Testis RNA Research Articles

Molecular cloning and partial characterisation of the mouse Cyp51 cDNA.

Northern analysis of the mouse testis and liver RNA show two types of lanosterol 14α-demethylase (CYP51) transcripts, somatic and a testis-specific transcript. At least two CYP51 mRNAs are expressed in liver and testis, while the smallest transcript is expressed only in testis. The partial mouse Cyp51 cDNA (1052 bp) was obtained by RT-PCR cloning and was also found in expressed sequence tag (EST) clones. Bacterial artificial chromosome (BAC) clones containing mouse Cyp51 gene were also found. The coding part of the partial mouse CYP51 is 93.9% homologous to the rat and 87.9% to the human CYP51 cDNAs. There is less than 2% variation between the CYP51 EST clones to the RT-PCR cloned partial cDNA and BAC clones.

The critical region of overlap defining theAZFa male infertility interval of proximal Yq contains three transcribed sequences

The position of deletion breakpoints in a series of fourAZFa male infertility patients has been refined using new markers derived from BAC clone DNA sequence covering the AZFa male infertility...

Structures and co-regulated expression of the genes encoding mouse cytosolic chaperonin CCT subunits.

The chaperonin-containing TCP-1 (CCT) is a hetero-oligomeric molecular chaperone that mediates protein folding in the cytosol of eukaryotes. Eight (or nine in testis) subunit species are assembled in the CCT hexadecamer complex. We have cloned seven CCT subunit genes, Cctb, Cctd, Ccte, Cctz-1, Cctz-2 (testis specific), Ccth and Cctq, from mouse genomic DNA libraries, in addition to the Ccta and Cctg genes reported previously, and the entire nucleotide sequences of these DNA clones were determined. These genes are approximately 15-20 kb in length except for Cctz-2 which is longer than 35 kb, and all the Cct genes consist of 11-16 exons. Primer extension analyses of testis RNA indicate one to several potential transcription start sites 50-150 bp upstream from the translation start codon of each Cct gene. There are several possible Sp1-binding sequences, but no obvious TATA box was observed around the potential start sites. From 5'-flanking regions to the first introns, the Cct genes are rich in CpG dinucleotides. In reporter gene assays using these regions, five of eight Cct genes showed strong transcriptional activity comparable with the combination of SV40 promoter and enhancer in HeLa cells. We also show, by Western and Northern blot analyses, that CCT expression levels vary widely among different tissues but the expression patterns are very similar among the eight subunit species. It is likely that expression levels of the eight different subunits are tightly co-regulated to maintain a constant ratio of these subunits which constitute the CCT hexadecamer complex with a fixed subunit arrangement.

Identification of Rabbit Reticulocyte E217K as a UBC7 Homologue and Functional Characterization of Its Core Domain Loop

The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.

Expression and Regulation of Transcription Factors GATA-4 and GATA-6 in Developing Mouse Testis1

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

Mea2/Golga3 gene is disrupted in a line of transgenic mice with a reciprocal translocation between Chromosomes 5 and 19 and is responsible for a defective spermatogenesis in homozygotes.

A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation. The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization. The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis, while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis. Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604 mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes.

Identification of a Novel Highly Conserved Gene in the Centromeric Part of the Major Histocompatibility Complex

A novel highly conserved gene designatedSacm2laliasAre1) has been identified and fine mapped in the centromeric part of the major histocompatibility complex in rat, human, and mouse.Sacm2lis closely linked to the ribosomal protein S18 geneRps18with a distance of about 450 bp between the respective translational start points. NumerousSacm2l-homologous EST sequences can be identified in the database. Northern blot experiments of ratSacm2lrevealed a transcript of 3 kb in each organ tested, and by RT-PCR differentially spliced products could be detected in testis RNA. The deduced amino acid sequence of the ratSacm2lgene shows a putative coiled-coil region and significant homology to a putativeCaenorhabditis elegansprotein and the yeast SAC2 protein.

