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Cell Structure and Function 2009Spermatogenesis in Drosophila commences with cell division of germline stem cells (GSCs) to produce male germline cells at the tip of the testis. However, molecular...
Spermatogenesis in Drosophila commences with cell division of germline stem cells (GSCs) to produce male germline cells at the tip of the testis. However, molecular mechanisms inducing division of male GSCs have not been reported. Insulin-like peptides are known to play an essential role in stimulation of proliferation and growth of somatic cells, and it has recently been reported that such peptides promote cell division in female Drosophila GSCs. However, their effects on male germline cells have not been characterized. We found that inhibition of insulin production and insulin signaling mutations resulted in decreased numbers of germline cells in Drosophila testes. GSC numbers were maintained in young mutant males, with a gradual decrease in abundance of GSCs with age. Furthermore, in mutants, fewer germline cysts originated from GSCs and a lower frequency of GSC division was seen. Insulin signaling was found to promote cell cycle progression of the male GSCs at the G(2)/M phase. The cell volume of spermatocytes increases up to 25 times before initiation of meiosis in Drosophila. We examined whether insulin signaling extrinsically induces the greatest cell growth in Drosophila diploid cells and found that spermatocyte growth was affected in mutants. The results indicate that in addition to its function in somatic cells, insulin signaling plays an essential role in cell proliferation and growth during male Drosophila gametogenesis and that sperm production is regulated by hormonal control via insulin-like peptides.
Topics: Animals; Cell Division; Drosophila; G2 Phase; Insulin; Male; Meiosis; Mutant Proteins; Signal Transduction; Spermatocytes; Spermatogenesis; Spermatozoa; Stem Cells
PubMed: 19384053
DOI: 10.1247/csf.08042 -
International Journal of Molecular... Aug 2021Spermatogenesis is a complicated process involving mitotically proliferating spermatogonial cells, meiotically dividing spermatocytes, and spermatid going through...
Spermatogenesis is a complicated process involving mitotically proliferating spermatogonial cells, meiotically dividing spermatocytes, and spermatid going through maturation into spermatozoa. The post-translational modifications of proteins play important roles in this biological process. -palmitoylation is one type of protein modifications catalyzed by zinc finger Asp-His-His-Cys (ZDHHC)-family palmitoyl -acyltransferases. There are 23 mammalian ZDHHCs that have been identified in mouse. Among them, is highly expressed in adult testis. However, the in vivo function of in mouse spermatogenesis and fertility remains unknown. In this study, we knocked out the gene by generating a 2609 bp deletion from exon 3 to exon 6 in mice. No differences were found in testis morphology and testis/body weight ratios upon deletion. Spermatogenesis was not disrupted in knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules. Nevertheless, knockout mice were male infertile. deficient spermatozoa exhibited multiple defects including abnormal morphology of sperm tails and heads, decreased motility, and disturbed acrosome reaction. All of these led to the inability of mutant sperm to fertilize oocytes in IVF assays. Taken together, our results support the fact that is a testis enriched gene dispensable for spermatogenesis, but is essential for sperm functions in mice.
Topics: Acrosome Reaction; Acyltransferases; Animals; Female; Fertilization; Male; Mice; Mice, Knockout; Sperm Motility; Spermatocytes; Spermatogenesis; Spermatozoa
PubMed: 34445597
DOI: 10.3390/ijms22168894 -
Genesis (New York, N.Y. : 2000) Dec 2014Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in...
Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events.
Topics: Animals; DNA Methylation; Female; Green Fluorescent Proteins; HSP70 Heat-Shock Proteins; Male; Meiosis; Mice; Mice, Transgenic; Promoter Regions, Genetic; Recombinant Proteins; Spermatids; Spermatocytes
PubMed: 25293348
DOI: 10.1002/dvg.22830 -
PLoS Genetics May 2019Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique...
Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. We find that two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. We identify two RNA-binding proteins that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. We propose that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.
Topics: Animals; DNA, Satellite; Drosophila Proteins; Drosophila melanogaster; Exons; Gene Expression Regulation, Developmental; Introns; Male; RNA-Binding Proteins; Signal Transduction; Spermatocytes; Spermatogenesis; Transcription, Genetic; Y Chromosome
PubMed: 31071079
DOI: 10.1371/journal.pgen.1008028 -
Development (Cambridge, England) Nov 2023During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed...
During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.
Topics: Animals; Male; Meiosis; Spermatogenesis; Prophase; Mitosis; Spermatocytes; Drosophila; Cyclin B; Drosophila Proteins
PubMed: 37882771
DOI: 10.1242/dev.201709 -
Journal of Anatomy Jul 2015Spermatogenesis is a series of complex processes to generate mature sperm, and various molecules play crucial roles in regulating these processes. Previous studies imply...
