Postmeiotic Stages Research Articles

Elucidating the Transcriptional States of Spermatogenesis-Joint Analysis of Germline and Supporting Cell, Mice and Human, Normal and Perturbed, Bulk and Single-Cell RNA-Seq.

In studying the molecular underpinning of spermatogenesis, we expect to understand the fundamental biological processes better and potentially identify genes that may lead to novel diagnostic and therapeutic strategies toward precision medicine in male infertility. In this review, we emphasized our perspective that the path forward necessitates integrative studies that rely on complementary approaches and types of data. To comprehensively analyze spermatogenesis, this review proposes four axes of integration. First, spanning the analysis of spermatogenesis in the healthy state alongside pathologies. Second, the experimental analysis of model systems (in which we can deploy treatments and perturbations) alongside human data. Third, the phenotype is measured alongside its underlying molecular profiles using known markers augmented with unbiased profiles. Finally, the testicular cells are studied as ecosystems, analyzing the germ cells alongside the states observed in the supporting somatic cells. Recently, the study of spermatogenesis has been advancing using single-cell RNA sequencing, where scientists have uncovered the unique stages of germ cell development in mice, revealing new regulators of spermatogenesis and previously unknown cell subtypes in the testis. An in-depth analysis of meiotic and postmeiotic stages led to the discovery of marker genes for spermatogonia, Sertoli and Leydig cells and further elucidated all the other germline and somatic cells in the testis microenvironment in normal and pathogenic conditions. The outcome of an integrative analysis of spermatogenesis using advanced molecular profiling technologies such as scRNA-seq has already propelled our biological understanding, with additional studies expected to have clinical implications for the study of male fertility. By uncovering new genes and pathways involved in abnormal spermatogenesis, we may gain insights into subfertility or sterility.

Understanding the role of P-type ATPases in regulating pollen fertility and development in pigeonpea.

The P-type ATPase superfamily genes are the cation and phospholipid pumps that transport ions across the membranes by hydrolyzing ATP. They are involved in a diverse range of functions, including fundamental cellular events that occur during the growth of plants, especially in the reproductive organs. The present work has been undertaken to understand and characterize the P-type ATPases in the pigeonpea genome and their potential role in anther development and pollen fertility. A total of 59 P-type ATPases were predicted in the pigeonpea genome. The phylogenetic analysis classified the ATPases into five subfamilies: eleven P1B, eighteen P2A/B, fourteen P3A, fifteen P4, and one P5. Twenty-three pairs of P-type ATPases were tandemly duplicated, resulting in their expansion in the pigeonpea genome during evolution. The orthologs of the reported anther development-related genes were searched in the pigeonpea genome, and the expression profiling studies of specific genes via qRT-PCR in the pre- and post-meiotic anther stages of AKCMS11A (male sterile), AKCMS11B (maintainer) and AKPR303 (fertility restorer) lines of pigeonpea was done. Compared to the restorer and maintainer lines, the down-regulation of CcP-typeATPase22 in the post-meiotic anthers of the male sterile line might have played a role in pollen sterility. Furthermore, the strong expression of CcP-typeATPase2 in the post-meiotic anthers of restorer line and CcP-typeATPase46, CcP-typeATPase51, and CcP-typeATPase52 in the maintainer lines, respectively, compared to the male sterile line, clearly indicates their potential role in developing male reproductive organs in pigeonpea.

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

Expression Profiling of MSP1 Gene Involved in Mega-gametophyte Development in Ploidy Series of Guinea Grass (Panicum maximum Jacq.)

