Effect of vitamin A deficiency (VAD) on mouse spermatogonial transcriptome profiles
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE23119 | GEO | 2010/08/03
SECONDARY ACCESSION(S): PRJNA131717
REPOSITORIES: GEO
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Project description:The objective of this study was to understand the genetic mechanisms of Vitamin-A-Deficiency (VAD)-induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that, in the postnatal testis, leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. In this study, we investigated the molecular basis of VAD on spermatogenesis in mice. We used adult Balb/C mice fed with a Control or VAD diet for an extended period of time (8-28 weeks) and selected two time points (18 and 25 weeks) for microarray analysis. To understand the effect of VAD on the spermatogonial stem cell transcriptome, we studied isolated pure populations of spermatogonia from control and vitamin-A-deficient mice from two representative time points (18 and 25 weeks) using Affymetrix GeneChip microarrays. We identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results establish a better understanding of the chronology and magnitude of the consequences of VAD on mouse testes and add to the current knowledge of the molecular regulatory mechanisms of germ cell development. Spermatogonia were isolated by the STATPUT procedure with minor modifications. VAD mice were sacrificed at 18 and 25 weeks of VAD treatment for spermatogonia isolation. Decapsulated testes from 2 mice were suspended in RPMI medium containing collagenase (0.6 mg/ml), Hyaluronidase (0.12 mg/ml) and DNAse I (1.25 mg/ml) and incubated at 37ºC for 30 minutes in a shaking water bath. The tissues were then allowed to come down the tube and the supernatant (containing interstitial cells) was removed. The pellet was incubated with 3 ml of 0.25% Trypsin /EDTA (GIBCO, Invitrogen, USA), in the presence of DNAse I (0.2 mg/ml) at 37ºC for 15 minutes in a shaking water bath. The dispersed cells were washed twice with RPMI medium containing 10% heat inactivated FBS to neutralize the protease activity, and filtered through a sterile 0.22 nylon to remove any undigested fragments. Cells of the dissociated seminiferous epithelium were then plated overnight in a 34ºC 3% CO2 incubator. The following day, germ cells, in suspension, were separated by sedimentation with use of a 2-4% BSA gradient. The cells were allowed to sediment for a standard period of 2.5 h, and fractions of 2-ml volume were collected. The cells of each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation. Purity of spermatogonia was estimated and was routinely higher than 90%. Total RNA was extracted from the isolated germ cells using TRIzol ® Reagent, and cleaned with RNeasy minicolumns. RNA content was determined by measurement of optical density at 260 nm. Only the RNA samples showing an OD 260/280 ratio higher than 1.8 were used for microarray hybridization. Raw expression values in Affymetrix CEL file format were generated by GeneChip Operating Software.
2010-08-03 | E-GEOD-23119 | biostudies-arrayexpress
Project description:Spermatogonial differentiation is a developmental process that is essential for spermatogenesis, but the molecular and cellular changes that germ cells must undergo to transition from undifferentiated spermatogonia to differentiating spermatogonia remain largely undefined. Retinoic acid (RA) is necessary and sufficient for spermatogonial differentiation. Using the postnatal mouse testis, we examine the transcriptome changes that accompany spermatogonial differentiation. Spermatogenesis was synchronized by administration of potent and selective RA synthesis inhibitor; as a result, testes contained only undifferentiated spermatogonia. Then, the inhibitor was discontinued, and mice were given a single dose of exogenous RA to initiate spermatogonial differentiation. We measured transcriptomes in FACS-enriched germ cells either before RA administration, when the cells correspond to Aal spermatogonia (and a minor contribution of spermatogonial stem cells) or at two points after RA administration, when the cells correspond to A1 or A3 differentiating spermatogonia. The results of this study reveal the full transcriptome changes accompanying spermatogonial differentiation in the mouse.
2023-07-03 | GSE213716 | GEO
Project description:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) and find that Dmrt6 plays a critical role in directing germ cells through the mitotic to meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing expression in inappropriate cell types of spermatogonial differentiation factors including SOHLH1, SOHLH2 and DMRT1 and the meiotic initiation factor STRA8 and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant spermatogonia can complete differentiation and enter meiosis, but they show defects in chromosome pairing, establishment of the XY body, and processing of recombination foci, and mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 ChIP-seq suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis. Six samples for RNA-Seq with three biological replicates in each group. Two samples for ChIP-Seq (one input and one ChIP).
