Expression of the third member of the serum amyloid A gene family in mouse adipocytes.

Three homologous genes that code for three related proteins comprise the serum amyloid A (SAA) family in the mouse. Endotoxin induces equally vigorous expression of mRNAs for the three SAA genes in liver. In extrahepatic tissues SAA1 and/or SAA2 mRNAs have been found only in kidney and intestine, however, SAA3 is expressed in all extrahepatic tissues thus far examined. This observation raised the question: is SAA3 mRNA expressed by a single cell system dispersed throughout all tissues, or by differentiated cells of each tissue? This question was explored in various tissues by in situ hybridization with a single-stranded cRNA probe specific for SAA3 mRNA. We found expression in the liver of SAA3 mRNA by other cells as well as by hepatocytes. A common feature among extrahepatic tissues was SAA3 mRNA expression in adipocytes. SAA3 mRNA was also found in two nonadipose cells, Leydig cells of the testis, and some of the cells located in parafollicular zones of the spleen.

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens.

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.

Localization of ribosomal DNA within oocytes of the house cricket, Acheta domesticus (Orthoptera: Gryllidae).

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.

Genetics of microtubule systems.

In most eucaryotes the tubulin genes comprise small multigene families with approximately equal numbers of genes for alpha- and beta-tubulin, the structural proteins of microtubules. The recent isolation of tubulin mutations in several species is proving to be a powerful tool for examining the structure and function of specific sets of microtubules. In Drosophila melanogaster, genetic analysis of a testis-specific beta-tubulin gene has shown that a single tubulin gene product may fulfill a number of different microtubule functions. In addition to tubulin mutations, mutations in other genes whose products are involved in the regulation or structure of specific microtubule arrays have also been isolated. The combination of analysis of both classes of mutations is beginning to allow a molecular description of the construction and function of three-dimensional cellular structures. In addition, such studies may also shed light on the evolutionary pressures that gave rise to and serve to maintain small families of genes encoding very similar proteins.

Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification.

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellvé, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.

Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor: purification and localization on surfaces of spermatogenic cells and sperm.

We have found that the rat testis contains a cell surface galactosyl receptor that is antigenically related to the minor species of rat liver asialoglycoprotein receptor (ASGP-r) and has binding affinity for galactose coupled to agarose. In immunoblotting experiments, rat testis galactosyl receptor (RTG-r) is recognized by antiserum raised against the minor ASGP-r species of rat liver (designated rat hepatic lectin-2/3, RHL-2/3). Antiserum raised against the major species RHL-1 does not recognize an antigenic protein equivalent to RTG-r. Triton X-100-extracted rat liver and testes preparations fractionated by affinity chromatography on galactose-agarose and resolved by SDS-PAGE under reducing conditions, show that rat liver contains both the major (RHL-1) and minor (RHL-2/3) ASGP-r species whereas rat testis displays only a receptor species comigrating with RHL-2/3. RTG-r was present throughout testicular development. The receptor was found in seminiferous tubules, cultured Sertoli and spermatogenic cells, and epididymal sperm. Indirect immunofluorescent studies show RHL-2/3-like immunoreactivity on the surface of Sertoli cell, meiotic prophase spermatocytes, spermatids, and epididymal sperm. In spermatids and sperm, the immunoreactivity is restricted to the plasma membrane overlying the dorsal portion of the head. Because of RTG-r has galactose binding affinity, is present on surfaces of Sertoli and developing meiotic and postmeiotic spermatogenic cells, and overlies a region of the intact acrosome on epididymal sperm, RTG-r may have a role in spermatogenesis and in events leading to sperm-egg recognition.

Near-total glutathione depletion and age-specific cataracts induced by buthionine sulfoximine in mice.

