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PubMed1994

Proenkephalin transgenic mice: a short promoter confers high testis expression and reduced fertility.

The regulation and possible function of the preproenkephalin gene in testis were studied in vivo in transgenic mice containing: (1) bases -193 to +210 of the human proenkephalin gene and an additional one kilobase of 3' proenkephalin flanking sequence driving expression of bacterial chloramphenicol acetyltransferase (CAT), and (2) the same promoter and flanking sequences driving expression of a rat proenkephalin cDNA. Five lines of mice, designated HEC1-5, expressed the first construct and 10, HER1-10, the second. Each HEC male and many HER males showed dramatic expression of the transgene in the testis, although much lower expression was observed in the brain and other enkephalin-producing tissues. High levels of expression in testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Altered enkephalin expression may affect testicular function. One founder, HER8, displayed grossly abnormal testicular morphology and was completely infertile. A second founder, HER6, had low sperm motility. Two offspring from other lines also displayed subnormal fertility. These studies support a role for specific promoter sequences in testis expression and may further support a significant role for proenkephalin in testicular function.

Animals↗
PubMed1995

Identification of osteopontin (OPN) mRNA and protein in the rat testis and epididymis, and on sperm.

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immunoblot analysis and immunocytochemistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis lambda gt11 cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). Immunoblot analysis with a monoclonal antibody against OPN detects the presence of immunoreactive polypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. Immunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstrated positive hybridization to Sertoli cell-enriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididymal spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed.

Animals↗
PubMed2004

Expression profiling of the developing testis in wild-type and Dazl knockout mice.

Genetic understanding of male-factor infertility requires knowledge of gene expression patterns associated with normal germ cell differentiation. The mouse is one of the best models of mammalian fertility due to its well-characterized genetics and the existence of many infertile mutants both naturally occurring and experimentally induced. We used cDNA microarrays firstly to investigate normal gene expression in the wild-type (wt) testis and secondly to gain a better insight into the effect of the disruption of the Dazl gene on spermatogenesis. We constructed a cDNA microarray from a subtracted and normalized adult testis library and focused on six developmental time-points during the initial synchronous wave of spermatogenesis. The results suggest that in the wild-type testis, 89.5% of genes on our chip change expression dramatically during the time-course. To identify patterns in the gene-expression data, a k-means clustering algorithm and principal component analysis were used. In the Dazl knockout testes, the majority of genes remain at baseline levels of expression, because absence of Dazl has a severe effect on cell-types present in the testis. Although in the prepubescent Dazl-null mice the final point reached in germ cell development is the leptotene-zygotene stage, the microarray results suggest that lack of Dazl expression has a detectable effect on the mRNA complement of germ cells as early as day 5 when only type A spermatogonia are present. Mol. Reprod. Dev. 67: 26-54, 2004.

Algorithms↗
PubMed2004

Detection of msj-1 gene expression in the frog, Rana esculenta testis, brain, and spinal cord.

MSJ-1 is member of the DnaJ/heat shock protein (Hsp) 40 chaperone protein family. It is present in mouse testis and spinal cord. In particular, MSJ-1 is localized in post-meiotic cells and in motoneurones of the ventral horns. To assess whether the role of this protein is evolutionarily conserved, we have investigated if msj-1 gene is expressed in the frog, Rana esculenta. Using reverse transcription-polymerase chain reaction (RT-PCR), a msj-1-like transcript was detected in testis, brain, and spinal cord. Homology ranging from 42.3 to 46.0% was found as compared with the mammalian counterparts. Muscle did not show any signal. By Western blot analysis, a signal of the predicted size of 30 kDa was evidenced in testis, brain, and spinal cord but not in ovary, heart, liver, kidney, and muscle. MSJ-1 fluctuations in the testis reveal that it appeared in concomitance with post-meiotic events during the annual sexual cycle, as shown in a previous study. The protein is localized in spermatids and is still retained in mature spermatozoa, where it has perinuclear and centriolar localization. MSJ-1 levels did not change in brain and spinal cord. Furthermore, in the brain MSJ-1 was mainly present in diencephalon and mesencephalon, while in spinal cord MSJ-1 was localized into several motoneurones of the cervical and thoracic tract. A putative role in vesicle trafficking is briefly discussed.

Animals↗
PubMed2006

Expression of the spermatogenic cell-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) in rat testis.

