Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times 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)     

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.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B).

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.     

Developmental expression and characterization of FS39, a testis complementary DNA encoding an intermediate filament-related protein of the sperm fibrous sheath.

Proteins immunologically related to intermediate filaments have been identified in the sperm fibrous sheath but remain uncharacterized. We isolated and characterized a novel intermediate filament-related protein (FS39) localized to the fibrous sheath of the sperm tail. We used Northern blot analysis to establish that FS39 is transcribed predominantly in the testis of mice >18-20 days old. At this age, spermatogenesis has proceeded to the development of the first round haploid spermatids. In situ hybridization revealed that FS39 mRNA is first detectable in late step 3 spermatids, is at its highest level during steps 9 and 10, and diminishes in steps 13 and 14. Western blot analysis identified a single protein of 39 kDa in mouse and rat testis and epididymis, suggesting the protein is conserved in rodents. Indirect immunofluorescence localized FS39 to the fibrous sheath of the sperm tail, and in testis sections expression was detected from step 13 and step 14 spermatids onward, indicating FS39 is under translational control. Southern blot analysis showed FS39 to be a single copy gene, and hybridization to human genomic DNA suggested that a human equivalent gene is present. These results demonstrate that FS39 is transcribed in testis tissue during the haploid phase of spermatogenesis, is present in mature sperm, and codes for a novel 39-kDa intermediate filament-related protein of the fibrous sheath.     

A testis-specific gene encoding a nuclear high-mobility-group box protein located in elongating spermatids.

A cDNA encoding a DNA-binding protein has been isolated by screening a mouse testicular expression cDNA library with a concatemer of a 12-bp putative protein-binding element present in the promoter of the testis-specific gene PGK-2. Sequence analysis of the isolated cDNA indicated the presence of an open reading frame that encodes a protein with two conserved DNA-binding motifs known as the high-mobility-group (HMG) boxes. Northern (RNA) blot analysis demonstrated that expression of the gene is restricted to the postpuberal testis. The DNA-binding activity and sequence specificity of the recombinant HMG protein were confirmed by DNA mobility shift assay using the initial concatemer of the PGK-2 promoter element as a probe as well as the wild-type or mutated versions of the 12-bp element within its natural sequence context. Immunocytochemical staining of adult testis sections with polyclonal antisera recognizing this recombinant HMG protein demonstrated that it is located predominantly in the nuclei of elongated spermatids at steps 9 and 10. These results suggest that this novel HMG box protein gene may be involved in the regulation of gene expression of the haploid male genome. The gene from which the cDNA was derived has been termed testis-specific HMG (tsHMG).     

Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatids

We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X = His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7-18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15-18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa.