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A novel member of the RBCC family, Trif, expressed specifically in the spermatids of mouse testis.

Members of the RING finger family are implicated in a variety of functions such as signal transduction, transcriptional regulation and other developmental processes. Using degenerate oligonucleotide primers corresponding to the RING domain, we isolated a novel RING finger gene from the mouse testis cDNA library, which was about 1.8 kb and was termed Trif (testis-specific ring finger). This deduced protein contains an N-terminal RING-finger, a B-box, and a C-terminal B-30.2-like domain, which make the Trif protein a member of the RING finger-B-box-coil-coil family. Northern blot analysis of adult multiple tissues indicated that Trif is expressed predominantly in the testis. Further analysis detected Trif transcripts in the testis from day 20 of the postnatal stage. In situ hybridization indicated that Trif is expressed in the round spermatids of the seminiferous tubules. These expression data suggest that Trif may play an important role in the regulation of spermatogenesis.

Amino Acid Sequence↗

Expression of a novel TGIF subclass homeobox gene, Tex1, in the spermatids of mouse testis during spermatogenesis.

The homeobox gene superfamily is highly conserved during evolution. These genes act as transcription factors during several important developmental processes through their 60 amino acid homeodomains that recognize and bind to the regulatory region of their target genes. We isolated a novel homeobox gene designated Tex1 from a mouse testis cDNA library by degenerate oligonucleotide polymerase chain reaction (PCR) screening. Three extra amino acid residues, AYP, are inserted between the first and second helices of the homeodomain, which makes Tex1 a TGIF subclass homeobox gene. Tex1 was expressed specifically in the testis as seen by both Northern blot and RT-PCR analysis. Tex1 expression is initiated at around day 20 of testes. Furthermore, the results of in situ hybridization analysis reveal that the expression of Tex1 in testis is restricted in the germ cells at spermatid stage. Based on these data, it is suggested that Tex1, a novel testis homeobox gene, may play a crucial role during spermatogenesis.

Amino Acid Sequence↗

Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis.

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

RU 3117 a steroidal compound with high affinity for sigma sites in rat testis membranes.

RU 3117 belongs to a new series of steroids which exhibited a high relative binding affinity (RBA) for (+)[3H]PPP sites in rat testis membranes; its RBA was about 40 times higher than that of progesterone. Furthermore, it is devoid of any binding to classical steroid receptors; therefore in order to study its binding parameters on rat testis membranes it was tritiated. [3H]RU 3117 bound at least two distinct sites with Ka values of 0.4 +/- 0.06 x 10(9) M(-1) and 1.3 +/- 0.2 x 10(7) M(-1). Using this marker, competition studies with cold haloperidol showed that a part of this binding was haloperidol-sensitive, whereas another part was haloperidol-resistant. Interestingly, progesterone described as a sigma ligand competes with [3H]RU 3117 binding, with an RBA of 1.6%. When haloperidol was preincubated (250 nM) with rat testis membranes, in order to mask the sigma sites, we observed that DTG (1,3-di-O-tolylguanidine) and haloperidol displayed a very low RBA (< 0.1%) and were not able totally to displace the [3H]RU 3117 binding up to 50 microM. Furthermore, benztropine exhibited a significant RBA of 19% but its displacement curve showed a plateau (500-50,000 nM). These results showed that part of the haloperidol-resistant sites was benztropine sensitive but another part was displaced neither by haloperidol nor by benztropine. The presence of these remaining binding sites was confirmed by preincubating a mixture of haloperidol and benztropine with testis membranes. Under these conditions, [3H]RU 3117 displayed a Ka of 1.0 +/- 0.01 x 10(7) M(-1), and we observed that these sites were recognized, up to now, only by the steroids RU 1968 and RU 54173 which are also devoid of any binding to classical nuclear steroid receptors.

Animals↗

The effects of progesterone, 4,16-androstadien-3-one and MK-434 on the kinetics of pig testis microsomal testosterone-4-ene-5alpha-reductase activity.