The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1,417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS (94% identity) and human GAPD2 (83% identity) isozymes. Northern blotting of rat tissue RNAs detected the 1.5 kb Gapds transcript in the testis and not in RNA from liver, spleen, epididymis, heart, skeletal muscle, brain, seminal vesicle, and kidney. The rat Gapds mRNA was first detected at day 29 of postnatal testis development, an age which coincides with the initial post-meiotic differentiation of round spermatids. When isolated rat spermatogenic cell RNA was probed for Gapds expression, transcripts were detected only in round spermatids and condensing spermatids, but not in pachytene spermatocytes, demonstrating haploid expression of the Gapds gene. However, immunohistochemical staining of rat testis sections with anti-GAPDS antisera did not detect GAPDS in round spermatids, but localized the protein only to stage XIII and later condensing spermatids as well as testicular spermatozoa, indicating that Gapds expression is translationally regulated. The current results are similar to those previously obtained for mouse GAPDS and human GAPD2, suggesting that reliable comparisons can be made between these species in toxicant screening and contraceptive development.

Amino Acid Sequence↗
PubMed2006

Proopiomelanocortin gene expression and beta-endorphin localization in the pituitary, testis, and epididymis of stallion.

Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), beta-endorphin (beta-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, beta-EP, is produced locally in the testis, thus playing effects in a paracrine/autocrine fashion. To investigate this hypothesis the POMC gene expression was analyzed using 3' RACE-PCR and Northern Blot approaches in the testis and epididimys of stallion; moreover, immunocytochemical localization for beta-EP was also performed through confocal laser microscopy. The immunofluorescence results showed a positive beta-EP reaction not only in cellular nest of pituitary but also in the testis and genital tract of stallion, which function could be related with sperm mobility. Such role seem not to be no dependent on the peptide synthesized locally, because the molecular biology approach demonstrated the presence of POMC transcript in the pituitary only. In fact the Northern Blot analysis showed the presence of a single POMC transcript in the pituitary while no signal was detected in the testis and epididimys. The same results were obtained by applied 3' RACE-PCR analysis. In conclusion, opioid-derived peptide beta-EP is present in the genital tract of stallion, but is not locally produced as in other mammalian, and nonmammalian models; its possible biological function at testicular level could be linked to a long-loop feed-back mechanisms.

Animals↗
PubMed2006

Identification, molecular cloning, and cellular distribution of the rat homolog of minichromosome maintenance protein 7 (MCM7) in the rat testis.

As part of a program to decipher the rat testicular proteome, we studied spermatogonia and identified numerous proteins including the human homolog of the Minichromosome Maintenance Protein 7 (MCM7). MCM7 has been implicated in DNA replication in various species, but had not been detected in the testis. Here we describe the cellular distribution of MCM7 transcripts and protein, and their testicular ontogenetic expression. The full-length coding region of the rat MCM7 was also characterized. Northern blot analyses showed that MCM7 transcripts are more abundant in the testis than other organs and confirmed the presence of the 2.4 kb MCM7 transcript at all ages studied. Interestingly, two additional transcripts of 3.2 and 1.6 kb were found from 26 days post partum onwards, when spermatocytes and spermatids accumulate within the tubules. This was confirmed in isolated cell types: the three MCM7 transcripts were observed in meiotic and post-meiotic germ cells. The 3.2 kb isoform has an extended 5' untranslated region (UTR) and the 1.6 kb transcript is the result of alternative splicing of five exons. Western blot and immunohistochemistry experiments evidenced abundant MCM7 in proliferating gonocytes and Sertoli cells in the fetal testis. In the adult testis, an intense signal was observed in spermatogonia and primary spermatocytes. We conclude that the Mcm7 is one example of genes that are differently transcribed and translated in somatic and spermatogenetic cells in mammals. Further work is required to determine the roles of MCM7 in spermatogonia and germ lineage.

Adenosine Triphosphatases↗
PubMed1997

Effect of replacement of the amino and the carboxyl termini of rat testis fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase with those of the liver and heart isozymes.

