Embryonic Testis Research Articles

Wilms tumor protein–dependent transcription of VEGF receptor 2 and hypoxia regulate expression of the testis-promoting gene Sox9 in murine embryonic gonads

Wilms tumor protein 1 (WT1) has been implicated in the control of several genes in sexual development, but its function in gonad formation is still unclear. Here, we report that WT1 stimulates expression of Kdr, the gene encoding VEGF receptor 2, in murine embryonic gonads. We found that WT1 and KDR are co-expressed in Sertoli cells of the testes and somatic cells of embryonic ovaries. Vivo-morpholino-mediated WT1 knockdown decreased Kdr transcripts in cultured embryonic gonads at multiple developmental stages. Furthermore, WT1 bound to the Kdr promoter in the chromatin of embryonic testes and ovaries. Forced expression of the WT1(-KTS) isoform, which functions as a transcription factor, increased KDR mRNA levels, whereas the WT1(+KTS) isoform, which acts presumably on the post-transcriptional level, did not. ChIP indicated that WT1(-KTS), but not WT1(+KTS), binds to the KDR promoter. Treatment with the KDR tyrosine kinase inhibitor SU1498 or the KDR ligand VEGFA revealed that KDR signaling represses the testis-promoting gene Sox9 in embryonic XX gonads. WT1 knockdown abrogated the stimulatory effect of SU1498-mediated KDR inhibition on Sox9 expression. Exposure to 1% O2 to mimic the low-oxygen conditions in the embryo increased Vegfa expression but did not affect Sox9 mRNA levels in gonadal explants. However, incubation in 1% O2 in the presence of SU1498 significantly reduced Sox9 transcripts in cultured testes and increased Sox9 levels in ovaries. These findings demonstrate that both the local oxygen environment and WT1, which enhances KDR expression, contribute to sex-specific Sox9 expression in developing murine gonads.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23.

SummaryPrimordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; however, a bioassay that supports their identity as transplantable germ cells has not been reported. Here, we adopted a xenotransplantation assay by transplanting single-cell suspensions of human and nonhuman primate embryonic Macaca mulatta (rhesus macaque) testes containing PGCs into the seminiferous tubules of adult busulfan-treated nude mice. We discovered that both human and nonhuman primate embryonic testis are xenotransplantable, generating colonies while not generating tumors. Taken together, this work provides two critical references (molecular and functional) for defining transplantable primate PGCs. These results provide a blueprint for differentiating pluripotent stem cells to transplantable PGC-like cells in a species that is amenable to transplantation and fertility studies.

Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation.

Gonad morphogenesis relies on the correct spatiotemporal expression of a number of genes that together fulfill the differentiation of the bipotential gonad into testes or ovaries. As such, the transcription factors WT1 and GATA4 are pivotal for proper gonadal development. Here we address the contributions of GATA4 and WT1 to the sex differentiation phase in testes and ovaries. We applied an ex vivo technique for cultivating gonads in hanging droplets of media that were supplemented with vivo-morpholinos to knockdown WT1 and GATA4 either alone or in combination at the same developmental stage. We show that WT1 is equally important for both, the initial establishment and the maintenance of the sex-specific gene expression signature in testes and ovaries. We further identified Foxl2 as a novel putative downstream target gene of WT1. Moreover, knockdown of WT1 reduced mRNA levels of several molecular components of the hedgehog signaling pathway in XY gonads, whereas Gata4 vivo-morpholino treatment increased transcripts of Dhh and Ptch1 in embryonic testes. The data suggest that for its proper function, WT1 relies on the correct expression of the GATA4 protein. Furthermore, GATA4 down-regulates several ovarian promoting genes in testes, such as Ctnnb1, Fst, and Bmp2, suggesting that this repression is required for maintaining the male phenotype. In conclusion, this study provides novel insights into the role of WT1 and GATA4 during the sex differentiation phase and represents an approach that can be applied to assess other proteins with as yet unknown functions during gonadal development.

Human foetal ovary shares meiotic preventing factors with the developing testis.

