A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.     

Effects of flutamide on testicular involution induced by an antagonist of gonadotrophin-releasing hormone and on stimulation of spermatogenesis by follicle-stimulating hormone in rats.

The effects of combined treatment with an antagonist of gonadotrophin-releasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450-500 micrograms/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT+FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.     

Prenatal exposure to antiandrogen flutamide affects androgen receptor (AR) expression in postnatal ovarian development in pig

The following study was undertaken to localize androgen receptors (ARs) in various structures of the porcine ovary after prenatal exposure to antiandrogen flutamide. In utero treatment by antiandrogens may have adverse effects on reproductive function in immature and adult animals. Flutamide was injected into pregnant swines between days 20 and 28 (GD20) or 80 to 88 (GD80) of gestation. The ovaries were collected from treated animals and from control ones (non-treated) at two different points of development: from immature and adult pigs. Immunoexpression of AR was determined for preantral and antral follicles and for stroma cells. Immunostaining showed that AR expression in immature animals was unaffected in the primary follicles, while in the preantral and antral follicles the AR level fluctuated depending on day of treatment as well as on analyzed tissue. In adult animals, the immunoexpression of AR slightly decreased in antral follicles independently on the day of flutamide treatment. Therefore, AR expression in postnatal life may be affected by in utero exposure to antiandrogen flutamide.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.     

A Transcriptome-Wide Screen for mRNAs Enriched in Fetal Leydig Cells: CRHR1 Agonism Stimulates Rat and Mouse Fetal Testis Steroidogenesis

Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (Crhr1, Gramd1b, Itih5, Vgll3, and Vsnl1) was verified by whole-mount in situ hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2) were tested for a steroidogenic function using ex vivo fetal testes treated with receptor agonists (CRH, PRL, and PROK2). While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10) nM concentration, increased expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to ex vivo rodent fetal testes, ≥10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. Crh mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis in vivo.     

Effect of ethinyl estradiol on the differentiation of mouse fetal testis

In an evaluation of the effect of ethinyl estradiol (EE) on the differentiation of fetal mouse testes, the ratio of the seminiferous tubular region to the testicular tissue region, the ratio of Sertoli cells to gonocytes in tubule cross sections, and the size of Leydig cells were determined by the Texture Analyse System (T.A.S., Leitz) in histological preparations of the testes. The testes were those of fetuses taken from dams given orally 0, 0.02, 0.2 or 2.0 mg/kg of body weight of EE in olive oil from day 11 through day 17 of gestation and killed at term. From experimental and the control testes, five sections were taken at 40-micron intervals. The areas of the seminiferous tubular region and the testicular region were determined and the Sertoli cells and gonocytes in tubule cross section were counted in each of the five sections. The diameters of 100 Leydig cells selected at random were averaged. These data were analyzed by Student's t test. The seminiferous tubular region was significantly increased in the testes treated with 0.02 mg/kg of EE and significantly decreased in those treated with 0.2 mg/kg of EE. The number of gonocytes per tubule cross section was significantly increased in the testes treated with 0.02 or 2.0 mg/kg of EE. The number of Sertoli cells per tubule cross section and the number of Sertoli cells per gonocyte were significantly decreased in the experimental testes. The size of the Leydig cells was significantly decreased in the testes treated with 0.2 mg/kg of EE. These findings suggest that prenatal exposure to EE before testicular differentiation affects tubular formation, the proliferation of fetal Sertoli cells, and Leydig cell differentiation, resulting in disturbances of spermatogenesis.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Fetal radiation exposure induces testicular cancer in genetically susceptible mice

