Germ Cell Gene Research Articles

206 Overexpression of germ cell genes DAZL, STRA8, and BOULE in bovine adipose tissue-derived mesenchymal stem cells for male germ cell derivation

Invitro gamete derivation from stem cells has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) may be suitable candidates for invitro gamete derivation given their differentiation capacity and their potential for cell therapy. DAZL (deleted in azoospermia-like) and BOULE (also called BOLL) encode RNA-binding proteins that control differentiation of germ cells; STRA8 (stimulated by RA-8) encodes a protein required for meiosis. Considering the crucial roles of these factors, the aim of the present study was to evaluate co-overexpression of different combinations of DAZL, STRA8, and BOULE on germ cell gene expression profile in adipose tissue-derived MSC (AT-MSC). AT-MSC were harvested from abattoir-derived male bovine fetuses (n=9; 7-8 months of gestation). The optimal concentration of Lipofectamine 2000 (1, 1.5, 2, 1.5ng µL−1; Invitrogen) was analysed in AT-MSC transfected with plasmid pSIN-EF2-Puro containing DAZL, STRA8, and BOULE coding sequences (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro). Then, AT-MSC were transfected either with plasmids containing DAZL, STRA8, and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro), DAZL and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro), DAZL (pSIN-EF2-DAZL-P2A-T2A-Puro), or empty plasmid at a concentration of 1ng µL−1 of Lipofectamine. Cell samples were obtained from each plasmid treatment and analysed for expression of housekeeping genes ACTB and GAPDH and germ cell genes DAZL, PIWIl2, STRA8, and BOULE by quantitative-PCR using relative values (Quantity) through the ΔΔCT formula. AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro using 1ng µL−1 of Lipofectamine achieved higher (P<0.05) expression of DAZL (13.3-fold) compared with cells transfected with empty vector. Moreover, AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro had higher (P<0.05) levels of DAZL mRNA (3.8-fold) compared with empty vector. Messenger RNA levels of STRA8 (1.4-fold) were only detected in AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro; PIWIl2 and BOULE were not detected in transfected or untransfected AT-MSC. In conclusion, bovine fetal AT-MSC are amenable for overexpression of germ cell markers DAZL and STRA8. Transfection with plasmid containing three germ cell genes (DAZL, STRA8, and BOULE) allowed overexpression of DAZL, whereas transfection with plasmid containing two germ cell genes (DAZL and STRA8) achieved overexpression of STRA8. This study was supported by Fondecyt grant 1191114, Government of Chile.

Retinoic acid-stimulated ERK1/2 pathway regulates meiotic initiation in cultured fetal germ cells.

In murine fetal germ cells, retinoic acid (RA) is an extrinsic cue for meiotic initiation that stimulates transcriptional activation of the Stimulated by retinoic acid gene 8 (Stra8), which is required for entry of germ cells into meiotic prophase I. Canonically, the biological activities of RA are mediated by nuclear RA receptors. Recent studies in somatic cells found that RA noncanonically stimulates intracellular signal transduction pathways to regulate multiple cellular processes. In this study, using a germ cell culture system, we investigated (1) whether RA treatment activates any mitogen-activated protein kinase (MAPK) pathways in fetal germ cells at the time of sex differentiation, and (2) if this is the case, whether the corresponding RA-stimulated signaling pathway regulates Stra8 expression in fetal germ cells and their entry into meiosis. When XX germ cells at embryonic day (E) 12.5 were cultured with RA, the extracellular-signal-regulated kinase (ERK) 1/2 pathway was predominantly activated. MEK1/2 inhibitor (U0126) treatment suppressed the mRNA expressions of RA-induced Stra8 and meiotic marker genes (Rec8, Spo11, Dmc1, and Sycp3) in both XX and XY fetal germ cells. Furthermore, U0126 treatment dramatically reduced STRA8 protein levels and numbers of meiotic cells among cultured XX and XY fetal germ cells even in the presence of RA. Taken together, our results suggest the novel concept that the RA functions by stimulating the ERK1/2 pathway and that this activity is critical for Stra8 expression and meiotic progression in fetal germ cells.

Investigation of VASA Gene and Protein Expression in Neonate and Adult Testicular Germ Cells in Mice In Vivo and In Vitro.

Objective We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures. Materials and Methods In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR). Results The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05). ConclusionThe VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.

Comparison of PLZF Gene Expression between Pluripotent Stem Cells and Testicular Germ Cells.

Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05). ConclusionThese results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is down- regulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.

Abnormal Meiosis Initiation in Germ Cell Caused by Aberrant Differentiation of Gonad Somatic Cell.

The interaction between germ cell and somatic cell plays important roles in germ cell development. However, the exact function of gonad somatic cell in germ cell differentiation is unclear. In the present study, the function of gonad somatic cell in germ cell meiosis was examined by using mouse models with aberrant somatic cell differentiation. In Wt1R394W/R394W mice, the genital ridge is absent due to the apoptosis of coelomic epithelial cells. Interestingly, in both male and female Wt1R394W/R394W germ cells, STRA8 was detected at E12.5 and the scattered SYCP3 foci were observed at E13.5 which was consistent with control females. In Wt1-/flox; Cre-ERTM mice, Wt1 was inactivated by the injection of tamoxifen at E9.5 and the differentiation of Sertoli and granulosa cells was completely blocked. We found that most germ cells were located outside of genital ridge after Wt1 inactivation. STRA8, SYCP3, and γH2AX proteins were detected in germ cells of both male and female Wt1-/flox; Cre-ERTM gonads, whereas no thread-like SYCP3 signal was observed. Our study demonstrates that aberrant development of gonad somatic cells leads to ectopic expression of meiosis-associated genes in germ cells, but meiosis was arrested before prophase I. These results suggest that the proper differentiation of gonad somatic cells is essential for germ cell meiosis.

Gonadotropin Regulation Testicular RNA Helicase, Two Decades of Studies on Its Structure Function and Regulation From Its Discovery Opens a Window for Development of a Non-hormonal Oral Male Contraceptive.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is member of the DEAD-box family of RNA helicases present in Leydig and germ cells. GRTH is the only family member regulated by hormones, luteinizing hormone, through androgen action. Male mice with knock-out of the GRTH gene are sterile, lack sperm with arrest at round spermatids. GRTH participates on the nuclear export and transport of specific mRNAs, the structural integrity of Chromatoid Bodies of round spermatids, where mRNAs are processed and stored, and in their transit to polyribosomes, where it may regulate translation of relevant genes. GRTH has a central role in the control of germ cell apoptosis and acts as negative regulator of miRNAs which regulate expression of genes involved in the progress of spermatogenesis. In Leydig cells, GRTH gene transcription is regulated by LH via autocrine actions of androgen/androgen receptor and has regulatory effects in steroidogenesis. In germ cells, androgen actions are indirect via receptors in Sertoli cells. Transgenic mice carrying GRTH 5′ flanking region-GFP permitted to discern regions in the gene which directs its expression upstream, in germ cells, and downstream in Leydig cells, and the androgen-regulated transcription at interstitial (autocrine), and germ cell (paracrine) compartments. Further evidence for paracrine actions of androgen/androgen receptor is their transcriptional induction of Germ Cell Nuclear Factor as requisite up-regulator of GRTH gene transcription in round spermatids, linking androgen action to two relevant germ cell genes essential for the progress of spermatogenesis. A missense mutation of R to H at amino acid 242 of GRTH found in 5.8% of a patient population with azoospermia causes loss of the cytoplasmic phospho-GRTH species with preservation of the non-phospho form in transfected cells. Mice with knock-in of the human mutation, lack sperm due to arrest at round spermatids. This model permits to discern the function of phospho-GRTH. The GRTH phospho-site resides at a Threonine structurally adjacent to the mutant site found in patients. Molecular modeling of this site elucidated the amino acids that form the GRTH/PKA interphase and provide the basis for drug design for use as male contraceptive.

