Expression of homeobox genes during spermatogenesis.
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Amino Acid Sequence↗
Biomedical subjects
D J Wolgemuth
Publications and source records attributed to D J Wolgemuth.
Cell lineage specificity of expression of the murine transforming growth factor beta 3 and transforming growth factor beta 1 genes.
We have cloned a murine transforming growth factor (TGF) beta 3 complementary (cDNA) from normal tissue by low stringency screening of a testicular cDNA library with a TGF beta 1 probe. The coding domain of this TGF beta 3 cDNA agrees completely with the sequence reported for the TGF beta 3 cDNA isolated from the AKR-2B cell line, but the testicular clone uses a distinct and unusual polyadenylation signal resulting in an altered 3' untranslated domain. Northern blot hybridization analysis of gonadal tissues showed that both TGF beta 3 and TGF beta 1 mRNAs are detectable in the mouse testis and ovary. A detailed analysis of TGF beta 3 and TGF beta 1 gene expression in normal and germ cell-deficient male mice showed that the somatic cell compartment of the mouse testis expresses the usual-sized transcripts for both genes. However, a smaller (1.8-kilobase) TGF beta 1 mRNA is expressed selectively in male germ cells, and expression of this transcript was constitutive throughout the spermatogenic stages examined. This result demonstrates a new pattern of TGF beta 1 gene expression, consistent with cell lineage-specific transcriptional regulation during spermatogenesis.
Amino Acid Sequence↗
Characterization and inducibility of hsp 70 proteins in the male mouse germ line.
The properties and inducibility of the heat shock protein 70 (hsp 70) gene products were examined during differentiation of mouse testicular cells by one and two-dimensional gel electrophoresis and immunoblotting. Low levels of the 72- and 73-kD heat shock proteins normally found in mouse cell lines were detected in the mouse testis. A novel isoform with a relative molecular mass of 73 kD (called 73T) was also observed, in the presence or absence of heat shock. 73T was shown to be produced by germ cells since it was not detected in testes from mutant mice devoid of germ cells. Furthermore, 73T was found only in adult mouse testicular cells, not in testes from animals that lack meiotic germ cells. 73T was synthesized in enriched cell populations of both meiotic prophase and postmeiotic cells, but was not inducible by in vitro heat shock. In the adult testis, low levels of the bona fide 72-kD heat-inducible (hsp72) were induced in response to elevated temperatures. In contrast, in testes from animals in which only somatic cells and premeiotic germ cells were present, there was a substantial induction of hsp 72. It is suggested that hsp 72 is inducible in the somatic compartment and possibly in the premeiotic germ cells, but not in germ cells which have entered meiosis and which are expressing members of the hsp 70 gene family in a developmentally regulated fashion.
Animals↗
Identification of an IL-4-inducible gene expressed in differentiating lymphocytes and male germ cells.
Interleukin 4 (IL-4) is a cytokine that is involved in the differentiation of B and T lymphocytes. In this report, we describe the identification of a novel gene, N.52, which was cloned from the murine pre-B cell line R8205 grown in the presence of IL-4 for 48 hr. Although N.52 expression is detectable at low levels in unstimulated R8205 cells, the level of N.52 dramatically increases after only 4 hr exposure to IL-4 and remains at a high level up to 48 hr. Although N.52 expression is low or absent in normal spleen B and T cells, its expression can be induced by the differentiation signals delivered by LPS in B cells and by Con A in T-cell hybrids. While N.52 mRNA is absent in all highly differentiated organs, it is detectable in stem cell harboring lymphoid tissues such as bone marrow, fetal liver, and thymus. Furthermore, N.52 mRNA is expressed at strikingly high levels in the testis, specifically in differentiating male germ cells. It is induced by differentiation signals triggered by the combination of cyclic AMP and retinoic acid in teratocarcinoma F9 cells. Taken together, these data suggest that N.52 is a developmentally regulated gene whose expression in cells of the immune and reproductive systems may be controlled by stimuli that induce differentiation.
