FIG 3 - uploaded by Romain Lambrot
Content may be subject to copyright.
Testosterone secretion by human fetal testes in organ culture. Explants of testes from 6-wk, between 6-and 7-wk, and greater than 7-wk-old fetuses were cultured for 4 d in control medium (A, B, and C). The media were changed every 24 h and their testosterone content was measured by RIA. Values are means SEM of four to 26 determinations. For explants from 7-to 12-wk fetuses (D), one piece of a testis from each fetus was cultured in control medium and the other one in medium supplemented with 100 ng/ml LH from 24 to 96 h. Data are expressed as a percentage of testosterone secretion at d 1 with the value of this day taken as 100%. Means SEM of three determinations are shown. *, P 0.05 in the paired statistical comparison with the corresponding control values. a, P 0.05; b, P 0.01; c, P 0.001 in the comparison with the first day of culture (ANOVA test).
Source publication
In human, the chronology of the testicular development has been extensively studied, but the factors implicated in the onset and the regulation of gametogenesis and steroidogenesis remain hardly known.
To identify these factors, we developed an organ culture system for human fetal testes recovered during the first trimester (6-12 wk) of gestation....
Contexts in source publication
Context 1
... cultured testes from fetuses at 6 -12 wk of development for 4 d in control medium, and we measured the daily tes- tosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A-C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 2.5 pg/testis per hour at d 1 to 36.4 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/ testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH in- creased the production of testosterone during the whole cul- ture (Fig. 3D), avoiding the Leydig cell ...
Context 2
... cultured testes from fetuses at 6 -12 wk of development for 4 d in control medium, and we measured the daily tes- tosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A-C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 2.5 pg/testis per hour at d 1 to 36.4 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/ testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH in- creased the production of testosterone during the whole cul- ture (Fig. 3D), avoiding the Leydig cell ...
Context 3
... cultured testes from fetuses at 6 -12 wk of development for 4 d in control medium, and we measured the daily tes- tosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A-C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 2.5 pg/testis per hour at d 1 to 36.4 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/ testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH in- creased the production of testosterone during the whole cul- ture (Fig. 3D), avoiding the Leydig cell ...
Context 4
... cultured testes from fetuses at 6 -12 wk of development for 4 d in control medium, and we measured the daily tes- tosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A-C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 2.5 pg/testis per hour at d 1 to 36.4 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/ testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH in- creased the production of testosterone during the whole cul- ture (Fig. 3D), avoiding the Leydig cell ...
Context 5
... cultured testes from fetuses at 6 -12 wk of development for 4 d in control medium, and we measured the daily tes- tosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A-C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 2.5 pg/testis per hour at d 1 to 36.4 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/ testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH in- creased the production of testosterone during the whole cul- ture (Fig. 3D), avoiding the Leydig cell ...
Similar publications
![Figure 1. Effect of B[a]P treatment on BW (A), testis weight (B), and...](profile/Seung-Lee-11/publication/51475160/figure/fig1/AS:667178651090948@1536079194091/Effect-of-BaP-treatment-on-BW-A-testis-weight-B-and-testosterone-concentrations_Q320.jpg)
![Figure 7. Effects of exposure to B[a]P (0, 0.001, 0.01, and 0.1...](profile/Seung-Lee-11/publication/51475160/figure/fig4/AS:667178651103235@1536079194160/Effects-of-exposure-to-BaP-0-0001-001-and-01-mg-kg-BW-on-LH-serum-levels-and_Q320.jpg)
Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is a ubiquitous environmental pollutant that is currently suspected of being an endocrine disruptor. The testis is an important target for PAHs, yet insufficient attention has been paid to their effects on steroidogenesis in Leydig cells.
We hypothesized that long-term exposure to low...
Citations
... In the fetal testis, Sox9 up-regulates the expression of Cyp26b1 to prevent ATRA entry into the testis, while in the fetal ovary, Foxl2 down-regulates the expression of Cyp26b1 to allow ATRA to enter the gonad and drive germ cell entry into meiosis (Kashimada et al., 2011, Nicol et al., 2018. Disruption of the fetal testicular retinoic acid concentration by introducing exogenous retinoic acid can impair cord formation and induce germ cell death (Livera et al., 2000, Spade et al., 2019a, Trautmann et al., 2008, Bowles et al., 2018, Lambrot et al., 2006. ...
Phthalic acid esters (phthalates) are a class of industrial chemicals that cause developmental and reproductive toxicity, but there are significant gaps in knowledge of phthalate toxicity mechanisms. There is evidence that phthalates disrupt retinoic acid signaling in the fetal testis, potentially disrupting control of spatial and temporal patterns of testis development. Our goal was to determine how a phthalate would interact with retinoic acid signaling during fetal mouse testis development. We hypothesized that mono-(2-ethylhexyl) phthalate (MEHP) would exacerbate the adverse effect of all-trans retinoic acid (ATRA) on seminiferous cord development in the mouse fetal testis. To test this hypothesis, gestational day (GD) 14 C57BL/6 mouse testes were isolated and cultured on media containing MEHP, ATRA, or a combination of both compounds. Cultured testes were collected for global transcriptome analysis after one day in culture and for histology and immunofluorescent analysis of Sertoli cell differentiation after three days in culture. ATRA disrupted seminiferous cord morphogenesis and induced aberrant FOXL2 expression. MEHP alone had no significant effect on cord development, but combined exposure to MEHP and ATRA increased the number of FOXL2-positive cells, reduced seminiferous cord number, and increased testosterone levels, beyond the effect of ATRA alone. In RNA-seq analysis, ATRA treatment and MEHP treatment resulted in differential expression of genes 510 and 134 genes, respectively, including 70 common differentially expressed genes (DEGs) between the two treatments, including genes with known roles in fetal testis development. MEHP DEGs included RAR target genes, genes involved in angiogenesis, and developmental patterning genes, including members of the homeobox superfamily. These results support the hypothesis that MEHP modulates retinoic acid signaling in the mouse fetal testis and provide insight into potential mechanisms by which phthalates disrupt seminiferous cord morphogenesis.
... RA plays a crucial role in initiating spermatogenesis, but its effects may be age dependent. RA supplement enhanced in vitro spermatogenesis in neonatal mouse testis tissues [195,197] but was found to be detrimental to fetal human [45,207] or mouse [195,208] testicular tissues in culture. FSH and luteinizing hormone (LH) play important roles in maturation of Sertoli cells and Leydig cells, respectively, which are required for germ cell development. ...
Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child, but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperm can be frozen and stored for long periods of time, thawed at a later date and, used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperm. When sperm and testicular tissues are not available for fertility preservation, induce pluripotent stem cells (iPSCs) derived from somatic cells such as blood or skin may provide an alternative path to produce sperm through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as experimental options to produce functional spermatids or sperm from immature cryopreserved testicular tissues or somatic cells.
... Limitations are mainly related to a relatively short culture period of up to two weeks as well as the need to culture small (~1 mm3) tissue fragments to avoid necrosis/apoptosis in the centre of the tissue. Advantages relate to the preservation of testicular morphology, spatial organisation, interactions between the different testicular cell types, paracrine signalling and endocrine function within the intact foetal testicular fragments [80][81][82][83][84][85]. This ensures maintenance of the foetal testis tissue to support continued germ cell development, differentiation of the somatic cells and endocrine function. ...
... This ensures maintenance of the foetal testis tissue to support continued germ cell development, differentiation of the somatic cells and endocrine function. This allows examination of functional outcomes following manipulations such as increased steroid hormone production observed when, for example, LH or hCG is added to the culture media [81,82,84]. Therefore, ex vivo culture approaches provide important advantages compared to in vitro culture of dissociated testicular cells and may be an attractive experimental option to examine human foetal testicular development and function following exposure to environmental chemicals and pharmaceutical drugs. ...
... This includes determining the effects of the phthalate MEHP (metabolite of DEHP) on human foetal testis tissue. No effects on testosterone production were found [82,84], but the number of germ cells were reduced as a result of increased apoptosis [84]. Also, the effects of bisphenols have been examined using this experimental approach. ...
