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.

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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.

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