Expression of murine H1 histone genes during postnatal development

Murine genes encoding the seven H1 histone isoforms H1.1–H1.5, H1° and H1t have been isolated and sequenced. We have established expression patterns of these genes in several tissues during postnatal development. For that analysis, RNase protection assay rather than Northern blot hybridization was used, since the sequences of these genes are highly similar and would cross-hybridize under Northern blot conditions. Expression patterns of H1.1 to H1.5 and H1° were determined in tissues of animals at days 5, 9 and 20 after birth and of adult mice. In addition, RNA was analyzed in three mouse cell lines (NIH3T3, P19, TM4). Transcription of the subtype genes H1.2 and H1.4 was found in all tissues and cell lines studied. The most varied expression patterns were obtained with the H1.1 subtype. H1.1 mRNA was found at high concentrations in thymus and spleen throughout development and in testis beginning with a low expression in 5-day-old animals and increasing levels in testis RNA from 9- and 20-day-old and adult mice. H1° mRNA was found primarily in highly differentiated tissues with concentrations decreasing from 5-day-old to adult animals.

Changes in Testicular Steroidogenic Acute Regulatory (STAR) Protein, Steroidogenic Enzymes and Testicular Morphology Associated with Increased Testosterone Secretion in Bulls Receiving The Luteinizing Hormone Releasing Hormone Agonist Deslorelin

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450 scc), 3β-hydroxysteroid dehydrogenase /Δ 5 → Δ 4 − isomerase (3β-HSD), and 17α-hydroxylase cytochrome P450/C 17–20 lyase (P450 17α) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin ( n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per μg total testis protein or RNA) of StAR protein, 3β-HSD, P450 scc, and mRNA for P450 17α in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin ( n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.

The olfactory adenylyl cyclase III is expressed in rat germ cells during spermiogenesis.

To identify the adenylyl cyclase (AC) genes expressed in mammalian germ cells, RT-PCR of testis and germ cell RNA was performed using degenerated primers based on the homologous region of the AC catalytic domain. This strategy yielded high-frequency amplification of a complementary DNA (cDNA) identical to type III AC (ACIII), a form previously identified as the major adenylyl cyclase expressed in the olfactory system. Ribonuclease protection studies confirmed that ACIII transcripts are present in germ cells, appear during the meiotic prophase, and accumulate during spermiogenesis. A Northern blot analysis performed on total testis RNA demonstrated the presence of a predominant transcript of 7.5 kb, suggesting that the ACIII expressed in germ cells may derive from a splicing variant different from the 4.5 kb transcripts expressed in somatic cells. To determine whether these RNAs are translated into a protein, Western blot analysis was performed using an antibody specific for the carboxyl terminus of ACIII. An immunoreactive protein of 170 kDa was detected in extracts from total testis and from germ cells. Immunofluorescence localization of this protein in the seminiferous tubules showed that ACIII was predominantly expressed in postmeiotic germ cells from round spermatids in the cap phase to maturing elongating spermatids. The ACIII antigen was located mostly on the acrosomal membrane rather than on the plasma membrane of developing spermatids. The spatial and temporal expression of ACIII in germ cells indicates a role of this AC in the acrosome formation. Together with the observation that members of the olfactory receptor family and an olfactory phosphodiesterase are expressed in spermatids, these findings suggest that a signal transduction system used in olfaction is also used during gamete development.

MSJ-1, a New Member of the DNAJ Family of Proteins, Is a Male Germ Cell-Specific Gene Product

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein–protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages andin situhybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.

Testis epidermal growth factor and spermatogenesis.

Epidermal growth factor (EGF) is a cytokine that promotes cell proliferation, regulates tissue differentiation, and modulates organogenesis. Although a rich source of EGF is the submaxillary gland, many tissues produce this cytokine, including the testis. Leydig cells are the principal source of EGF in the testis. On attainment of sexual maturation the germ cells, primarily spermatocytes and round spermatids, form EGF with the onset of spermatogenesis. EGF appears to be involved in the development of the testis and in spermatogenesis. The expression of the EGF gene in rat testis was determined by the application of the RT-PCR method and testis RNA as substrate. The results suggest that EGF produced by Leydig cells and germ cells may modulate spermatogenesis as an autocrine and/or paracrine factor.

Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain ( DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, g HDHC7 and g MDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.

The Detection of Thyrotropin-Releasing Hormone (TRH) and TRH Receptor Gene Expression in Siberian Hamster Testes

Rao, J. N., L. Debeljuk, A. Bartke, Y.-P. Gao, J. F. Wilber and P. Feng. The detection of thyrotropin releasing hormone (TRH) and TRH receptor gene expression in Siberian hamster testes. Peptides 18(8) 1217–1222, 1997.—Thyrotropin-releasing hormone (TRH) from the hypothalamus is the major regulator of TSH synthesis and secretion. Most recently, TRH and TRH receptors (TRH-R), as well as their mRNAs, have been identified in rat testis. To expand our knowledge on the testicular TRH and TRH receptor gene expression in different species, in the present study the mRNA levels of testicular TRH and TRH-R were investigated in Siberian hamsters. To further localize the cellular sites of the gene expression, the animal model was treated with a single injection of ethylene dimethane sulfonate (EDS) (IP, 80 mg/kg body weight), a compound known as to specifically eliminate testicular Leydig cells. The elimination of Leydig cells induced by EDS treatment was confirmed by histological studies of the testis sections and by serum hormonal analyses, which showed a dramatic reduction of serum testosterone (T) levels and significantly elevated serum LH concentrations. Messenger RNA levels of TRH and TRH-R in the testes were determined by Northern blot analyses quantitated with densitometry scanning. The results showed that specific TRH-R mRNA, 3.8 kb in size, was identified in Siberian hamster testes and the mRNA levels were significantly elevated in the EDS-treated testes compared to the controls ( p < 0.01). Testicular TRH mRNA was also detected; however, no significant differences in TRH mRNA levels were found between EDS-treated and control groups. The size of TRH mRNA was characterized as about 1.2 kb in hamster testes, which was smaller than that observed in the rat hypothalamus (1.6 kb) and in the rat testis (2.0 kb). Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis. No hybridization signal for TRH mRNA was detected by RNase protection assay, when a rat TRH riboprobe was applied to hamster testis RNA, suggesting the limited homology of TRH gene sequences between these two species. Our results demonstrate that both TRH and TRH-R genes are expressed in Siberian hamster testes, and a significant increase of TRH-R mRNA levels occurs in the Leydig cell eliminated hamster testes. Unlike the rat testicular TRH mRNA mainly detected in Leydig cells, in hamster TRH mRNA could also be detected in other testicular compartment.

Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function.

Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.

Exons Lost and Found: UNUSUAL EVOLUTION OF A SEMINAL VESICLE TRANSGLUTAMINASE SUBSTRATE

The GP1G gene codes for three of the four abundant androgen-regulated secretory proteins produced by the guinea pig seminal vesicle. Sequencing of the entire 6.3-kilobase gene and comparison with other mammalian seminal vesicle secretory protein genes reveals a common three-exon, two-intron organization. However, significant sequence similarity between this group of genes is largely limited to their 5'-flanking regions and first exons, which code almost exclusively for signal peptides in each case. The first intron of GP1G does contain a region with high similarity to the coding exon of a human seminal vesicle secretory protein gene, semenogelin II. The 3' half of the GP1G gene appears to share a common ancestry with the human SKALP/elafin gene. Sequences related to the elafin promoter, coding, untranslated regions, and introns are clearly identifiable within the GP1G sequence. The elafin gene codes for a serine protease inhibitor and is expressed in a variety of different human tissues. To determine if the GP1G gene was also active outside of the seminal vesicle, RNA from a variety of guinea pig tissues was hybridized to a GP1G cDNA probe. At least three novel RNA bands hybridizing to the GP1G probe were detected in testis RNA samples, and GP1G-related mRNAs were also found in other tissues. These data suggest that these seminal vesicle secretory proteins may have functional roles outside the reproductive system.

