Mouse Male Germ Cells Research Articles

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Methylation of the cyclin A1 promoter correlates with gene silencing in somatic cell lines, while tissue-specific expression of cyclin A1 is methylation independent.

Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

The mouse homolog of Drosophila Vasa is required for the development of male germ cells

Restricted expression of a mouse Vasa homolog gene (Mvh) expression is first detected in primordial germ cells (PGCs) after colonization of the genital ridges. Subsequently, Mvh is maintained until postmeiotic germ cells are formed. Here, we demonstrate that male mice homozygous for a targeted mutation of Mvh exhibit a reproductive deficiency. Male homozygotes produce no sperm in the testes, where premeiotic germ cells cease differentiation by the zygotene stage and undergo apoptotic death. In addition, the proliferation of PGCs that colonize homozygous male gonads is significantly hampered, and OCT-3/4 expression appears to be reduced. These results indicate that the loss of Mvh function causes a deficiency in the proliferation and differentiation of mouse male germ cells.

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.

Trichlorfon induces spindle disturbances in V79 cells and aneuploidy in male mouse germ cells.

In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40-120 microg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 microg/ml trichlorfon, whereas at 120 microg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.

Two distinct forms of the 64,000 Mr protein of the cleavage stimulation factor are expressed in mouse male germ cells.

Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3' ends but are efficiently polyadenylated. To determine whether the 64,000 Mr protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000 Mr form, we found a related approximately 70,000 Mr protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the approximately 70,000 Mr CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000 Mr form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000 Mr CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene.

Acrylamide causes preimplantation abnormalities in embryos and induces chromatin-adducts in male germ cells of mice

Acrylamide, a known male postmeiotic germ cell mutagen, caused a dose-dependent increase in the frequency of morphologic abnormalities in preimplantation embryos. Single-cell eggs, growth retardation, and blastomere lysis were detected after paternal treatment with acrylamide (10 to 50 mg/kg, 5 d). The major effects were seen at weeks 1 to 3 after male treatment, with the highest level of abnormalities at the first week (>90% vs. 5% in control). The frequency of abnormal four-day embryos was similar to preimplantation loss assessed at 15 to 16 d p.c. A >100-fold elevation of chromatin adducts in sperm was observed during 1st and 2nd week after treatment, after which adduct levels decreased to baseline level. However, morphologic defects in embryos are not fully explained by the spermatid adduct curve. These findings demonstrate the effects of paternal exposure to acrylamide on preimplantation development and indicate a potential risk to the offspring of men exposed to acrylamide.

Griseofulvin-induced aneuploidy and meiotic delay in male mouse germ cells: detected by using conventional cytogenetics and three-color FISH

Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). The frequencies of diploidies were 0.01–0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy.

Genomic structure and chromosomal localization of the mouse Hsf2 gene and promoter sequences.

The mouse heat shock factor 2 (HSF2) cDNA was previously cloned by homology to HSF1, the heat shock factor involved in the cellular response to stress [Sarge, K.D., Zimarino, V., Holm, K., Wu, C., Morimoto, R.I., Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5 (1991) 1902-1911]. HSF2 is active in restricted cell types during pre- and post-implantation stages of development, and only in male germ cells of adult mice. However, the function of this factor remains elusive. We report here the cloning of the mouse Hsf2 gene and its genomic structure. We show that the gene is composed of 13 exons of variable sizes spanning at least 43kb in the genome. The transcription start site has been determined, and upstream sequences with promoter activity have been identified by their ability to direct the expression of a luciferase reporter gene in transfected cells. A preliminary analysis of the proximal promoter sequence determined that the TATA box is absent, but that a GC-rich region with several potential binding sites for transcription factors is present. The gene has been mapped to mouse chromosome 10 by in-situ hybridization on metaphase chromosomes.

Role of heat shock protein HSP70-2 in spermatogenesis.

The HSP70 heat-shock proteins are molecular chaperones that assist other proteins in their folding, transport and assembly into complexes. Most of these proteins are either constitutively expressed or their expression is induced by heat shock and other stresses. However, two members of the Hsp70 family (HSP70-2 and HSC70T in mice) are regulated developmentally and expressed specifically in spermatogenic cells. The HSP70-2 protein is synthesized during the meiotic phase of spermatogenesis and is abundant in pachytene spermatocytes. The knockout approach was used to determine whether HSP70-2 is a chaperone for proteins involved in meiosis. Male mice lacking HSP70-2 were infertile while females lacking HSP70-2 were fertile. Spermatogenic cell development was arrested in prophase of meiosis I at the G2-M-phase transition and late pachytene spermatocytes were eliminated by apoptosis, resulting in an absence of spermatids. HSP70-2 is required for Cdc2 to form a heterodimer with cyclin B1, suggesting that it is a chaperone necessary for the progression of meiosis in the germ cells of male mice. HSP70-2 is also associated with the synaptonemal complex and desynapsis is disrupted in male mice lacking this protein. Homologues of HSP70-2 are present in the testes of many animals, suggesting that the role of this spermatogenic cell chaperone is conserved across phyla.

In vivo dominant lethal effect of pyrimethamine in male mouse germ cells.

Pyrimethamine is used for treatment of malaria and toxoplasmosis. The embryotoxicity and clastogenicity of pyrimethamine is known and our aim was to investigate its dominant lethal effect in vivo. For this purpose, we used three groups of Swiss-albino male mice and a control group. We injected males with doses of 16, 32 or 64 mg/kg pyrimethamine and housed them with 10 females/male for each mating interval. Females were sacrificed and their uteri were evaluated for dominant lethality. As a result of this study we found that pyrimethamine induced dominant lethal mutations in the third, fourth and sixth weeks at the 64 mg/kg dose level, without the effect being dose-dependent. We conclude that pyrimethamine is a suspected germ cell mutagen.

Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining

DNA double-strand break (DSB) processing was studied in mouse testicular extracts using a defined DSB created by cleaving supercoiled pUC12 DNA at a unique site as the substrate, and analysing the processed DNA by gel electrophoresis. Our results demonstrated that enzymatic activity in the extracts promoted multimerization of DNA and suppressed its circularization. This was distinctly different from T4 DNA ligase activity in the control and therefore the process must be more complex than simple ligation. Efficiency of this end-to-end joining was ATP and Mg2+-dependent and was much higher with cohesive (especially with 5′) than with blunt ends. On recleaving, the joining was predominantly faithful, especially for cohesive ends; but a detectable fraction of DNA had undergone end-processed joining causing junctional deletions, mostly with blunt ends. Redigestion of end-joined products from time course experiments established that the end-deleted joining occurred concurrent to the faithful joining. Junctional segments were cloned and their restriction analysis confirmed the presence of large deletions from both the sides. These results suggested the association of an end-processing activity (exonuclease/helicase+flap endonuclease) along with the end-joining ligase(s). Suppression of end-edited joining on lowering the reaction temperature to 17° or 14°C, despite efficient faithful joining, indicated that this enzymatic activity is retarded at low temperature. Though the efficiency and fidelity of joining were termini-dependent, the orientation of joining was random. Lack of preference for homologous ends (H:H or T:T), as well as efficient joining of heterologous DNA (pUC12/pBR322) having two different blunt termini, showed that the end joining could occur independent of extensive/terminal homology. Retention of radioactivity on end joining of (α-32P)dCTP end-filled cohesive termini, and lack of their junctional cleavability, apparently due to restriction site duplication, suggested direct double strand ligation. Thus it is demonstrated that mouse male germ cells possess an efficient DNA end-joining activity, involving either a major pathway of precise joining, or a minor end-deleted joining, and it seems to be achieved by a multienzymatic complex as suggested also for somatic cells by others. These results show that mammalian male germ cells that are proficient in homologous recombination utilize nonhomologous end-joining (NHEJ) mechanism for DSB processing and therefore NHEJ is a conserved, universal pathway for the vital function of DSB repair.