Hybrid seeds are playing important role in increasing agricultural production. However, process of hybrid seed development involves high cost for most of the major crops. Under such situation apomixis can play crucial role in creation of one-time hybrid and maintain it for multiple generations. Apomixis and polyploidy are interrelated with each other and expression of apomixis completely relies on polyploidy. Guinea grass (Panicum maximum Jacq.) provides ideal system for research on apomixis and polyploidy because of its extensive diversity in morphological, cytological, biochemical, and molecular characteristics. The experiment was carried out in the eight-ploidy series i.e. 3x, 4x, 5x, 6x, 7x, 8x, 9x, 11x and one obligate sexual Guinea grass genotype SRP75. Considering role of MSP1 gene in apomeiosis, its expression was studied in guinea grass at three developmental stages of spikelet viz., pre-meiotic, meiotic and post-meiotic. Findings of this study highlight the complex regulation of MSP-1 expression during different developmental stages and across various ploidy levels in guinea grass. At pre-meiotic stage, except 4x, downregulation pattern of MSP1 was observed in all the ploidy levels. But opposite was observed in the post-meiotic stage.

Identification of two genes essential for basidiospore formation during the postmeiotic stages in Pleurotus ostreatus

A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro.

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO2 incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced maturation of spermatogonial cells from prepubertal mice in vitro is enhanced by testosterone.

Spermatogenesis is the complicated process of sperm generation. During this process, spermatogonial cells proliferate and differentiate via meiotic and post-meiotic stages to produce mature sperm. This process is under the regulation of testicular autocrine/paracrine factors. In addition, endocrine factors are crucial to complete spermatogenesis. We aimed to localize granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR) in testicular cells and further evaluate its involvement in the development of spermatogenesis in vitro. Weisolated cells from seminiferous tubule cells of seven-day-old mice and cultured them in vitro using a methylcellulose culture system (MCS), in the presence of GM-CSF and/or testosterone for four weeks. The cells were then examined for markers of different stages of spermatogenesis by immunofluorescence staining and/or qPCR analyses. Our results revealed the presence of GM-CSF and GM-CSFR in testicular cells (premeiotic and meiotic cells as well as somatic cells; Leydig and Sertoli cells). We further demonstrated the development of colonies/spheroids in the MCS which contained pre-meiotic, meiotic, and post-meiotic cells. The addition of GM-CSF to the MCS significantly increased the percentage of pre-meiotic and meiotic cells compared to control. Furthermore, the addition of GM-CSF and testosterone together significantly increased the percentage of cells in the post-meiotic stage compared to the addition of each separately. In conclusion, our results indicate that testicular cells express GM-CSF/GM-CSFR, and that GM-CSF is involved in the development of different stages of spermatogenesis in vitro. Furthermore, testosterone enhances the development of spermatogenic cells and potentiates the effect of GMCSF on the development of post-meiotic cells. These findings provide evidence that GM-CSF and testosterone are involved in the development of spermatogenesis in vitro and in vivo. In brief: Testicular somatic and germ cells express GM-CSF and GM-CSFR. Our study suggests that testicular GM-CSF is involved in the development of spermatogenesis, which is potentiated by testosterone.

Isolation and characterisation of promoters from mouse genome to drive post-meiotic germ cell-specific robust gene expression for functional genomics studies

The generation of spermatozoa from developing germ cells through mitotic and meiotic divisions is a highly regulated and complex process. Any defect in this process, may lead to subfertility/infertility. The role of different transcripts (mRNA/miRNA/lncRNA) in regulation of the pre-meiotic, meiotic, and post-meiotic stages of spermatogenesis are being proposed based on various multiomics based approaches. Such differential gene-expression is regulated by promoter elements that are activated in a stage specific manner. To determine the role of these differentially expressed transcripts in the process of meiosis, a robust post-meiotic germ cell specific promoter is required. In the present study, we have isolated and characterized the expression of the mouse Proacrosin, SP10, and ELP promoters for driving post-meiotic germ cell specific gene-expression. Promoter regions of all these 3 genes were isolated and cloned to generate mammalian expression vector. The transgene expression in post-meiotic germ cells was assessed in mice using the testicular electroporation method in vitro as well as in vivo, using above promoters. It was also validated in goat seminiferous tubules, in vitro. We have also carried out a comparative analysis of the strength of these promoters to confirm their robustness that indicated Proacrosin to be the most robust promoter that can be useful for diving post-meiotic germ cells specific gene-expression. These promoters can be used to alter gene-expression specifically in post-meiotic germ cells for deciphering the role(s) of germ cell genes in spermatogenic progression or for expressing various genome editing tools for engineering the germ cell genome to understand basis of subfertility/infertility.

Novel Aspects of cAMP-Response Element Modulator (CREM) Role in Spermatogenesis and Male Fertility.

Spermatogenesis is a very complex process with an intricate transcriptional regulation. The transition from the diploid to the haploid state requires the involvement of specialized genes in meiosis, among other specific functions for the formation of the spermatozoon. The transcription factor cAMP-response element modulator (CREM) is a key modulator that triggers the differentiation of the germ cell into the spermatozoon through the modification of gene expression. CREM has multiple repressor and activator isoforms whose expression is tissue-cell-type specific and tightly regulated by various factors at the transcriptional, post-transcriptional and post-translational level. The activator isoform CREMτ controls the expression of several relevant genes in post-meiotic stages of spermatogenesis. In addition, exposure to xenobiotics negatively affects CREMτ expression, which is linked to male infertility. On the other hand, antioxidants could have a positive effect on CREMτ expression and improve sperm parameters in idiopathically infertile men. Therefore, CREM expression could be used as a biomarker to detect and even counteract male infertility. This review examines the importance of CREM as a transcription factor for sperm production and its relevance in male fertility, infertility and the response to environmental xenobiotics that may affect CREMτ expression and the downstream regulation that alters male fertility. Also, some health disorders in which CREM expression is altered are discussed.

Jasmonic acid-mediated stress responses share the molecular mechanism underlying male sterility induced by deficiency of ZmMs33 in maize

Plant male reproduction is a fine-tuned developmental process that is susceptible to stressful environments and influences crop grain yields. Phytohormone signaling functions in control of plant normal growth and development as well as in response to external stresses, but the interaction or crosstalk among phytohormone signaling, stress response, and male reproduction in plants remains poorly understood. Cross-species comparison among 514 stress-response transcriptomic libraries revealed that ms33-6038, a genic male sterile mutant deficient in the ZmMs33/ZmGPAT6 gene, displayed an excessive drought stress-like transcriptional reprogramming in anthers triggered mainly by disturbed jasmonic acid (JA) homeostasis. An increased level of JA appeared in ZmMs33-deficient anthers at both meiotic and post-meiotic stages and activated genes involved in JA biosynthesis and signaling as well as genes functioning in JA-mediated drought response. Excessive accumulation of JA elevated expression level of a gene encoding a WRKY transcription factor that activated the ZmMs33 promoter. These findings reveal a feedback loop of ZmMs33-JA-WRKY-ZmMs33 in controlling male sterility and JA-mediated stress response in maize, shedding light on the crosstalk of stress response and male sterility mediated by phytohormone homeostasis and signaling.

P-420 Involvement of Interluekin-6 (IL-6) in the development of different stages of spermatogenesis in vitro, using spermatogonial cells isolated from normal and busulfan-treated immature mice.

Abstract Study question Can Interluekin-6 (IL-6) induce spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer IL-6 induced the development of spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to premeiotic, meiotic and post-meiotic cells in vitro. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. These microenvironmental factors are modified following pathological condition, such as chemotherapy, which may lead to subfertility or sterility. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro; however, this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used as normal or were intraperitoneally (i.p) injected with busulfan (45 mg/kg) to isolate cells from their seminiferous tubules (STs). Cells were enzymatically isolated from the STs of the mice and were cultured in methylcellulose culture system (MCS), as a 3-dimensinal in vitro system. Fresh media without (CT) or with IL-6 were added to the cultures from the beginning, and after two weeks. The cultures were determined after 4 weeks Participants/materials, setting, methods Isolated cells from the STs were cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF and in the presence/absence of IL-6 (1,10 or 100 pg/ml) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/organoids were examined microscopically and quantified for the development of cells of the different stages of spermatogenesis and Sertoli cells functional markers by immunofluorescence staining (IF) and/or qPCR analyses. Main results and the role of chance Addition of IL-6 to MCS that contained isolated cells from STs of normal immature mice significantly increased the development of premeiotic cells (VASA), meiotic (BOULE) and meiotic/postmeiotic cells (ACROSIN) as examined by specific IF and/or qPCR analyses. Addition of IL-6 also differently affected the expression levels of Sertoli cell functionality markers (androgen receptor, androgen binding protein, transferrin, GDNF, FSHR). Furthermore, addition of IL-6 to isolated cells from STs of busulfan-treated immature mice also increased the development of VASA-, BOULE- and ACROSIN-positive cells (as examined by IF and qPCR analyses). However, the effect of IL-6 was more potent in the development of different stages of spermatogenesis in in vitro cultures that contained isolated cells from STs of normal compared busulfan-treated mice. Also, addition of IL-6 distinctly affected the expression levels of Sertoli cell functional markers in cultures of normal and busulfan-treated mice. Limitations, reasons for caution This in-vitro culture used animal models. The bio-activity of the developed post-meiotic needs to be confirmed Wider implications of the findings These findings indicate the possible involvement of IL-6 in the development of spermatogenesis in vitro. This effect could be directly on spermatogonial cells and/or through Sertoli cells. Our result could suggest IL-6 as a potential factor to be used in developing future in vitro therapeutic strategies for male fertility preservation Trial registration number not applicable

Reproductive Suppression Caused by Spermatogenic Arrest: Transcriptomic Evidence from a Non-Social Animal

Reproductive suppression is an adaptive strategy in animal reproduction. The mechanism of reproductive suppression has been studied in social animals, providing an essential basis for understanding the maintenance and development of population stability. However, little is known about it in solitary animals. The plateau zokor is a dominant, subterranean, solitary rodent in the Qinghai-Tibet Plateau. However, the mechanism of reproductive suppression in this animal is unknown. We perform morphological, hormonal, and transcriptomic assays on the testes of male plateau zokors in breeders, in non-breeders, and in the non-breeding season. We found that the testes of non-breeders are smaller in weight and have lower serum testosterone levels than those of breeders, and the mRNA expression levels of the anti-Müllerian hormone (AMH) and its transcription factors are significantly higher in non-breeder testes. Genes related to spermatogenesis are significantly downregulated in both meiotic and post-meiotic stages in non-breeders. Genes related to the meiotic cell cycle, spermatogenesis, flagellated sperm motility, fertilization, and sperm capacitation are significantly downregulated in non-breeders. Our data suggest that high levels of AMH may lead to low levels of testosterone, resulting in delayed testicular development, and physiological reproductive suppression in plateau zokor. This study enriches our understanding of reproductive suppression in solitary mammals and provides a basis for the optimization of managing this species.

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation.

Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.

O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p < 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p < 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable

O-215 Embryo developmental competence of de novo male gametes in a novel three-dimensional culture

