Andrew J. Childs

Retinoic acid signalling and the control of meiotic entry in the human fetal gonad

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8–9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis.

Do Phthalates Affect Steroidogenesis by the Human Fetal Testis? Exposure of Human Fetal Testis Xenografts to Di-n-Butyl Phthalate

CONTEXT Phthalates are ubiquitous environmental chemicals. Fetal exposure to certain phthalates [e.g. di-n-butyl phthalate (DBP)] causes masculinization disorders in rats, raising concern for similar effects in humans. We investigated whether DBP exposure impairs steroidogenesis by the human fetal testis. OBJECTIVE The aim of the study was to determine effects of DBP exposure on testosterone production by normally growing human fetal testis xenografts. DESIGN Human fetal testes (14-20 wk gestation; n=12) were xenografted into castrate male nude mice that were treated for 4-21 d with vehicle, or 500 mg/kg·d DBP, or monobutyl phthalate (active metabolite of DBP); all mice were treated with human chorionic gonadotropin to mimic normal human pregnancy. Rat fetal testis xenografts were exposed for 4 d to DBP as a positive control. MAIN OUTCOME MEASURES Testosterone production was assessed by measuring host serum testosterone and seminal vesicle (SV) weights at termination, plus testis gene expression (rats). RESULTS Human fetal testis xenografts showed similar survival (∼80%) and total graft weight (8.6 vs. 10.1 mg) in vehicle and DBP-exposed hosts, respectively. Serum testosterone (0.56 vs. 0.64 ng/ml; P>0.05) and SV weight (67.2 vs. 81.9 mg; P>0.05) also did not differ. Exposure to monobutyl phthalate gave similar results. In contrast, exposure of rat fetal xenografts to DBP significantly reduced SV weight and testis Cyp11a1/StAR mRNA expression and lowered testosterone levels, confirming that DBP exposure can inhibit steroidogenesis in xenografts, further validating the negative findings on testosterone production in the human. CONCLUSIONS Exposure of human fetal testes to DBP is unlikely to impair testosterone production as it does in rats. This has important safety and regulatory implications.

BMP Signaling in the Human Fetal Ovary is Developmentally Regulated and Promotes Primordial Germ Cell Apoptosis

Primordial germ cells (PGCs) are the embryonic precursors of gametes in the adult organism, and their development, differentiation, and survival are regulated by a combination of growth factors collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the growth factor composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long‐term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)1 and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP‐induced apoptosis in the ovary, as in other systems. These data reveal for the first time growth factor regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells. STEM CELLS 2010;28:1368–1378

Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation

BACKGROUND Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development. METHODS Human fetal testes (at 9 weeks, n = 4 and 14–18 weeks gestation, n = 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls. RESULTS Xenografts showed >75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P = 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4+) into pre-spermatogonia (VASA+) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls. CONCLUSIONS Human fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Deletion of the pluripotency-associated Tex19.1 gene causes activation of endogenous retroviruses and defective spermatogenesis in mice

As genetic information is transmitted through successive generations, it passes between pluripotent cells in the early embryo and germ cells in the developing foetus and adult animal. Tex19.1 encodes a protein of unknown function, whose expression is restricted to germ cells and pluripotent cells. During male spermatogenesis, Tex19.1 expression is highest in mitotic spermatogonia and diminishes as these cells differentiate and progress through meiosis. In pluripotent stem cells, Tex19.1 expression is also downregulated upon differentiation. However, it is not clear whether Tex19.1 has an essential function in germ cells or pluripotent stem cells, or what that function might be. To analyse the potential role of Tex19.1 in pluripotency or germ cell function we have generated Tex19.1−/− knockout mice and analysed the Tex19.1−/− mutant phenotype. Adult Tex19.1−/− knockout males exhibit impaired spermatogenesis. Immunostaining and histological analysis revealed defects in meiotic chromosome synapsis, the persistence of DNA double-strand breaks during meiosis, and a loss of post-meiotic germ cells in the testis. Furthermore, expression of a class of endogenous retroviruses is upregulated during meiosis in the Tex19.1−/− testes. Increased transposition of endogenous retroviruses in the germline of Tex19.1−/− mutant mice, and the concomitant increase in DNA damage, may be sufficient to disrupt the normal processes of recombination and chromosome synapsis during meiosis and cause defects in spermatogenesis. Our results suggest that Tex19.1 is part of a specialised mechanism that operates in the germline to repress transposable genetic elements and maintain genomic stability through successive generations.

Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression

Ovarian germ cell survival is dependent upon the formation of primordial follicles, which occurs during fetal life in the human. Activin contributes to germ cell proliferation and survival at this time. SMADs2 and 3 are central elements in the activin signalling pathway and thus indicate sites of activin action. We have investigated the expression and localisation of SMADs2 and 3 in the fetal ovary between 14 and 20 weeks gestation, i.e. preceding and during primordial follicle formation. SMAD3 mRNA expression increased 1.9 fold (P=0.02). SMAD2 and 3 proteins were localised by immunofluorescence to the nuclei of three distinct populations of somatic cells: (a) stromal cells between clusters of germ cells; (b) some somatic cells intermingled with activin beta A-expressing germ cells; (c) pre-granulosa cells surrounding primordial follicles. Germ cells did not express SMAD2 or 3. Activin A increased and follistatin decreased phosphorylation of SMAD2/3 in vitro, and activin increased SMAD2 and decreased KITLG mRNA expression. It therefore appears that somatic cells are the targets for activin signalling in the developing ovary. The effects of activin on germ cells are indirect and include mediation by the kit ligand/c-Kit pathway, rather than being an autocrine germ cell effect.

