Late Germ Cells Research Articles

Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model ofether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

Comparison of the efficacy of bone morphogenetic protein-4 on in vitro differentiation of murine adipose and bone marrow mesenchymal stem cells into primordial germ cells.

Background and purpose:In vitro development of functional gametes from pluripotent stem cells is a promising prospect to treat infertility. Mesenchymal stem cells with a high degree of plasticity and less tumorigenicity are a reliable source of stem cells for the generation of gametes. The present study aimed to compare the differentiation potential in the mesenchymal stem cells that are derived from bone marrow (BMDMSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) into germ cells in a culture medium containing bone morphogenic protein-4 (BMP-4).Experimental approach:In this study, MSCs were isolated from both bone marrow and adipose tissue of murine samples. To further verify the nature of the harvested stem cells, their multipotency and surface marker were examined. The identified stem cells were cultured in a medium supplemented with 0 and 25 ng/mL of BMP-4 for 4 days. Flow cytometry analysis, immunofluorescence staining, and real RT-PCR were used to assess the expression levels in germ cell-specific biomarkers (Mvh, Dazl, Stra8, and Scp3).Findings/Results:CD44+, CD45-, CD31-, BMD-MSCs, and AD-MSCs showed to be capable of differentiating to osteo-adipogenic lineages. The flow cytometry, immunofluorescence, and RT-PCR results indicated that early germ cell markers (Mvh and Dazl) were expressed in both types of cells but they were significantly higher in BMD-MSCs than AD-MSCs.Conclusion and implications:Based on our results, the addition of exogenous BMP4 to the culture medium could differentiate BMD-MSCs and AD-MSCs into primordial germ cells, but it is inadequate to further develop into late germ cells in vitro. Moreover, the results revealed that, although AD-MSCs were easier to collect and had faster growth and proliferation rates than BMD-MSCs, the BMD-MSCs were better capable of differentiation into primordial germ cells. They may serve to be considered a more suitable source of MSC for in vitro generation of gametes than AD-MSCs.

In vitro derived female hPGCLCs are unable to complete meiosis in embryoid bodies

The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs.

Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells.

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3' UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

A Novel Dual Immunostain to Characterize Sloughed Cells in Testicular Biopsies for Infertility.

Evaluation of testicular biopsies from azoospermic men requires recognition of phases of germ cell maturation as organized architecturally within the seminiferous tubule, as well as distinguishing the inability to generate mature spermatozoa (germ cell aplasia or maturation arrest) from normal spermatogenesis, which may be associated with a reversible obstruction. While traditional fixatives (eg, Bouin solution) provide exquisite nuclear detail and preserve the architectural integrity of the seminiferous tubule, formalin fixation yields biopsies with relatively poor nuclear detail and frequent luminal sloughing of cells, making it difficult to assess sperm maturation. One clone of the anti-DOG1 antibody was recently found to be expressed in late (postspermatogonial) germ cells. We developed a dual stain including DOG1 and SF-1 to mark late germ cells and Sertoli cells, respectively, in both sloughed and intact cells. Consecutive testicular biopsies (N=28) from men with azoospermia were classified by hematoxylin and eosin morphology and stained with a dual SF-1 (Perseus)/DOG1 (Cell Marque) immunohistochemical stain. Histologic patterns included normal spermatogenesis (5 cases), hypospermatogenesis (5 cases), late maturation arrest (2 cases), Sertoli cell only pattern (15 cases), and extensive tubular hyalinization (1 case). Architectural disruption of seminiferous tubules with sloughing of cells into the lumens was noted in all biopsies, at least focally. SF-1 (nuclear) was expressed in sloughed Sertoli cells; DOG1 (cytoplasmic) in sloughed postspermatogonial germ cells (spermatocytes and spermatids). This resulted in two distinct immunophenotypes: SF-1(+)/DOG1(-) sloughed cells in cases with the Sertoli cell only pattern and SF-1(+)/DOG1(+) sloughed cells in all other histologic patterns (normal spermatogenesis, hypospermatogenesis, and maturation arrest). Because the rate of sperm retrieval is lower in men with the Sertoli cell only pattern, this immunohistochemical stain may assist pathologists in the proper interpretation of testicular biopsies, allowing better-informed decision making by patients and clinicians regarding the subsequent use of assisted reproductive technologies.

Gonadal, body color, and genotoxic alterations in Lithobates catesbeianus tadpoles exposed to nonylphenol.

Endocrine disrupting chemicals are one of the most important factors contributing to worldwide amphibian decline. The 4-nonylphenol (NP) is a degradation product of several compounds, such as detergents and pesticides, affecting the aquatic environment. Here, we test whether treatment with NP has an effect on developing ovarian tissue, nuclear abnormalities in erythrocytes, and body darkness in pre-metamorphic tadpoles of the bullfrog Lithobates catesbeianus. Tadpoles were exposed for 14days to three different concentrations of NP (1, 10, and 100μg/L) besides the control group, which was maintained only with water. After determining body coloration, animals were euthanized and gonads and blood were collected and processed for histology and genotoxic analysis. Even though most animals were females, intersex tadpoles were observed in control and treated groups and there were no males in any group. The highest concentration of NP showed an increase in atretic oocytes, but the area corresponding to somatic compartment and early and late germ cells were not affected. Furthermore, all treated groups presented higher amount of nuclear abnormalities in erythrocytes and body darkening when compared with the control group. These results suggest that NP causes genetic damage and morphological alterations in L. catesbeianus tadpoles by disrupting oogenesis, inducing genotoxicity and increasing body coloration. Its effects on gonadal development could cause future impairments in reproduction, while its deleterious effects on genotoxicity and body pigmentation could be used as a biomarker of effect to this compound.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. Here we report that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.

Stem Cell Therapy for Male Infertility: A Review

Infertility affects 15% of couples in reproductive age world-wide and male factor is solely responsible in about 50% of the cases and contributory in 30-40% of cases. Spermatogonial stem cells (SSCs) are small self-renewing cells found in the basal compartment of seminiferous tubules where they form the foundation of spermatogenesis and are required for the continuous production of sperm. Transplantation of spermatogonial stem cells results in a donor derived sperm production and fertility in rodent and non-rodent species like Goat. Transplantation of cryopreserved spermatogonial stem cells could help oncology patients undergoing radiotherapy or chemotherapy by prior isolation of the SSCs and transplantation after treatment. Induced pluripotent stem cells also have the ability to differentiate into late stage germ cells. The efficacy and safety of SSCs transplantation showed that offspring produced did not show any morphological differences or alteration of genetic material but is most effective through assisted reproduction and better on young/immature Animals. The transfer of germ cells through micro-injection of seminiferous tubules and cannulation of efferent ducts is most effective on rodent testis while injection into the rete testis guided by ultrasound was reported to be the best technique in higher Animals (Bulls and Apes). Researches are still ongoing to get a safe and successful method of SSCs transplantation with no or less side effects on humans.Keywords: Cryopreserved, Pluripotent, self-renewing, Spermatogonia, and Spermatogenesis

Does mouse embryo primordial germ cell activation start before implantation as suggested by single-cell transcriptomics dynamics?

Does primordial germ cell (PGC) activation start before mouse embryo implantation, and does the possible regulation of the DNA (cytosine-5-)-methyltransferase 3-like (Dnmt3l) by transcription factor AP-2, gamma (TCFAP2C) have a role in this activation and in the primitive endoderm (PE)-epiblast (EPI) lineage specification? A burst of expression of PGC markers, such as Dppa3/Stella, Ifitm2/Fragilis, Fkbp6 and Prdm4, is observed from embryonic day (E) 3.25, and some of them, together with the late germ cell markers Zp3, Mcf2 and Morc1, become restricted to the EPI subpopulation at E4.5, while the dynamics analysis of the PE-EPI transitions in the single-cell data suggests that TCFAP2C transitorily represses Dnmt3l in EPI cells at E3.5 and such repression is withdrawn with reactivation of Dnmt3l expression in PE and EPI cells at E4.5. In the mouse preimplantation embryo, cells with the same phenotype take different fates based on the orchestration between topological clues (cell polarity, positional history and division orientation) and gene regulatory rules (at transcriptomics and epigenomics level), prompting the proposal of positional, stochastic and combined models explaining the specification mechanism. PGC specification starts at E6.0-6.5 post-implantation. In view of the important role of DNA methylation in developmental events, the cross-talk between some transcription factors and DNA methyltransferases is of particular relevance. TCFAP2C has a CpG DNA methylation motif that is not methylated in pluripotent cells and that could potentially bind on DNMT3L, the stimulatory DNA methyltransferase co-factor that assists in the process of de novo DNA methylation. Chromatin-immunoprecipitation analysis has demonstrated that Dnmt3l is indeed a target of TCFAP2C. We aimed to assess the timing of early preimplantation events and to understand better the segregation of the inner cell mass (ICM) into PE and EPI. We designed a single-cell transcriptomics dynamics computational study to identify markers of the PE-EPI bifurcation in ICM cells through searching for statistically significant (using the Student's t-test method) differently expressed genes (DEGs) between PE and EPI cells from E3.5 to E4.5. The DEGs common for E3.5 and E4.5 were used as the markers defining the steady states. We collected microarray and next-generation sequencing transcriptomics data from public databases from bulk populations and single cells from mice at E3.25, E3.5 and E4.5. The results are based on three independent single-cell transcriptomics data sets, with a fold change of 3 and P-value <0.01 for the DEG selection. The dynamics analysis revealed new transitory E3.5 and steady PE and EPI markers. Among the transitory E3.5 PE markers (Dnmt3l, Dusp4, Cpne8, Akap13, Dcaf12l1, Aaed1, B4galt6, BC100530, Rnpc3, Tfpi, Lgalsl, Ckap4 and Fbxl20), several (Dusp4, Akap13, Cpn8, Dcaf12l1 and Tfpi) are related to the extracellular regulated kinase pathway. We also identified new transitory E3.5 EPI markers (Sgk1, Mal, Ubxn2a, Atg16l2, Gm13102, Tcfap2c, Hexb, Slc1a1, Svip, Liph and Mier3), six new stable PE markers (Sdc4, Cpn1, Dkk1, Havcr1, F2r/Par1 and Slc7a6os) as well as three new stable EPI markers (Zp3, Mcf2 and Hexb), which are known to be late stage germ cell markers. We found that mouse PGC marker activation starts at least at E3.25 preimplantation. The transcriptomics dynamics analyses support the regulation of Dnmt3l expression by TCFAP2C. Since the regulation of Dnmt3l by TCFAP2C is based on computational prediction of DNA methylation motifs, Chip-Seq and transcriptomics data, functional studies are required to validate this result. We identified a collection of previously undescribed E3.5-specific PE and EPI markers, and new steady PE and EPI markers. Identification of these genes, many of which encode cell membrane proteins, will facilitate the isolation and characterization of early PE and EPI populations. Since it is so well established in the literature that mouse PGC specification is a post-implantation event, it was surprising for us to see activation of PGC markers as early as E3.25 preimplantation, and identify the newly found steady EPI markers as late germ cell markers. The discovery of such early activation of PGC markers has important implications in the derivation of germ cells from pluripotent cells (embryonic stem cells or induced pluripotent stem cells), since the initial stages of such derivation resemble early development. The early activation of PGC markers points out the difficulty of separating PGC cells from pluripotent populations. Collectively, our results suggest that the combining of the precision of single-cell omics data with dynamic analysis of time-series data can establish the timing of some developmental stages as earlier than previously thought. Not applicable. This work was supported by grants DFG15/14 and DFG15/020 from Diputación Foral de Gipuzkoa (Spain), and grant II14/00016 from I + D + I National Plan 2013-2016 (Spain) and FEDER funds. The authors declare no conflict of interest.

Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss).

Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a, cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reverse-transcriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens up-regulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion.

Spatial and temporal translational control of germ cell mRNAs mediated by the eIF4E isoform IFE-1.

Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation.

BMP4 can generate primordial germ cells from bone‐marrow‐derived pluripotent stem cells

Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro.

Bone morphogenetic protein 4 is an efficient inducer for mouse embryonic stem cell differentiation into primordial germ cell

Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation.

Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro

Pluripotent epiblast stem cells (EpiSCs) derived from postimplantation embryos exhibit properties that are characteristically different when compared with pluripotent embryonic stem cells (ESCs) derived from mouse blastocysts. However, EpiSCs are relatively less well characterised compared with ESCs. In particular, the relationship between EpiSCs and primordial germ cells (PGCs) is unknown, and is worthy of investigation because PGCs originate from postimplantation epiblast cells in vivo. We show that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal. These PGCs generated in vitro show appropriate transcriptional and epigenetic reprogramming events and are able to develop further into late germ cells. Notably, the PGCs can, in turn, be induced to undergo dedifferentiation into pluripotent embryonic germ cells (EGCs), which resemble ESCs and not the EpiSC from which they are derived. Our observations demonstrate intrinsic reprogramming during specification of PGCs that results in the erasure of epigenetic memory of EpiSCs following reactivation of the X-chromosome, DNA demethylation and re-expression of key pluripotency genes. This study provides novel insights into the nature and properties of EpiSCs, and introduces an in vitro model system that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells.

024. Post meiotic gene transcripts — sense, antisense, or nonsense

The mature spermatid is a highly specialised cell type whose structure and physiology have evolved to convey a haploid genome intact through a relatively inimical environment to find, interact with and achieve fertilisation of a mature female gamete, thus to produce a healthy diploid embryo. It is logical to assume that these highly specialist functions are the product of a unique set of genes or transcripts, expressed in late germ cell stages. It is equally logical to see the protein products of these genes as likely targets for a post meiotic approach to male contraception. Indeed many such spermatocyte- and spermatid-specific transcripts have been identified by differential cloning approaches. Some transcripts appear to represent novel sperm-specific genes, some represent sperm-specific alternative splice products, or alternatively initiated transcripts. However, for many such transcripts, there are features that lend doubt to the notion that they are truly functional in the context of sperm physiology. Many transcripts derive from undefined, TATA-less promoters. Some gene products have no legible open reading frame. Some transcripts are even produced as antisense molecules. Some appear as functional transcripts, but are not translated. Some appear to be highly species specific. Some appear to be functionally redundant, when tested in gene ablation experiments. The male gamete is under extreme selection pressure. It is therefore plausible that these apparently aberrant transcripts may have a function beyond that of conventionally generating physiologically relevant proteins, as in most somatic cells. This presentation reviews current ideas about the sperm transcriptome and presents various hypotheses to help us understand the mechanisms and purpose of post meiotic gene expression.