How can pre-meiotic germ cells persist in the human foetal ovary? Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. N/A. Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes

ABSTRACTAn expressional lack of fibroblast growth factor 9 (FGF9) would cause male-to-female sex reversal in the mouse, implying the essential role of FGF9 in testicular organogenesis and maturation. However, the temporal expression of FGF9 and its receptors during testicular development remains elusive. In this study, immunohistochemistry was used to identify the localization of FGF9 and its receptors at different embryonic and postnatal stages in mice testes. Results showed that FGF9 continuously expressed in the testis during development. FGF9 had highest expression in the interstitial region at 17–18 d post coitum (dpc) and in the spermatocytes, spermatids and Leydig cell on postnatal days (pnd) 35–65. Regarding receptor expression, FGFR1 and FGFR4 were evenly expressed in the whole testis during the embryonic and postnatal stages. However, FGFR2 and FGFR3 were widely expressed during the embryonic testis development with higher FGFR2 expression in seminiferous tubules at 16–18 dpc and higher FGFR3 expression in interstitial region at 17–18 dpc. In postnatal stage, FGFR2 extensively expressed with higher expression at spermatids and Leydig cells on 35–65 pnd and FGFR3 widely expressed in the whole testis. Taken together, these results strongly suggest that FGF9 is correlated with the temporal expression profiles of FGFR2 and FGFR3 and possibly associated with testis development.

Prenatal exposure to dexamethasone disturbs sex-determining gene expression and fetal testosterone production in male embryos

Prenatal stress is known to cause intrauterine fetal growth retardation, and is also associated with various long-term effects in the form of metabolic and neurodevelopmental diseases in adults. Many of the diseases associated with prenatal stress exhibit a sex bias. Perturbations and vulnerability to prenatal stress are often more profound in males, but the mechanisms responsible for this relationship are not clear. We have previously shown that administration of the synthetic glucocorticoid, dexamethasone (Dex), at embryonic days 7.5, 8.5, and 9.5, induces embryonic growth restriction in a sex-dependent manner in a mouse model. Here we examined the effect of prenatal exposure to Dex on gonadal development. During male gonadal development, sex-determining genes, such as Sry, Sox9, and other downstream genes, were found to be dysregulated in response to prenatal Dex, whereas the genes for the ovarian pathway were affected to a lesser degree in females. In addition, fetal testosterone concentrations were decreased by prenatal exposure to Dex, in parallel with reduced numbers of 3β-hydroxysteroid dehydrogenase (3β-HSD)-positive cells in the embryonic testis. These results show that prenatal exposure to Dex differentially influences male versus female on the gene expression and hormone production during sex determination. We believe these studies provide valuable insights into possible mechanisms responsible for sex-specific responses to prenatal stress.

Vascular endothelial growth factor A: just one of multiple mechanisms for sex-specific vascular development within the testis?

Testis development from an indifferent gonad is a critical step in embryogenesis. A hallmark of testis differentiation is sex-specific vascularization that occurs as endothelial cells migrate from the adjacent mesonephros into the testis to surround Sertoli-germ cell aggregates and induce seminiferous cord formation. Many in vitro experiments have demonstrated that vascular endothelial growth factor A (VEGFA) is a critical regulator of this process. Both inhibitors to VEGFA signal transduction and excess VEGFA isoforms in testis organ cultures impaired vascular development and seminiferous cord formation. However, in vivo models using mice which selectively eliminated all VEGFA isoforms: in Sertoli and germ cells (pDmrt1-Cre;Vegfa(-/-)); Sertoli and Leydig cells (Amhr2-Cre;Vegfa(-/-)) or Sertoli cells (Amh-Cre;Vegfa(-/-) and Sry-Cre;Vegfa(-/-)) displayed testes with observably normal cords and vasculature at postnatal day 0 and onwards. Embryonic testis development may be delayed in these mice; however, the postnatal data indicate that VEGFA isoforms secreted from Sertoli, Leydig or germ cells are not required for testis morphogenesis within the mouse. A Vegfa signal transduction array was employed on postnatal testes from Sry-Cre;Vegfa(-/-) versus controls. Ptgs1 (Cox1) was the only upregulated gene (fivefold). COX1 stimulates angiogenesis and upregulates, VEGFA, Prostaglandin E2 (PGE2) and PGD2. Thus, other gene pathways may compensate for VEGFA loss, similar to multiple independent mechanisms to maintain SOX9 expression. Multiple independent mechanism that induce vascular development in the testis may contribute to and safeguard the sex-specific vasculature development responsible for inducing seminiferous cord formation, thus ensuring appropriate testis morphogenesis in the male.

