Wild-type Testes Research Articles

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish.

The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified an adad1 mutant zebrafish line that develops as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using nanos2 RNA FISH and a label retaining cell (LRC) assay, and found that the mutant testes had fewer LRCs and nanos2-expressing cells compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that the RNA regulatory protein Adad1 is required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

Combined analysis of mRNA-miRNA from testis tissue in Tibetan sheep with different FecB genotypes.

Testis size is important for identifying breeding animals with adequate sperm production. The aim of this study was to survey the expression profile of mRNA and miRNA in testis tissue from rams carrying different FecB genotypes, including the wild-type and heterozygous genotypes in Tibetan sheep. Comparative transcriptome profiles for ovine testes were established for wild-type and heterozygote Tibetan sheep by next-generation sequencing. RNA-seq results identified 3,910 (2,034 up- and 1,876 downregulated) differentially expressed (DE) genes and 243 (158 up- and 85 downregulated) DE microRNAs (miRNAs) in wild-type vs heterozygote sheep, respectively. Combined analysis of mRNA-seq and miRNA-seq revealed that 20 miRNAs interacted with 48 true DE target genes in wild-type testes compared to heterozygous genotype testes. These results provide evidence for a functional series of genes operating in Tibetan sheep testis. In addition, quantitative real-time PCR analysis showed that the expression trends of randomly selected DE genes in testis tissues from different genotypes were consistent with high-throughput sequencing results.

WIN18,446 enhances spermatogonial stem cell homing and fertility after germ cell transplantation by increasing blood-testis barrier permeability.

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.

SATINN: an automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants.

The mammalian testis is a complex organ with a cellular composition that changes smoothly and cyclically in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. We developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses residual learning to train convolutional neural networks (CNNs) to classify nuclei from seminiferous tubules into seven distinct cell types with an accuracy of 81.7%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 12 distinct tubule stages with 57.3% direct accuracy and 94.9% within ±1 stage. We further describe numerous cell- and tubule-level statistics that can be derived from wild-type testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially resolved genomic and proteomic technologies. The source code to reproduce the results described here and a SATINN standalone application with graphic-user interface are available from http://github.com/conradlab/SATINN. Supplementary data are available at Bioinformatics online.

Lhx2 in germ cells suppresses endothelial cell migration in the developing ovary

LIM-homeobox genes play multiple roles in developmental processes, but their roles in gonad development are not completely understood. Herein, we report that Lhx2, Ils2, Lmx1a, and Lmx1b are expressed in a sexually dimorphic manner in mouse, rat, and human gonads during sex determination. Amongst these, Lhx2 has female biased expression in the developing gonads of species with environmental and genetic modes of sex determination. Single-cell RNAseq analysis revealed that Lhx2 is exclusively expressed in the germ cells of the developing mouse ovaries. To elucidate the roles of Lhx2 in the germ cells, we analyzed the phenotypes of Lhx2 knockout XX gonads. While the gonads developed appropriately in Lhx2 knockout mice and the somatic cells were correctly specified in the developing ovaries, transcriptome analysis revealed enrichment of genes in the angiogenesis pathway. There was an elevated expression of several pro-angiogenic factors in the Lhx2 knockout ovaries. The elevated expression of pro-angiogenic factors was associated with an increase in numbers of endothelial cells in the Lhx2−/− ovaries at E13.5. Gonad recombination assays revealed that the increased numbers of endothelial cells in the XX gonads in absence of Lhx2 was due to ectopic migration of endothelial cells in a cell non-autonomous manner. We also found that, there was increased expression of several endothelial cell-enriched male-biased genes in Lhx2 knockout ovaries. Also, in absence of Lhx2, the migrated endothelial cells formed an angiogenic network similar to that of the wild type testis, although the coelomic blood vessel did not form. Together, our results suggest that Lhx2 in the germ cells is required to suppress vascularization in the developing ovary. These results suggest a need to explore the roles of germ cells in the control of vascularization in developing gonads. Preprint version of the article is available on BioRxiv at https://doi.org/10.1101/2022.03.07.483280.

Lrh1 can help reprogram sexual cell fate and is required for Sertoli cell development and spermatogenesis in the mouse testis.

The mammalian nuclear hormone receptors LRH1 (NR5A2) and SF1 (NR5A1) are close paralogs that can bind the same DNA motif and play crucial roles in gonadal development and function. Lrh1 is essential for follicle development in the ovary and has been proposed to regulate steroidogenesis in the testis. Lrh1 expression in the testis is highly elevated by loss of the sex regulator Dmrt1, which triggers male-to-female transdifferentiation of Sertoli cells. While Sf1 has a well-defined and crucial role in testis development, no function for Lrh1 in the male gonad has been reported. Here we use conditional genetics to examine Lrh1 requirements both in gonadal cell fate reprogramming and in normal development of the three major cell lineages of the mouse testis. We find that loss of Lrh1 suppresses sexual transdifferentiation, confirming that Lrh1 can act as a key driver in reprogramming sexual cell fate. In otherwise wild-type testes, we find that Lrh1 is dispensable in Leydig cells but is required in Sertoli cells for their proliferation, for seminiferous tubule morphogenesis, for maintenance of the blood-testis barrier, for feedback regulation of androgen production, and for support of spermatogenesis. Expression profiling identified misexpressed genes likely underlying most aspects of the Sertoli cell phenotype. In the germ line we found that Lrh1 is required for maintenance of functional spermatogonia, and hence mutants progressively lose spermatogenesis. Reduced expression of the RNA binding factor Nxf2 likely contributes to the SSC defect. Unexpectedly, however, over time the Lrh1 mutant germ line recovered abundant spermatogenesis and fertility. This finding indicates that severe germ line depletion triggers a response allowing mutant spermatogonia to recover the ability to undergo complete spermatogenesis. Our results demonstrate that Lrh1, like Sf1, is an essential regulator of testis development and function but has a very distinct repertoire of functions.

NLRP3 inflammasome is involved in testicular inflammation induced by lipopolysaccharide in mice.

Systemic inflammation induced by infection, which is associated with testicular inflammation, predisposes males to subfertility. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome was identified as a key mediator of inflammation, and excessive activation of the NLRP3 inflammasome was shown to contribute to the pathogenesis of a wide variety of diseases. However, the mechanisms underlying infectious inflammation in the testis remain unclear. We investigated the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the role of the NLRP3 inflammasome in murine testes. We performed in vivo and in vitro studies using an LPS-induced model of NLRP3 inflammasome activation and testicular inflammation. Intraperitoneal administration of LPS significantly impaired sperm motility in the epididymis of wild type (WT) and NLRP3-knockout (KO) mice. LPS administration stimulated interleukin (IL)-1β production and secretion in the testes of WT mice, and these adverse effects were improved in the testes of NLRP3-KO mice. LPS administration also stimulated neutrophil infiltration as well as its chemoattractant C-C motif chemokine ligand 2 (CCL2) in WT testes, which were suppressed in NLRP3-KO testes. In in vitro cell culture, treatment with LPS and NLRP3 inflammasome activation significantly induced IL-1β and CCL2 secretion from WT but not NLRP3-KO testicular cells. Taken together, our results suggest that testicular cells have the potential to secrete IL-1β and CCL2 in an NLRP3 inflammasome-dependent manner and that these cytokines from the testis may further exacerbate testicular function, resulting in subfertility during infectious diseases.

Single-Cell RNA Sequencing Defines the Regulation of Spermatogenesis by Sertoli-Cell Androgen Signaling.

Androgen receptor (AR) signaling is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which AR acts between male germ cells and somatic cells during spermatogenesis have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking AR in Sertoli cells (SCARKO) and single-cell transcriptomic sequencing (scRNA-seq), the cell specific targets of AR action as well as the genes and signaling pathways that are regulated by AR are being identified. In this study, we collected scRNA-seq data from wild-type (WT) and SCARKO mice testes at p20 and identified four somatic cell populations and two male germ cell populations. Further analysis identified that the distribution of Sertoli cells was completely different and uncovered the cellular heterogeneity and transcriptional changes between WT and SCARKO Sertoli cells. In addition, several differentially expressed genes (DEGs) in SCARKO Sertoli cells, many of which have been previously implicated in cell cycle, apoptosis and male infertility, have also been identified. Together, our research explores a novel perspective on the changes in the transcription level of various cell types between WT and SCARKO mice testes, providing new insights for the investigations of the molecular and cellular processes regulated by AR signaling in Sertoli cells.

