Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Isolation and properties of somatic and testicular cytochromes c from rat tissues

Somatic (c_s) and a testis-specific (c_{t I}) cytochromes c were purified to homogeneity from rat tissues (heart, liver, kidney, and testis). The purification procedure involved (1) homogenization of tissues at pH 4.5, (2) treatment with methanol-chloroform solvents, (3) hydroxylapatite column chromatography, (4) carboxymethyl-cellulose column chromatography, and (5) Sephacryl S-200 gel filtration. The isolated cytochromes c were free from polymeric and other “modified” forms, and did not bind CO, azide, or cyanide. The absorption maxima and the molecular weights of both cytochromes c_s and c_{t I} were identical. The ratio of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$ for cytochromes c_s averaged 1.28. The unique properties of cytochrome c_{t I}, compared to somatic cytochrome c, were as follows: (1) different elution profiles from hydroxylapatite and carboxymethyl-cellulose column chromatography experiments, (2) less basic intrinsic molecular charge shown by the slow mobility in native polyacrylamide gel electrophoresis, (3) probable asymmetric molecular shape as evidenced from gel filtration experiments, (4) significantly higher millimolar extinction coefficient values (33.6 at 549.5 nm), (5) a low ratio (1.04) of $\text{A}{}_{\mathrm{<mtext>549.5\; </mtext><mtext>nm</mtext>}}\text{(}\text{reduced}\text{)}\text{A}{}_{\mathrm{<mtext>280\; </mtext><mtext>nm</mtext>}}\text{(}\text{oxidized}\text{)}$, and (6) difference of about 20 amino acid residues per mole.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.     

The identification and characterization of a testis-specific cDNA during spermatogenesis

Using bioinformatics and experimental validation, we obtained a cDNA (named srsf) which was exclusively expressed in the mouse testes. RT-PCR analysis showed that srsf mRNA was not expressed in the gonad during the sex determination period or during embryogenesis. In developing mouse tests, srsf expression was first detected on post-natal day 10, reached its highest level on day 23, and then reduced to and remained at a moderate level throughout adulthood. In situ hybridization analysis demonstrated that srsf mRNA was expressed in pachytene spermatocytes and round spermatids in the testes. The predicted protein contains one RNA-binding domain (RBD) and a serine-arginine rich domain (RS), which are characterized by some splicing factors of SR family members. These findings indicate that srsf may play a role during spermatogenesis.     

Stage-specific enhanced expression of mitochondrial fusion and fission factors during spermatogenesis in rat testis

The mitochondrial fusion factors mitofusins 1 and 2 (Mfn1 and Mfn2) and the fission factor dynamin-related protein 1 (Drp1) were found to be highly expressed in the pubertal and adult rat testis by Northern blot analysis. Immunohistochemistry using specific antisera to Mfn2 and Drp1 revealed a pronounced expression of the fusion and fission factors in the round and elongating spermatids in the seminiferous tubules, suggesting that at precise steps of spermiogenesis (i.e., steps 8-12), spermatid mitochondria are rapidly homogenized by frequent fusion and division. Although physiological relevance of this phenomenon remains to be clarified, a role is proposed for it as an effective means of achieving complete and homogeneous ubiquitination of mitochondria, which has recently been demonstrated to be a mechanism for the elimination of paternal mitochondria during fertilization, based on the fact that the timing of expression of Mfn2 and Drp1 coincides well with that reported for a spermatid-specific ubiquitin-conjugating enzyme.     

Quantitation of testicular and somatic cytochromes c in testis and somatic tissues from developing rats

By combining chromatographic and spectral procedures, simple and quantitative assays for somatic cytochrome c (cyt cs) and testicular cytochrome c (cyt ct) in crude animal tissue extracts were developed. Using this assay procedure, limited developmental studies of cyt ct and cs were performed with tissue extracts of 27-, 58-, and 85-day-old rats. Specific contents of cyt cs in somatic tissues (i.e., micrograms of cyt c/g of tissue) of these three age groups did not show significant variations. However, the amounts of both cyt ct and cs in testis were markedly increased as the rats approached maturity. Increasing cyt ct/cyt cs ratios as the rat developed to maturity suggest that expression of cyt ct is preferentially required for specific function of testis. Application of both molecular biological techniques and this assay (for holo-cyt ct) should be useful to study the overall regulation of the expression of cyt ct in testis.     