Background:
Male reproductive development in mammals can be divided into a gonadal formation phase followed by a hormone-driven differentiation phase. Failure of these processes may result in Differences in Sex Development (DSD), which may include abnormalities of the male reproductive tract, including cryptorchidism, hypospadias, infertility, and testicular germ cell cancer (TGCC). These disorders are also considered to be part of a testicular dysgenesis syndrome (TDS) in males. Whilst DSDs are considered to result primarily from genetic abnormalities, the development of TDS disorders is frequently associated with environmental factors.
Summary:
In this review, we will discuss the development of the male reproductive system in relation to DSD and TDS. We will also describe the experimental systems, including studies involving animals and human tissues or cells that can be used to investigate the role of environmental factors in inducing male reproductive disorders. We will discuss recent studies investigating the impact of environmental chemicals (e.g., phthalates and bisphenols), lifestyle factors (e.g., smoking) and pharmaceuticals (e.g., analgesics) on foetal testis development. Finally, we will describe the evidence, involving experimental and epidemiologic approaches, for a role of environmental factors in the development of specific male reproductive disorders, including cryptorchidism, hypospadias, and TGCC. Key Messages: Environmental exposures can impact the development and function of the male reproductive system in humans. Epidemiology studies and experimental approaches using human tissues are important to translate findings from animal studies and account for species differences in response to environmental exposures.
... Human fetal embryos at 6.3 weeks of gestation were obtained from pregnant women referred to the Department of Gynecology and Obstetrics at the 9 Antoine Béclere hospital (Clamart, France) for legally induced abortions in the first trimester of pregnancy, as previously described (Lambrot et al., 2006). All women provided written informed consent for scientific use of the fetal tissues. ...
Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.
This article has an associated ‘The people behind the papers’ interview.
... We next asked about the role of RAR in the somatic cells of fetal testes. Testosterone production by fetal Leydig cells is crucial for the proper masculinization of the embryo and has previously been reported to be altered following RA exposure in the developing rat and human testes [22,33]. Therefore, we cultured 12.5 dpc mouse fetal testes in the presence of exogenous RA and measured testosterone production. ...
... Using an organ culture of mouse fetal testes, we demonstrated that RA increases testosterone production. This RA action on steroidogenesis is opposite to that which we described previously, indicating that RA decreases testosterone production in the developing rat testis, but is similar to that reported in human fetal testes [22,33]. RA effects on steroidogenesis are thus highly variable. ...
In female mammals, germ cells enter meiosis in the fetal ovaries, while in males, meiosis is prevented until postnatal development. Retinoic acid (RA) is considered the main inducer of meiotic entry, as it stimulates Stra8 which is required for the mitotic/meiotic switch. In fetal testes, the RA-degrading enzyme CYP26B1 prevents meiosis initiation. However, the role of endogenous RA in female meiosis entry has never been demonstrated in vivo. In this study, we demonstrate that some effects of RA in mouse fetal gonads are not recapitulated by the invalidation or up-regulation of CYP26B1. In organ culture of fetal testes, RA stimulates testosterone production and inhibits Sertoli cell proliferation. In the ovaries, short-term inhibition of RA-signaling does not decrease Stra8 expression. We develop a gain-of-function model to express CYP26A1 or CYP26B1. Only CYP26B1 fully prevents STRA8 induction in female germ cells, confirming its role as part of the meiotic prevention machinery. CYP26A1, a very potent RA degrading enzyme, does not impair the formation of STRA8-positive cells, but decreases Stra8 transcription. Collectively, our data reveal that CYP26B1 has other activities apart from metabolizing RA in fetal gonads and suggest a role of endogenous RA in amplifying Stra8, rather than being the initial inducer of Stra8. These findings should reactivate the quest to identify meiotic preventing or inducing substances.
... Ge et al. (2006) 2006 Human Retinoic acid is a potential regulator of both male germ cell differentiation and steroidogenesis in fetal (6-12 weeks of age) testis. Lambrot et al. (2006) ...
The term “Puberty”, socially known as “Adolescence” is the transitional period from juvenile life to adulthood with functional maturation of gonads and genital organs. In this process, some remarkable developmental changes occur in morphology, physiology, and behavior leading to reproductive competence. Despite sufficient levels of gonadotropins (luteinizing hormone [LH] and follicle‐stimulating hormone [FSH]), robust spermatogenesis is not initiated during infancy in primates due to the immaturity of testicular Sertoli cells. Recent studies suggest that developmental competence augmenting functional activities of receptors for androgen and FSH is acquired by Sertoli cells somewhere during the prolonged hypo‐gonadotropic juvenile period. This juvenile phase is terminated with the re‐awakening of hypothalamic Kisspeptin/Neurokinin B/Dynorphin neurons which induce the release of the gonadotropin‐releasing hormone leading to reactivation of the hypothalamo‐pituitary‐testicular axis at puberty. During this period of pubertal development, FSH and LH facilitate further maturation of testicular cells (Sertoli cells and Leydig cells) triggering robust differentiation of the spermatogonial cells, ensuing the spermatogenic onset. This review aims to precisely address the evolving concepts of the pubertal regulation of hormone production with the corresponding cooperation of testicular cells for the initiation of robust spermatogenesis, which can be truly called “testicular puberty.” The review aims to precisely address the evolving concepts of the pubertal regulation of hormone production with the corresponding maturation of testicular cells for the initiation of robust spermatogenesis.
... Retinoic acid (RA) is well known to influence stem cell differentiation 45 and steroidogenesis 46,47 . RA also stimulates testosterone secretion from human fetal testis organ culture over a short period of time 48 . cAMP, as a second messenger, is known to induce steroidogenesis in a number of steroidogenic cell lines 23 . ...
Leydig cells (LCs) are the primary source of testosterone in the testis, and testosterone deficiency caused by LC functional degeneration can lead to male reproductive dysfunction. LC replacement transplantation is a very promising approach for this disease therapy. Here, we report that human adipose derived stem cells (ADSCs) can be differentiated into Leydig‐like cells using a novel differentiation method based on molecular compounds. The isolated human ADSCs expressed positive CD29, CD44, CD59 and CD105, negative CD34, CD45 and HLA‐DR using flow cytometry, and had the capacity of adipogenic and osteogenic differentiation. ADSCs derived Leydig‐like cells (ADSC‐LCs) acquired testosterone synthesis capabilities, and positively expressed LC lineage‐specific markers LHCGR, STAR, SCARB1, SF‐1, CYP11A1, CYP17A1, HSD3B1 and HSD17B3 as well as negatively expressed ADSC specific markers CD29, CD44, CD59 and CD105. When ADSC‐LCs labelled with lipophilic red dye (PKH26) were injected into rat testes which were selectively eliminated endogenous LCs using ethylene dimethanesulfonate (EDS, 75 mg/kg), the transplanted ADSC‐LCs could survive and function in the interstitium of testes, and accelerate the recovery of blood testosterone levels and testis weights. These results demonstrated that ADSCs could be differentiated into Leydig‐like cells by few defined molecular compounds, which might lay the foundation for further clinical application of ADSC‐LC transplantation therapy.
... Retinoic acid (RA) is well known to influence stem cell differentiation 45 and steroidogenesis 46,47 . RA also stimulates testosterone secretion from human fetal testis organ culture over a short period of time 48 . cAMP, as a second messenger, is known to induce steroidogenesis in a number of steroidogenic cell lines 23 . ...
Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.
... The early differentiation of these cells must be independent of LH, since the onset of testicular androgen production precedes the secretion of LH by the fetal pituitary [9]. It has been shown that retinoic acid has potential to stimulate steroidogenesis in the developing hFLC by upregulating expression of CYP11A1, CYP17A1 and StAR [11]. In the present study we observed significant activation of the expression of the same steroidogenic enzymes in the human fetal testes at GW11-12 compared to GW8-9, which was associated with an increase in the amount of androgens and total steroids, strongly suggesting rapid and intensive differentiation of hFLC at the end of first trimester. ...