The specific inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO), although relatively nontoxic in adult mice, induces severe glutathione depletion and age-specific pathological changes when repeatedly administered to male suckling mice. Dense cataracts developed when mice aged 9 to 12 days were given a series of injections of L-BSO, despite excellent survival and the absence of other significant long-term effects. By contrast, similar treatment of mice aged 14 to 17 days, although slightly less effective in reducing glutathione levels, resulted frequently in death, hind-leg paralysis, or impaired spermatogenesis, but did not produce cataracts. Administration of L-BSO to preweanling mice provides a novel model system for the induction of cataracts by depletion of lens glutathione and may enable the study of critical functions of glutathione in the lens and other growing tissues during early postnatal development.

Eggshell thinning in Japanese quail fed mercuric chloride.

The eggs produced by developing Japanese quail (Coturnix coturnix japonica) fed 1 to 8 parts of mercury per million as mercuric chloride for 10 weeks have thinned shells. Total amounts of mercury in tissues were quite proportional to the dosage and were higher in males than in females. Methylation of mercury was not observed.

Starfish gonad: action and chemical identification of spawning inhibitor.

The gamete-shedding substance obtained from the radial nerves induces spawning when it is applied to the gonads of mature starfish in vivo and in vitro. A substance that inhibits the action of this spawning factor is present in both ovary and testis; it has been isolated from testis of Asterina pectinifera and chemically identified as L-glutamic acid.

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

Androgen binding proteins of testis, epididymis, and plasma in man and monkey.

Androgen-binding protein (ABP) has been found in the cytosol of testicular and epididymal homogenates of several sub-primate species. In those species which had the plasma androgen binding protein, testosterone-estradiol-binding globulin (TeBG), ABP and TeBG were found to be physically similar. We investigated the possibility that ABP might exist in monkey and man using the cytosol of testicular and epididymal homogenates and aspirates obtained by direct micropuncture of the rete testis. In polyacrylamide gel electrophoresis, pH 7.8, testicular and epididymal cytosols of monkey and man were found to contain several binding proteins of different size and net charge that bind dihydrotestosterone. These binding proteins were either indistinguishable from TeBG or could be related to TeBG as size and/or charge isomers. No ABP was detectable in up to 200 mul of monkey rete testis fluid obtained by direct micropuncture, though ABP is detectable in as little as 5 mul of rat rete testis fluid. The data suggest that the ABP's detected in the testicular and epididymal cytosols in monkey and man represent isomeric forms of plasma TeBG, and their presence in testicular cytosol most likely derives from blood contamination.

Studies on zinc deficiency: changes in trace elements and enzyme activities in tissues of zinc-deficient rats.

Zinc content of testes, bones, esophagus, kidneys, and muscles was decreased, whereas iron content was increased in the testes of zinc-deficient rats compared to restrictedly fed control rats. Histochemical enzyme determinations revealed reduced activities of certain enzymes in the testes, bones, esophagus, and kidneys. In the testes, lactic dehydrogenase (LDH), malic dehydrogenase (MDH), alcohol dehydrogenase (ADH), and NADH diaphorase; in the bones, LDH, MDH, ADH, and alkaline phosphatase; in the esophagus, MDH, ADH, and NADH diaphorase; and in the kidneys, MDH and alkaline phosphatase were decreased in zinc-deficient rats compared to restrictedly fed controls. Succinic dehydrogenase (SDH) revealed no significant changes under the conditions of our experiments in various groups of rats that were investigated. In a "repleted" group of rats, content of zinc in testes and bones increased significantly, compared to the deficient group. The iron content of the testes decreased after repletion with zinc. In the testes, bones, esophagus, and kidneys, the activities of various enzymes increased after repletion with zinc. Inasmuch as the major manifestations of zinc deficiency syndrome in the rat include growth retardation, testicular atrophy, and esophageal parakeratosis, our results suggest that the content of zinc in the above tissues most likely controls the physiological processes through the formation of zinc-dependent enzymes.

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.

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.

Radioimmunological determination of cellular retinol-binding protein in human tissue extracts.