MP40-04 EMBRYONIC TESTIS CONTAINS VASCULAR ENDOTHELIAL PRECURSOR CELLS- A NEW INSIGHT

You have accessJournal of UrologyPediatrics: Testis & Varicoceles, Stones1 Apr 2015MP40-04 EMBRYONIC TESTIS CONTAINS VASCULAR ENDOTHELIAL PRECURSOR CELLS- A NEW INSIGHT Pankaj Dangle, Elina Mukherjee, and Sunder Sims-Lucas Pankaj DanglePankaj Dangle More articles by this author , Elina MukherjeeElina Mukherjee More articles by this author , and Sunder Sims-LucasSunder Sims-Lucas More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1186AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Vascular formation in the testis is a highly integrative process involving complex vascular networks. Defective vascular formation in the testis may lead to undescended hypoplastic testis. There are two types of vascular processes that happen concurrently to form a functional vascular system in the testis, angiogenesis and vasculogenesis. Although significant work has been performed interrogating sprouting angiogenesis, little is known about vascular precursors in the testis and the role they play in patterning this organ. In this study we hypothesize that the testis contains endothelial progenitor cells that are important for the development of the vascular networks and overall formation of the testis. METHODS The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments. Linage tracing experiments were performed by breeding Tbx18cre, which we know to be expressed in the testis with TdTomato reporter mice. These testes were dissected at various developmental stages ages (E15, E18 and postnatal day 21) and the tissue processed for either fluorescently activate cell sorting (FACS) or immunohistochemistry. RESULTS From the embryonic testis we observed a subset of Tbx18 derived cells that gave rise to a subset of the developing vasculature. Furthermore, when we performed immunohistochemistry, were able to co-localize the Tbx18 derived cells with vascular markers (PECAM, Endomucin, Erg). CONCLUSIONS Our data shows that the developing testis contains a subset of endothelial progenitors. These findings suggest that vasculogenesis may play an important role in patterning the testis. Future directions Our future goals are to determine the role of vasculogenic progenitors in mouse models of undescended hypoplastic testis. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e464 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pankaj Dangle More articles by this author Elina Mukherjee More articles by this author Sunder Sims-Lucas More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of Culture in Simulated Microgravity on the Development of Mouse Embryonic Testes.

All known organisms develop and evolve in the presence of gravitational force, and it is evident that gravity has asignificant influence on organism physiology and development. Microgravity is known to affect gene expression, enzyme activity, cytoskeleton organization, mitotic proliferation and intracellular signaling. The aim of the present study was to study some aspects of the development in vitro of mouse embryonic testes in simulated microgravity. Testes from mouse embryos (12.5-16.5days post coitum, d.p.c.) were cultured in simulated microgravity and standard static culture conditions. The microgravity condition was provided by aRotary Cell Culture System (RWV) bioreactor, an apparatus designated for 3D tissue and small organ cultures. After 48 hof the culture in the RWV, testis morphology and size was evaluated. The first observation was that the culture in the RWV bioreactor had abeneficial effect on the testis growth and on the survival of germ cells in comparison to static 2D culture methods. Moreover, we found, that RWV culture caused disorganization the gonadal tissues, namely of the testis cords. The results suggest that the maintenance of testis cord could be sensitive to microgravity. We hypothesize that while the effect on testis growth is due to abetter nutrient and oxygen supply, the testis cord's disorganization might depend on the microgravity conditions simulated by the bioreactor. Considering the complexity of the processes involved in the formation of the testis cords and their dynamic changes during the embryo fetal period, further studies are needed to identify the causes of such effect.