The prevalence of testicular germ cell tumors (TGCT), a common solid tissue malignancy in young men, has been annually increasing at an alarming rate of 3%. Since the majority of testicular cancers are derived from germ cells at the stage of transformation of primordial germ cell (PGC) into gonocytes, the increase has been attributed to maternal/fetal exposures to environmental factors. We examined the effects of an estrogen (diethylstilbestrol, DES), an antiandrogen (flutamide), or radiation on the incidence of testicular germ cell tumors in genetically predisposed 129.MOLF-L1 (L1) congenic mice by exposing them to these agents on days 10.5 and 11.5 of pregnancy. Neither flutamide nor DES produced noticeable increases in testis cancer incidence at 4 weeks of age. In contrast, two doses of 0.8-Gy radiation increased the incidence of TGCT from 45% to 100% in the offspring. The percentage of mice with bilateral tumors, weights of testes with TGCT, and the percentage of tumors that were clearly teratomas were higher in the irradiated mice than in controls, indicating that irradiation induced more aggressive tumors and/or more foci of initiation sites in each testis. This radiation dose did not disrupt spermatogenesis, which was qualitatively normal in tumor-free testes although they were reduced in size. This is the first proof of induction of testicular cancer by an environmental agent and suggests that the male fetus of women exposed to radiation at about 5-6 weeks of pregnancy might have an increased risk of developing testicular cancer. Furthermore, it provides a novel tool for studying the molecular and cellular events of testicular cancer pathogenesis.     

Effects of pre- and post- natal exposure to flutamide on connexin 43 expression in testes and ovaries of prepubertal pigs

The aim of this study was to show whether connexin43 (C×43) expression in gonads is affected by an anti-androgen action. To perform this test, pigs were prenatally (on gestational days 20–28 and 80–88; GD20, GD80) and postnatally (on days 2–10 after birth; PD2) exposed to flutamide, which was given in five doses every second day and its effect was observed in prepubertal gilts and boars. Morphology and expression of C×43 was investigated in testes and ovaries by means of routine histology, immunohistochemistry, Western blotting, and RT-PCR. In boars exposed to flutamide varying degrees of seminiferous tubule abnormality, including reduced number of Sertoli cells, tubules with severely dilated lumina and multinucleated germ cells were observed, whereas in gilts, the administration of flutamide at GD20 resulted in delayed folliculogenesis. Only follicles at the preantral stage were observed. Qualitative analysis of immunohistochemical staining for C×43 was confirmed by quantitative image analysis, where the staining intensity was expressed as relative optical density of diaminobenzidine deposits. After flutamide exposure, statistically significant increase in C×43 signal intensity was observed between interstitial tissue of GD20 and control pigs (**P<0.01), between seminiferous tubules of PD2 and control boars (**P<0.01) and between theca cells of GD80, of PD2 and control gilts (**P<0.01). In contrast, statistically significant decrease in C×43 signal intensity was found between granulosa cells of GD20, of PD2 and control gilts (**P<0.01 and *P<0.05, respectively) and between theca cells of GD20 and control gilts (**P<0.01). Since we demonstrated changes in gonad morphology and in the expression of C×43 at the level of protein of prepubertal boars and gilts, it seems possible that flutamide, through blocking androgen action, causes delayed gonadal maturation in later postnatal life and, among other factors, may be involved in the regulation of C×43 gene expression in pig gonads.Key words: C×43 gene expression, testis, ovary, flutamide, prepubertal pigs.     

Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl phthalate

BACKGROUND: Gestational exposure to di-n-butyl phthalate (DBP), a ubiquitous environmental contaminant, has been shown to interfere with the development of the male reproductive tract by acting as an antiandrogen. This study was conducted to identify the critical days for the abnormal development of the male reproductive tract, specifically the testis and epididymis. METHODS: Timed-pregnant Sprague-Dawley rats were dosed with DBP at 500 mg/kg/day on gestation day (GD) 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, or 19 and 20 (GD 0=plug day). Anogenital distance (AGD) was measured on postnatal day (PND) 1 and 13, while areloa number was recorded on PND 13 only. After weaning, males were allowed to mature to PND 90 at which time they were necropsied. Areloa number and AGD were recorded and testes, epididymides, seminal vesicles, prostate gland, kidneys, and liver weighed. Blood serum was collected and assayed for total testosterone concentration. RESULTS: There were no observable effects on litter size, sex ratio, serum testosterone concentration, or mortality of pups. Statistically significant permanent reductions in AGD were seen in males exposed prenatally to DBP on GD 15 and 16 or GD 18 and 19. On PND 13, areola were present in males exposed to DBP on GD 15 and 16, 16 and 17, 17 and 18, and 19 and 20. However, permanent retention occurred only in males after DBP exposure on GD 16 and 17. Exposure to DBP on only GD 17 and 18 elicited a reduction in epididymal weights; while exposure on only GD 16 and 17 caused a significant increase in the weights of the testes due to edema. In this study, epididymal and testicular malformations were most prevalent after exposure to DBP on any gestational day. Epididymal malformations, characterized by agenesis of various regions and small or flaccid testes were significantly increased in DBP-exposed males only on GD 16 and 17. CONCLUSIONS: These findings suggest that 2-day DBP exposure is highly detrimental to the developing reproductive tract of the male fetus and the critical window for abnormal development is GD 16-18.     