Variations in mitochondrial size and ultrastructure during germ cell development

Summary Size variations and ultrastructural changes in mitochondria of developing germ cells of the female hamster were analyzed. Mitochondria in oogonia of foetus and newborn were elongate with transverse cristae. During pre-dictyate meiotic prophase they became small, rounded, and electron-dense with pleomorphic cristae. These changes were largely reversed when dictyate was reached. Maximum mitochondrial size and complexity of cristae were reached just at the beginning of the phase of rapid oocyte growth, and thereafter declined. As mitochondrial size and number of cristae decreased in the rapidly enlarging oocyte, the ratio of length to width increased, as did electron density of the matrix, until the formation of an antrum within the follicle. After antrum formation, the mitochondria again became more rounded and cristae were seldom seen. An attempt is made to correlate changes of mitochondrial morphology with other events occurring during oogenesis.The author wishes to thank the Department of Anatomy, University of Dundee, for financial support and for the use of the AEI EM 801 electron microscope     

The relationship between the nuage and the chromatoid body during spermatogenesis in the Rat

Summary Cytoplasmic structures ultrastructurally similar to the nuage are present in the cytoplasm of all spermatogenic cells in adult rats. The nuage is a discrete organelle which should not be confused with the chromatoid body. In step 7–8 spermatids transient contact is established between the nuage and the chromatoid body. This indicates a very specific recognition of the nuage by the chromatoid body. It is suggested that the nuage and the chromatoid body are separate cell organelles the functions of which are somehow related to each other.     

Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fractionation of somatic and sperm chromatin during spermatogenesis in fish

A new approach is suggested for studying changes in the interactions of protein with DNA in the cells. Measurements of the buoyant density of chromatin were performed in somatic cells and in cells undergoing meiosis in fish. During the process of spermatogenesis some of the somatic histones on the DNA are replaced by a new class of proteins; consequently, the mature sperm contains a unique type of protein having a low mol. wt and a high proportion of arginine.The chromatin obtained from mature sperm is composed of a single component with a density of 1.48–1.49 g/cm³ as measured by CsCl equilibrium sedimentation. On the other hand, somatic cells contain chromatin with lower densities. Chromatin obtained from erythrocytes contains a single component with a density of 1.41–1.42 g/cm³ while liver chromatin shows two components; a main component with a density of 1.45–1.46 and a more heterogeneous component with a lighter density (1.32–1.35). There is a correlation between the buoyant density of the chromatin, the type of its basic proteins and the level of biosynthetic activity in the cells.     

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Organelle DNA and male fertility variation in Solanum spp. and interspecific somatic hybrids

Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspecific somatic hybridization in plants. To evaluate such variability and correlate it with nuclear-cytoplasmic interactions leading to male sterility in Solanum spp., we examined progeny of male-sterile and male-fertile somatic hybrids between Solanum tuberosum (tbr), the common potato, and S. commersonii (cmm), a wild species showing sexual incongruity with tbr, for fertility and organelle DNA composition. Uniform male-fertile and male-sterile progenies were obtained by selfing the male-fertile hybrid and crossing the male-sterile ones, indicating maternal inheritance of the fertility phenotype. The two fusion partners were only slightly differentiated in the plastidial genome. MtDNA polymorphism between the species was greater, although its extent varied with the genomic region investigated. All somatic hybrids had non-parental organelle genomes, with reassorted organelles and/or rearranged mitochondria (i.e., cmm-specific bands for some regions and tbr-specific bands for others). Mitochondria reassorted independently from chloroplasts. Most hybrids showed the cmm cpDNA hybridization pattern, indicating non-random transmission of chloroplasts. Most male-sterile hybrids showed preferential inheritance of tbr mtDNA fragments. The male-fertile somatic hybrid clone had predominantly cmm mtDNA fragments. This result suggests that a tbr-derived region involved in nuclear-cytoplasmic incompatibility and male sterility has been lost by rearrangement; however, no clear correlation between a specific mitochondrial region and male sterility has been found so far. Received: 3 January 1999 / Accepted: 20 February 1999     

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment

Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs.     

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.     

Cellular localization of mRNAs for retinoic acid receptor-alpha, cellular retinol-binding protein, and cellular retinoic acid-binding protein in rat testis: evidence for germ cell-specific mRNAs

In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.