Spermatogenesis is a series of complex processes to generate mature sperm, and various molecules play crucial roles in regulating these processes. Previous studies imply a possibility that a transcriptional factor Pax6, a key player of brain and sensory organ development, could be involved in spermatogenesis, but neither expression nor function of Pax6 in the adult testis has been examined yet. In the present study, we described for the first time Pax6 expression dynamics in the adult mouse testis. Using cell-type-specific markers, the expression of Pax6 was detected in 67.0% of promyelocytic leukemia zinc finger (Plzf)-positive type A spermatogonia. The expression of Pax6 was also observed in p63-positive spermatocytes and round spermatids. We did not detect any expression of Pax6 in Sox9-positive Sertoli cells or in elongated spermatids and mature sperm. High-resolution analyses revealed that Pax6 formed a single dot-like structure during mid-phase of the pachytene spermatocyte. This dot-like structure co-localized with γH2A.X demarcating XY body, a domain in which X and Y chromosomes are silenced and compartmentalized. These results may suggest a novel role of Pax6 in spermatogenesis.
Topics: Animals; Eye Proteins; Homeodomain Proteins; Male; Mice; Mice, Inbred C57BL; PAX6 Transcription Factor; Paired Box Transcription Factors; Repressor Proteins; Sertoli Cells; Spermatocytes; Spermatogenesis; Spermatogonia; Testis
PubMed: 26032914
DOI: 10.1111/joa.12318 -
Environmental Toxicology Mar 2017Di(2-ethylhexyl) phthalate (DEHP), a plasticizer of synthetic polymers, is a well-known endocrine disrupting chemical (EDC) and reproductive toxicant. Addressing the...
Di(2-ethylhexyl) phthalate (DEHP), a plasticizer of synthetic polymers, is a well-known endocrine disrupting chemical (EDC) and reproductive toxicant. Addressing the unclear mechanism of DEHP-induced reproductive dysfunction, this study used GC-2spd cells to investigate the molecular mechanism involved in the DEHP-induced toxicity in the male reproductive system. The results indicated that the apoptotic cell death was significantly induced by DEHP exposure over 100 μM. Furthermore, DEHP treatment could induce oxidative stress in GC-2spd cells involving in the decrease of superoxide dismutase (SOD) activity (200 μM) and glutathione peroxidase (GSH-Px) activity (50 and 100 μM). In addition, DEHP induction also caused the elevated ratios of Bax/Bcl-2, release of cytochrome c and decomposition of procaspase-3 and procaspase-9 in GC-2spd cells. Taken together, our work provided the evidence that DEHP exposure might induce apoptosis of GC-2spd cells via mitochondria pathway mediated by oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1055-1064, 2017.
Topics: Animals; Apoptosis; Caspase 3; Caspase 9; Cells, Cultured; Diethylhexyl Phthalate; Endocrine Disruptors; Male; Mitochondria; Oxidative Stress; Plasticizers; Signal Transduction; Spermatocytes
PubMed: 27416487
DOI: 10.1002/tox.22304 -
PloS One 2015Evidence from previous studies suggests that the male reproductive system can be disrupted by fetal or neonatal exposure to diethylstilbestrol (DES). However, the...
Evidence from previous studies suggests that the male reproductive system can be disrupted by fetal or neonatal exposure to diethylstilbestrol (DES). However, the molecular basis for this effect remains unclear. To evaluate the effects of DES on mouse spermatocytes and to explore its potential mechanism of action, the levels of DNA methyltransferases (DNMTs) and DNA methylation induced by DES were detected. The results showed that low doses of DES inhibited cell proliferation and cell cycle progression and induced apoptosis in GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. Furthermore, global DNA methylation levels were increased and the expression levels of DNMTs were altered in DES-treated GC-2 cells. A total of 141 differentially methylated DNA sites were detected by microarray analysis. Rxra, an important component of the retinoic acid signaling pathway, and mybph, a RhoA pathway-related protein, were found to be hypermethylated, and Prkcd, an apoptosis-related protein, was hypomethylated. These results showed that low-dose DES was toxic to spermatocytes and that DNMT expression and DNA methylation were altered in DES-exposed cells. Taken together, these data demonstrate that DNA methylation likely plays an important role in mediating DES-induced spermatocyte toxicity in vitro.
Topics: Animals; Apoptosis; Carcinogens; Cell Cycle; Cell Line, Transformed; Cell Proliferation; Cell Survival; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Diethylstilbestrol; Gene Expression Profiling; Gene Expression Regulation; Male; Mice; Microarray Analysis; Molecular Sequence Annotation; Myosins; Protein Kinase C-delta; Retinoid X Receptor alpha; Spermatocytes; DNA Methyltransferase 3B
PubMed: 26588706
DOI: 10.1371/journal.pone.0143143 -
BMC Cell Biology Aug 2007Loopin-1 is an abundant, male germ line specific protein of Drosophila melanogaster. The polyclonal antibody T53-F1 specifically recognizes Loopin-1 and enables its...
BACKGROUND
Loopin-1 is an abundant, male germ line specific protein of Drosophila melanogaster. The polyclonal antibody T53-F1 specifically recognizes Loopin-1 and enables its visualization on the Y-chromosome lampbrush-like loop named kl-3 during primary spermatocyte development, as well as on sperm tails. In order to test lampbrush-like loop evolutionary conservation, extensive phase-contrast microscopy and immunostaining with T53-F1 antibody was performed in other drosophilids scattered along their genealogical tree.
RESULTS
In the male germ line of all species tested there are cells showing giant nuclei and intranuclear structures similar to those of Drosophila melanogaster primary spermatocytes. Moreover, the antibody T53-F1 recognizes intranuclear structures in primary spermatocytes of all drosophilids analyzed. Interestingly, the extent and conformation of the staining pattern is species-specific. In addition, the intense staining of sperm tails in all species suggests that the terminal localization of Loopin-1 and its orthologues is conserved. A comparison of these cytological data and the data coming from the literature about sperm length, amount of sperm tail entering the egg during fertilization, shape and extent of both loops and primary spermatocyte nuclei, seems to exclude direct relationships among these parameters.
CONCLUSION
Taken together, the data reported strongly suggest that lampbrush-like loops are a conserved feature of primary spermatocyte nuclei in many, if not all, drosophilids. Moreover, the conserved pattern of the T53-F1 immunostaining indicates that a Loopin-1-like protein is present in all the species analyzed, whose localization on lampbrush-like loops and sperm tails during spermatogenesis is evolutionary conserved.
Topics: Animals; Antibodies; Biological Evolution; Drosophila Proteins; Drosophila melanogaster; Drosophilidae; Immunohistochemistry; Male; Nuclear Proteins; Spermatocytes; Spermatogenesis; Y Chromosome
PubMed: 17697358
DOI: 10.1186/1471-2121-8-35 -
Andrology Mar 2021MicroRNAs play a crucial role in the regulation of spermatogenesis. For example, miR-128-3p expression is known to decrease significantly after testicular hyperthermia,...
BACKGROUND
MicroRNAs play a crucial role in the regulation of spermatogenesis. For example, miR-128-3p expression is known to decrease significantly after testicular hyperthermia, but the regulatory effect of this change on the spermatogenesis damage caused by heat stress remains unclear.
OBJECTIVES
This study aimed to verify whether the target gene of miR-128-3p is MAPK14, which affects spermatogenic cell proliferation and apoptosis under testicular hyperthermia.
MATERIALS AND METHODS
Mouse testis and GC2 spermatocyte cell line heat stress models were established. miR-128-3p expression before and after heat stress was analyzed by reverse transcription polymerase chain reaction. MAPK14 and p-MAPK14 expression was detected by Western blot, and cell apoptosis was analyzed by Annexin V-FITC/PI. Subsequently, miR-128-3p inhibitors and mimics were used to interfere with spermatocytes before and after heat stress, respectively, for correlation detection.
RESULTS
Compared with the control group, the heat stress group showed decreased miR-128-3p expression, increased p-MAPK14 expression, and decreased cell proliferation activity. In the GC2-spd cell line in vitro, miR-128-3p inhibitors were found to upregulate p-MAPK14 expression, reduce cell proliferation activity, and increase apoptosis, consistent with the results obtained in the heat treatment alone. Furthermore, miR-128-3p mimics transfected in the GC2 cells after heat stress reduced p-MAPK14 expression, alleviated the decrease in cell proliferation, and decreased the apoptosis level.
CONCLUSIONS
The downregulation of miR-128-3p expression plays an important role in spermatogenesis damages after testicular hyperthermia, which is probably attributable to the activation of the MAPK signaling pathway. Downregulated miR-128-3p expression induces the apoptosis and inhibits the proliferation of spermatogenic cells by promoting MAPK14 phosphorylation.
Topics: Animals; Apoptosis; Cell Line; Enzyme Activation; Gene Expression Regulation, Developmental; Heat-Shock Response; Male; Mice; Mice, Inbred ICR; MicroRNAs; Mitogen-Activated Protein Kinase 14; Spermatocytes; Spermatogenesis; Testis
PubMed: 33089633
DOI: 10.1111/andr.12923