The enzyme 3-oxo-steroid: NADP+ 4-oxidoreductase (EC 1.3.1.22; 5alpha-reductase) was assayed in testicular microsomes of pigs of 3, 20 and 24 weeks of age. The activity was very low in 3-week-old animals and approximately 10-fold higher in 5- and 6-month-old pigs. The pH optimum was 6.3 in 6-month-old animals, 5.7 in 5-month-old animals, but could not be reliably determined in 3-week-old animals. The kinetic parameters for 5alpha-reductase in testis microsomes from 6-month-old animals were; K((m)(app)), 8.0 micromol/l, V((max)(app)), 6.7 nmoles/90 min/mg protein. Progesterone was a competitive inhibitor of testosterone 5alpha-reduction with an apparent K((i)(app)) of 0.86 micromol/l. However, 4,16-androstadien-3-one (dienone), which undergoes 5alpha-reduction in the biosynthesis of the pheromonally active 16-androstenes, was a comparatively poor inhibitor with a K((i)(app)) of 4.9 micromol/l. Similarly, MK434, which is a selective inhibitor of the human type 2 5alpha-reductase, but which inhibits both types 1 and 2 in the rat, was also a poor competitive inhibitor of testosterone 5alpha-reductase in the pig testis (K((i)(app)), 3.1 micromol/l). It would appear from these studies that the pig testis microsomal 5alpha-reductase corresponds to a type 1 isozyme that is not capable of reducing dienone other than under conditions where the dienone concentration would be in considerable excess of testosterone. It is, therefore, probable that substrate-specific 5alpha-reductases exist in the pig testis for the 5alpha-reduction of testosterone and dienone.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.

Androgens↗

The amphibian testis as model to study germ cell progression during spermatogenesis.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.

Acrosome Reaction↗

Testis-specific expression of the rat histone Hlt gene in transgenic mice.

The testis-specific histone Hlt gene is transcribed only in testis. The appearance of testis-specific nuclear proteins that bind to a unique promoter sequence element designated Hlt/TE located between the H1/AC box and the H1/CCAAT box correlates with the onset of transcription of the Hlt gene during the meiotic cell cycle. In order to determine whether sequences flanking the rat Hlt gene are sufficient to confer tissue-specific expression in vivo, a 6859 bp EcoRI restriction fragment of genomic DNA containing the rat histone Hlt gene has been microinjected into mouse embryos. S1 nuclease protection analysis has shown that the descendants of the resulting transgenic mice express the rat gene in the proper tissue and at the proper meiotic cell cycle stage. Furthermore, when populations of mouse testis cells were prepared by centrifugal elutriation, only the fraction enriched in pachytene primary spermatocytes had a significant steady-state level of rat Hlt mRNA. Although the copy-number of the transgene was variable in these animals, rat Hlt mRNA levels in high copy-number animals never exceeded 2.6 times the level in normal rat testes. The appearance of appropriate meiotic cell cycle-specific transcription indicates the importance of the conserved promoter sequence elements between the two species.

Animals↗

Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis.

A hamster sperm 26 kDa protein (P26h) is strikingly homologous with mouse lung carbonyl reductase (MLCR) and is highly expressed in the testis, but its physiological functions in the testis are unknown. We show that recombinant P26h resembles NADP(H)-dependent MLCR in the tetrameric structure, broad substrate specificity, inhibitor sensitivity, and activation by arachidonic acid, but differs in a preference for NAD(H) and high efficiency for the oxidoreduction between 5alpha-androstane-3alpha,17beta-diol (k(cat)/K(M) = 243 s(-1) mM(-1)) and 5alpha-dihydrotestosterone (k(cat)/K(M) = 377 s(-1) mM(-1)). The replacement of Ser38-Leu39-Ile40 in P26h with the corresponding sequence (Thr38-Arg39-Thr40) of MLCR led to a switch in favor of NADP(H) specificity, suggesting the key role of the residues in the coenzyme specificity. While the P26h mRNA was detected only in the testis of the mature hamster tissues, its enzyme activity was found mainly in the mitochondrial fraction of the testis and in the nuclear fraction of the epididymis on subcellular fractionation, in which a mitochondrial enzyme, isocitrate dehydrogenase, exhibited a similar distribution pattern. The enzyme activity of P26h in the two tissue subcellular fractions was effectively solubilized by mixing with 1% Triton X-100 and 0.2 M KCl, and enhanced more than 10-fold. The enzymes purified from the two tissue fractions exhibited almost the same structural and catalytic properties as those of the recombinant P26h. These results suggest that P26h mainly exists as a tetrameric dehydrogenase in mitochondria of testicular cells and plays a role in controlling the intracellular concentration of a potent androgen, 5alpha-dihydrotestosterone, during spermatogenesis, in which it may be incorporated in mitochondrial sheaths of spermatozoa.

