Sertoli cell development and function in an animal model of testicular dysgenesis syndrome.

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.

The origins and time of appearance of focal testicular dysgenesis in an animal model of testicular dysgenesis syndrome: evidence for delayed testis development?

A testicular dysgenesis-like syndrome is induced in rats by fetal exposure to di(n-butyl) phthalate (DBP). A key feature of this is the formation of focal dysgenetic areas comprising malformed seminiferous cords/tubules and intratubular Leydig cells (ITLC), but how and why these arise remains unclear. The present study has used combinations of cell-specific markers and immunohistochemistry to address this. The results show that focal dysgenetic areas and ITLC first appear postnatally at 4-10 days of age, but this only occurs in treatment groups in which formation of fetal Leydig cell aggregation is induced between e17.5 and e21.5. Extreme variability in the formation and size of the Leydig cell aggregates probably accounts for the equally extreme variation in occurrence and size of focal dysgenetic areas postnatally. DBP-induced fetal Leydig cell aggregation traps Sertoli and other cells within the aggregates, but it is unclear why this happens nor why cords fail to form prenatally in these cell mixtures but do elsewhere in the fetal testis. The present studies show that differentiation of the fetal Leydig cells is drastically delayed at e15.5 after DBP exposure, which may be indicative of a wider delay in testis cell development and organisation, and this might account for some of the unexplained findings.

Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human.

BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15-19 weeks of gestation) were cultured for 24-48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5-20.5) DBP treatment. In vitro, MBP (10(-3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(-3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2-7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.

Role of androgens in fetal testis development and dysgenesis.

This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels.

In utero exposure to di(n-butyl) phthalate and testicular dysgenesis: comparison of fetal and adult end points and their dose sensitivity.

BACKGROUND: Fetal exposure of male rats to di(n-butyl) phthalate (DBP) induces reproductive disorders similar to those in human testicular dysgenesis syndrome (TDS), including infertility, cryptorchidism, focal "dysgenetic areas," and Sertoli cell-only tubules in the adult testis. Humans are widely exposed to DBP, but at much lower levels than those causing adverse effects in rats. OBJECTIVES: The objective of this study was to evaluate end points affected by DBP action in rats in fetal and adult life that are relevant to human TDS, and to compare their dose sensitivity. METHODS: Pregnant rats were gavaged daily with corn oil (control) or with 4, 20, 100, or 500 mg/kg DBP. We examined adult end points of TDS (infertility, cryptorchidism) and indicators within the fetal testis of dysgenesis [abnormal Leydig cell (LC) aggregation, multinucleated gonocytes (MNGs)], as well as conditions that may result from these indicators in adulthood (occurrence of focal dysgenetic areas). Fetal testis weight and testicular testosterone levels were also evaluated. RESULTS: The fetal end points analyzed (testicular testosterone levels, abnormal LC aggregation, occurrence of MNGs) were most sensitive to disruption by DBP, as all were significantly affected at a dose of 100 mg/kg/day DBP, with a trend toward effects occurring at 20 mg/kg/day DBP; adult end points were affected consistently only by 500 mg/kg/day DBP. CONCLUSIONS: The fetal end points we evaluated can be objectively quantified and may prove helpful in evaluating the health risk of exposure to DBP and other phthalates, as well as identifying DBP-sensitive fetal events that have adult consequences/end points that are identifiable in human TDS.

Acute and long-term effects of in utero exposure of rats to di(n-butyl) phthalate on testicular germ cell development and proliferation.