Fru 6-P,2-kinase:Fru 2,6-Pase is a bifunctional enzyme, consisting of highly conserved catalytic domains and variable regulatory domains. The regulatory domains reside in either the N- or the C-terminus, depending upon the isozyme. The rat testis enzyme (RT2K) lacks the regulatory domain, but the rat liver and the bovine heart enzymes contain phosphorylation site(s) in the N- and the C-termini, respectively. In order to determine whether the regulatory domains can be swapped, we have constructed mutant enzymes in which the N- or the C-terminal tail of the testis enzyme was replaced with that of either the liver or the heart enzyme. The substitution with the N-terminus of the liver enzyme (RLN-RT2K) resulted in a small change in the kinetic properties of Fru 6-P,2-kinase, but that with the heart enzyme increased the KFru 6-P 18-fold without affecting the Vmax. The substitution with the C-terminus of the heart enzyme had little effect. The phosphorylation of RLN-RT2K increased KFru 6-P fivefold as in the liver enzyme but did not affect the Fru 2,6-Pase, unlike the liver enzyme. All these mutant enzymes were more thermally labile than the wild type testis enzyme. RLN-RT2K was more sensitive to the denaturant. These results suggest that the N-terminus of the liver enzyme could interact with the kinase domain of the testis enzyme, regulating the kinase activity but was unable to affect the phosphatase domain. These differences could be explained by the large differences in net charges of the terminal tails.

Allosteric Regulation↗
PubMed2001

Comparative squalene synthase gene expression in mouse liver and testis.

RNA from various mouse organs was analyzed by Northern hybridization to determine the response of squalene synthase (SQS) mRNA to dietary cholesterol, or lovastatin and cholestyramine, administration. Two size-classes of highly abundant mouse SQS (mSQS) mRNAs of approximately 1.9 and 2.0 kb were found in testis. These transcripts were unresponsive to sterol regulation. A single size-class of liver mSQS mRNA of approximately 1.9 kb was sterol-regulated. Studies using primer extension and 5' rapid amplification of cDNA ends (RACE) indicated that the size differences in liver and testis mSQS transcripts were due to variations in the lengths of the 5' untranslated regions (UTRs). The longest testis 5' UTR extended approximately 106 nt 5' of the primary transcription initiation site in liver of mice fed lovastatin and cholestyramine. These results suggest that tissue-specific promoter elements control the transcriptional regulation of the promoters for the mSQS gene in liver and testis.

5' Untranslated Regions↗
PubMed1996

Expression of multiple c-kit receptor messenger ribonucleic acid transcripts during postnatal development of the rat testis.

The c-kit protooncogene is a transmembrane tyrosine kinase receptor expressed during gametogenesis. Using the polymerase chain reaction (PCR), we have identified the c-kit receptor mRNA transcripts in the rat testis and studied their expression during postnatal development of the testis. Five different transcripts were identified using sets of primers encoding within the extracellular domain. Two transcripts were obtained from primer sets encoding regions within the cytoplasmic domain and the primer set encoding the entire length of the c-kit receptor. We have compared the levels of expression of these transcripts on different days during postnatal development. The level of expression of a particular transcript varied depending upon the developmental stage of the testis. In summary, our results suggest that multiple forms of mRNAs exist for the c-kit receptor in the rat testis, and they are regulated differentially during postnatal development.

Aging↗
PubMed1997

Foreign gene expression in the mouse testis by localized in vivo gene transfer.

In order to attain foreign gene expression in vivo in the testis of living mice, chloramphenicol acetyltransferase (CAT), firefly luciferase and bacterial lacZ reporter genes were transfected by microparticle bombardment and electroporation. The results showed that CAT reporter gene was expressed in a dose-dependent fashion. The X-gal staining showed that in some spermatogenic-like cells, the bacterial lacZ gene was also expressed by in vivo electroporation, but not by in vivo microparticle bombardment. The possibility of in vivo gene transfer to the spermatogenic cells of the mouse testis was further confirmed by the fact that the CAT reporter gene expression was testis-specific when driven by the mouse-protamin 1 promoter. It was concluded, therefore, that in vivo microparticle bombardment and, especially, electroporation provide convenient and efficient means of gene transfer to the testis of living mice.

Animals↗
PubMed1997

Cloning and functional expression of a second new aquaporin abundantly expressed in testis.