Amino Acid Sequence↗
Transgenic mice overexpressing the mouse homoeobox-containing gene Hox-1.4 exhibit abnormal gut development.
The mouse homoeobox-containing genes exhibit temporally and spatially specific patterns of expression in embryonic and adult tissues and are thought to be important in regulation of development and cellular differentiation, perhaps by mechanisms analogous to homoeotic genes in Drosophila melanogaster. There has been no direct demonstration that expression of these mammalian genes can affect developmental processes, however. Hox-1.4, like other mouse homoeobox-containing genes, has been shown to be expressed in specific regions of the mid-gestation embryo, but is unique in that its highest level of expression in the adult animal is restricted to developing male germ cells. We have introduced a construct carrying the mouse Hox-1.4 gene into the germ line of mice to begin to identify the cis-acting elements required for proper expression and to assess the consequences of increasing Hox-1.4 gene expression. The construct was designed to produce normal Hox-1.4 protein from transcripts that are distinguishable from the products of the endogenous gene. The integrated transgene seemed to exhibit the appropriate tissue specificity of expression, but transcript levels were elevated in certain tissues, particularly the embryonic gut. This overexpression correlated with changes in the normal developmental program of the gut, resulting in an inherited abnormal phenotype known as megacolon.
Animals↗
P150c-abl is detected in mouse male germ cells by an in vitro kinase assay and is associated with stage-specific phosphoproteins in haploid cells.
We have utilized c-abl antibodies and an in vitro kinase assay to identify the protein products of the c-abl gene in mouse testis. Although the testis contains high levels of a unique c-abl mRNA, along with lower amounts of two c-abl mRNAs common to somatic cells, we detected only a single polypeptide of approximately 150 kd, indistinguishable by our methods from P150c-abl observed in murine tissues and cell lines. P150c-abl was also detected in enriched populations of germ cells, including late stage spermatids which contain the highest levels of the novel c-abl transcript. In mature testis, and specifically in late spermatids, P150c-abl co-precipitated with phosphoproteins of approximately 74 kd which were labeled during the in vitro kinase assay. These proteins were phosphorylated predominantly on serine and their phosphopeptide maps differed from that of P150c-abl. The P74 phosphoproteins were not found in association with P150c-abl in germ cells at earlier developmental stages, nor in other tissues or cell lines examined.
Amino Acids↗
Evidence for the involvement of the proto-oncogene c-mos in mammalian meiotic maturation and possibly very early embryogenesis.
The c-mos proto-oncogene exists as a maternal mRNA in mammalian oocytes, in that it has been shown to accumulate in mouse oocytes during the growth phase and to be present at high levels in fully grown oocytes. The function of c-mos during the subsequent development of the oocytes and embryos was examined by determining the fate of the oocyte c-mos mRNAs by in situ hybridization and Northern blot hybridization analysis. A substantial decrease in the levels of c-mos transcripts was observed in oocytes undergoing meiotic maturation. By the two-cell stage, levels of c-mos transcripts dropped to below the limits of detection using in situ hybridization. c-mos transcripts remained undectable through the blastocyst stage of embryogenesis. Analysis of meiotic maturation in vitro permitted finer temporal resolution of the initial drop in c-mos levels. Between approximately 7 and 17 h of culture, the amount of c-mos mRNA fell to 18-43% of the levels found in the fully grown oocyte. This interval corresponds to the progression of meiotic maturation from metaphase I to metaphase II. Our in vivo studies showed that ovulation per se is not the stimulus for the drop in c-mos transcript levels, since preovulatory metaphase II oocytes exhibited this decline to a degree comparable to that of ovulated metaphase II oocytes. The development specificity of c-mos transcript levels suggests a role of this putative serine kinase in the meiotic maturation of mammalian germ cells.
Bucladesine↗
Translational regulation of the novel haploid-specific transcripts for the c-abl proto-oncogene and a member of the 70 kDa heat-shock protein gene family in the male germ line.
