DAZL Protein Research Articles

Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations

To improve the mechanistic screening of reproductive toxicants in chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

A novel cancer-germline gene DAZL promotes progression and cisplatin resistance of non-small cell lung cancer by upregulating JAK2 and MCM8

Germline-specific genes are usually activated in cancer cells and drive cancer progression; such genes are called cancer-germline or cancer-testis genes. The RNA-binding protein DAZL is predominantly expressed in germ cells and plays a role in gametogenesis as a translational activator or repressor. However, its expression and role in non-small cell lung cancer (NSCLC) are unknown. Here, mining of RNA-sequencing data from public resources and immunohistochemical analysis of tissue microarrays showed that DAZL was expressed exclusively in testis among normal human tissues but ectopically expressed in NSCLC tissues. Testis and NSCLC cells expressed the shorter and longer transcript variants of the DAZL gene, respectively. Overexpression of the longer DAZL transcript promoted tumor growth in a mouse xenograft model. Silencing of DAZL suppressed cell proliferation, colony formation, migration, invasion, and cisplatin resistance in vitro and tumor growth in vivo. Quantitative proteomic analysis based on tandem mass tag and Western blot analysis showed that DAZL upregulated the expression of JAK2 and MCM8. RNA-binding protein immunoprecipitation assays showed that DAZL bound to the mRNA of JAK2 and MCM8. The JAK2 inhibitor fedratinib attenuated the oncogenic outcomes induced by DAZL overexpression, whereas silencing MCM8 counteracted the effects of DAZL overexpression on cisplatin-damaged DNA synthesis and half-maximal inhibitory concentration of cisplatin. In conclusion, DAZL was identified as a novel cancer-germline gene that enhances the translation of JAK2 and MCM8 to promote NSCLC progression and resistance to cisplatin, respectively. These findings suggest that DAZL is a potential therapeutic target in NSCLC.

In-vitro generation of follicle-like structures from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells

BackgroundThe objective of this study was to induce the differentiation of human theca stem cells (hTSCs) into germ cell-like cells (hGCLCs) and assess their developmental progression following in vitro 3D culture with ovarian somatic cells within the follicle-like structures. To achieve this, the hTSCs were isolated from small antral follicles of three patients of varying ages and were then seeded in a differentiation medium for 40 days. The differentiated hGCLCs were subsequently aggregated with somatic ovarian cells (cumulus cells and hTSCs) in a ratio of 1:10 and cultured in a growth medium in a suspension culture dish. In addition to examining the morphologies, sizes, and viabilities of the differentiated hGCLCs, this study also analyzed the expression of DAZL and GDF9 proteins within the follicle-like structures.ResultsAfter 12 days, the hTSCs began to differentiate into hGCLCs, with their shapes changing from spindle-shaped to spherical. The sizes of hGCLCs increased during the differentiation period (from 25 μm to 50 μm). The survival rate of the hGCLCs after differentiation and in vitro development in primordial follicle-like structures was 54%. Unlike hTSCs, which did not express the DAZL protein, the hGCLCs and follicle-like structures successfully expressed DAZL protein (P-value < 0.05). However, hGCLCs poorly expressed the GDF9 protein. Further, the culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value < 0.05).ConclusionIn conclusion, our study demonstrated that 3D cultures with ovarian somatic cells in follicle-like structures caused the successful differentiation of reproducible hGCLCs from hTSCs derived from three patients of different ages. Moreover, this method not only enhanced the in vitro development of hGCLCs but also presented a novel approach for co-culturing and developing in vitro oocyte like cells, ultimately leading to the production of artificial follicles.

A Novel, Efficient Method to Isolate Chicken Primordial Germ Cells from Embryonic Blood Using Cell Culture Inserts.

Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the SSEA-1 and DAZL proteins and retained the ability to migrate in vivo. Overall, PGCs separated using cell culture inserts/CEF adhesion method retain their stem cell characteristics and migration ability. PGCs also exhibit good proliferation efficiency, making them suitable for subsequent transgenic experiments or genetic resource preservation.

P-429 Transcriptomic heterogeneity within cortical ovarian follicle pool in child and adult

Abstract Study question What are the transcriptomic differences between child and adult ovarian follicles in the human cortex? Summary answer Our results suggest that human ovarian cortical follicles show marked differences before and after puberty, with the greatest differences being present in the secondary stage. What is known already The ovaries and their main functional structures, follicles, are important for determining the reproductive health. Follicle growth and maturation are controlled by multiple mechanisms, including the hypothalamus pituitary ovarian axis and local paracrine signalling. Although dormant primordial follicles are activated to grow even in infancy, all growing follicles before the first menarche are bound to undergo atresia. Follicle growth activation in infancy and adulthood remains poorly understood. Additionally, child and adult follicles behave differently during in vitro culture suggesting differing biology, raising many questions e.g., about their suitability for fertility preservation. Study design, size, duration Ovarian cortical tissues from children were collected through fertility preservation program at the Helsinki Children’s hospital (Finland). Families of underage patients signed an informed written consent. Adult ovarian tissue was collected from gender reassignment patients at the Helsinki University hospital and Karolinska university hospital after informed written consent. Follicles at different maturation stages were isolated from ovarian cortex for single follicle transcriptomic analysis. Freshly-fixed cortical biopsies (3x3x1 mm3) were used for validation of transcriptomic analysis. Participants/materials, setting, methods Single viable follicles (n = 120, 60/age group) were mechanically and enzymatically isolated from frozen-thawed ovarian tissue from four adults (age &amp;gt;18 yrs) and five children (age 1-11 yrs) and processed for RNA-sequencing. Individual follicles were lysed, prepared for RNA libraries following Smart-Seq2 protocol and sequenced using NextSeq 500 platform. After quality assessment, 109 follicles were analysed (Adult n = 54, Child n = 55). DDX4, AMH, FOXL2 and DAZL transcripts and proteins localization were evaluated via immunodetection and RNA-FISH assay. Main results and the role of chance Principal component analysis (PCA) of all sequenced follicles demonstrated an expected timely progression of their stages based on maturation, and an overall similarity between child and adult. Interestingly, PCA displayed two distinct clusters of follicles in both adult and child samples. The first group contained only growing follicles and, after differentially expressed gene (DEG) analysis, demonstrated lower expression of traditional oocyte markers (group 1). The second group consisted of primordial and growing follicles and had lower expression of traditional granulosa marker (group 2). Group-related differential expression of main oocyte (DDX4, DAZL) and granulosa (FOXL2, AMH) marker genes were validated in tissue sections with RNA-FISH. Downstream analyses focused on group 2 follicles. When child and adult follicles were compared to each other, the highest number of DEGs were found in the secondary stage (primordial n = 32, primary n = 14, secondary n = 387). Pathway analysis of DEG displays enrichment of oestrogen-related related pathways in adult secondary follicles. Furthermore, we analysed DEGs between the follicular stages per age group, finding high number of DEGs between primordial and primary follicles (Adult n = 1663, Child n = 1914). The majority of those genes are unique to the age group (common=512). Limitations, reasons for caution Adult ovarian tissue is obtained from gender reassignment patients, who receive androgen treatments prior to surgery. Child ovarian tissue was collected from fertility preservation programme where some patients were exposed to chemotherapy prior to tissue collection. Wider implications of the findings The differences between child and adult follicles encourage further investigation of the suitability of child ovarian tissue in current fertility preservation protocols. Understanding transcriptomic changes during early folliculogenesis and reasons for follicle degeneration can help tailor protocols for in vitro growth of follicles. Trial registration number not applicable

O-237 In-vitro generation of primordial follicle-like structure from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells

Abstract Study question Does human ovarian somatic cells, improve the development of human germ cell-like cells (hGCLCs) derived from the human theca stem cell (hTSCs) after co-culture? Summary answer 1. Yes, morphological similarities, size, and expression of germ and oocytes-specific proteins in developed hGCLCs in primordial follicle-like structure indicate the increase of in-vitro development. What is known already 1. Somatic stem cells of the ovary can differentiate into primordial germ cell-like cells in appropriate in vitro conditions. The ovarian somatic cells secrete growth factors that can lead to further development. In ovarian dysfunction, follicle growth may be abnormal due to defects in the function of the oocyte or somatic cells. Replacement of defective oocytes or somatic cells, using the follicle-like structure technique, may “save” follicle growth. However, finding an optimal way to create a kind of co-culture that is economically viable and beneficial is a challenge. Study design, size, duration hTSCs which were differentiated into human germ cell-like cells hGCLCs after 12 days, were combined with ovarian somatic cells to assess the effects of ovarian somatic cells on the developmental potential of hGCLC after being placed in the structure named “primordial follicle-like structure”. Participants/materials, setting, methods 1. The hTSCs were isolated from antral follicles of transgender individuals. Isolated hTSCs were seeded in the differentiation medium (DMEM-F12 containing 5% human follicular fluid and 5% FBS) for 40 days. The differentiated hGCLCs aggregated with somatic cells (cumulus cells and hTSCs) in a ratio of 1 to 10 and cultured in a growth medium (αMEM containing 3mg/ml BSA, 1% ITS, 0.005 IU/ml FSH and 0.11 mg/ml ascorbic acid) in suspension culture dish for 11 days. Main results and the role of chance 1. After 12 days, hTSCs began to differentiate into hGCLCs and their morphology changed from spindle-shaped to round. The size of hGCLCs increased during the differentiation period (from 25-25 μm to 50 μm). The survival of hGCLC cells after differentiation and development in vitro in primordial follicle-like structures was 54%. Unlike hTSCs, hGCLCs and hGCLCs developed with ovarian somatic cells expressed the DAZL protein well (P-value&amp;lt;0.05), hGCLCs poorly expressed the GDF9 protein. The culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value&amp;lt;0.05). Limitations, reasons for caution 1. So far, there have been few studies of three-dimensional culture in the primordial follicle-like structure, all of which were performed on the oocyte. The lack of previous studies on the development of hGCLCs was a limitation that led us to set up the study from the beginning. Wider implications of the findings 1. The hTSCs can differentiate to hGCLCs. In the presence of ovarian somatic cells, hGCLCS expressed the oocyte-specific markers which could not be expressed before. It seems that the combination of ovarian somatic cells can simulate primordial follicle structure and lead to more development of hGCLCs. Trial registration number 25

Identification and expression analysis of Dazl homologue in Cynops cyanurus.

The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

Testes of DAZL null neonatal sheep lack prospermatogonia but maintain normal somatic cell morphology and marker expression.

Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem, we disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9-mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL null male neonatal sheep lacked germ cells on histological sections and showed greatly reduced germ cell markers. Somatic cells within their testes were morphologically intact and expressed normal levels of lineage-specific markers, suggesting that the germ cell niche remained intact. This extends the DAZL mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute germline transmitters in rapid livestock improvement.

Mammalian germ cells are determined after PGC colonization of the nascent gonad

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

DAZL Regulates Germ Cell Survival through a Network of PolyA-Proximal mRNA Interactions

SUMMARYThe RNA binding protein DAZL is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in Dazl-null mice are unknown. Here, we mapped transcriptome-wide DAZL-RNA interactions in vivo, revealing DAZL binding to thousands of mRNAs via polyA-proximal 3′ UTR interactions. In parallel, fluorescence-activated cell sorting and RNA-seq identified mRNAs sensitive to DAZL deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that DAZL post-transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes that are critical for germ cell proliferation and survival. In addition, we provide evidence that polyA sequences have key roles in specifying DAZL-RNA interactions across the transcriptome. Our results reveal a mechanism for DAZL-RNA binding and illustrate that DAZL functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

Human Ovarian Theca-Derived Multipotent Stem Cells Have The Potential to Differentiate into Oocyte-Like Cells In Vitro.