Alcohol Oxidoreductases↗

Diversity of sequences of polyadenylated cytoplasmic RNA from rainbow trout (Salmo gairdnerii) testis and liver.

We have compared the sequence complexity and diversity of polyadenylated cytoplasmic RNA derived from two differentiated trout tissues: liver and testis. The kinetics of hybridization of polyadenylated RNA from each of these tissues with complementary DNA synthesized by reverse transcriptase revealed three abundance classes for liver RNA, the first comprising 4 sequences, the second 120, and the third, 20 000; in contrast, testis RNA showed only two abundance classes containing 6 and 6100 different RNA sequences, respectively and of average length 6 x 10(5) daltons. The extent of overlapping among those two RNA populations was further studied by performing heterologous annealing reactions between cDNA and a vast excess of mRNA. Liver mRNA was complementary to 80% of the testis cDNA. Conversely, testis mRNA reacted with only 25% of the liver cDNA. Experiments with fractionated cDNA probes indicated that the unshared sequences belonged mainly to the less frequent, most complex, class of mRNAs.

Animals↗

Bovine epidermal fatty acid-binding protein: determination of ligand specificity and cellular localization in retina and testis.

The fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Using an antibody against testis lipid-binding protein, a member of the FABP family, a protein was identified from bovine retina and testis that coeluted with exogenously added docosahexaenoic acid during purification. Amino acid sequencing and subsequent isolation of its cDNA revealed it to be nearly identical to a bovine protein expressed in the differentiating lens and to be the likely bovine homologue of the human epidermal fatty acid-binding protein (E-FABP). From quantitative Western blot analysis, it was estimated that bovine E-FABP comprised 0.9%, 0.1%, and 2.4% of retina, testis, and lens cytosolic proteins, respectively. Binding studies using the fluorescent probe ADIFAB indicated that this protein bound fatty acids of differing levels of saturation with relatively high affinities. Kd values ranged from 27 to 97 nM. In addition, the protein was immunolocalized to the Müller cells in the retina as well as to Sertoli cells in the testis. The location of bovine E-FABP in cells known to be supportive to other cell types in their tissues and the ability of E-FABP to bind a variety of fatty acids with similar affinities indicate that it may be involved in the uptake and transport of fatty acids essential for the nourishment of the surrounding cell types.

Amino Acid Sequence↗

Identification of human CDV-1R and mouse Cdv-1R, two novel proteins with putative signal peptides, especially highly expressed in testis and increased with the male sex maturation.

Human systemic carnitine deficiency (SCD) is a hereditary disease caused by the mutation of OCTN2 and has the characteristics of cardiac hypertrophy. Previous studies based on JVS mouse, an animal model of this disease, showed that Cdv-1 was highly expressed in ventricles of normal mouse, but was remarkably down-regulated in JVS mouse and can be up-regulated to normal level by breeding carnitine, which suggested Cdv-1 was possibly involved in cardiac hypertrophy caused by carnitine deficiency. In this study, the expression of human CDV-1, a homolog of mouse Cdv-1, was undetectable in heart by northern hybridization. The inconsistent expression levels of human CDV-1 and mouse Cdv-1 in heart implied that cardiac hypertrophy in human SCD might not be associated with the abnormal expression of CDV-1. Interestingly, another long transcripts of the gene, Cdv-1R/CDV-1R, were cloned in the present study, in mouse and human, respectively. This long transcript predominantly expressed in both human and mouse testis and its expression level was increased with testis development. Furthermore, we proved that the open reading frame of Cdv-1R/CDV-1R spans the exons 2 approximately 19 instead of exons 9 approximately 19; and the peptide encoded by CDV-1R was composed of 676 amino acids containing a putative signal peptide instead of 414 amino acids described previously. In addition, it was proved that the expression level of Cdv-1R in JVS mouse testis was as high as that in normal mouse testis, and both were not regulated by carnitine.

Alternative Splicing↗

Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis.

The transcription factor GATA-1 is a fundamental regulator of genes in haematopoietic cell lineages and belongs to a family of factors that bind to the consensus sequence WGATAR. The GATA motif was originally identified in cis-regulatory regions of globin and other erythroid-specific genes, but the range of genes controlled by the GATA factors has since expanded. Members of the GATA transcription factor family share a conserved zinc-finger DNA-binding domain, but the expression profile of each GATA factor is distinct. Here we show that a testis form of murine (m)GATA-1 messenger RNA is transcribed from a promoter located 5' to the erythroid first exon, and the remaining exons (which encode the mGATA-1 protein) are used in common by both testis and erythroid transcripts. We use an anti-mGATA-1 monoclonal antibody to show that the factor expressed in erythroid cells is the same as that found in the seminiferous tubules of murine testis. The GATA-1-expressing cells in 10-week-old testis were found only in contact with the basement membrane of seminiferous tubules, suggesting that GATA-1 regulates genes during the earliest stages of spermatogenesis.