The onset of steroidogenesis in human fetal testes (HFT) during the first trimester is poorly investigated. One important unresolved question is the ontogeny of steroidogenic enzymes and formation of steroidogenic pathways in the HFT at early pregnancy. Our aim was to explore steroidogenesis, the expression of steroidogenic enzymes and their maturation in the HFT at gestational weeks (GW) 8-12. Steroids in the HFT were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the HFT at GW8-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that the HFT at GW8-9 produced low level of testosterone via the Δ⁴ pathway and progesterone was the major steroid found in the testicular tissue. In contrast, more mature Leydig cells from the HFT at GW11-12 synthesized high levels of androgens via the Δ⁵ pathway. We also observed a significant upregulation of the expression of StAR, CYP11A1, CYP17A1 and its accessory proteins, P450 oxidoreductase (POR) and cytochrome b5 in the HFT at GW11-12 compared to GW8-9. Altogether, our data suggest that that human fetal Leydig cells differentiate rapidly at the end of the first trimester by acquiring capacity to express high levels of steroidogenic enzymes and switch from the Δ⁴ to the Δ⁵ pathways to synthesize high levels of androgens due to maturation of the CYP17-POR-b5 complex.
... Cyp26b1 levels in the testis are directly under the control of the pro-testis developmental signaling factors Sry and Sox9 (Kashimada et al., 2011;Li et al., 2014), indicating that metabolism of RA is required for testis development. Following RA exposure in vitro, or in systems in which CYP26B1 is knocked out or inhibited in vivo, fetal testis development is disrupted, resulting in decreased Leydig cell function, inappropriate meiotic entry in germ cells, and loss of seminiferous cord structure, with some variation by species (Cupp et al., 1999;Hogarth et al., 2015;Jorgensen et al., 2015;Lambrot et al., 2006;Livera et al., 2000Livera et al., , 2001Livera et al., , 2004Trautmann et al., 2008). The disorganized testicular phenotype caused by all-trans retinoic acid (ATRA) exposure ex vivo is associated with enhanced expression of the ovarian development gene, Wnt4, and the anti-testis transcription factor, Nr0b1, as well as an increase in the number of FOXL2-positive Sertoli cells, indicating aberrant induction of signaling for ovary developmental processes in the testis (Spade et al., 2018). ...
Humans are universally exposed to low levels of phthalate esters (phthalates), which are used to plasticize polyvinyl chloride. Phthalates exert adverse effects on the development of seminiferous cords in the fetal testis through unknown toxicity pathways. To investigate the hypothesis that phthalates alter seminiferous cord development by disrupting retinoic acid signaling in the fetal testis, gestational day 15 fetal rat testes were exposed for 1-3 days to 10-6 M all-trans retinoic acid (ATRA) alone or in combination with 10-6 to 10-4 M mono-(2-ethylhexyl) phthalate (MEHP) in ex vivo culture. As previously reported, exogenous ATRA reduced seminiferous cord number. This effect was attenuated in a concentration-dependent fashion by MEHP co-exposure. ATRA and MEHP-exposed testes were depleted of DDX4-positive germ cells but not Sertoli cells. MEHP alone enhanced the expression of the retinoic acid receptor target Rbp1 and the ovary development-associated genes Wnt4 and Nr0b1, and suppressed expression of the Leydig cell marker, Star, and the germ cell markers, Ddx4 and Pou5f1. In co-exposures, MEHP predominantly enhanced the gene expression effects of ATRA, but the Wnt4 and Nr0b1 concentration-responses were non-linear. Similarly, ATRA increased the number of cells expressing the granulosa cell marker FOXL2 in testis cultures, but this induction was attenuated by addition of MEHP. These results indicate that MEHP can both enhance and inhibit actions of ATRA during fetal testis development and provide evidence that retinoic acid signaling is a target for phthalate toxicity in the fetal testis.
