Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.     

Selenium-independent epididymis-restricted glutathione peroxidase 5 protein (GPX5) can back up failing Se-dependent GPXs in mice subjected to selenium deficiency

We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs.     

Epididymal dysfunction initiated by the expression of simian virus 40 T-antigen leads to angulated sperm flagella and infertility in transgenic mice

We have generated two transgenic mouse lines (GPX5-Tag1 and GPX5-Tag2) by expressing the Simian virus 40 large and small T-antigens under a 5-kb promoter of the murine glutathione peroxidase 5 (GPX5) gene. In GPX5-Tag1 mice, with a high level of T-antigen expression, severe dysplasia was found in the epididymis and seminal vesicles. These mice also developed adrenal and prostate tumors, and spermatogenesis was disrupted. In GPX5-Tag2 mice, with a lower level of T-antigen expression, the only histological change was the slightly hyperplastic epithelium in the initial segment of the epididymidis and in the seminal vesicles. Despite normal mating behavior, these mice were infertile. The most conspicuous feature of the sperm was angulation of the flagellum, which appeared during epididymal transit, probably due to the observed reduction in the osmotic pressure of cauda epididymidal fluid. The angulation did not affect the motility or kinematic parameters of the sperm, but the sperm were also incapable of fertilization in vitro. The lack of expression of several genes specific for the initial segment suggests that in the GPX5-Tag2 mice the transgene expression brings about a differentiation arrest in this part of epididymis. This novel mouse line provides a model for epididymal dysfunction leading to defects in posttesticular sperm maturation and infertility.     

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.     

Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.     

Expression, immunolocalization and sperm-association of a protein derived from 24p3 gene in mouse epididymis

The cDNA sequence for 24p3 protein in ICR mouse epididymal tissue was determined by PCR using primers designed according to the cDNA sequence derived from 24p3 protein in mouse uterine tissue. In the present study, 24p3 protein was immunolocalized in the epithelial cells and lumen of mouse epididymis. Both immunoblot analysis for protein and northern blot analysis for mRNA level showed a declining gradient of 24p3 expression from the caput to caudal region of the epididymis. The 24p3 protein was undetectable in the testis. These findings suggest that the 24p3 protein is a caput-initiated secretory protein in the mouse epididymis. A postnatal study revealed that 24p3 gene expression occurred in mice at the age of 14 days, before the completion of epididymal differentiation. This expression remained at a constant level until epididymal differentiation was completed. We also found that the secreted 24p3 protein interacted predominantly with the acrosome of caudal spermatozoa. Our findings suggest that the epididymal 24p3 protein is a caput-initiated and sperm-associated gene product and may be important in the reproductive system.     

Expression of an endogenous murine leukemia virus-related proviral sequence is androgen regulated and primarily restricted to the epididymis/vas deferens and oviduct/uterus.

A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.     

Pem homeobox gene regulatory sequences that direct androgen-dependent developmentally regulated gene expression in different subregions of the epididymis

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.     

Subcellular compartmentalization of glutathione peroxidase 1 allelic isoforms differentially impact parameters of energy metabolism

Specific genetic variations in the gene for the selenium-containing antioxidant protein glutathione peroxidase 1 (GPX1) are associated with the risk of a variety of common diseases, including cancer, diabetes, and cardiovascular disorders. Two common variations have been focused upon, one resulting in leucine or proline at codon 198 and another resulting in 5, 6, or 7 alanine repeats were previously shown to affect the distribution of GPX1 between the cytoplasm and mitochondria. Human MCF7 cells engineered to exclusively express GPX1 with five alanine repeats at amino terminus and proline at codon 198 (A5P) and seven alanine repeats at amino terminus and leucine at codon 198 (A7L), as well as derivatives targeted to the mitochondria by the addition of a mitochondrial localization sequence (mA5P and mA7L) were used to assess the consequences of the expression of these proteins on the cellular redox state and bioenergetics. Ectopic expression of A5P and A7L reduced the levels of reactive oxygen species, and the mitochondrially targeted derivatives exhibited better activity in these assays. Bioenergetics and mitochondrial integrity were assessed by measuring mitochondrial membrane potential, oxygen consumption, adenosine triphosphate (ATP) levels, and the levels of lactate dehydrogenase. The results of these assays indicated distinctively, and sometimes opposing, patterns with regard to differences between the consequences of the expression of A5P, A7L, mA5P, and mA7L. These data provide new information on the consequences of differences in the primary structure and cellular location of GPX1 proteins and contribute to the understanding of how these effects might contribute to human disease.     

Improved technique for detection of enhanced green fluorescent protein in transgenic mice.