Multiple roles of the prostaglandin D2 signaling pathway in reproduction.

Prostaglandins signaling molecules are involved in numerous physiological processes. They are produced by several enzyme-limited reactions upon fatty acids, which are catalyzed by two cyclooxygenases and prostaglandin synthases. In particular, the prostaglandins E2 (PGE2), D2 (PGD2), and F2 (PGF2 α) have been shown to be involved in female reproductive mechanisms. Furthermore, widespread expression of lipocalin- and hematopoietic-PGD2 synthases in the male reproductive tract supports the purported roles of PGD2 in the development of both embryonic and adult testes, sperm maturation, and spermatogenesis. In this review, we summarize the putative roles of PGD2 signaling and the roles of both PGD2 synthases in testicular formation and function. We review the data reporting the involvement of PGD2 signaling in the differentiation of Sertoli and germ cells of the embryonic testis. Furthermore, we discuss the roles of lipocalin-PGD2 synthase in steroidogenesis and spermatogenesis, in terms of lipid molecule transport and PGD2 production. Finally, we discuss the hypothesis that PGD2 signaling may be affected in certain reproductive diseases, such as infertility, cryptorchidism, and testicular cancer.

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development.

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.

Microanatomical Study of Embryonic Gonadal Development in Japanese Quail (Coturnix japonica).

Gonadal development of quail embryos was examined histologically using histological and histochemical methods. In the present study, quail embryos were studied at various stages of incubation period based on phases of gonadogenesis. Germ cell migration was observed on day 3-4 but gonadal differentiation and gonadal function were observed on day 6–8 and day 11–14, respectively. During germ cell migration, quail primordial germ cells (qPGCs) were successfully detected in both left and right genital ridges as well as the dorsal mesentery by lectin histochemistry. Unexpectedly, qPGCs-like cells were found next to the neural tube by Mallory-AZAN stain. During gonadal differentiation, embryonic sex can be distinguished histologically since day 8 of incubation. Embryonic testis exhibited a thin cortex, whereas embryonic ovary exhibited a thick cortex. Testicular cord formation was found in the medulla of embryonic testes while the lacunae and fat-laden cells were found in the medulla of embryonic ovary during gonadal function. This is the first report on a comparison of phases of gonadogenesis and histochemical study of quail embryonic gonads in both sexes.

Epistasis between IGF2R and ADAMTS19 polymorphisms associates with premature ovarian failure

