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

GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is Regulated by a Novel NOBOX Transcript

During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations

Co-localisation of GDF9 with activin βA but not DAZL or BOLL prior to follicle formation.

<p>(A) Double immunohistochemistry of 18 week fetal ovary stained for GDF9 (green) and activin βA (red), thus in the merged image co-expression is yellow. Unstained germ cells are indicated with arrows. Counterstain is TOPRO. (B) Triple fluorescent immunohistochemistry for GDF9 (green), DAZL (blue) and BOLL (red) in 20 week human fetal ovary with DAPI as counterstain (grey). Split channel and merged images in (A) and (B) are shown as are merged images of non-immune serum negative control (NEG). Scale bars are 20μm. (C) Nuclear diameters of DAZL, BOLL and GDF9 stained germ cells indicates that GDF9 positive cells are significantly larger (p<0.001) than DAZL but not BOLL expressing cells (bars indicate mean ± sem). (D) Higher magnification merged image of GDF9/DAZL/BOLL immunohistochemistry showing one large primordial follicle is positive for both GDF9 and DAZL but other follicles are positive only for DAZL.</p

Structure of the human <i>NOBOX</i> gene and expression of exons in the human fetal ovary.

<p>(A) Database analysis of human <i>NOBOX</i> transcripts identified 3 possible transcripts for human NOBOX. Only those exons marked in black have been confirmed previously at the experimental level [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119819#pone.0119819.ref041" target="_blank">41</a>]. Exon and intron sizes are indicated and primers used for RT-PCR are shown above each exon. (B) Agarose gel analysis of RT-PCR products using primer pairs as indicated above the lanes. (+) and (-) indicate RT+ and RT- fetal ovary cDNA template. Size marker is the 100bp ladder (Promega) where the 500bp band is more intense. The arrow indicates the position of the weak band identified with the 2Fa + 3Rb primer pair. (C) Structure of the mouse <i>Nobox</i> locus for comparison with the human sequences.</p

Expression of human NOBOX enhances expression from promoters containing NBEs in luciferase assays.

<p>Luciferase assays were performed on HEK293 cells transfected with a selection of promoter-luciferase plasmids containing (<i>GDF9</i> and <i>ZP3</i>) or lacking (<i>ZP2</i>) a putative NOBOX Binding Element (NBE) in combination with either NOBOX expression plasmid or empty vector. Data represent the mean (±sem) activity from at least 5 separate experiments. Statistically significant differences are denoted by asterisks.</p