroacetic acid exerts qualitative effects on rat sperm: implications for a novel biomarker.
Disubstituted haloacid by-products of drinking water disinfection such as dibromoacetic acid and dichloroacetic acid have been shown to perturb spermatogenesis and fertility in adult male rats. In the present study we sought to establish whether equi... expand abstractmolar exposure to bromochloroacetic acid (BCA), a prevalent by-product in finished drinking water, is also capable of disrupting these endpoints, and if so to determine whether the novel biomarker of fertility (SP22) would be correlated with subfertility induced by testicular toxicity. A dose range finding study indicated that body weight was not affected by exposure to 14 daily doses of 72 mg/kg BCA while numerous male reproductive parameters were altered, including decreases in the number and progressive motility of cauda epididymal sperm. In addition, there was an increased incidence of delayed spermiation in the testes of males exposed to 72 mg/kg BCA. In the definitive study, exposures ranged from 8 to 72 mg/kg, the fertility of cauda epididymal sperm was evaluated by in utero insemination, and the two-dimensional profile of cauda sperm membrane proteins was evaluated quantitatively. The morphology of both caput and cauda epididymal sperm was altered by 72 mg/kg BCA. The fertility of cauda epididymal sperm, the percentages of progressively motile sperm and progressive tracks, and two sperm membrane proteins (SP22 and SP9) were decreased significantly by each BCA exposure. While the two sperm proteins and the two measures of progressive motility were each significantly correlated with fertility, only one of these measures (i.e., SP22) had an r value of greater than 0.5. When data for SP22 and fertility were fit to a nonlinear model, r(2) was 0.84. Using this exposure paradigm, the no-observed-effect level for BCA is less than 8 mg/kg. Moreover, SP22 may be useful in predicting compromised fertility after exposure to by-products of drinking water disinfection. collapse abstract
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Journal of andrology 23(1)
Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro.
We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 tran... expand abstractscript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization. collapse abstract
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Journal of andrology 22(5)
Dibromoacetic acid, a prevalent by-product of drinking water disinfection, compromises the synthesis of specific seminiferous tubule proteins following both in vivo and in vitro exposures.
Dibromoacetic acid (DBA) is a by-product of drinking water disinfection that alters spermatogenesis in adult male rats. To identify a mechanism by which DBA alters spermatogenesis, seminiferous tubules representing specific groups of spermatogenic st... expand abstractages were exposed either in vivo or in vitro, and structural and functional consequences were evaluated. Seminiferous tubules representing stages I-V, VI-VIII, and IX-XIV were isolated from testes of adult rats and cultured overnight in conditions of reduced oxygen and temperature. For in vivo exposures, seminiferous tubules were recovered from animals that had received 250 mg/kg DBA via gavage for 5 days. For in vitro exposures, 180 and 600 microM concentrations were tested; these concentrations bracket the concentration of DBA observed within the testis following in vivo exposure. Protein synthesis was evaluated by 35S-methionine labeling overnight and quantitative analysis of radiolabeled proteins in mini, 2-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Radio-inert cultures were processed for light and electron microscopy. Morphologicaf evaluation indicated that all spermatogenic stages of the seminiferous tubules from control animals were well maintained during the isolation and culture period. Although no treatment-related lesions were observed following in vivo exposure, histological alterations were observed at the lowest in vitro exposure. There was a significant diminution (P < .05) in the synthesis of 4 cytosolic proteins following both in vivo and in vitro exposures. Diminution in these proteins was restricted to stages I-V and IX-XIV of spermatogenesis, suggesting that proteins involved in the early stages of spermiogenesis are uniquely sensitive to DBA exposure. Because histology and protein synthesis were affected by relevant in vitro exposures, this indicates that DBA is capable of altering spermatogenesis directly. collapse abstract
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Journal of andrology 22(4)
Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells.
Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, co... expand abstractmbining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used. collapse abstract
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Biology of reproduction 2001 Sep; 65(4)
Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate.
Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Ley... expand abstractdig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg(-1) kg(-1) day(-1) DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg(-1) kg(-1) day(-1) DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17beta-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21-48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg(-1) kg(-1) day(-1) and 10 mg(-1) kg(-1) day(-1), respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 28 days (Postnatal Days 62-89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2000 Nov; 58(2)
The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat.