Changes in Lectin Binding Patterns of Mouse Male Germ Cells (Gonocytes) during Prespermatogenesis.

The distribution of sugar residues in gonocytes of the differentiating mouse testis was examined by light microscopy using 22 different kinds of lectins. Characteristic binding patterns of sWGA, VVA, and LEA in gonocytes were observed during prespermatogenesis. sWGA preferentially bound to the cytoplasm and plasma membrane of gonocytes on 16.5 days post coitus (dpc). Its reaction decreased thereafter and almost disappeared on 1.5 days post partum (dpp), but reaction reappeared on 4.5 dpp and continued until 6.5 dpp. The VVA reaction was recognized in a few gonocytes on 0.5 dpp, and remained strong until 6.5 dpp. LEA reacted strongly in the plasma membrane and cytoplasm of gonocytes from 0.5 dpp to 6.5 dpp. The present study indicates that sWGA, VVA, and LEA are useful markers for gonocytes, and the appearance or disappearance of sWGA and VVA may be related to the differentiation of gonocytes during prespermatogenesis.

Radiation sensitivity in male mouse germ cells

Radiation sensitivity in male mouse germ cells

Autonomous cell death of mouse male germ cells during fetal and postnatal period.

Germ cell degeneration is common in mammalian testes during the developmental as well as the adult period. To investigate the extent and mechanisms of male germ cell death during fetal and neonatal life, the testes of mice at various fetal and postnatal ages extending from 13 days of gestation to 7 wk after birth were examined by electron microscopy and/or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Electron microscopy revealed that the number of cells with typical features of spermatogenic cell apoptosis was highest at 13 days of gestation, coinciding with the time of immigration of primordial germ cells into gonads. A second peak was observed around 10-13 days after birth when the first wave of spermatogenesis had started and active spermatogonial proliferation was present. Surprisingly, we found a significant number of dying cells around birth, which exhibited morphological features of necrotic death. In agreement with the results of electron microscopy, TUNEL staining revealed that the dying germ cells present around birth were TUNEL negative, while positive nuclei were abundant in the lumen of seminiferous tubules of testes of 10- to 13-day-old mice. To investigate the mechanisms of induction of germ cell death, we examined the expression of Fas antigen immunohistochemically using rabbit antiserum raised against synthetic peptides for part of mouse Fas antigen. We found that among various developmental stages investigated, positive immunostaining for Fas antigen was present between 17 days of gestation and 1 day after birth, with the most intensive staining occurring on 17 days of gestation. Therefore, Fas-induced pathways may be implicated in embryonic male germ cell death, not prepubertal spermatogenic cell death.

Colcemid and econazole do not show aneugenicity in male mouse germ cells.

Colcemid (COM) and econazole (EZ) were tested for induction of mitotic arrest and C-mitotic effects in mouse bone marrow cells and for meiotic delay and induction of hyperploidy in mouse spermatocytes after single injection. Doses of 1 and 3 mg/kg COM were used and bone marrow and spermatocytes were sampled at 2, 6, 10, 14 and 18 h. For EZ a dose of 120 mg/kg and intervals of 6, 10, 14 and 18 h were chosen. At 2 h after COM treatment of bone marrow cells the mitotic index and C-mitotic effect were highest, then both decreased from 6 to 18 h. At 18 h the mitotic indices decreased to or significantly below the control level at doses of 1 and 3 mg/kg respectively, whereas the corresponding frequencies of C-mitotic cells were still significantly higher than in the controls. EZ also induced mitotic arrest and C-mitotic effects in mouse bone marrow cells. In contrast to COM, the effects of EZ were highest at 18 h after treatment. COM and EZ caused disturbances in progression from the first to second meiotic division, however, after COM treatment the ratios of MMII to MMI were significantly below the control. In contrast, after EZ treatment the ratios were significantly higher than in the controls. Such differences may result from the different mechanisms by which COM and EZ act on cell cycle progression. COM and EZ did not induce non-disjunction under the experimental conditions. However, EZ induced structural chromosomal aberrations.