Restore natural fertility of Kitw/Kitwv mouse with nonobstructive azoospermia through gene editing on SSCs mediated by CRISPR-Cas9

BackgroundMale infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established.MethodsWe choose Kitw/Kitwv mouse as a research model and try to develop a new treatment strategy and “cure” its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kitw/Kitwv mouse and propagated in vitro. The C to T point mutation on Kitwv site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis.ResultsHealthy offsprings with wild type Kit gene or Kitw mutation were obtained through natural mating 4 months after SSC transplantation.ConclusionIn this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.

CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

Histone demethylase Kdm2a regulates germ cell genes and endogenous retroviruses in embryonic stem cells.

Aim: To investigate the function of Kdm2a in embryonic stem cells (ESCs). Materials & methods: Expression profile analysis after Kdm2a knockout. Analysis of Kdm2a, H3K4me3 and H3K27me3 ChIP-seq data in ESCs. qPCR analysis and ChIP-qPCR analysis of epigenetic changes after Kdm2a loss. Results:Kdm2a was dispensable for pluripotency maintenance in ESCs. Kdm2a genomic binding profile was positively correlated with that of H3K4me3, Zfx and Tet1. Kdm2a directly regulated germ cell genes in primordial germ cell-like cells. Kdm2a loss led to the reduced expression of endogenous retrovirus IAPEy and resulted in the gain of H3K36me2 and loss of H3K4me3 on IAPEy. Conclusion: Kdm2a regulates germ cell genes and endogenous retroviruses in ESCs possibly through demethylating H3K36me2 and influencing H3K4me3 deposition.

Intraflagellar transport protein 74 is essential for spermatogenesis and male fertility in mice†.

Intraflagellar transport protein 74 (IFT74) is a component of the core intraflagellar transport complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axonemes in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 and sperm-associated antigen 16L proteins were also not changed. However, the processed A-Kinase anchor protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.

SAT-227 Diet-Induced Obesity and Prenatal Androgen Exposure Reprogram the Fetus and Cause Transgenerational PCOS-Like Phenotypic Changes in Adult Female Offspring

The fetal life is a critical window of developmental patterning affecting growth trajectories before birth and can influence the risk of reproductive and metabolic disease in adulthood. Maternal obesity and elevated prenatal androgens are two prominent potential exposures which may affect the development of the embryo and its postnatal life. If in utero androgen exposure with or without maternal obesity increase female offspring susceptibility for transgenerational inheritance of reproductive and metabolic disease has not been investigated. Therefore we fed female mice high-fat high sucrose (HFHS) or control diet (CD) diet for 6 weeks prior mating to induce obesity and exposed pregnant female mice to prenatal androgen exposure with dihydrotestosterone (DHT) or vehicle during the embryonic day (E) 16.5-E18.5. At weaning, first, second and third generation (F1, F2, and F3) female offspring from DHT exposed mothers with diet-induced obesity had longer anogenital distance (AGD), a marker of in utero androgen exposure, and disturbed estrous cycle compared with offspring from vehicle treated mothers fed CD. F1 and F3 female offspring from DHT exposed mothers and great-grand mothers with diet-induced obesity weighed more compared to offspring from vehicle-treated obese mothers, and F3 female offspring from great-grand DHT exposed mothers had more fat mass assessed by EchoMRI. Additionally, F1 and F3 female offspring from DHT exposed mothers and great-grand mothers with or without diet-induced obesity displayed a lower respiration exchange ratio and reduced energy expenditure than offspring from vehicle-treated mothers fed CD assessed by the metabolic cages. Finally, mice were superovulated to collect MII oocytes for single-cell RNA sequencing. Our preliminary analysis reveal that genes related to preimplantation embryonic imprinting are differentially expressed in F1, F2, and F3 female offspring from DHT exposed mothers, grandmothers, and great-grandmothers fed CD, and in mice from mothers with diet-induced obesity. These results demonstrate that in utero androgen exposure and diet-induced obesity contribute to transgenerational effects on reproductive and metabolic phenotypes in adult female offspring, involving altered expression of preimplantation imprinting genes in germ cells.

Dynamic transcriptome profiles within spermatogonial and spermatocyte populations during postnatal testis maturation revealed by single-cell sequencing.

Spermatogenesis is the process by which male gametes are formed from a self-renewing population of spermatogonial stem cells (SSCs) residing in the testis. SSCs represent less than 1% of the total testicular cell population in adults, but must achieve a stable balance between self-renewal and differentiation. Once differentiation has occurred, the newly formed and highly proliferative spermatogonia must then enter the meiotic program in which DNA content is doubled, then halved twice to create haploid gametes. While much is known about the critical cellular processes that take place during the specialized cell division that is meiosis, much less is known about how the spermatocytes in the “first-wave” in juveniles compare to those that contribute to long-term, “steady-state” spermatogenesis in adults. Given the strictly-defined developmental process of spermatogenesis, this study explored the transcriptional profiles of developmental cell stages during testis maturation. Using a combination of comprehensive germ cell sampling with high-resolution, single-cell-mRNA-sequencing, we have generated a reference dataset of germ cell gene expression. We show that discrete developmental stages of spermatogenesis possess significant differences in the transcriptional profiles from neonates compared to juveniles and adults. Importantly, these gene expression dynamics are also reflected at the protein level in their respective cell types. We also show differential utilization of many biological pathways with age in both spermatogonia and spermatocytes, demonstrating significantly different underlying gene regulatory programs in these cell types over the course of testis development and spermatogenic waves. This dataset represents the first unbiased sampling of spermatogonia and spermatocytes during testis maturation, at high-resolution, single-cell depth. Not only does this analysis reveal previously unknown transcriptional dynamics of a highly transitional cell population, it has also begun to reveal critical differences in biological pathway utilization in developing spermatogonia and spermatocytes, including response to DNA damage and double-strand breaks.

Gonadoblastoma Y locus genes expressed in germ cells of individuals with dysgenetic gonads and a Y chromosome in their karyotypes include DDX3Y and TSPY.