Androgens in relation to prenatal development and postnatal inversion of the gubernacula in rats.

Exposure of male rats to the anti-androgen flutamide during fetal life, from day 10 after conception to the day of birth, allowed quantitatively unaltered development of the gubernacula. Apparently, androgens play no important role or no role at all in their growth. Castration of newborn male rats did not interfere with the inversion during further postnatal life of the gubernacula to create the muscular parts of the scrotum (cremaster muscles). Prenatal exposure to flutamide, followed by castration immediately after birth, also allowed gubernacular inversion and cremaster muscle growth. Neonatal administration of testosterone, after castration at birth, did not enhance gubernacular inversion or promote cremaster muscle growth in infancy or during adulthood. Apparently, postnatal gubernacular inversion and cremaster muscle growth are independent not only of androgens, but also of all testis hormones. Neonatal administration of the potent androgen 5 alpha-dihydrotestosterone propionate suppressed gonadotrophin secretion and, in intact males, inhibited testicular growth. Administration from the day of birth to day 33 delayed testicular descent and enhanced growth of the genital apparatus, but did not affect the size of the cremaster muscles. These experiments indicate that androgens are not involved in the processes that create the cavities into which testes descend to acquire their full reproductive potential.     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.     

Evaluation of the developmental toxicity of lenalidomide in rabbits

BACKGROUND: Lenalidomide, a thalidomide analog, is indicated for treatment of patients with deletion-5q myelodysplastic syndromes or multiple myeloma. NZW rabbits were used because of sensitivity to thalidomide's teratogenicity. METHODS: Range-finding and pulse-dosing studies preceded a full developmental toxicity study in New Zealand white (NZW) rabbits (25/group) given lenalidomide (0, 3, 10, or 20 mg/kg/day) or thalidomide (180 mg/kg/day) by stomach tube on gestation days (GD) 7-19. Clinical signs, body weights, and feed consumption were recorded daily from GD 7. On GD 29, standard maternal necropsy, uterine content, and fetal evaluations were carried out. RESULTS: In all studies, thalidomide was selectively toxic to development. In the pulse-dosing study, lenalidomide did not affect development at 100 mg/kg/day. Increases in C(max) and AUC(0-24 hr) values for lenalidomide were slightly less than dose-proportional; lenalidomide occurred in the fetuses. At 10 and 20 mg/kg/day, lenalidomide was maternally toxic (reduced body weight gain and feed consumption; at 20 mg/kg/day, weight loss and one abortion). Developmental toxicity at 10 and 20 mg/kg/day included reduced fetal body weights and increased postimplantation losses and fetal variations (morbidity/purple-discolored skin, undeveloped intermediate lung lobe, irregular nasal-frontal suture, and delayed metacarpal ossification). Thalidomide selectively reduced fetal body weight, increased postimplantation loss and caused characteristic limb and other dysmorphology. CONCLUSIONS: The maternal and developmental NOAELs for lenalidomide are 3 mg/kg/day. Unlike thalidomide, lenalidomide affected embryo-fetal development only at maternally toxic dosages, confirming that structure-activity relationships may not predict maternal or developmental effects. No fetal malformations were attributable to lenalidomide.