Expression of activin beta C subunit mRNA in reproductive tissues.

A cDNA encoding the potential activin beta C subunit was produced from human testis RNA using reverse transcription and PCR and then used as a probe in Northern blot analysis of poly(A)+ RNA. A transcript of approximately 1.8 kb was evident in human ovary, placenta and testis samples. A transcript of this size was also detected in adult rat caput and cauda epididymis, and adult and day-15 testis and adult spleen, while adult liver contained a single transcript of approximately 2.1 kb. Poly(A)+ RNA from primary spermatocytes but not from round spermatids contained the 1.8 kb activin beta C mRNA. These findings highlight the need for further studies to determine the physiological role(s) of activin beta C in a wide variety of tissues.

Spermatocyte-specific expression of the gene for mouse testis-specific transcription elongation factor S-II

Previously, we characterized a rat cDNA for testisspecific transcription elongation factor S-II (SII-T1) (Q. Xu et al., J. Biol. Chem. 269, 3100–3103 (1994)). Here, we isolated a 335-bp fragment of the cDNA for mouse SII-T1, and used it to examine the expression of the SII-T1 gene in the testis by in situ hybridization. The results indicated that the SII-T1 gene is expressed exclusively in spermatocytes, showing no appreciable expression in spermatogonia, spermatids, or Leydig cells. RTPCR experiments using testis RNA from WI W v mutant mice also suggested that SII-T1 is a specific transcription elongation factor essential for spermatogenesis.

Expression of cytochrome P450 17alpha-hydroxylase/C17-20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens.

Testosterone is required for normal development of the male reproductive tract. Synthesis of testosterone occurs in the Leydig cells and is dependent upon the expression of several enzymes, including cytochrome P450 17alpha-hydroxylase/C17-20-lyase (P450c17), which is highly regulated within the testis. The aim of the present study was to investigate whether maternal exposure to estrogenic chemicals was able to affect Leydig cell function in the developing male fetus at the time of masculinization. Pregnant rats were injected sc with diethylstilbestrol (DES; 100 or 500 micrograms/kg), 4-octylphenol (OP; 100 or 600 mg/kg), or vehicle (oil, control) on days 11.5 and 15.5 postcoitum. Doses were chosen to reflect the reported estrogenic potency of the chemicals in vitro. On day 17.5, fetal testes were fixed before performing in situ hybridization and immunocytochemistry, used for extraction of RNA, or homogenized in phosphate buffer for determination of 17alpha-hydroxylase enzyme activity. There was no difference between fetuses from control and treated mothers in either the overall histology of the testes or the apparent number of Leydig cells, as determined by immunocytochemistry with an antibody directed against 3beta-hydroxysteroid dehydrogenase. However, there was a consistent and striking reduction in the amount of P450c17 detected by immunocytochemistry in testes from the groups given the higher dose of DES or OP. These observations were supported by measurement of 17alpha-hydroxylase activity, which was significantly reduced compared with that in controls (6.25 +/- 0.65 pmol/testis x min) in fetuses from animals treated with 100 micrograms/kg DES (4.27 +/- 0.39; P < 0.05), 500 micrograms/kg DES (1.4 +/- 0.47; P < 0.001), or 600 micrograms/kg OP (4.25 +/- 0.33; P < 0.05). RT-PCR and in situ hybridization revealed that these changes were mirrored by reductions in P450c17 messenger RNA in testes from fetuses from treated mothers compared with control levels. In conclusion, maternal treatment with either a potent sythetic estrogen (DES) or a putative environmental estrogen (OP) results in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells. These results, therefore, provide a mechanism by which inappropriate exposure of the fetus to estrogenic chemicals might have an adverse effect on fetal steroid synthesis and masculinization.