Postmeiotic Male Germ Cells Research Articles

Characterization of the testes-specific pim-1 transcript in rat.

The pim-1 proto-oncogene encodes a serine/threonine protein kinase and is expressed in cells of hematolymphoid origin and in the germ cell lineages. In somatic cells, the pim-1 gene is expressed as a 2.8 kb transcript while a shorter sized transcript (2.3 kb) is expressed in rat testes. We have determined that the shorter testes-specific pim-1 transcript arises through the use of an alternate polyadenylation signal present in the 3' untranslated region of the gene. This alternate polyadenylation event results in the removal of an A/U-rich regulatory element located in the 3' untranslated region of the pim-1 gene. This A/U-rich motif has been shown by a number of laboratories to destabilize the transcripts of genes that contain this sequence. Consistent with these findings, we have demonstrated that the shortened testes-specific pim-1 transcript is more stable than the longer A/U-rich containing somatic transcript. We suggest that the functional significance of different sized pim-1 transcripts may be directly related to their different stabilities and that the greater stability of the testes-specific transcript may be essential for the translational delay observed in post-meiotic male germ cells.

Validation studies with the micronucleus test for early spermatids of rats. A tool for detecting clastogenicity of chemicals in differentiating spermatogonia and spermatocytes

Male Wistar rats were given a single i.p. injection with different doses of ethylnitrosourea, mitomycin C, methyl methanesulphonate, cyclophosphamide or vincristine sulphate. Clastogenic damage induced in differentiating spermatogonia and spermatocytes was measured by counting micronuclei in derived early spermatids. At dose levels not resulting in cell death of resting spermatocytes, all chemicals--with the exception of vincristine--induced most of the damage in G1- and S-phase of primary spermatocytes (also called resting, pre-leptotene or pre-meiotic spermatocytes). However, at doses causing death of G1- and S-phase spermatocytes, high frequencies of micronuclei may be observed in early spermatids derived from spermatocytes treated in diplotene, diakinesis and MI and II. This is exemplified by our results with ethylnitrosourea. In our experience, the most sensitive stage of primary spermatocyte development (i.e. G1- and S-phase cells) can best be sampled 20 days after treatment. This is the optimal time interval for demonstrating the clastogenic potential of low or moderate doses of a test chemical in meiotic male germ cells of rats. The optimal sampling time for the detection of typical spindle poisons remains to be established. In general, at low or moderate dose levels, smaller or negligible amounts of chromosomal damage were induced in differentiating spermatogonia, in spermatocytes in meiotic prophase and in dividing primary or secondary spermatocytes. For obvious reasons, the micronucleus test for early spermatids cannot be used to detect clastogens which act exclusively on postmeiotic male germ cells.

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.

Allele- and haploid-specific product generated by alternative splicing from a mouse t complex responder locus candidate

Mouse t haplotypes represent a variant form of chromosome 17 that has evolved the ability to propagate through natural populations by the phenomenon of 'transmission ratio distortion' (TRD), in which heterozygous +/t males transmit their t-carrying chromosome to 95% or more of their offspring. Although multiple t-associated loci have a role in expression of this phenotype, only one--the t complex responder (Tcr) locus--is responsible for determining which of the two homologues of chromosome 17 will be transmitted at a high ratio. A candidate gene (Tcp-10b) for Tcr that is expressed in both meiotic and post-meiotic male germ cells has been cloned. But for this candidate gene to function as the haploid effector of TRD, the t-allele of this gene (Tcp-10bt) must express a unique product in a haploid-specific manner. Here we show that a change in the splicing pattern of Tcp-10bt transcripts occurs during sperm differentiation. This change results in a unique allele-specific and haploid-specific transcript which could encode a variant polypeptide that would fulfil the conditions required of the Tcr effector of TRD.

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

Expression of the proto-oncogene int-1 is restricted to postmeiotic male germ cells and the neural tube of mid-gestational embryos

The int-1 proto-oncogene is transcriptionally activated in mammary tumors by mouse mammary tumor virus insertion mutations and is normally expressed only in adult mouse testes and mid-gestational embryos. We have used anatomical dissection of embryos, germcell fractionation, peripuberal expression studies, and spermatogenesis mutants to identify more precisely the tissues and cells that contain int-1 RNA. In the testis, int-1 RNA is detected only in postmeiotic germ cells undergoing differentiation from round spermatids into mature spermatozoa. In embryos 11–15 days after conception, expression of the gene is restricted to the developing central nervous system in regions of the neural tube other than the telencephalon. Our findings suggest that int-1 mediates developmental events at these two sites.