Expression of the c-abl proto-oncogene in the mouse testis is characterized by the production of a unique 4.7-kb transcript present in germ cells that have entered the haploid phase of spermatogenesis. A similar developmental stage specificity of expression is observed for a member of the 70-kDa heat-shock protein (hsp 70) gene family. A unique-sized hsp 70 transcript (T-hsp 70) is produced in haploid spermatids and is stable throughout spermatogenesis. In the present study, we examined the regulation of expression of these genes by examining their association with polyribosomes. The germ cell-specific c-abl and T-hsp 70 mRNA variants were both associated with the polysomal fractions of mouse testis cells, suggesting that they are functional mRNAs. However, both c-abl and T-hsp 70 mRNAs were also found in the ribonucleoprotein particle fractions. The distribution of these mRNAs in both the polysomal and nonpolysomal fractions is comparable to that seen for the mRNA of protamine-1, a gene whose expression in the testis is known to be regulated at the level of translation. In contrast, transcripts from the beta-tubulin gene were seen predominantly in the polyribosomal fractions. These findings suggest that translation of the novel c-abl and T-hsp 70 transcripts is confined to subpopulations of testicular cells.
Animals↗
Identification and sequence analysis of a new member of the mouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line.
A unique member of the mouse HSP70 gene family has been isolated and characterized with respect to its DNA sequence organization and expression. The gene contains extensive similarity to a heat shock-inducible HSP70 gene within the coding region but diverges in both 3' and 5' nontranslated regions. The gene does not yield transcripts in response to heat shock in mouse L cells. Rather, the gene appears to be activated uniquely in the male germ line. Analysis of RNA from different developmental stages and from enriched populations of spermatogenic cells revealed that this gene is expressed during the prophase stage of meiosis. A transcript different in size from the major heat-inducible mouse transcripts is most abundant in meiotic prophase spermatocytes and decreases in abundance in postmeiotic stages of spermatogenesis. This pattern of expression is distinct from that observed for another member of this gene family, which was previously shown to be expressed abundantly in postmeiotic germ cells. These observations suggest that specific HSP70 gene family members play distinct roles in the differentiation of the germ cell lineage in mammals.
Animals↗
Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus laevis egg extracts.
An in vitro sperm activation system was used to study nuclear swelling-chromatin decondensation and DNA synthesis; processes that occur in vivo following fertilization. Lysolecithin-permeabilized human sperm were incubated in Xenopus laevis egg extract and examined by using phase-contrast light microscopy, electron microscopy, and autoradiography. During a 3-hour incubation, the activated sperm nuclear chromatin underwent a decondensation-recondensation cycle during which DNA was synthesized. This also occurred when egg extract was given a 3-hour preincubation before the addition of the sperm, suggesting that the factor(s) required for initiating the decondensation-recondensation cycle is associated with the sperm. Because both nuclear swelling and DNA synthesis were found to be reproducible and quantifiable, we studied the effect of various agents on the two processes, characterizing the critical component(s) in the egg extract that induces these events. EGTA was found to have no effect on the induced nuclear swelling or DNA synthesis that occurs in the activated sperm. Freezing and thawing the extract or treating the extract with aphidicolin also had no effect on subsequent nuclear swelling; however, the DNA synthesis activity was blocked. Sperm incubated in extract treated with alkaline phosphatase (AP) had both nuclear swelling and DNA synthesis blocked. However, if the sperm were pretreated with DTT, and then incubated with the AP-treated extract, only the DNA synthesis activity of the extract was blocked. When the extract was treated with serine protease inhibitors (PMSF, soybean trypsin inhibitor, or alpha-2-macroglobulin), nuclear swelling occurred; however, DNA synthesis was blocked. These data suggest that phosphoproteins are involved in one or more of the activation events and that a serine protease(s) is involved in the synthesis of DNA.
Alkaline Phosphatase↗
Distinct developmental patterns of c-mos protooncogene expression in female and male mouse germ cells.