This study investigated effects of in utero exposure [embryonic day (e)13.5-e21.5] to di(n-butyl) phthalate (DBP) on fetal gonocytes and postnatal germ cell (GC) development in rats and focused on changes (delayed development) relevant to the postulated origins of human carcinoma-in situ cells. DBP treatment resulted in both early (e15.5-e17.5) and late (e19.5-e21.5) effects on gonocytes. The former involved delayed entry of proliferating gonocytes into quiescence, as indicated by prolongation/overexpression of octamer-binding transcription factor 3/4 and retinoblastoma protein phosphorylated at Ser 807/811 and Ki67 plus a 2- to 4-fold increase in gonocyte apoptosis. The late effect of DBP was to induce a greater than 10-fold increase in occurrence of multinucleated gonocytes. GC numbers in DBP-exposed males were reduced (P < 0.01) by 37, 53, 79, and 80% at e21.5 and postnatal d (d) 4, 8, and 15, respectively, with recovery to normal in scrotal testes between postnatal d 25 and 90. The DBP-induced decrease in GC numbers at d 4-8 was associated with delayed exit from quiescence, as indicated by retinoblastoma protein expression and a 28% reduction (P < 0.001) in GC proliferation index at d 6, although the latter was increased by 84% at d 25. The postnatal GC changes were associated with the early, but not late, effects of DBP on gonocytes as short-term DBP treatment from e19.5 to e20.5, induced multinucleated gonocytes as effectively as did treatment from e13.5 to e20.5, but did not reduce GC numbers on d 4. In conclusion, fetal DBP exposure delays normal GC development in both fetal (as early as e15.5) and postnatal life with the possibility of consequences for fertility.

Cellular origins of testicular dysgenesis in rats exposed in utero to di(n-butyl) phthalate.

Foetal exposure of male rats to di(n-butyl) phthalate (DBP) induces testicular changes similar to testicular dysgenesis syndrome in humans, including the formation of focal 'dysgenetic areas' within post-natal testes, surrounded by otherwise normal tubules exhibiting complete spermatogenesis. We hypothesize that these dysgenetic areas form when Sertoli (and other) cells are 'trapped' during the abnormal formation of large Leydig cell (LC) clusters in foetal life and by post-natal day (d) 4 these groups of intermingled cells attempt to form seminiferous tubules. It is likely that the malformed tubules resulting correspond to the dysgenetic areas evident in later life. This also provides a plausible explanation for the occurrence of LCs within seminiferous cords/tubules in or bordering the dysgenetic areas. In our previous studies intratubular LCs (ITLCs) were identified by immunostaining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD), the definitive LC cytoplasmic marker. However, the possibility remained that the 'presumptive' ITLCs were in fact Sertoli cells that had aberrantly gained the ability to express 3beta-HSD. Therefore, the aim of the present study was to fully characterize the ITLCs induced by in utero DBP exposure in d25 rats using a number of LC- (3beta-HSD, P450 side-chain cleavage enzyme, insulin-like factor 3, oestrogen receptor alpha) and Sertoli cell- (vimentin, Wilm's tumour-1) specific markers. Our results show that ITLCs express all four LC-specific markers but do not express either of the Sertoli cell markers. It is therefore concluded that the ITLCs are bona fide LCs that are abnormally located within the seminiferous tubules of DBP-exposed rats in post-natal life.

Abnormal Leydig Cell aggregation in the fetal testis of rats exposed to di (n-butyl) phthalate and its possible role in testicular dysgenesis.

Fetal exposure of male rats to di (n-butyl) phthalate (DBP) induces testicular changes remarkably similar to testicular dysgenesis syndrome in humans; these include induction of focal areas of dysgenetic tubules in otherwise normal testes. In searching for the fetal origins of the latter, we used image analysis to show that exposure to 500 mg/kg DBP [embryonic day (E)13.5-20.5)] caused abnormal aggregation of Leydig cells centrally in the fetal testis. This aggregation was not due to increase in Leydig cell number, and Leydig cell size was significantly reduced in DBP-exposed animals, as were testosterone levels and immunoexpression of P450 side-chain cleavage enzyme. The Leydig cell aggregates did not exhibit evidence of focal proliferation at E17.5-19.5. Using confocal microscopy and Leydig (3beta-hydroxysteroid dehydrogenase) and Sertoli (anti-Mullerian hormone) cell-specific markers, we show that fetal Leydig cell aggregates in DBP-exposed animals trap isolated Sertoli cells within them at E21.5. These areas of intermingled cells are still apparent on postnatal d 4, after cessation of DBP treatment, when they may form misshapen seminiferous cords that trap (intratubular) Leydig cells within them. These centrally located dysgenetic tubules contain germ cells in early puberty, but by adulthood they are Sertoli cell only, implying that presence of intratubular Leydig cells interferes with spermatogenesis. It is concluded that DBP-induced fetal Leydig cell aggregation may be a key event in formation of focal dysgenetic areas in the testis, and identification of the mechanisms underlying these events may give new insights into the fetal origins of testicular dysgenesis syndrome disorders in the human.