Abstract Study question Are de novo gametes generated from mouse embryonic stem cells (mESCs) in a novel three-dimensional (3D) culture system capable of supporting full preimplantation development? Summary answer A novel 3D culture system yielded de novo male gametes capable of fertilizing an oocyte and supporting full preimplantation development. What is known already In regenerative medicine, several 3D culture systems have been shown to be capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids, in the mouse model. However, a heterologous transplantation technique is required to obtain functional gametes. Study design, size, duration mESCs were first cultured on a gelatin-coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that normal spermatogenesis occurs in 30 days, utilization of the de novo gametes was planned for days 15, 22, 29, and 36. Participants/materials, setting, methods mESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF, and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF, and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage), and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance Expression of DAZL in 20% and VASA in 15% of the cells at day 3 demonstrated progression into spermatogenesis. On day 10, DAZL and VASA were assessed again, revealing increases to 45% and 18%. A small proportion of cells expressed post-meiotic biomarkers, BOULE (1%) and acrosin (2%). On day 15, VASA expression plateaued at 17%, BOULE expression peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased slightly to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, acrosin expression remained stable at 7%, and BOULE expression dropped to 2%. On day 36, VASA was still expressed at 13%, and few cells expressed acrosin (1%); there was complete loss of BOULE expression. Our mouse ICSI control achieved 89.2% fertilization and 77.8% blastocyst rates. De novo gametes were injected into oocytes on days 15, 22, 29, and 36, achieving fertilization rates of 35.0%, 61.1%, 81.8%, and 75.0%, respectively. Correspondently, hatching blastocysts were obtained at rates of 5.0%, 16.7%, 36.4%, and 8.3%, respectively. Limitations, reasons for caution Despite the ability to fertilize normally, the blastulation rate remained suboptimal. Most importantly, the ability to generate live offspring still needs to be documented. Wider implications of the findings Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allo-/xeno-geneic transplantation. If reproducibility and the ability to obtain healthy offspring are confirmed, this method may represent a tool for achieving neogametogensis in mammals. Trial registration number N/A

Gene regulation network analyses of pistil development in papaya

BackgroundThe pistil is an essential part of flowers that functions in the differentiation of the sexes and reproduction in plants. The stigma on the pistil can accept pollen to allow fertilization and seed development. Papaya (Carica papaya L.) is a dioecious plant, where female flowers exhibit normal pistil, while the male flowers exhibit aborted pistil at a late stage of pistil development.ResultsThe developmental stages of papaya pistil were analyzed after first dividing it into slices representing the primordium stage 1 (S1), the pre-meiotic stages S2, post-meiotic stage S3, and the mitotic stage S4. The SS scoring algorithm analysis of genes preferentially expressed at different stages revealed differentially expressed genes between male and female flowers. A transcription factor regulatory network for each stage based on the genes that are differentially expressed between male and female flowers was constructed. Some transcription factors related to pistil development were revealed based on the analysis of regulatory networks such as CpAGL11, CpHEC2, and CpSUPL. Based on the specific expression of genes, constructed a gene regulatory subnetwork with CpAGL11-CpSUPL-CpHEC2 functioning as the core. Analysis of the functionally enriched terms in this network reveals several differentially expressed genes related to auxin/ brassinosteroid signal transduction in the plant hormone signal transduction pathway. At the same time, significant differences in the expression of auxin and brassinosteroid synthesis-related genes between male and female flowers at different developmental stages were detected.ConclusionsThe pistil abortion of papaya might be caused by the lack of expression or decreased expression of some transcription factors and hormone-related genes, affecting hormone signal transduction or hormone biosynthesis. Analysis of aborted and normally developing pistil in papaya provided new insights into the molecular mechanism of pistil development and sex differentiation in dioecious papaya.

An exocyst component, Sec5, is essential for ascospore formation in Bipolaris maydis.

In this study, we identified Sec5 in Bipolaris maydis, a homologue of Sec5 in Saccharomyces cerevisiae and a possible exocyst component of the fungus. To examine how Sec5 affects the life cycle of B. maydis, we generated null mutant strains of the gene (Δsec5). The Δsec5 strains showed a strong reduction in hyphal growth and a slight reduction in pathogenicity. In sexual reproduction, they possessed the ability to develop pseudothecia. However, all ascospores were aborted in any of the asci obtained from crosses between Δsec5 and the wild-type. Our cytological study revealed that the abortion was caused by impairments of the post-meiotic stages in ascospore development, where ascospore delimitation and young spore elongation occur.

Conserved and non‐conserved triggers of 24‐nucleotide reproductive phasiRNAs in eudicots

Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre-meiotic 21-nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non-canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a lineage-specific trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275-derived 24-nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24-nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24-nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs.

The Presence of Colony-Stimulating Factor-1 and Its Receptor in Different Cells of the Testis; It Involved in the Development of Spermatogenesis In Vitro.

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2–12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2–3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2–12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.