Which Y genes mapped to the 'Gonadoblastoma Y (GBY)' locus on human Y chromosome are expressed in germ cells of individuals with some Differences of Sexual Development (DSD) and a Y chromosome in their karyotype (DSD-XY groups)? The GBY candidate genes DDX3Y and TSPY are expressed in the germ cells of DSD-XY patients from distinct etiologies: patients with mixed gonadal dysgenesis (MGD) and sex chromosome mosaics (45,X0/46,XY; 46,XX/46,XY); patients with complete androgen insensitivity (CAIS), patients with complete gonadal dysgenesis (CGD; e.g. Swyer syndrome). A GBY locus was proposed to be present on the human Y chromosome because only DSD patients with a Y chromosome in their karyotype have a high-although variable-risk (up to 55%) for germ cell tumour development. GBY was mapped to the proximal part of the short and long Y arm. TSPY located in the proximal part of the short Y arm (Yp11.1) was found to be a strong GBY candidate gene. It is expressed in the germ cells of DSD-XY patients with distinct etiologies but also in foetal and pre-meiotic male spermatogonia. However, the GBY region extends to proximal Yq11 and therefore includes probably more than one candidate gene. Protein expression of the putative GBY candidate gene in proximal Yq11, DDX3Y, is compared with that of TSPY in serial gonadal tissue sections of 40 DSD-XY individuals from the three DSD patient groups (MGD, Complete Androgen Insensitivity Syndrome [CAIS], CGD) with and without displaying malignancy. Expression of OCT3/4 in the same tissue samples marks the rate of pluripotent germ cells. A total of 145 DSD individuals were analysed for the Y chromosome to select the DSD-XY subgroup. PCR multiplex assays with Y gene specific marker set score for putative microdeletions in GBY Locus. Immunohistochemical experiments with specific antisera mark expression of the GBY candidate proteins, DDX3Y, TSPY, in serial sections of the gonadal tissue samples; OCT3/4 expression analyses in parallel reveal the pluripotent germ cell fraction. Similar DDX3Y and TSPY protein expression patterns were found in the germ cells of DSD-XY patients from each subgroup, independent of age. In CAIS patients OCT3/4 expression was often found only in a fraction of these germ cells. This suggest that GBY candidate proteins are also expressed in the non-malignant germ cells of DSD-XY individuals like in male spermatogonia. Variation of the expression profiles of GBY candidate genes in the germ cells of some DSD-XY individuals suggests distinct transcriptional and translational control mechanisms which are functioning during expression of these Y genes in the DSD-XY germ cells. Their proposed GBY tumour susceptibility function to transform these germ cells to pre-malignant GB/Germ Cell Neoplasia in Situ (GB/GCNIS) cells seems therefore to be limited and depending on their state of pluripotency. These experimental findings are of general importance for each individual identified in the clinic with DSD and a Y chromosome in the karyotype. To judge their risk of germ cell tumour development, OCT3/4 expression analyses on their gonadal tissue section is mandatory to reveal the fraction of germ cells still being pluripotent. Comparative expression analysis of the GBY candidate genes can be helpful to reveal the fraction of germ cells with genetically still activated Y chromosomes contributing to further development of malignancy if at high expression level. This research project was supported by a grant (01GM0627) from the BMBF (Bundesministerium für Bildung und Forschung), Germany to P.H.V. and B.B. The authors have no competing interests.

Differential Proliferation Effects after Short-Term Cultivation of Mouse Spermatogonial Stem Cells on Different Feeder Layers.

Objective Spermatogonial stem cells (SSCs) provide the cellular basis for sperm production transforming the male’s genetic information to the next generation. We aimed to examine the effect of different feeder layer on proliferation of SSCs. Materials and Methods In this experimental study, we compared the in vitro effects of the co-culture of mouse SSCs with mouse embryonic fibroblasts (MEFs), sandos inbred mice (SIM) embryo-derived thioguanine- and ouabain- resistant (STO) feeders, and neonate and adult testicular stroma cell (TSC) feeders on the efficiency of mouse SSC proliferation and colony formation. Cells were cultivated on top of MEFs, STO, and neonate and adult TSCs feeder layers for 30 days. The number and diameter of colonies and also the number of cells were evaluated during day 7, 15, 25, and 30 of culture. The mRNA expression of germ cells and somatic cells were analyzed. Results In our study, we observed a significant difference in the proliferation rates and colony size of SSCs among the groups, especially for MEFs (P<0.05). SSCs can proliferate on MEFS, but not on STO, neonate or adult TSCs. Using immunocytochemistry by KI67 the proliferative activities of SSC colonies on MEFs were confirmed. The results of Fluidigm real-time polymerase chain reaction (RT-PCR) showed a high expression of the germ cell genes the promyelocytic leukemia zinc finger protein (PLZF), deleted in azoospermia-like (DAZL), octamer-binding transcription factor 4 (OCT4), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4 or VASA) in SSCs, and a low expression of these genes in the feeder layers. Furthermore, we observed a higher expression of vimentin and integrin-B1 in feeder layers than in SSCs (P<0.05). ConclusionBased on the optimal effect of MEFs for better colonization of SSCs, these feeder cells seem to be appropriate candidates for SSC cultures prior to transplantation. Therefore, it is suggested using these feeder cells for SSC cultivation.