2014-10-31 | E-GEOD-60440 | biostudies-arrayexpress
Project description:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) and find that Dmrt6 plays a critical role in directing germ cells through the mitotic to meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing expression in inappropriate cell types of spermatogonial differentiation factors including SOHLH1, SOHLH2 and DMRT1 and the meiotic initiation factor STRA8 and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant spermatogonia can complete differentiation and enter meiosis, but they show defects in chromosome pairing, establishment of the XY body, and processing of recombination foci, and mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 ChIP-seq suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis.
2014-10-31 | GSE60440 | GEO
Project description:Little is known of the fundamental processes governed by epigenetic mechanisms in the supplier cells of spermatogenesis, the spermatogonial stem cells (SSCs). The histone H3 lysine demethylase KDM1A is expressed in spermatogonia. We hypothesized that KDM1A serves in transcriptional regulation of SSCs and fertility. Using a conditional deletion of Kdm1a [conditional knockout (cKO)] in mouse spermatogonia, we determined that Kdm1a is essential for spermatogenesis as adult cKO males completely lack germ cells. Analysis of postnatal testis development revealed that undifferentiated and differentiating spermatogonial populations form in Kdm1a-cKO animals, yet the majority fail to enter meiosis. Loss of germ cells in the cKO was rapid with none remaining by postnatal day (PND) 21. To gain insight into the mechanistic implications of Kdm1a ablation, we isolated PND 6 spermatogonia enriched for SSCs and analyzed their transcriptome by RNA sequencing. Loss of Kdm1a was associated with altered transcription of 1206 genes. Importantly, differentially expressed genes between control and Kdm1a-cKO animals included those that are essential for SSC and progenitor maintenance and spermatogonial differentiation. The complete loss of fertility and failure to establish spermatogenesis indicate that Kdm1a is a master controller of gene transcription in spermatogonia and is required for SSC and progenitor maintenance and differentiation.
2019-02-16 | GSE126607 | GEO
Project description:A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference of bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid-induced differentiation. To accommodate this elevated energy demand, retinoic acid signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. In addition, inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway blocked the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon retinoic acid-induced differentiation by both RNA-seq analyses and quantitative proteomics. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration which is required for spermatogonial proliferation and differentiation.
2021-09-09 | PXD019136 | Pride
Project description:Defective germline reprogramming in Miwi2- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises due to defective germline reprogramming rather than a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia the intracisternal-A particle (IAP) family of endogenous retroviruses is de-repressed, but in contrast to meiotic cells DNA damage is not observed. Instead we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring host genes. In summary, defective reprogramming deregulates the spermatogonial transcriptome that may underlie spermatogonial dysfunction.
2018-03-09 | E-MTAB-6202 | biostudies-arrayexpress
Project description:Sry is sufficient to induce testis formation and subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth due to germ cell autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here we demonstrated that the testicular somatic environment of XX/Sry males is defective in the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, entering meiosis and differentiating into the round spermatid stage. XY donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, thereby preventing further progress beyond the elongated spermatid stage. In contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed missing expression of several Y-linked genes and alterations in the expression profile of genes associated with spermatogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, supporting our hypothesis that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice. Keywords: comparative genomic hybridization
2007-07-15 | GSE5319 | GEO
Project description:Despite the high incidence of male infertility, about 70% of infertile men do not receive a causative diagnosis. To gain insights into the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (crypto group), we combined single cell RNA sequencing (>30.000 cells), proteome, and histomorphometric analyses of testicular tissues. We found major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4+ State 0 cells). We also observed a transcriptional switch within the spermatogonial compartment driven by the increased and prolonged expression of the transcription factor EGR4. Intriguingly, EGR4-regulated genes included the chromatin-associated transcriptional repressor UTF1, which was downregulated. Histomorphometrical analyses showed that these transcriptional changes were mirrored at the protein level and accompanied by a change in the chromatin structure of spermatogonia. This resulted in a reduction of Adark spermatogonia - characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are at a disadvantage especially in cases of gonadotoxic damage as they have less cells to help repopulate the testis. We hypothesize that the more relaxed chromatin status of spermatogonia is dependent on decreased UTF1 expression caused by EGR4 activation. These identified regulators of the spermatogonial compartment will be highly interesting targets to uncover genetic causes of male infertility.
2021-11-02 | PXD023979 | Pride
Project description:Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by Retinoic Acid 8 (Stra8), an RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage due to failure to complete pre-meiotic DNA replication. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of Transcription Elongation Regulator-1 Like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.
2021-05-05 | GSE173873 | GEO