A new member of water channels has been identified from rat testis. This gene, termed aquaporin 8 (AQP8), encoded a 263-amino-acid protein that contained the conserved NPA motifs of MIP family proteins. AQP8 has amino acid sequence identity with other aquaporins (approximately 35%) and highest with a plant water channel, AQP-gamma TIP (39%), suggesting that AQP8 is a unique member in mammalian aquaporins. The expression of AQP8 in Xenopus oocytes stimulated the osmotic water permeability (Pr) 8.5 folds. The increase of Pr was inhibited with 0.3 mM mercury chloride by 55%, which was reversed with mercaptoethanol. The Arrhenius activation energy for the stimulated water permeability was low (5.1 kcal/mol). AQP8 did not facilitate glycerol transport. Northern blot analysis revealed a 1.5-kb transcript of AQP8 abundantly in testis and slightly in liver. In situ hybridization of testis revealed the expression of AQP8 mRNA in all stages of spermatogenesis from primary spermatocytes to spermatids in seminiferous tubules. Together with previously cloned AQP7, AQP8 may also play an important role in spermatogenesis. The unexpected complexity of the presence of two aquaporins in testis may call for the further analysis of the role of aquaporins in the reproduction biology.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of a DEG/ENaC sodium channel cDNA from human testis.

The number of members of the recently defined DEG/ENaC sodium channel superfamily is increasing. Their importance in Na transport, taste perception, acid sensing, and mechanotransduction has been implicated. We have cloned a new member of this superfamily from human testis, which was named hTNaC1 (for human testis sodium channel 1). The hTNaC1 has 532 amino acid residues with two hydrophobic transmembrane domains. It has the highest identity (82%) with a rat H(+)-gated Na channel specific for sensory neurons (DRASIC) and a low identity (29%) with an epithelial isoform (alpha-ENaC) of this superfamily. Northern blot of human tissues revealed its selective expression in testis (7 kb) and absence in other tissues. The identification of a new member of Na channel specifically expressed in testis will expand the role of this channel family to the reproduction physiology.

Acid Sensing Ion Channels↗
PubMed1999

A 1.3-kb upstream 5' region of the rat phosphoglycerate mutase m gene confers testis and skeletal muscle-specific expression in transgenic mice.

Spermatogenesis is a complex process that occurs in successive mitotic, meiotic and post-meiotic phases and involves a highly regulated selective gene-expression pattern. However, this process has not been well characterised at the gene expression level due to the absence of germinal cell lines. We previously demonstrated that the rat skeletal muscle-specific gene for the glycolytic enzyme phosphoglycerate mutase is also specifically expressed in meiotic and haploid male germ cells from testis (12). To analyse the promoter elements that regulate the transcription of the phosphoglycerate mutase m gene (pgam-m)during spermatogenesis, we developed transgenic mice for a construct containing 1.3 kb from the pgam-m promoter linked to the Escherichia coli LacZ gene. RNA analysis by retrotranscription and PCR amplification of transgene expression showed transcriptional activity in the testis with a pattern during testis development that was identical to the endogenous gene. The transgene was also active in skeletal muscle but not in the adult heart in all the transgenic lines analysed. Collectively, these studies demonstrate that the 1.3 kb pgam-m promoter contains sufficient sequences to specify temporally regulated testis-specific expression as well as skeletal-muscle expression.

Animals↗
PubMed2001

Cloning and expression of activator of CREM in testis in human testicular tissue.

Activator of cAMP-responsive element modulator (CREM) in testis (ACT) has recently been found in the mouse testis where it activates CREM, a transcription factor essential for the differentiation of spermatids into mature spermatozoa. The importance of CREM in human spermatogenesis prompted us to examine whether ACT was also present in the human testis. Western blot analysis, performed with an anti-mouse ACT serum, showed the presence of a single immunoreactive band of a size similar to murine ACT. A library screening resulted in the isolation and characterization of the complete cDNA which showed 88% homology with the mouse counterpart. The human ACT gene is composed of five coding exons, being the first untranslated, and the mRNA spans 835 nucleotides coding for a 284 amino acid protein. Expression studies by RT-PCR confirmed that ACT is present in normal human testis. The human ACT gene is localized on the chromosome 6.

Amino Acid Sequence↗
PubMed1997

Developmental testis-specific regulation of mRNA levels and mRNA translational efficiencies for TATA-binding protein mRNA isoforms.

