Hazel L. Kinnell

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.

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

Development of steroid signaling pathways during primordial follicle formation in the human fetal ovary.

CONTEXT Ovarian primordial follicle formation is critical for subsequent human female fertility. It is likely that steroid, and especially estrogen, signaling is required for this process, but details of the pathways involved are currently lacking. OBJECTIVE The aim was to identify and characterize key members of the steroid-signaling pathway expressed in the second trimester human fetal ovary. DESIGN We conducted an observational study of the female fetus, quantifying and localizing steroid-signaling pathway members. SETTING The study was conducted at the Universities of Aberdeen, Edinburgh, and Glasgow. PATIENTS/PARTICIPANTS Ovaries were collected from 43 morphologically normal human female fetuses from women undergoing elective termination of second trimester pregnancies. MAIN OUTCOME MEASURES We measured mRNA transcript levels and immunolocalized key steroidogenic enzymes and steroid receptors, including those encoded by ESR2, AR, and CYP19A1. RESULTS Levels of mRNA encoding the steroidogenic apparatus and steroid receptors increased across the second trimester. CYP19A1 transcript increased 4.7-fold during this period with intense immunostaining for CYP19A detected in pregranulosa cells around primordial follicles and somatic cells around oocyte nests. ESR2 was localized primarily to germ cells, but androgen receptor was exclusively expressed in somatic cells. CYP17A1 and HSD3B2 were also localized to oocytes, whereas CYP11A1 was detected in oocytes and some pregranulosa cells. CONCLUSIONS The human fetal ovary expresses the machinery to produce and detect multiple steroid signaling pathways, including estrogenic signaling, with the oocyte acting as a key component. This study provides a step-change in our understanding of local dynamics of steroid hormone signaling during the key period of human primordial follicle formation.

Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles

In the mammalian ovary a small number of follicles are steadily recruited from the quiescent pool to undergo development. Follicle loss, maintenance and growth are strictly controlled by complex molecular interactions including the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway. Stimulation of PI3K promotes phosphorylation of Akt resulting in follicle survival and activation of growth whereas this pathway is suppressed by the actions of the phosphatase and tensin homologue (PTEN). The aim of this study was to determine the effect of dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (bpV), a reversible inhibitor of PTEN, on the activation, survival and development of human ovarian follicles in vitro. Biopsied ovarian tissue fragments were obtained from 17 women aged 23–46 years and exposed to 1 µM bpV(HOpic) (n = 146) or control medium (n = 128) for 24 h. Media were then replaced with control medium and all tissue incubated for a further 5 days. Ovarian tissue from each treatment group was fixed after the initial 24 h culture period and phosphorylated Akt was quantified by western blotting. After 6 days incubation all tissue fragments were inspected under light microscopy and any secondary follicles ≥100 µm isolated. Isolated follicles were cultured individually in control medium supplemented with 100 ng/ml recombinant human activin A. Tissue fragments without follicles suitable for isolation were fixed and processed for histological and immunohistochemical analysis. During 6 days culture, follicle activation occurred in tissue samples from both treatment groups but with significantly more follicles progressing to the secondary stage of development in the presence of 1 µM bpV(HOpic) compared with control (31 versus 16%; P < 0.05). Increased activation was associated with increased Akt phosphorylation and increased nuclear export of FOXO3. However isolated and cultured follicles that had been exposed to bpV(HOpic) showed limited growth and reduced survival compared with follicles from control fragments (P < 0.05). This study demonstrates that inhibition of PTEN with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles.

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.

GPR50 interacts with neuronal NOGO-A and affects neurite outgrowth

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signalling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. We sought to understand the function of one such orphan, G protein-coupled receptor 50 (GPR50), through identification of protein interactors. GPR50 was previously discovered as a candidate gene for psychiatric illness and lipid metabolism. Here, we identified neurite outgrowth inhibitor NOGO-A as an interacting partner of GPR50 by yeast two-hybrid studies. We confirmed the interaction in mammalian cells and found an enrichment of both Gpr50 and neuronal Nogo-A at the synapse. In contrast to neuronal NOGO-A overexpression, overexpression of GPR50 increased neurite length and filopodia- and lamellipodia-like structures in differentiated Neuroscreen-1 cells. The results are markedly similar to a recent study in Nogo-A KO mice and support the involvement of GPR50 in mental disorders with links to several disease mechanisms.

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.

Fetal Cyclophosphamide Exposure Induces Testicular Cancer and Reduced Spermatogenesis and Ovarian Follicle Numbers in Mice

Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼70% of control and induced atrophic seminiferous tubules in ∼30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan.

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.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.