The protooncogene c-mos is expressed in murine reproductive tissues, producing transcripts of 1.7 and 1.4 kilobases in testis and ovary, respectively. In situ hybridization analysis of c-mos expression in histological sections of mouse ovaries revealed that oocytes are the predominant if not exclusive source of c-mos transcripts. c-mos transcripts accumulate in growing oocytes, increasing 40- to 90-fold during oocyte and follicular development. c-mos transcripts were also detected in male germ cells and are most abundant after the cells have entered the haploid stage of spermatogenesis. This developmentally regulated pattern of c-mos expression in oocytes and spermatogenic cells suggests that the c-mos gene product may have a function in normal germ-cell differentiation or early embryogenesis.
Animals↗
Differential expression of the mouse homeobox-containing gene Hox-1.4 during male germ cell differentiation and embryonic development.
Hox-1.4 is a mouse homeobox-containing gene (initially identified as HBT-1), whose expression appears to be testis-specific in the adult animal. Examination of Hox-1.4 transcripts in RNA from testes of mutant mice deficient in germ cells confirms that Hox-1.4 expression within the testis is germ cell-specific. Enriched populations of spermatogenic cells were used to localize the expression of Hox-1.4 specifically to germ cells that have entered into and progressed beyond the meiotic prophase stage of differentiation and to demonstrate the presence of two different size Hox-1.4 transcripts. Examination of RNA from teratocarcinoma cell cultures and mouse embryos at 10.5-16.5 days of gestation demonstrated the presence of several Hox-1.4 transcripts, which are larger than those present in germ cells. In the midgestation fetus, Hox-1.4 expression is most abundant in the spinal cord.
Alleles↗
Expression of the proopiomelanocortin gene is developmentally regulated and affected by germ cells in the male mouse reproductive system.
Proopiomelanocortin (POMC), a major pituitary product, is also present in the adult mouse testis. We have shown previously that POMC mRNAs are most abundant in a subpopulation of Leydig cells associated with tubules in specific stages of the cycle of the seminiferous epithelium. In the present study, we examined the expression of the gene encoding POMC during testicular development and in other tissues of the male reproductive system. We also analyzed the effects of cellular interactions on POMC gene expression in the testis. Blot-hybridization analysis revealed that POMC transcripts of approximately equal to 800 nucleotides were present in enriched populations of meiotic prophase spermatocytes and in caput epididymis but were absent in cauda epididymis and vas deferens. POMC transcripts were present in fetal testis (day 17 of gestation to newborn), could not be detected in prepuberal testis (days 7-8 postpartum), but reappeared in the adult testis. No difference in the size or abundance of POMC transcripts was seen in testes from mouse mutant strains in which spermatogenesis is arrested in early spermiogenesis. In contrast, POMC transcripts were virtually undetectable in testes that are devoid of germ cells. These results emphasize the importance of interactions between germ cells and interstitial cells and the regulation of the POMC gene in the mammalian testis.
Animals↗
Differential expression in murine somatic and germinal tissues of transcripts homologous to an abundant embryonal-carcinoma-cell mRNA.
EC1, a clone from a cDNA library of embryonal-carcinoma (EC) cells, was used to study the expression of the gene(s) homologous to this sequence in murine germ cells and somatic tissues. Northern-blot analysis was used to determine the size and relative abundance of ECl transcripts in EC cells, ovary, testis, liver, spleen, brain, thymus, and bone marrow. The stage-specific expression of EC1-related transcripts in meiotic cells was examined in enriched populations of testicular cells (meiotic prophase spermatocytes, early and late spermatids, and residual bodies). All tissues and cells examined contained mRNAs of approximately 1.3 and approximately 3.6 kb which were homologous to EC1. In RNA from liver and thymus, a faint, lower-molecular-weight transcript of 0.75 kb was seen. A unique pattern of expression was observed in male germ cells. In cells that had completed meiosis, two additional transcripts of 1.0 and 4.4 kb were visualized, as well as high levels of expression of the 1.3- and 3.6-kb species. Only the 1.3- and 3.6-kb transcripts were detected in ovarian RNAs. The high level and stage specificity of expression of EC1-related mRNAs in germ cells suggest that this gene may be important in meiosis or in germ-cell maturation.