Early spermatids contain roughly 1000-fold more TATA-binding protein (TBP) mRNA than do somatic cells. The appearance of TBP-overexpressing spermatids in the developing testis is accompanied by a large increase in whole-organ levels of total RNA and of poly(A)+ RNA per cell. Whereas somatic cells initiate transcription of TBP mRNA at a single promoter/first exon (exon 1C), in adult testis, two additional major promoter/first exons (1D and 1E) are used. We have examined the expression of the somatic and testis-specific TBP mRNA isoforms during rodent testis development. In juvenile testes TBP mRNAs containing either exon 1C or exon 1D, but none containing exon 1E, are detected. At 21 days of age, all TBP mRNA isoforms begin to overaccumulate. The onset of TBP mRNA overaccumulation is marked first by an increase in levels of polysomal TBP mRNA, and later by accumulation of mRNP-associated TBP mRNA. In adult testes, only 30% of the total TBP mRNA is engaged by polysomes; the remainder is sequestered as mRNP particles. All of the TBP mRNA isoforms in adults exist both as free mRNP particles and as polysomes; however, the fraction in polysomes varies from 60% (exon 1C) to 10% (exon 1E). This suggests that sequences within the first exons alter the probability that the mRNA will either assemble into polysomes or into translationally inactive mRNP particles.

Animals↗
PubMed2002

The T complex distorter 2 candidate gene, Dnahc8, encodes at least two testis-specific axonemal dynein heavy chains that differ extensively at their amino and carboxyl termini.

Homozygosity for the t haplotype allele of the testis-specifically expressed axonemal dynein heavy chain (axDHC) gene, Dnahc8, has been linked to male sterility resulting from aberrant sperm motility. However, the near absence of Dnahc8 expression has been associated with male sterility resulting from an early breakdown in sperm flagellar development. Although axDHCs are integral participants in flagellar motility, a role in flagellar morphogenesis has never been attributed to a member of this highly conserved gene family. To gain a better understanding of this presumed novel role for Dnahc8, we have studied the organization and expression of full-length Dnahc8(+) and Dnahc8(t) transcripts. Our results demonstrate the existence of at least two alternatively spliced, testis-specific Dnahc8 mRNAs transcribed from both the + and t alleles. A highly expressed isoform encodes a protein with significant homology nearly throughout to the gamma heavy chain of the Chlamydomonas axonemal outer arm dynein, while a more poorly expressed isoform codes for a protein whose sequence diverges significantly from that of other axDHCs at both its N and C termini. While in situ hybridization studies demonstrate that both mRNA species accumulate exclusively in mid to late spermatocytes, each isoform shows spatial independence. Additional experiments demonstrate the existence of a testis-expressed mRNA with no significant open reading frame, a portion of which is antisense to the 5'-untranslated region of the highly divergent Dnahc8 isoform. The cumulative data imply that Dnahc8 may have acquired functional plasticity in the testis through the tightly controlled expression of both typical and unusual isoforms.

Amino Acid Sequence↗
PubMed1998

Autoregulation of estrogen and androgen receptor mRNAs and downregulation of androgen receptor mRNA by estrogen in primary cultures of lizard testis cells.

Steroid hormones regulate many developmental and physiological processes via specific receptors whose number can be up- or downregulated. The regulation of estrogen (ER) and androgen (AR) receptor mRNAs in primary cultures of lizard testis is described. The high degree of homology between the probes used and the receptor mRNAs in lizard testis was consistent with the high-stringency hybridisation conditions and the molecular size of ER mRNAs (7.4 and 4.5 kb) and AR mRNA (9.5 kb). Primary cultures of testis cells revealed a time- and drug-dependent relationship between ER and AR mRNAs. 17beta-oestradiol (E) autoregulated ER mRNA and downregulated AR mRNA. The antiestrogen ICI 164,384 reversed the latter effect. Cycloheximide (Cy), to inhibit protein synthesis, in combination with E, impaired the AR mRNA expression. Testosterone (T) autoregulated the expression of its own receptor mRNA whereas this effect was reversed by both flutamide (F) and Cy. Dose-response experiments showed that low concentrations of steroids (E or T 10(-12) M) increased ER or AR mRNA levels, respectively. These results suggest that both estrogen and androgen may autoregulate the expression of their own receptor mRNAs. Since in lizard testis androgens are significantly involved in meiosis and spermiogenesis and E dramatically impairs the AR mRNA expression, the latter effect may be key in regulating certain phases of reproduction.

Animals↗