Comparison of Germ Cell Gene Expressions in Spontaneous Monolayer versus Embryoid Body Differentiation of Mouse Embryonic Stem Cells toward Germ Cells.

BackgroundGenetic and morphologic similarities between mouse embryonic stem cells (ESCs) and primordial germ cells (PGCs) make it difficult to distinguish differentiation of these two cell types in vitro. Using specific GC markers expressed in low level or even not expressed in ESCs- can help recognize differentiated cells in vitro. We attempted to differentiate the mouse ESCs into Gc-like cells spontaneously in monolayer and EB culture method.Materials and MethodsIn this experimental study, we attempted to differentiate ESCs, Oct4-GFP OG2, into GC-like cells (GCLCs) spontaneously in two different ways, including: i. Spontaneous differentiation of ESCs in monolayer culture as (SP) and ii. Spontaneous differentiation of ESCs using embryoid body (EB) culture method as (EB+SP). During culture, expression level of four GC specific genes (Fkbp6, Mov10l1, Riken and Tex13) and Mvh, Scp3, Stra8, Oct4 were evaluated.ResultsIn both groups, Mov10l1 was down-regulated (P=0.3), while Tex13 and Riken were up-regulated (P=0.3 and P=0.04, respectively). Fkbp6 and Stra8 were decreased in EB+SP and they were increased in SP group, while no significant difference was determined between them (P=0.1, P=0.07). Additionally, in SP group, gene expression of Mvh and Scp3 were up-regulated and they had significant differences compared to EB+SP group (P=0.00 and P=0.01, respectively). Oct4 was down-regulated in the both groups. Flow-cytometry analysis showed that mean number of Mvh-positive cells in the SP group was significantly greater compared to ESCs, EB+SP and EB7 groups (P=0.00, P=0.01, and P=0.3, respectively).ConclusionThese findings showed that ESCs were differentiated into GCLCs in both group. But spontaneous dif- ferentiation of ESCs into GCLCs in SP group (monolayer culture) compared to EB+SP (EB culture methods) has more ability to express GCs markers.

Inhibition of ovarian development and instances of sex reversal in genotypic female sablefish (Anoplopoma fimbria) exposed to elevated water temperature

This study determined high temperature effects on ovarian development in a marine groundfish species, sablefish (Anoplopoma fimbria), with potential application in sex reversal or sterilization for aquaculture. Monosex female (XX-genotype) sablefish larvae (∼30 mm) were randomly divided into three groups and exposed to control (15.6 °C ± 0.8 °C), moderate (20.4 °C ± 0.5 °C), or high (21.7 °C ± 0.5 °C) temperatures for 19 weeks. Treated fish were then tagged and transferred to ambient seawater (11.2 °C ± 2.3 °C) for one year to determine whether temperature effects on reproductive development were maintained post-treatment. Fish were periodically sampled for gonadal histology, gene expression and plasma 17β-estradiol (E2) analyses to assess gonadal development. Short-term (4-week) exposure to elevated temperatures had only minor effects, whereas longer exposure (12–19 weeks) markedly inhibited ovarian development. Fish from the moderate and high treatment groups had significantly less developed ovaries relative to controls, and mRNA levels for germ cell (vasa, zpc) and apoptosis-associated genes (p53, casp8) generally indicated gonadal degeneration. The high treatment group also had significantly reduced plasma E2 levels and elevated gonadal amh gene expression. After one year at ambient temperatures, however, ovaries of moderate and high treatment fish exhibited compensatory recovery and were indistinguishable from controls. Two genotypic females possessing immature testes (neomales) were observed in the high treatment group, indicating sex reversal had occurred (6% rate). These results demonstrate that extreme elevated temperatures may inhibit ovarian development or trigger sex reversal. High temperature treatment is likely not an effective sterilization method but may be preferable for sablefish neomale broodstock production.

DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads.