LIN28 is selectively expressed by primordial and pre-meiotic germ cells in the human fetal ovary.

Germ cell development requires timely transition from primordial germ cell (PGC) self-renewal to meiotic differentiation. This is associated with widespread changes in gene expression, including downregulation of stem cell-associated genes, such as OCT4 and KIT, and upregulation of markers of germ cell differentiation and meiosis, such as VASA, STRA8, and SYCP3. The stem cell-expressed RNA-binding protein Lin28 has recently been demonstrated to be essential for PGC specification in mice, and LIN28 is expressed in human germ cell tumors with phenotypic similarities to human fetal germ cells. We have therefore examined the expression of LIN28 during normal germ cell development in the human fetal ovary, from the PGC stage, through meiosis to the initiation of follicle formation. LIN28 transcript levels were highest when the gonad contained only PGCs, and decreased significantly with increasing gestation, coincident with the onset of germ cell differentiation. Immunohistochemistry revealed LIN28 protein expression to be germ cell-specific at all stages examined. All PGCs expressed LIN28, but at later gestations expression was restricted to a subpopulation of germ cells, which we demonstrate to be primordial and premeiotic germ cells based on immunofluorescent colocalization of LIN28 and OCT4, and absence of overlap with the meiosis marker SYCP3. We also demonstrate the expression of the LIN28 target precursor pri-microRNA transcripts pri-LET7a/f/d and pri-LET-7g in the human fetal ovary, and that expression of these is highest at the PGC stage, mirroring that of LIN28. The spatial and temporal restriction of LIN28 expression and coincident peaks of expression of LIN28 and target pri-microRNAs suggest important roles for this protein in the maintenance of the germline stem cell state and the regulation of microRNA activity in the developing human ovary.

Activation of the aryl hydrocarbon receptor by a component of cigarette smoke reduces germ cell proliferation in the human fetal ovary

Fetal life is a critical time for female fertility, when germ cells complete proliferation, initiate meiosis and ultimately form the lifetime stock of primordial follicles. Female fertility may be reduced by in utero exposure to cigarette smoke, which contains ligands for the aryl hydrocarbon receptor (AhR). The AhR is a critical regulator of ovarian germ cell survival in mice; thus activation of this receptor in the ovaries of fetuses exposed to maternal cigarette smoke in utero may provide a mechanism by which female fertility is reduced in later life. We have therefore investigated AhR expression in the human fetal ovary, and examined the effects of an AhR ligand present in cigarette smoke, on germ cells in human fetal ovaries cultured in vitro. The results showed that AHR mRNA expression increased 2-fold between first and late second trimester (P = 0.008). AhR protein was confined to germ cells at all gestations, but varied from expression in most germ cells during the first trimester, to only patchy expression by clusters of germ cells at later gestations. Culture of human fetal ovaries with the AhR ligand 9,10-dimethyl-1,2-benzanthracene-3,4-dihydrodiol (DMBA-DHD; a component of cigarette smoke) did not affect germ cell number in vitro, but significantly reduced the proportion of proliferating germ cells by 29% (as assessed by phospho-histone H3 staining (P = 0.04)). Germ cell apoptosis was not significantly affected. These results reveal that germ cells in the human fetal ovary express AhR from the proliferative stage of development through entry into meiosis and beyond, and demonstrate that AhR ligands found in cigarette smoke have the capacity to impair human fetal ovarian germ cell proliferation.

Modelling germ cell development in vitro

Germ cells have a critical role in mediating the generation of genetic diversity and transmitting this information across generations. Furthermore, gametogenesis is unique as a developmental process in that it generates highly-specialized haploid gametes from diploid precursor stem cells through meiosis. Despite the importance of this process, progress in elucidating the molecular mechanisms underpinning mammalian germ cell development has been retarded by the lack of an efficient and reproducible system of in vitro culture for the expansion and trans-meiotic differentiation of germline cells. The dearth of such a culture system has rendered the study of germ cell biology refractory to the application of new high-throughput technologies such as RNA interference, leaving in vivo gene-targeting approaches as the only option to determine the function of genes believed to be involved in gametogenesis. Recent reports detailing the derivation of gametes in vitro from stem cells may provide the first steps in developing new tools to solve this problem. This review considers the developments made in modelling germ cell development using stem cells, and some of the challenges that need to be overcome to make this a useful tool for studying gametogenesis and to realize any future clinical application.

The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation

Interactions between germ cells and surrounding somatic cells are central to ovarian development as well as later function. Disruption of these interactions arising from abnormalities in either cell type can lead to premature ovarian failure (POF). The forkhead transcription factor FOXL2 is a candidate POF factor, and mutations in the FOXL2 gene are associated with syndromic and non-syndromic ovarian failure. Foxl2-deficient mice display major defects in primordial follicle activation with consequent follicle loss, and earlier roles in gonadal development and sex determination have also been suggested. However, despite its importance no data presently exist on its expression in the developing human ovary. Expression of FOXL2 mRNA was demonstrated in the human fetal ovary between 8 and 19 weeks gestation, thus from soon after sex determination to primordial follicle development. Expression in the ovary was higher after 14 weeks than at earlier gestation weeks and was very low in the fetal testis at all ages examined. Immunolocalization revealed FOXL2 expression to be confined to somatic cells, both adjacent to germ cells and those located in the developing ovarian stroma. These cells are the site of action of oocyte-derived activin signalling, but in vitro treatment of human fetal ovaries with activin failed to reveal any regulation of FOXL2 transcription by this pathway. In summary, the expression of FOXL2 in somatic cells of the developing human ovary before and during follicle formation supports a conserved and continuing role for this factor in somatic/germ cell interactions from the earliest stages of human ovarian development.