Aging↗

Identification and expression of epidermal growth factor gene in mouse testis.

Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig-cells.

Animals↗

Identification of gamma1 subunit of GABA(A) receptor in rat testis.

The isoform type of gamma subunits of GABA(A) receptor is a molecular determinant of its pharmacological characteristics. At present, the existence of GABA(A) receptor in mammalian sperm is still a controversy. By using degenerate primers designed according to highly conserved region in all three gamma (gamma1, gamma2 and gamma3) subunits cloned in rat brain, we performed reverse transcription polymerase chain reaction (RT-PCR) to examine the expression pattern of gamma subunits of GABA(A) receptor in rat testis. Only one 370 bp fragment was obtained from RT-PCR in rat testis and sequencing results showed that it represented gamma1 subunit, but not gamma2 or gamma3 subunit. Using the cloned fragment as probe, a 3.8 kb transcript which in size as same as gamma1 subunit in rat brain was detected in rat testis mRNA by performing Northern blot assay. Furthermore, results of in situ hybridization assay confirmed that gamma1 subunit was expressed in round spermatids and spermatozoa, maybe also in secondary spermatocyte. These evidences proved that gamma1 subunit of GABA(A) receptor is exclusively expressed in rat testis and this feature may be the structural basis of the specific function of GABA(A) receptors in sperm acrosome reaction.

Animals↗

Studies on the biosynthesis of 16-dehydro steroids. The metabolism of [4-14C]pregnenolone by boar adrenal and testis tissue in vitro.

1. The metabolism of [4-(14)C]pregnenolone in vitro by boar adrenocortical and testis tissue has been studied. 2. Boar testis tissue formed three labelled Delta(16)-steroids, 5alpha-androst-16-en-3alpha-ol, 5alpha-androst-16-en-3beta-ol and androsta-4,16-dien-3-one. In adrenal tissue very much smaller yields of the same metabolites were obtained. 3. Both tissues produced labelled progesterone, androst-4-ene-3,17-dione and testosterone in varying quantities. The amount of progesterone was about 120 times greater in the adrenal tissue. In testis tissue dehydroepiandrosterone was found only in small quantity. 4. A pathway is suggested for the biosynthesis of Delta(16)-steroids from pregnenolone in boar testis tissue. The possibility that progesterone may be an intermediate is discussed.

Adrenal Glands↗

High-affinity binding of oestradiol-17beta by cytosols from testis interstitial tissue, pituitary, adrenal, liver and accessory sex glands of the male rat.

The specificity of the binding of oestradiol-17beta by cytoplasmic fractions of several tissues of the male rat was investigated. 1. Agar-gel electrophoresis, Sephadex chromatography, adsorption by dextran-coated charcoal and sucrose-gradient centrifugation were used to estimate the binding capacity and specificity. The four different methods all gave similar results for the capacity of the specific oestradiol-17beta-binding macromolecules in the testis. 2. The presence of a specific saturable binding protein with a sedimentation coefficient of 8S was demonstrated in liver, adrenal, pituitary, prostate, epididymis and testis interstitial tissue. The highest concentration of oestradiol-17beta-binding macromolecules was found in testis interstitial tissue (0.12pmol/mg of protein) and in the pituitary (0.075pmol/mg of protein). 3. The oestradiol-17beta receptor in the testis cytosol showed the characteristics of a protein with respect to Pronase treatment and temperature sensitivity. In competition experiments with different steroids the receptor showed a high affinity for oestradiol-17beta, a moderate affinity for diethylstilboestrol and oestradiol-17alpha and a low affinity for oestrone, oestriol, testosterone and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one). 4. The wide distribution of oestradiol-17beta receptors in the male rat is in apparent contradiction to the current concept of the specificity of steroid-hormone action. Further research is required to investigate a possible physiological meaning of the presence of specific receptors in the different tissues.

Adrenal Glands↗

Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis.

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.

Amino Acid Sequence↗