Do single nucleotide polymorphisms (SNPs) or synergistic interactions between SNPs and diplotypes within the insulin-like growth factor 2 receptor (IGF2R) and ADAM metallopeptidase with thrombospondin type 1 motif, 19 (ADAMTS19), contribute to premature ovarian failure (POF)? Synergistic interactions were detected between SNPs, including a non-synonymous SNP, and diplotypes within IGF2R and ADAMTS19 which may contribute to POF; however, there was no correlation with POF in a single SNP model after Bonferroni correction. IGF2R regulates free IGF2 level, which is involved in steroidogenesis in bovine granulosa cells. ADAMTS19 expression is higher in the murine embryonic ovary than in the embryonic testis during sexual differentiation, and an ADAMTS19 SNP (rs246246) showed a possible association with POF in a genome-wide association study in Caucasian women. This study analyzed interactions between SNPs and diplotypes within IGF2R and ADAMTS19 as well as SNPs within the two genes. In Stage I, a total of 120 patients with POF and 152 female controls were recruited. All patients were diagnosed with POF at the CHA hospital in Seoul, Korea, and were recruited between 1994 and 2004. The 152 controls were recruited from Chungju, Korea, as part of another study that was conducted from April 2002 to March 2004. For Stage II, we obtained genotype data for an additional 1641 female controls, recruited in Ansung and Ansan from 2001 to 2008, from the Korean Genome Epidemiology Study (KoGES). In Stage I, the GoldenGate assay with VeraCode technology was used to genotype SNPs in IGF2R and ADAMTS19. In Stage II, we obtained genotype data for IGF2R and ADAMTS19 using Affymetrix Genome-Wide Human SNP array 5.0 and imputed data by the IMPUTE program from the KoGES. To identify POF-associated SNPs, logistic regression analysis in an additive model was performed using the PLINK tool. Synergistic interactions between SNPs and diplotypes within IGF2R and ADAMTS19 were analyzed by logistic regression analysis in three alternative models. In Stage I, 13 combinations of SNPs showed significant synergistic interactions after Bonferroni correction [the strongest association had odds ratio (OR) = 5.77, 95% confidence interval (CI): 2.26-14.75, P = 0.00025]. In Stage II and combined analyses, two and four combinations, respectively, of the significant results in Stage I showed significant synergistic interactions after Bonferroni correction. For interactions between diplotypes in block 2 of IGF2R and block 3 of ADAMTS19 in Stage I, we found 17 synergistic interactions with P < 0.0001, but there was no significant interaction after Bonferroni correction. In Stage II and combined analyses, we found that three and seven combinations in the same blocks, respectively, showed significant synergistic interactions after Bonferroni correction (strongest association: OR = 4.12, 95% CI: 2.22-7.62, P = 6.74E-06). The sample size for patients with POF in this study was small but, compared with recent reports describing associations between SNPs and POF and considering the low prevalence of POF (1%), the sample size is considered to be reasonable. These results should be confirmed in large-scale studies involving different ethnic groups. Our results may ultimately provide predictive markers for women at a high risk of POF. This study was supported by grants from Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education (2009-0093821, 2011-0010637). There are no competing interests.

Ubiquitin meets PIWI protein

Ubiquitin meets PIWI protein

The critical importance of ovarian angiogenesis

The vascular system develops through two distinct pathways known as vasculogenesis and angiogenesis. The former involves formation of vascular networks from endothelial progenitor cells (Kasmeyer et al. 2009), while angiogenesis is the extension of new blood vessels from pre-existing ones (Robinson et al. 2009). The establishment and optimal functioning of the vasculature is an essential component throughout the body and the ovary is no exception. Blood vessel growth is tightly regulated requiring the timely balance between proand antiangiogenic growth factors as well as the complex, intimate interplay between endothelial cells and various other cell types (e.g. immune cells and pericytes). In this Research Front on ovarian angiogenesis, we present four reviews and one original article that reveal the latest research