Phthalate esters (PE) such as DEHP are high production volume plasticizers used in vinyl floors, food wraps, cosmetics, medical products, and toys. In spite of their widespread and long-term use, most PE have not been adequately tested for transgener... expand abstractational reproductive toxicity. This is cause for concern, because several recent investigations have shown that DEHP, BBP, DBP, and DINP disrupt reproductive tract development of the male rat in an antiandrogenic manner. The present study explored whether the antiandrogenic action of DEHP occurs by (1) inhibiting testosterone (T) production, or by (2) inhibiting androgen action by binding to the androgen receptor (AR). Maternal DEHP treatment at 750 mg/kg/day from gestational day (GD) 14 to postnatal day (PND) 3 caused a reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. As a consequence, anogenital distance (AGD) on PND 2 was reduced by 36% in exposed male, but not female, offspring. By GD 20, DEHP treatment also reduced testis weight. Histopathological evaluations revealed that testes in the DEHP treatment group displayed enhanced 3ss-HSD staining and increased numbers of multifocal areas of Leydig cell hyperplasia as well as multinucleated gonocytes as compared to controls at GD 20 and PND 3. In contrast to the effects of DEHP on T levels in vivo, neither DEHP nor its metabolite MEHP displayed affinity for the human androgen receptor at concentrations up to 10 microM in vitro. These data indicate that DEHP disrupts male rat sexual differentiation by reducing T to female levels in the fetal male rat during a critical stage of reproductive tract differentiation. collapse abstract
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Biology of reproduction 2000 Feb; 62(3)
A metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane, reduces testosterone biosynthesis in rat leydig cells through suppression of steady-state messenger ribonucleic acid levels of the cholesterol side-chain cleavage enzyme.
Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testo... expand abstractsterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1, 1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450(scc)) and cholesterol utilization. The reduced steady-state mRNA level for P450(scc) in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds. collapse abstract
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Critical reviews in toxicology 1999 Feb; 29(2)
Rodent Leydig cell tumorigenesis: a review of the physiology, pathology, mechanisms, and relevance to humans.
Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has... expand abstract increased in recent years because of its common occurrence in rodent bioassays and the importance in assessing the relevance of this tumor type to humans. To date, there have been no comprehensive reviews to identify all the compounds that have been shown to induce LCTs in rodents or has any review systematically evaluated the epidemiology data to determine whether humans were at increased risk for developing LCTs from exposure to these agents. This review attempts to fill these deficiencies in the literature by comparing the cytology and ontogeny of the LC, as well as the endocrine and paracrine regulation of both normal and tumorigenic LCs. In addition, the pathology of LCTs in rodents and humans is compared, compounds that induce LC hyperplasia or tumors are enumerated, and the human relevance of chemical-induced LCTs is discussed. There are plausible mechanisms for the chemical induction of LCTs, as typified by agonists of estrogen, gonadotropin releasing hormone (GnRH), and dopamine receptors, androgen receptor antagonists, and inhibitors of 5alpha-reductase, testosterone biosynthesis, and aromatase. Most of these ultimately involve elevation in serum luteinizing hormone (LH) and/or LC responsiveness to LH as proximate mediators. It is expected that further work will uncover additional mechanisms by which LCTs may arise, especially the role of growth factors in modulating LC tumorigenesis. Regarding human relevance, the pathways for regulation of the hypothalamo-pituitary-testis (HPT) axis of rats and humans are similar, such that compounds that either decrease testosterone or estradiol levels or their recognition will increase LH levels. Hence, compounds that induce LCTs in rats by disruption of the HPT axis pose a risk to human health, except for possibly two classes of compounds (GnRH and dopamine agonists). Because GnRH and prolactin receptors are either not expressed or are expressed at very low levels in the testes in humans, the induction of LCTs in rats by GnRH and dopamine agonists would appear not to be relevant to humans; however, the potential relevance to humans of the remaining five pathways of LCT induction cannot be ruled out. Therefore, the central issue becomes what is the relative sensitivity between rat and human LCs in their response to increased LH levels; specifically, is the proliferative stimulus initiated by increased levels of LH attenuated, similar, or enhanced in human vs. rat LCs? There are several lines of evidence that suggest that human LCs are quantitatively less sensitive than rats in their proliferative response to LH, and hence in their sensitivity to chemically induced LCTs. This evidence includes the following: (1) the human incidence of LCTs is much lower than in rodents even when corrected for detection bias; (2) several comparative differences exist between rat and human LCs that may contribute, at least in part, to the greater susceptibility of the rat to both spontaneous and xenobiotic-induced LCTs; (3) endocrine disease states in man (such as androgen-insensitivity syndrome and familial male precocious puberty) underscore the marked comparative differences that exist between rats and man in the responsiveness of their LC's to proliferative stimuli; and (4) several human epidemiology studies are available on a number of compounds that induce LCTs in rats (1,3-butadiene, cadmium, ethanol, lactose, lead, nicotine) that demonstrate no association between human exposure to these compounds and induction of LC hyperplasia or adenomas. (ABSTRACT TRUNCATED) collapse abstract
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