The Evolution of the Endozepine-like Peptide (ELP) in the Mammalian Testis

The endozepine-like peptide (ELP) is a testis-specific isoform of the acyl-CoA binding protein (ACBP) and shares the latter's peptide motif for binding mid-long chain acyl-CoA groups. ELP is expressed both as mRNA and protein at high levels in the testes of a wide range of mammals, including rodents, carnivores and ruminants. However, the ELP gene is progressively inactivated through primate evolution, with no protein detectable in a range of primates studied, including human. In nonprimate species, ELP is expressed in very late postmeiotic germ cell stages only, such that its function in these species is probably associated with the metabolism of the mature spermatozoon. Current research is looking at both the function of the ELP protein and the haploid regulation of the gene.

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.

The germline: familiar and newly uncovered properties.

Transplantations of both Drosophila pole cells and mouse primordial germ cells suggest that the pregonadal germline of these two organisms is not pluripotent. In mouse PGCs, however, the potential for activating pluripotency is clearly present, as seen in the relative case of deriving EG cells from migratory-stage PGCs. EG cells are derived from altering the in vitro growth conditions of PGCs by the addition of one factor. Conceivably, straying of PGCs in vivo could also lead to novel fates if they migrated into a suitable environment. Aberrant PGC migration underlies models for the origin of several tumor types. How are migratory-stage PGCs prevented from adopting alternative fates in wild-type development? One solution is to link accurate migration into the gonads with germ cell survival. One mechanism of accomplishing this is through the action of pleiotrophic factors such as Steel. Steel is both a proliferation and migration factor; sterility in Steel mutants is due to poor germ cell survival and improper migration. Networking of migratory PGCs is another mechanism to reduce the chances of individual germ cells straying from the migration path. Postmigratory germ cells apparently undergo another restriction on potency--novel imprinting. Assuming that EG methylation profiles accurately reflect modifications made in their founder PGCs, then "erased" pluripotent EG lines suggest that PGCs can be diverted from the germline lineage. Direct assays on PGCs show that the unmethylated phenotype at Igf2r region 2 is characteristic of late germ cells. The maintenance of germline-like methylation with a switch in PGC fate is somewhat analogous to the situation in lag mutants from Volvox, which retain large gonidial-sized cells that can nevertheless initiate somatic differentiation. Just as it is not possible to readjust cell size with a subsequent change in gonidial differentiation, it may be difficult, without gametogenesis, to reimpose methylation modifications once they have been erased. In addition, the methylation status of some sites/loci may be inconsequential to developmental gene activity in germ cells or EG cells. Recent studies indicate that the methylation imprint at Igf2r is not interpreted at functional level until embryogenesis (51). In summary, we have reviewed some distinctive properties of the development of germ cells, from their early segregation, mitotic and meiotic profile, sex determination, to their migration and sequestration into gonads. Some constraints on development of the germline likely serve to maintain its integrity until maturation, when the gametic genomes must be capable of initiating embryonic development. To accomplish this ultimate goal, germ cells in some organisms may retain a bias towards pluripotency throughout development.(ABSTRACT TRUNCATED AT 400 WORDS)

A Novel, Testis-Specific Member of the Cellular Lipophilic Transport Protein Superfamily, Deduced from a Complimentary Deoxyribonucleic Acid Clone1

A novel member of the cellular lipophilic transport protein superfamily was identified after an antiserum raised against cellular retinoic acid-binding protein (CRABP) was found also to contain antibodies against another 15-kDa protein present in the cytosol of pubertal and adult rat testis. These antibodies were used to screen a rat testis cDNA expression library and isolate a 561-bp clone containing a full open reading frame from which the sequence of a novel 132 amino acid protein was deduced. The protein has 58% amino acid sequence identity to bovine myelin P2, 58% identity to murine adipocyte lipid-binding protein, and 40% identity to rat CRABP. Although the endogenous ligand has not yet been identified, conservation of residues involved in the binding of carboxylate groups suggests that the ligand is a fatty acid or an acidic retinoid. Tissue-specific expression was examined by Northern analysis and immunolocalization and appears to be restricted to late germ cells within the testis and epididymis. Immunostaining was first detectable in mid-pachytene spermatocytes and increased in intensity as these cells progressed to elongated spermatids, suggesting that this testis lipid-binding protein has a specific role in sperm development.