and current hypotheses on the key stages of vascular development in the ovary. Remarkably little was known about how the vasculature in the fetal ovary is formed, until recently. In the first paper of this Research Front, the latest insights are highlighted byMcFee and Cupp (2012). Intriguingly, ovarian vasculature in the embryo appears to develop through a vasculogenic process rather than the typical angiogenesis that occurs in embryonic testes. Moreover, the vascular patterning is preferentially aligned along the neuronal network template and thus could be influenced by neuronal growth factors (Anderson et al. 2002). McFee and Cupp (2012) also suggest that the ability of germ cell/oocytes to proliferate, develop and form primordial follicles is potentially influenced by fetal ovarian medulla blood supply since these cells lack a direct blood supply. Indeed, follicle assembly initially originates near to the medullary vasculature (Sawyer et al. 2002). This would imply that the inappropriate development of an ovarian vasculature could result in improper prenatal folliculogenesis. This could then adversely affect fertility of both women and animals. The VEGF system is very complex with the existence of numerous proand anti-angiogenic isoforms, receptors and signalling partners (Gabhann and Popel 2008).McFee and Cupp (2012) provide a scholarly account of their importance in the regulation of fetal ovarian development. They speculate that the temporal differences in expression of pro-angiogenic ‘a’ and anti-angiogenic ‘b’ VEGFA isoforms might explain the different patterns of vascularisation in the fetal ovary and testes (McFee andCupp 2012). Interestingly, Qiu et al. (2012) recently showed that overexpression of VEGF165b reduced follicular development and number of ovulations and more importantly, this was associatedwith lower fertility inmice. Collectively, this further emphasises that optimal ovarian function requires an appropriate balance between proand anti-angiogenic factors. It is well established that hypoxia-induced expression of VEGFA plays a pivotal role in stimulating angiogenesis during tumour development. However, the exact role of hypoxia in ovarian angiogenesis has remained elusive for a long time. In the second review of this Research Front, Meidan et al. (2012) discuss the role of the transcription factor, hypoxia inducible factor 1p/b (HIF1p/b), which is critical for ovulation in mice (Kim et al. 2009). The current evidence indicates that HIF1pis upregulated and translocates to the nucleus of luteinising granulosa cell during the ovulatory window although this upregulation is only short-lived. It is likely that, at least in part, the LH surge upregulates the HIF1p in luteinising follicular cells (van den Driesche et al. 2008). This feature of HIF1p regulation could be unique to the ovary. It is also increasingly apparent that any induction of HIF1pstimulates VEGFA expression in luteal cells (Zhang et al. 2011). What is less well known is the role of hypoxia and/or HIF1pin the regulation of FGF2. This certainly requires investigation since FGF2 and HIF1p expression profiles are closelymatched in the bovine CL (Robinson et al. 2007; Nishimura and Okuda 2010). Furthermore, it is becoming increasing apparent that FGF2 is a key factor controlling endothelial cell sprouting during the follicle-luteal transition in cattle (Laird et al. 2012). Accurate measurement of tissue hypoxia is challenging and will continue to hinder the elucidation of the role hypoxia plays in ovarian angiogenesis. The use of novel, live imaging positron emission tomography (PET) technologies using biomarkers such as F-labelled fluoromisonidazole (F-MISO) as used in tumour biology (Mendichovszky and Jackson 2011) will help to address this challenge. Furthermore, such technologiesmight increase our understanding of the role of hypoxia in disorders such as luteal deficiency. It has been long-recognised that ovulation has numerous characteristics of an inflammatory response. However, the role of immune cells in the regulation of ovarian angiogenesis has been neglected, until recently and these findings are highlighted by Shirasuna et al. (2012) in the third review. While there are some definite species differences, there is a pool of evidence that immune cells (e.g. neutrophils, macrophages and/or lymphocytes) infiltrate into the developing CL. This is likely due to CSIRO PUBLISHING