Chromosome-type aberrations in postmeiotic male germ cells of mice administered trimethylphosphate

Chromosome-type aberrations in postmeiotic male germ cells of mice administered trimethylphosphate

The role of feeding rejection in Drosophila mutation assays

The frequencies of sex-linked recessive lethal mutations recovered in the male post-meiotic germ cells of Drosophila after feeding on solutions containing a mutagen (either 1 mM methyl methanesulfonate, MMS or 2 mM ethyl methanesulfonate, EMS) and 5-bromo-2-deoxyuridine (BUdR) were significantly lower than the frequencies observed after treatment with the mutagen alone. In an attempt to explain the apparent ‘protective’ effect of BUdR, the feeding behavior of the flies was monitored for differences in the uptake of the mutagen-containing solution in the presence and absence of BUdR. This was accomplished by measuring the uptake of [ 14C]sucrose. The results indicated that the uptake of the feeding solution is inhibited by the presence of the selected concentration of BUdR (1.0 or 32.5 mM). Such a reduced uptake of the mutagen could alone account for the reduction in mutational yields noticed in treatments containing mutagen + BudR compared to the ones with the mutagen alone. These results emphasize the need to monitor the feeding behavior of flies in experiments involving adult feeding.

Studies on mutagen-sensitive strains of Drosophila melanogaster: IV. Modificaiton of genetic damage induced by X-irradiation of spermatozoa and spermatids in N 2 or O 2 by mei-9a, mei-41 D5 and mus(1)101D1

The influence of defects in DNA repair processes on X-ray-induced genetic damage in post-meiotic male germ cell stages of Drosophila melanogaster was studied using the ‘maternal effects approach’. Basc males were irradiated in N 2, air or O 2 either as 48-h-old pupae (to sample spermatids) or as 3–4-day-old adults (to sample mature spermatozoa) and mated to females of 3 repair-deficient strains ( mei-9 a: excision-repair-deficient; mei-41 D5 : post-replication-repair-deficient; mus(1)101 D1: post-replication-repair-deficient and impaired in DNA synthesis). Simultaneous controls involving mating of males to repair-proficient females ( mei +) were run. The frequencies of sex-linked recessive lethals and of autosomal translocations were determined following standard genetic procedures. The responses elicited in the different crosses with repair-deficient females were compared with those in mei + crosses. The main findings are the following: (1) with mei-9 females, the frequencies of recessive lethals are higher after irradiation of spermatids in N 2, but not after irradiation in air of O 2 (relative to those in the mei + crosses); this result is different from that obtained in earlier work with spermatozoa, in which cell stage, higher yields of recessive lethals were obtained after irradiation of males in either N 2 or air; (ii) in the mei-9 crosses, there are no significant differences in response (relative to mei +) after irradiation of either spermatozoa or spermatids in O 2; (iii) the translocation frequencies in the mei-9 crosses are similar to those in the mei + crosses, irrespective of the treated germ cell stage or the irradiation atmosphere; (iv) irradiation of either spermatozoa or spermatids in N 2, air or O 2 does not result in any differential recovery of recessive lethals in the mei-41 relative to mei + crosses; (v) irradiation of spermatids in N 2 and of spermatozoa in air leads to a higher recovery of translocations in the mei-41 crosses; and (vi) after irradiation of spermatids or spermatozoa in any of the gaseous atmospheres, the frequencies of recessive lethals and of translocations are lower in the mus-101 crosses. The differences in responses (between cell stages, in different gaseous atmospheres and with different repair-deficient females) are explained on the basis of both qualitative and quantitative differences in the composition of the initial lesions and the extent to which their repair may be affected by the defects present in the different repair-deficient females. Several discrepancies between expectations based on biochemical results and the genetic results are pointed out. Thus it is made clear that not all the genetic results can be easily explained on the basis of the available biochemical information. On the basis of these and other data from experiments on maternal effects with the repair-deficient mutants, a scheme is presented. This scheme envisages the existence of at least 6 classes of qualitatively different types of initial lesions and of at least as many repair pathways through which these lesions are processed.

Formation of chromatid-type aberrations with procarbazine hydrochloride in postmeiotic male germ cells of mice

Incomplete combustion of kerosene heater, and fuel gas and liquefied petroleum gas-burner emissions produces indoor pollutants that may be carcinogenic. The incomplete-combustion products from each type of appliance were therefore collected by adsorption on about 3 g of XAD-2 resin, and were extracted with benzene—methanol as a solvent for determination and identification of mutagens in the Salmonella-microsome test system.Benzene—methanol extracts of the particulates generated by a heater and two burners showed extreme mutagenicity for strains TA97 and TA98 without S9 mix. Based on the results of analysis, a combination of high performance liquid chromatography (h.p.l.c.) and gas chromatography (GC), about 40–80% of the direct-acting mutagenicity in each crude extract showed the same h.p.l.c. and GC retention times as dinitropyrenes (1,3-, 1,6- and 1,8-isomers), and 1-nitropyrene. Moreover, other nitroarenes, 2-nitrofluorene, 1,5- and 1,8-dinitronaphthalene, and 4,4′-dinitrobiphenyl, were detectable in almost all samples, but their contribution to the mutagenicity of each extract was very low.Kerosene heaters were found to generate small amounts (0.2 ng/h) of dinitropyrenes, which are potential mutagens/carcinogens, only after 1 h of operation.

Studies on mutagen-sensitive strains of Drosophila melanogaster III. A comparison of the mutagenic sensitivities of the ebony (UV and X-ray sensitive) and Canton-S (wild-type) strains to MMS, ENU, DEB, DEN and 2,4,6-Cl 3-PDMT

The sensitivity of male germ cells of ebony (UV and X-ray sensitive) and Canton-S (wild-type) flies to the induction of sex-linked recessive lethals by 3 direct-acting mutagens (methyl methanesulphonate (MMS), N-ethyl- N-nitrosourea (ENU) and 1,2:3,4-diepoxybutane (DEB) and 2 chemicals that require metabolic activation ( N-nitrosodiethylamine (DEN) and 1-(2,4,6-trichlorophenyl)-3-3-dimethyltriazene (2,4,6-CI 3-PDMT) was investigated. In addition, experiments were carried out to examine whether mutational lesions induced by MMS, ENU and DEN in mature spermatozoa of males were processed differently by the oocytes of ebony and Canton-S flies (maternal effect studies). With all 3 direct-acting mutagens, the mutational responses of the post-meiotic male germ cells of the ebony and Canton-S flies were similar. After DEN treatment however, the ebony males responded with significantly lower frequencies of sex-linked recessive lethals in their pre- and post-meiotic germ cells. Measurements of microsomal P-450 enzymes revealed no detectable differences between the 2 strains with respect to either total cytochrome P-450 content or benzo[ a]pyrene hydroxylation activity; the p-nitroanisole demethylation activity however, was lower in the ebony than in Canton-S flies. With 2,4,6-C1 3-PDMT, the spermatids of ebony males responded with higher frequencies of recessive lethals than those of Canton-S males. The maternal effect studies provided no evidence for any differential processing of mutational lesions in the Canton-S and ebony females. The contrasting responses observed with DEN and 2,4,6-C1 3-PDMT have been explained on the basis of the assumption that in the ebony flies, there is an increased bioactivation of the triazene and a less efficient activation of DEN to their respective mutagenic metabolites, relative to the situation obtained in the Canton-S flies.

Relationship between chromosome aberrations at the first cleavage metaphases and postimplantation loss in dominant lethal mutations induced by isopropyl methanesulfonate.

A high dose (200mg/kg) of isopropyl methanesulfonate (iPMS) induced a high frequency of postimplantation loss in postmeiotic male germ cell stages, but the frequency of eggs with chromosome aberrations observed at the first cleavage metaphases was very low. Therefore, we suggest that the postimplantation loss in dominant lethal mutations induced by iPMS is mainly attributable to the alteration of subchromosome material at the first cleavage metaphases.

Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[ a]pyrene in postmeiotic germ cells of male mice

Benzo[ a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatoza. In contrast to the dominant-lethal effects observed the study showed no detectable increase in hertiable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.

Difference in the ratio of dominant-lethal mutations to heritable translocations produced in mouse spermatids and fully mature sperm after treatment with triethylenemelamine (TEM).

The relative induction of dominant-lethal mutations and heritable translocations in triethylenemelamine-treated postmeiotic germ cells of mice was determined depending on the stage treated. Males were mated either 11.5-14.5 days after treatment (middle spermatids) or less than 2.5 hours after treatment (fully mature sperm). Results clearly showed that, even through similar levels of dominant-lethal mutations were induced in fully mature sperm and in middle spermatids, the frequency of heritable translocations induced in mature sperm was markedly lower than that induced in middle spermatids. This observation was used, together with earlier ones, to suggest a mechanism by which dominant-lethal mutations and heritable translocations are produced following chemical treatment of male postmeiotic germ cels.