Germ cells are not essential for sexual dimorphism of gonads in common carp, C. carpio L.

Common carp is the fourth most cultivated fish species in the world. Sexually dimorphic growth of common carp makes its sex differentiation an important research topic. The main objective of this study was to investigate the role of germ cells in sex differentiation of common carp. We generated the germ cell-deficient common carp through knocking down dead end gene with a morpholino antisense oligonucleotide during the embryogenesis. In the common carp without germ cells, we found about half of the gonads (type I gonads) contain granulosa cells and theca cells, which were the same with somatic cells we observed in the wild-type ovaries. The remaining half of the gonads (type II gonads) contain Sertoli cells and Leydig cells, which were the same with somatic cells in the wild-type testes. Furthermore, the expression of sex differentiation related genes was detected in the gonads without germ cells. It was found cyp19a1a was mainly expressed in the type I gonads, and the cyp19a1a mRNA level is much higher in each type I gonad than in the type II gonad. While dmrt1 was mainly expressed in the type II gonads, and the dmrt1 mRNA level is much higher in each type II gonad than in the type I gonad. Our comprehensive analysis showed that the gonadal structure and expression of genes related with sex differentiation were sexually dimorphic in the germ cell-deficient carp. These findings are valuable for discovering the underlying mechanism of common carp sex differentiation which is crucial for cultivating monosexual carp for aquaculture.

The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum.

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.

NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

SummaryDuring murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity through the repression of Rheb in a post-transcriptional manner.

Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis.

BackgroundRetinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis.MethodsSperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR.ResultsRai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups.ConclusionIn summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

Transcriptional changes revealed genes and pathways involved in the deficient testis caused by the inhibition of alkaline ceramidase (Dacer) in Drosophila melanogaster.

Sphingolipids are ubiquitous structural components of eukaryotic cell membranes which are vital for maintaining the integrity of cells. Alkaline ceramidase is a key enzyme in sphingolipid biosynthesis pathway; however, little is known about the role of the enzyme in the male reproductive system of Drosophila melanogaster. To investigate the impact of alkaline ceramidase (Dacer) on male Drosophila, we got Dacer deficiency mutants (MUs) and found they displayed apparent defects in the testis's phenotype. To profile the molecular changes associated with this abnormal phenotype, we performed de novo transcriptome analyses of the MU and wildtype (WT) testes; and revealed 1239 upregulated genes and 1102 downregulated genes. Then, six upregulated DEGs (papilin [Ppn], croquemort [Crq], terribly reduced optic lobes [Trol], Laminin, Wunen-2, collagen type IV alpha 1 [Cg25C]) and three downregulated DEGs (mucin related 18B [Mur18B], rhomboid-7 [Rho-7], CG3168) were confirmed through quantitative real-time polymerase chain reaction in WT and MU samples. The differentially expressed genes were mainly associated with catalytic activity, oxidoreductase activity and transmembrane transporter activity, which significantly contributed to extracellular matrix-receptor interaction, fatty acids biosynthesis as well as glycine, serine, and threonine metabolism. The results highlight the importance of Dacer in the reproductive system of D. melanogaster and provide valuable resources to dig out the specific biological functions of Dacer in insect reproduction.

Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway.

Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual's lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSCself-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.

Normal fertility in male mice lacking ADAM32 with testis-specific expression

The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.

A threshold dosage of estrogen for male-to-female sex reversal in the Glandirana rugosa frog.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17β (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