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as reporter proteins in studies carried out in vitro and in vivo. In the present study, several detection techniques for the enhanced green fluorescent protein (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash preparations, vibratome sections, frozen sections) were evaluated. As a model we used transgenic mice expressing EGFP under the control of a 5.0-kb fragment of the glutathione peroxidase isoenzyme 5 protein promoter (GPX5-EGFP) or under a 3.8-kb fragment of the cysteine rich protein-1 promoter (CRISP1-EGFP). In the GPX5-EGFP mice, expression of EGFP was observed in the distal part of the caput epididymis, while the CRISP1 promoter directed EGFP expression in the tubular compartment of the testis. Among the various tissue preparation procedures tested, the best morphological and histological preservation, and reproducibility in EGFP detection, were obtained using frozen sections after a slow tissue-freezing protocol developed in the present study. After slow tissue freezing, specimens of testis and epididymis could be stored at -70 degrees C for at least six weeks without any affect on EGFP fluorescence. Hence, the method developed offers the possibility to analyze EGFP fluorescence in tissues several weeks after specimen collection. The sensitivity achieved was equal to that found in immunohistochemistry, applying biotin-streptavidin-FITC detection. Confocal microscopy is known to have the advantage that fluorescence can be detected from cells in different layers. This was found to be important as regards detecting EGFP fluorescence because the fluorescence was destroyed at the cut surfaces of sections produced by either vibratome or cryomicrotome.     

Comparative sequence and expression analyses of African green monkey (Cercopithecus aethiops) TNPO3 from CV-1 cells

Transportin 3 (TNPO3 or TRN-SR2) is a key host cellular factor involved in the early steps of several lentiviral replications. In the present study, we cloned the TNPO3 gene from CV-1 cells of African green monkey (AGM) using a homologue based cloning technology, analyzed the sequence, and evaluated the cellular expression of the proteins by western blotting and immunostaining assays. DNA sequencing of TNPO3 showed homologies of 99 % with human, rhesus monkey, chimpanzee, and baboon; the predicted protein sequence differed in only one amino acid (leucine in place of methionine). The deduced sequence revealed that AGM is phylogenetically related to human, chimpanzee, rhesus monkey, orangutan and baboon rather than bovine, rate and mouse. Western blot analysis demonstrated immunoreactive proteins in both the cytoplasmic and nuclear fractions. A similar expression pattern was observed in human and baby hamster cells. The specific detection of TNPO3 was also confirmed in the cytoplasm and nucleus by immunostaining. The present findings conclusively demonstrate that AGM-TNPO3 is genetically and physiologically almost identical with that of humans and could be a good candidate for HIV and AGM research as well as an ideal system for a TNPO3 vaccine trial.     

Evidence for sulfatase and 17beta-hydroxysteroid dehydrogenase type 1 activities in equine epididymis and uterus

Our previous work showed that stallion testis produces high amounts of estrogens which are subsequently found in the ejaculate. These estrogens are mainly synthesized by testicular aromatase, and the major estrogen produced is estrone sulfate (E1S). The objective of this study was to investigate the potential role of E1S as a source of estrogens in the male and female horse reproductive tracts by determining whether both estrone sulfatase (Sulf) and 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1) activities were present in equine testes, epididymis and uterus. We assessed E1S bioconversion into estrone (E1) and estradiol (E2) in these tissues. Both Sulf and 17beta-HSD1 activities were well detected in the cauda epididymis and uterus. Additionally, Sulf activity was present in the distal corpus of the epididymis, and 17beta-HSDI in the proximal corpus. In contrast, aromatase gene expression, measured as an internal control of endogenous estrogen production, had high activity only in the testis. We found that seminal E1S of testicular origin can be metabolized to E2, especially in the cauda epididymis and uterus. Because E2 appears to play a major role in male and female reproduction, we propose that the bioconversion of seminal E1S could affect male and female fertility.     

Bovine RNase MRP cleaves the divergent bovine mitochondrial RNA sequence at the displacement-loop region

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves mitochondrial RNA from the origin of leading-strand DNA synthesis contained within the displacement-loop region. Bovine mitochondrial DNA maintains the typical gene content and order of mammalian mitochondrial DNAs but differs in the nature of sequence conservation within this displacement-loop regulatory region. This markedly different sequence arrangement raises the issue of the degree to which a bovine RNase MRP would reflect the physical and functional properties ascribed to the enzymes previously characterized from mouse and human. We find that bovine RNase MRP exists as a ribonucleoprotein, with an RNA component of 279 nucleotides that is homologous to that of mouse or human RNase MRP RNA. Characterization of the nuclear gene for bovine RNase MRP RNA showed conservation of sequence extending 5 of the RNase MRP RNA coding sequence, including the presence of a cis-acting element known to be important for the expression of some mitochondrial protein-coding nuclear genes. Bovine or mouse RNase MRP cleaves a standard mouse mitochondrial RNA substrate in the same manner; each also cleaves a bovine mitochondrial RNA substrate identically. Since bovine and mouse RNase MRPs process both bovine and mouse substrates, we conclude that the structural features of the mitochondrial RNA substrate required for enzymatic cleavage have been well conserved despite significant overall primary sequence divergence. Inspection of the bovine RNA substrate reveals conservation of only the most critical portion of the primary sequence as indicated by earlier studies with mouse and human RNase MRPs. Interestingly, a principal cleavage site in the bovine mitochondrial RNA substrate is downstream of the promoter located at the leading-strand mitochondrial DNA replication origin. Correspondence to: D.J. Dairaghi     

The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.