The distribution of randomly recovered X-ray-induced sex-linked genetic effects in Drosophila melanogaster.

Cytogenetic analysis of more than 1500 randomly recovered lethal X chromosomes derived from 2000 and 3000 r X-ray exposures of post-meiotic male germ cells has made possible a plot of the distribution in different regions of the X chromosome of: (1) gene mutations associated with cytologically normal chromosomes, (2) mutations associated with chromosomal rearrangement breakpoints, (3) deficiencies, and (4) rearrangement breakpoints whether or not they are associated with mutations. The distribution of point mutations, vital loci and rearrangement breakpoints in different regions of the X chromosome is not proportional to either the number of bands or the relative DNA content. Further, the density of vital loci (those capable of mutating to a lethal allele) is quite different in some regions as compared to others. For example, vital loci in the 3AB region, which has been thoroughly studied by Judd and others, are at least as numerous as bands; whereas, the 3CD region, equally long, has only two vital loci. Other regions densely populated with vital loci include 1B, 1F-2A, 10A, 11A, and 19EF; sparsely populated regions include 6EF and 10B-10E. It seems reasonable to conclude that the recovered X-ray-induced mutants available for analysis do not represent a random sample of those initially induced in the exposed male germ cells.

Mutagenicity of acrylonitrile in mouse

The capacity of acrylonitrile (ACN) to produce chromosome aberrations in mammals was studied in somatic and germ cells of male NMRI mice. Induction of chromosome aberrations was followed in bone marrow cells 6, 18, 24, 48 and 72 h after an i.p. injection of 20 or 30 mg/kg acrylonitrile, and polychromatic erythrocytes were examined for the presence of micronuclei 24, 30 and 48 h after injection. The dominant lethal test served to detect chromosome aberrations in meiotic and postmeiotic male germ cells. All the tests utilized yielded negative results, so that it may be concluded that acute treatment with acrylonitrile has no clastogenic effects on male mouse cells in vivo.

Decrease of gamma-ray-induced mutations by visible light in Drosophila.

The effects of visible light post-treatment on the spectrum and frequencies of gamma-ray-induced mutations at five specific loci and of macrodeletions of the X chromosome in post-meiotic male germ cells in mutant c(3)G17 of Drosophila melanogaster were studied. It was found that post-treatment by light decreased the yield of chromosome rearrangements to the extent of 1.5 to 2 times, but not that of point mutations.

Repair in fertilized eggs of mice and its role in the production of chromosomal aberrations.

The fertilized egg may influence the yield of dominant-lethal mutations produced from chemical treatment of male postmeiotic germ cells to a small or large extent depending upon the mutagen used and the competence of the egg to repair the premutational lesions induced. The strain of females has little influence on the yield of dominant-lethal mutations induced by triethylenemelamine or ethyl methane-sulfonate in spermatids and spermatozoa, but it has a large influence in the case of isopropyl methanesulfonate. In addition to this difference, triethylenemelamine and ethyl methanesulfonate induce high levels of heritable translocations at these germ cell stages whereas isopropyl methanesulfonate is practically ineffective, even though doses of these chemicals produced comparable levels of dominant-lethal mutations. These differences between ethyl methanesulfonate and triethylenemelamine on one hand and isopropyl methanesulfonate on the other were hypothesized to be a function of the types of chromosomal lesions present at the time of repair activity and whether or not chromosomal aberrations were already fixed at the time of postfertilization pronuclear DNA synthesis.

RELATIVE RATES AT WHICH DOMINANT-LETHAL MUTATIONS AND HERITABLE TRANSLOCATIONS ARE INDUCED BY ALKYLATING CHEMICALS IN POSTMEIOTIC MALE GERM CELLS OF MICE

There is a close relationship between the rates at which dominant lethal mutations and heritable translocations are induced by ethyl methanesulfonate (EMS) or triethylenemelamine (TEM) in male postmeiotic germ cells. This relationship does not hold for isopropyl methanesulfonate (IMS), which induced only negligible frequencies of heritable translocations at doses that induced high levels of dominant lethal mutations. Nor does IMS behave like EMS and TEM in the degree to which eggs of different stocks of females repair premutational lesions that are carried in the sperm-large differences between stocks for IMS treatment and small differences for EMS or TEM treatment. These dissimilarities between IMS and the other two alkylating chemicals are postulated to be attributable to differences in the types of lesions present at the time of repair activity and to whether or not chromosomal aberrations are already fixed prior to postfertilization pronuclear DNA synthesis.