STUDY QUESTIONDoes loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?SUMMARY ANSWERDMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.WHAT IS KNOWN ALREADYTesticular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.STUDY DESIGN, SIZE, DURATIONTo determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4–6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8–20 weeks gestation; n = 12) in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3–4), or xenografting (n = 5) into immunocompromised mice for 4–6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.MAIN RESULTS AND THE ROLE OF CHANCEDMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8–11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12–20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype.LIMITATIONS, REASONS FOR CAUTIONHuman fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings.WIDER IMPLICATIONS OF THE FINDINGSOur findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest.

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.

Evaluation of specific germ cell genes expression in mouse embryonic stem cell-derived germ cell like cells treated with bone morphogenetic protein 4 in vitro

Bone morphogenetic protein 4 (BMP4) is a significant signaling molecule that involves in initiating of differentiation and performs multifunctional effects on embryonic stem cells (ESCs) and embryos. The goal of the present study was to evaluate an in vitro differentiation model of mouse embryonic stem cells into germ cells, using BMP4. in this experimental study, we used Oct4-GFP mouse ESCs to form embryoid body (EB) aggregations for two days. Then, single cells from EB were cultured for four days with BMP4. Using MTT assay and gene expression levels for evaluation of Mvh and Riken by real-time RT-PCR of six concentrations, 12.5 ng/ ml BMP4 was determined as an optimized dose. Then, the expression level of Fkbp6, Mov10l1, 4930432K21Rik, Tex13, Mvh, Scp3, Stra8, Oct4 were evaluated. Flow cytometry and immunostaning were used to confirm the findings of the real-time RT-PCR. In the +BMP4 group, the genes encoding Riken (p≤0.001) and Mvh (p≤0.001) were found to be increased with significant differences compared with the control group. Mov10l1 (p=0.22), Tex13 (p=0.10), Fkbp6 (p=0.90), Scp3 (p=0.61) and Stra8 (p=0.08) were up-regulated without significance differences compared with control group. Flow cytometry analysis showed that the mean number of Mvh-positive cells in the +BMP4 group was greater when compared with ESCs, -BMP4 and EB groups (p=0.03, p≤0.001, p=0.02, respectively). Down-regulation of Oct4, expression of germ cells genes and meiosis markers expression raise this hypothesis that ESCs were differentiated by BMP4, and may be introduced into the first meiosis as germ cell-like cells.

Mare and Stallion Effects on Blastocyst Production in a Commercial OPU-ICSI Program

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa). Out of the series of RA doses (2, 4, 8, 16, 20 and 30 μM), 16 μM RA for 8 day culture interval was found to induce highest expression of PGC- and meiosis-associated genes like DAZL, VASA, SYCP3, MLH1, TNP1/2 and PRM2, while mature germ cell genes like BOULE and TEKT1 (Spermatocyte markers), GDF9 and ZP2 (Oocyte markers) showed higher expression at 2 μM RA dose, suggesting functional concentration-gradient of RA activity. Immunocytochemistry revealed expression of germ lineage-specific markers like: c-KIT, DAZL and VASA (PGC-markers); SYCP3, MLH1 and PROTAMINE1 (Meiotic-markers); ACROSIN and HAPRIN (Spermatocyte-markers); and GDF9 and ZP4 (Oocyte-markers) in optimally differentiated embryoid bodies (EBs) and adherent cultures. We observed significantly reduced (p < 0.05) concentration of 5-methyl-2-deoxycytidine in RA-differentiated EBs which is suggestive of the occurrence of methylation erasure. FACS analysis of optimally differentiated cultures detected 3.07% haploid cell population, indicating completion of meiosis. Oocyte-like structures (OLS) were obtained in adherent differentiated cultures. They had a big nucleus and a zona pellucida (ZP4) coat. They showed progression through 2-cell, 4-cell, 8-cell, morula and blastocyst-like structures upon extended culture beyond 14 days.