A sensitive and specific radioimmunoassay was developed for human cellular retinol-binding protein (CRBP) using antibodies raised in rabbits against purified human liver CRBP. The assay could determine concentrations of CRBP in extracts down to 8 micrograms/liter. CRBP could be demonstrated in all tissues investigated except in serum, and the same immunoreactive CRBP seemed to be present in all tissues. The highest concentrations were found in the gonads. In the gastrointestinal tract, the small intestine had a higher concentration than did stomach or colon. Muscle and skin had low concentrations of CRBP.

Analysis of DNA adducts in putative premalignant hepatic nodules and nontarget tissues of rats during 2-acetylaminofluorene carcinogenesis.

Exposure of rats to a standard four-cycle feeding regimen of 0.06% 2-acetylaminofluorene (AAF) results in the formation of putatively premalignant hepatic nodules, but the types and magnitude of DNA adducts formed in these nodules has not been previously examined. By using a sensitive 32P-adduct assay (R. C. Gupta, Cancer Res., 45: 5656-5662, 1985), we analyzed the DNA adduct lesions in individual hepatic nodules at various times during and after exposure to AAF. Kidney, spleen, and testis were included as nontarget tissues. No qualitative difference was observed in the DNA adducts found in hepatic nodules and nontarget tissues; however, quantitative differences occurred. At least one unknown and two known (dG-C8-AF and dG-N2-AAF) DNA adducts were detected, with dG-C8-AF being predominantly (96-98%) formed, in all tissues examined. At the end of the first three weeks of AAF feeding, the concentration of the deacetylated adduct dG-C8-AF in liver (223 fmol/micrograms DNA) was found to be about 2, 6, and 5 times higher than in kidney, spleen, and testis, respectively. The concentration of the N2-acetylated adduct in liver (4.5 fmol/micrograms DNA) was 4-fold higher than in kidney and strikingly higher (51- and 42-fold, respectively) than in spleen and testis. At the end of the fourth feeding cycle, total DNA adducts measured in the hepatic nodules ranged from 30-100 fmol/micrograms DNA, while the "surrounding liver," kidney, spleen, and testis showed 235, 218, 62, and 28 fmol adducts/micrograms DNA, respectively. Sixty days following the cessation of AAF, the binding in both the persistent nodules and liver had decreased to 7% of their respective levels measured at the end of the fourth cycle, while adducts in kidney, spleen, and testis were 32%, 18% and 19%. After 88 days, the binding levels in the nontarget tissues declined further, but no additional adduct removal occurred in the nodules. Our data indicate that (a) although the metabolic apparatus for activation of AAF is diminished in the hepatic nodules, a significant level of adduct formation occurs in the cells of this putative, premalignant lesion, and (b) unlike in the nontarget tissues, repair processes in the premalignant nodules may not be operative several weeks after the cessation of AAF exposure.

Nuclear magnetic resonance investigations of human neoplastic and abnormal nonneoplastic tissues.

A large number of samples of human neoplastic and abnormal nonneoplastic tissues were studied by nuclear magnetic resonance spectrometry in order to evaluate the possible role of this technique in the diagnosis of cancer. The spin-lattice magnetic relaxation times (T1) of abnormal nonneoplastic tissue were longer, in many instances, than those of malignant tumors from similar sites, preventing recognition of the tumors in this manner. The evidence for the nonspecific nature of the prolongation of T1 in abnormal tissue is reviewed, and additional limitations of this technique in the diagnosis of cancer are indicated.

Identification of the protein product of the c-mos proto-oncogene in mouse testes.

The mouse c-mos proto-oncogene RNA is expressed primarily in mouse gonadal tissues and embryos. Until now, the c-mos protein has not been identified. Utilizing two different site-directed affinity purified anti-peptide antibodies, we have identified a 43 kDa c-mos protein in mouse testes and in germ cell preparations derived from testes. This 43 kDa testicular protein was found to be structurally related to a bacterially expressed c-mos protein by peptide mapping. Immunoblots of whole mouse sections were employed to establish that the c-mos protein is expressed primarily in the testes.

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.