Cdc14a has a role in spermatogenesis, sperm maturation and male fertility

Cdc14a is an evolutionarily conserved dual-specific protein phosphatase, and it plays different roles in different organisms. Cdc14a mutations in human have been reported to cause male infertility, while the specific role of Cdc14a in regulation of the male reproductive system remains elusive. In the present study, we established a knockout mouse model to study the function of Cdc14a in male reproductive system. Cdc14a−/− male mice were subfertile and they could only produce very few offspring. The number of sperm was decreased, the sperm motility was impaired, and the proportion of sperm with abnormal morphology was elevated in Cdc14a−/− mice. When we mated Cdc14a−/− male mice with wild-type (WT) female mice, fertilized eggs could be found in female fallopian tubes, however, the majority of these embryos died during development. Some empty spaces were observed in seminiferous tubule of Cdc14a−/− testes. Compared with WT male mice, the proportions of pachytene spermatocytes were increased and germ cells stained with γH2ax were decreased in Cdc14a−/− male mice, indicating that knockout of Cdc14a inhibited meiotic initiation. Subsequently, we analyzed the expression levels of some substrate proteins of Cdc14a, including Cdc25a, Wee1, and PR-Set7, and compared those with WT testes, in which the expression levels of these proteins were significantly increased in Cdc14a−/− testes. Our results revealed that Cdc14a−/− male mice are highly subfertile, and Cdc14a is essential for normal spermatogenesis and sperm function.

Heavy ion radiation-induced DNA damage mediates apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway in mouse spermatogonia.

The risk of exposure to ionizing radiation (IR) environments has increased with the development of nuclear technology. IR exposure induces excessive apoptosis of the spermatogonia, which leads to male infertility. Spermatogonia apoptosis may be involved in ribosomal stress triggered by DNA damage following exposure to IR because ribosomal proteins (RPs) directly interact with mouse double minute 2 homolog (MDM2) to induce apoptosis. This study aimed to use comparative proteomics and transcriptomics approach to screen the differential RPs and ribosomal mRNAs in mouse testes following high linear energy transfer (LET) carbon ion radiation (CIR). The expression of ribosomal large subunit protein 27a (Rpl27a) decreased at both protein and mRNA levels in the spermatogonia in vivo. After 6h of CIR, the immunofluorescence signal of 8-oxo-dG and phosphorylated ataxia-telangiectasia-mutated protein (ATM)/histone H2Ax increased, but that of Rpl27a decreased in the spermatogonia of p53 wild-type and knockout mouse testes. Moreover, the nucleolin was scattered throughout the nucleoplasm after CIR. These results suggested that CIR-induced DNA damage might trigger ribosomal stress, and the reduction in the expression of Rpl27a was associated with DNA damage in the spermatogonia. Similarly, in vitro, the immunofluorescence signal of 8-oxo-dG increased in the GC-1cells after CIR. Moreover, the expression of Rpl27a was regulated by DNA damage because the co-transfection of ATM and Rpl27a or inhibition of ATM-treated CIR could restore the expression of Rpl27a. Furthermore, the reduction in the expression of Rpl27a led to weakened binding of E2F transcription factor 1 (E2F1) and p53 to MDM2, causing p53 activation and E2F1 degradation in p53 wild-type and knockdown GC-1cells. This study proposed that heavy ion radiation-induced DNA damage mediated spermatogonia apoptosis via the Rpl27a-Rpl5-MDM2-p53/E2F1 signaling pathway. The results provided the underlying molecular mechanisms of spermatogonia apoptosis following exposure to high LET radiation.

Single-cyst transcriptome analysis of Drosophila male germline stem cell lineage.

The Drosophila male germline stem cell (GSC) lineage provides a great model to understand stem cell maintenance, proliferation, differentiation and dedifferentiation. Here, we use the Drosophila GSC lineage to systematically analyze the transcriptome of discrete but continuously differentiating germline cysts. We first isolated single cysts at each recognizable stage from wild-type testes, which were subsequently applied for RNA-seq analyses. Our data delineate a high-resolution transcriptome atlas in the entire male GSC lineage: the most dramatic switch occurs from early to late spermatocyte, followed by the change from the mitotic spermatogonia to early meiotic spermatocyte. By contrast, the transit-amplifying spermatogonia cysts display similar transcriptomes, suggesting common molecular features among these stages, which may underlie their similar behavior during both differentiation and dedifferentiation processes. Finally, distinct differentiating germ cell cyst samples do not exhibit obvious dosage compensation of X-chromosomal genes, even considering the paucity of X-chromosomal gene expression during meiosis, which is different from somatic cells. Together, our single cyst-resolution, genome-wide transcriptional profile analyses provide an unprecedented resource to understand many questions in both germ cell biology and stem cell biology fields.

Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice

The blood-testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11-deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11-deficient testes, those from WT or Cldn11-deficient testes regenerated sperm in Cldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3/5 regenerated endogenous spermatogenesis in Cldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.