Animals↗
Differential expression of the c-abl proto-oncogene and the homeo box-containing gene Hox 1.4 during mouse spermatogenesis.
Mammalian spermatogenesis is a complex developmental process. Spermatozoa, like ova, are uniquely capable of supporting embryonic development. Our approach to understanding this process is to identify genes whose developmental pattern of expression suggests that they may play a role in spermatogenesis. Experiments on the cellular oncogene c-abl and the homeo box-containing gene Hox-1.4 indicate that these genes may be important for male germ cell development. Both genes produce testis-specific transcripts that are present in particular cellular populations of the adult testis. Their developmental specificity, however, is different: c-abl is haploid-specific, whereas Hox-1.4 is expressed in the germ cells as soon as they have entered meiosis. Future studies will focus on examining the protein products of these genes and their function in testicular cells.
Amino Acid Sequence↗
Developmental-stage-specific expression of the hsp70 gene family during differentiation of the mammalian male germ line.
Mouse somatic tissues contain low levels of transcripts homologous to the heat shock-inducible and cognate members of the heat shock protein 70 (hsp70) gene family. An abundant, unique sized hsp70 mRNA of 2.7 kilobases (kb) is present in testes in the absence of exogenous stress. Its expression is restricted to germ cells and is developmentally regulated. The 2.7-kb transcript first appears during the haploid phase of spermatogenesis and is stable throughout the morphogenic stages of spermiogenesis. A 2.7-kb hsp70 mRNA is present in rat and human testes. These observations suggest that a member of the hsp70 gene family plays a role in the development of the mammalian male germ cell lineage.
Age Factors↗
Isolation of a mouse cDNA coding for a developmentally regulated, testis-specific transcript containing homeo box homology.
A clone, pHBT-1, containing sequences homologous to Drosophila homeo boxes has been isolated from a mouse testis cDNA library. The sequence is 80% homologous at the DNA level and 88% homologous at the amino acid level to the homeo box sequence of the Antennapedia gene of Drosophila. Sequences flanking the 3' end of the homeo box are highly diverged from other murine homeo box-containing genes characterized to date. RNA blot hybridization analysis of mouse testis poly(A)+ RNA revealed transcripts of approximately 1.4 kb in length. Within the limits of sensitivity of detection of Northern blot analysis, no transcripts were seen in any of the adult somatic tissues examined. Other tissues that contain stem cells, namely those of the hemopoietic system, also lacked detectable amounts of HBT-1 transcripts. HBT-1 transcripts were limited to male germ cell-containing tissues, since RNAs from juvenile and adult ovaries did not contain detectable amounts of the 1.4-kb transcripts. Expression of the HBT-1 gene was not detected in embryonic testes, nor in tests of neonatal animals which contain germ cells up to the Type B stage of spermatogonial development. A role for the expression of the HBT-1 gene in the meiotic stages of male germ cell differentiation is postulated.
Animals↗
Genetic and cytogenetic localisation of the homeo box containing genes on mouse chromosome 6 and human chromosome 7.
Probes from the m6 homeo box cluster were mapped to mouse chromosome 6 by somatic cell genetics, in situ hybridisation, and by a Mus spretus--Mus musculus backcross mapping system. In addition, the testis-specific homeo box containing cDNA, clone, HBT-1, has been mapped using the same back-cross system and the B X D recombinant inbred strain set. Close genetic and physical linkage between the m6 cluster and HBT-1 was demonstrated, positioning these sequences to the same local cluster of homeo box containing genes. The map location of this cluster between IgK and Tcrb coincides with the morphological mutation hypodactyly (Hd). Synteny has been observed between a region of mouse chromosome 6 and the long arm of human chromosome 7 encompassing the markers Cpa, Tcrb and Try-1. Here we localise human sequences hybridising to the mouse m6 probes to the short arm of chromosome 7, breaking the region of synteny.
Animals↗