Vascular contributions to early ovarian development: potential roles of VEGFA isoforms

Vascularisation is an essential component of ovarian morphogenesis; however, little is known regarding factors regulating the establishment of vasculature in the ovary. Angiogenesis involving extensive endothelial cell migration is a critical component of vessel formation in the embryonic testis but vasculogenic mechanisms appear to play a prominent role in ovarian vascularisation. Vasculature has a strong influence on the formation of ovarian structures, and the early developmental processes of ovigerous cord formation, primordial follicle assembly and follicle activation are all initiated in regions of the ovary that are in close association with the highly vascular medulla. The principal angiogenic factor, vascular endothelial growth factor A (VEGFA), has an important role in both endothelial cell differentiation and vascular pattern development. Expression of VEGFA has been localised to ovigerous cords and follicles in developing ovaries and an increased expression of pro-angiogenic Vegfa isoform mRNA in relation to anti-angiogenic isoform mRNA occurs at the same time-point as the peak of primordial follicle assembly in perinatal rats. Elucidation of specific genes that affect vascular development within the ovary may be critical for determining not only the normal mechanisms of ovarian morphogenesis, but also for understanding certain ovarian reproductive disorders.

Sox9 and Sox8 Are Required for Basal Lamina Integrity of Testis Cords and for Suppression of FOXL2 During Embryonic Testis Development in Mice1

The sex-determining gene Sry and its target gene Sox9 initiate the early steps of testis development in mammals. Of the related Sox genes Sox8, Sox9, and Sox10, all expressed during Sertoli cell differentiation, only inactivation of Sox9 before the sex determination stage at Embryonic Day 11.5 (E11.5) causes XY sex reversal, while Sox9 inactivation after this stage has no effect on testis cord differentiation. We have previously shown that both Sox9 and Sox8 are essential for maintaining testicular function in post-E14.0 Sertoli cells. To gain insight into the molecular and cellular processes underlying the abnormal development of Sox9 and Sox8 mutant testes, we performed a detailed developmental study of embryonic and neonatal stages. We observe a progressive disruption of the basal lamina surrounding the testis cords that starts at E17.5 and already at E15.5 reduced expression levels of collagen IV, collagen IXa3 and testatin, structural components of the basal lamina, and the extracellular matrix transcriptional regulator Scleraxis. Lineage tracing reveals that mutant Sertoli cells delaminate from testis cords and are present as isolated cells between remaining cords. Also, Sox10 expression is strongly reduced in the absence of Sox9 and/or Sox8. Finally, we document increasing expression of the ovarian marker FOXL2 in mutant cords starting at E15.5, indicating progressive transdifferentiation of mutant Sertoli cells. This study shows that Sox9 and Sox8 maintain integrity of the basal lamina to prevent testis cord disintegration and that both factors actively suppress the ovarian program during early testis development.

Metformin exposure affects human and mouse fetal testicular cells

Metformin is a drug used in the treatment of diabetes and of some disorders related to insulin resistance, such as polycystic ovary syndrome. Gestational diabetes can cause complications for both mother and child, and some studies have shown a beneficial effect of metformin during pregnancy without an increase in perinatal complications. However, the effects on the gonads have not been properly studied. Here we investigated the effect of metformin administered during pregnancy on the development and function of the fetal testis. A dual approach in vitro and in vivo using human and mouse models was chosen. Cultures of human and murine organotypic testes were made and in vivo embryonic testes were analysed after oral administration of metformin to pregnant mice. In human and mouse organotypic cultures in vitro, metformin decreased testosterone secretion and mRNA expression of the main factors involved in steroid production. In vitro, the lowest observed effect concentration (LOEC) on testosterone secretion was 50 µM in human, whereas it was 500 µM in mouse testis. Lactate secretion was increased in both human and mouse organotypic cultures with the same LOEC at 500 µM as observed in other cell culture models after metformin stimulation. In vivo administration of metformin to pregnant mice reduced the testicular size of the fetal and neonatal testes exposed to metformin during intrauterine life. Although the number of germ cells was not affected by the metformin treatment, the number of Sertoli cells, the nurse cells of germ cells, was slightly yet significantly reduced in both periods (fetal period: P = 0.007; neonatal period: P = 0.03). The Leydig cell population, which produces androgens, and the testosterone content were diminished only in the fetal period at 16 days post-coitum. This study showed a potentially harmful effect of metformin treatment on the development of the fetal testis and should encourage future human epidemiological studies.

Pattern and Density of Vascularization in Mammalian Testes, Ovaries, and Ovotestes

According to the classical paradigm, the vasculature of the embryonic testis is more dense and complex than that of the ovary, but recent studies based on whole-mount detection of Caveolin-1 (CAV1) as an endothelial cell marker, have suggested that the level of ovarian vascularization is higher than previously assumed. However, this new hypothesis has been neither tested using alternative methodology nor investigated in other mammalian species. In this paper, we have studied the vascularization process in the gonads of males and females of two mammalian species, the mouse (Mus musculus) and the Iberian mole (Talpa occidentalis). Our results show that the pattern of testis vascularization is very well conserved among mammals, including both pre- and postnatal stages of development and, at least in the mole, it is conserved irrespectively of whether the testicular tissue is XY or XX. We have shown that CAV1 is present not only in endothelial cells but also in prefollicular oocytes and in an ovarian population of somatic cortical cells. These data clearly establish that: (1) according to the classical hypothesis, the degree of vascularization of the developing ovary is lower than that of the testis, (2) ovarian vascularization is also evolutionarily conserved as it occurs similarly both in moles and in mice, and (3) that the degree of vascular development of the mammalian ovary is age-dependent increasing significatively at puberty. The expression of CAV1 in the ovary of most animal taxa, from nematodes to mammals, strongly suggests a role for this gene in the female meiosis.