Objective In this study, we have examined human theca stem cells (hTSCs) in vitro differentiation capacity into human oocyte like cells (hOLCs). Materials and Methods In this interventional experiment study, hTSCs were isolated from the theca layer of small antral follicles (3-5 mm in size). Isolated hTSCs were expanded and cultured in differentiation medium, containing 5% human follicular fluid, for 50 days. Gene expressions of PRDM1, PRDM14, VASA, DAZL, OCT4, ZP1, 2, 3 GDF9, SCP3 and DMC1 were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on days 0, 18, and 25 after monoculture as well as one week after co-culture with human granulosa cells (hGCs). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were immune-localized in oocyte-like structures. Results After 16-18 days, the color of the medium became acidic. After 25 days, the cells started to differentiate into round-shaped cells (20-25 µm diameter). One week after co-culturing with hGCs, the size of the round cells increased 60 to70 µm and convert to hOLCs. However, these growing cells expressed some primordial germ cell (PGC)- and germ cell genes (PRDM1, PRDM14, VASA, DAZL, and OCT4) as well as oocyte specific genes (ZP1, 2, 3 and GDF9), and meiotic-specific markers (SCP3 and DMC1). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were present in hOLCs. ConclusionTo sum up, hTSCs have the ability to differentiate into hOLCs. This introduced model paved the way for further in vitro studies of the exact mechanisms behind germ cell formation and differentiation. However, the functionality of hOLCs needs further investigation.

Age-related and photoperiodic variation of the DAZ gene family in the testis of the Syrian hamster (Mesocricetus auratus).

SummaryThe Deleted in AZoospermia (DAZ) gene family regulates the development, maturation and maintenance of germ cells and spermatogenesis in mammals. The DAZ family consists of two autosomal genes, Boule and Dazl (Daz-like), and the Daz gene on chromosome Y. The aim of this study was to analyze the localization of DAZL and BOULE during testicular ontogeny of the seasonal-breeding Syrian hamster, Mesocricetus auratus. We also evaluated the testicular expression of DAZ family genes under short- or long-photoperiod conditions. In the pre-pubertal and adult testis, DAZL protein was found mainly in spermatogonia. BOULE was found in the spermatogonia from 20 days of age and during the pre-pubertal and adult period it was also detected in spermatocytes and round spermatids. DAZL and BOULE expression in spermatogonia was strictly nuclear only in 20-day-old hamsters. We also detected the novel mRNA and protein expression of BOULE in Leydig cells. In adult hamsters, Dazl expression was increased in regressed testis compared with non-regressed testis and DAZL protein expression was restricted to primary spermatocytes in regressed testis. These results show that DAZL and BOULE are expressed in spermatogonia at early stages in the Syrian hamster, then both proteins translocate to the cytoplasm when meiosis starts. In the adult regressed testis, the absence of DAZL in spermatogonia might be related to the decrease in germ cell number, suggesting that DAZ gene family expression is involved in changes in seminiferous epithelium during photoregression.

Reproductive system development in male and female horse embryos and fetuses: Gonadal hyperplasia revisited

In horses, pregnancy is characterized by high levels of maternal estrogens that are produced largely by the interstitial tissue inside the gonads of the offspring, associated with a physiological gonadal hyperplasia, that is uncommon in other species. However, a detailed structural-functional understanding of the early stages of gonadal development and hyperplasia has remained elusive in horse pregnancy because of the lack of substantial data. The goal of this study was to describe the genital organs' development in 19 early horse embryos and fetuses (days 20–140 of gestation) of both sexes by means of anatomy, histology, stereology, and immunohistochemistry, with a specific focus on gonadal hyperplasia and interstitial tissue development. Gonadal hyperplasia with similar amounts of interstitial cells was observed in both sexes, but only during the early stage of development (days 40–90). Surprisingly, a higher degree of hyperplasia, characterized by larger amounts of interstitial cell-rich areas, was seen in fetal ovaries from 90 days of gestation onwards. Another novel aspect was that parallel to the hyperplasia of the interstitial cells, a much more precocious and pronounced differentiation of germinal cells was seen in the ovary, characterized by an earlier peak and decrease of DAZL and OCT protein immune markers. In conclusion, a reduced degree of hyperplasia and interstitial tissue in the fetal testis after 90 days of gestation suggests the existence of a more efficient mechanism regarding the synthesis of estrogen precursors as a structural or physiological difference between both fetal sexes, which warrants further investigation.

BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

Breast cancer amplified sequence 2 (BCAS2) is involved in multiple biological processes, including pre-mRNA splicing. However, the physiological roles of BCAS2 are still largely unclear. Here we report that BCAS2 is specifically enriched in spermatogonia of mouse testes. Conditional disruption of Bcas2 in male germ cells impairs spermatogenesis and leads to male mouse infertility. Although the spermatogonia appear grossly normal, spermatocytes in meiosis prophase I and meiosis events (recombination and synapsis) are rarely observed in the BCAS2-depleted testis. In BCAS2 null testis, 245 genes are altered in alternative splicing forms; at least three spermatogenesis-related genes (Dazl, Ehmt2 and Hmga1) can be verified. In addition, disruption of Bcas2 results in a significant decrease of the full-length form and an increase of the short form (lacking exon 8) of DAZL protein. Altogether, our results suggest that BCAS2 regulates alternative splicing in spermatogonia and the transition to meiosis initiation, and male fertility.

Dazl is a critical player for primordial germ cell formation in medaka.

The DAZ family genes boule, daz and dazl have conserved functions in primordial germ cell (PGC) migration, germ stem cell proliferation, differentiation and meiosis progression. It has remained unknown whether this family is required for PGC formation in developing embryos. Our recent study in the fish medaka (Oryzias latipes) has defined dnd as the critical PGC specifier and predicted the presence of additional factors essential for PGC formation. Here we report that dazl is a second key player for medaka PGC formation. Dazl knockdown did not prevent PGC formation even in the absence of normal somatic structures. It turned out that a high level of Dazl protein was maternally supplied and persisted until gastrulation, and hardly affected by two antisense morpholino oligos targeting the dazl RNA translation. Importantly, microinjection of a Dazl antibody remarkably reduced the number of PGCs and even completely abolished PGC formation without causing detectable somatic abnormality. Therefore, medaka PGC formation requires the Dazl protein as maternal germ plasm component, offering first evidence that dazl is a critical player in PGC formation in vivo. Our results demonstrate that antibody neutralization is a powerful tool to study the roles of maternal protein factors in PGC development in vivo.

Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis.

Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl.

Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells.

Dynamic intracellular localization of Dazl protein during Xenopus germline development.

Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.

Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.

Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin− HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and β-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA-E-cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and β-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells.

Oocyte polarity requires a Bucky ball-dependent feedback amplification loop

In vertebrates, the first asymmetries are established along the animal-vegetal axis during oogenesis, but the underlying molecular mechanisms are poorly understood. Bucky ball (Buc) was identified in zebrafish as a novel vertebrate-specific regulator of oocyte polarity, acting through unknown molecular interactions. Here we show that endogenous Buc protein localizes to the Balbiani body, a conserved, asymmetric structure in oocytes that requires Buc for its formation. Asymmetric distribution of Buc in oocytes precedes Balbiani body formation, defining Buc as the earliest marker of oocyte polarity in zebrafish. Through a transgenic strategy, we determined that excess Buc disrupts polarity and results in supernumerary Balbiani bodies in a 3'UTR-dependent manner, and we identified roles for the buc introns in regulating Buc activity. Analyses of mosaic ovaries indicate that oocyte pattern determines the number of animal pole-specific micropylar cells that are associated with an egg via a close-range signal or direct cell contact. We demonstrate interactions between Buc protein and buc mRNA with two conserved RNA-binding proteins (RNAbps) that are localized to the Balbiani body: RNA binding protein with multiple splice isoforms 2 (Rbpms2) and Deleted in azoospermia-like (Dazl). Buc protein and buc mRNA interact with Rbpms2; buc and dazl mRNAs interact with Dazl protein. Cumulatively, these studies indicate that oocyte polarization depends on tight regulation of buc: Buc establishes oocyte polarity through interactions with RNAbps, initiating a feedback amplification mechanism in which Buc protein recruits RNAbps that in turn recruit buc and other RNAs to the Balbiani body.