Perspectives on fish gonadotropins and their receptors.

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.

Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development.

Early expression of estrogen receptors (esr) and their role in regulating early expression of cyp19a1b encoding brain aromatase were examined in the brain of zebrafish. Using in toto hybridization and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), a significant increase in the expression of esr1, esr2a, and esr2b was observed between 24 and 48 hours postfertilization (hpf). In toto hybridization demonstrated that esr2a and esr2b, but not esr1, are found in the hypothalamus. Using real-time RT-PCR, an increase in cyp19a1b mRNAs occurs between 24 and 48 hpf, indicating that expression of cyp19a1b is temporally correlated with that of esr. This increase is blocked by the pure anti-estrogen ICI182,780. Furthermore, E2 treatment of cyp19a1b-GFP (green fluorescent protein) transgenic embryos results in appearance of GFP expression in the brain as early as 25 hpf. These results indicate that basal expression of cyp19a1b expression in the brain of developing zebrafish most likely relies upon expression of esr that are fully functional before 25 hpf.

DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss.

DMRT1 has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. To gain insight on its implication for fish gonadal differentiation, we cloned a DMRT1 homologue in the rainbow trout, Oncorhynchus mykiss (rtDMRT1), and showed that this gene is expressed during testicular differentiation, but not during ovarian differentiation. After 10 days of steroid treatment, expression was shown to be decreased in estrogen-treated male differentiating gonads but not to be restored in androgen-treated differentiating female gonads. This clearly reinforces the hypothesis of an important implication for DMRT1 in testicular differentiation in all vertebrates. In the adults a single 1.5 kb transcript was detected by Northern blot analysis in the testis, and its expression was found to be sustained throughout spermatogenesis and declined at the end of spermatogenesis (stage VI). Along with this expression in the testis we also detected by reverse transcriptase-polymerase chain reaction a slight expression in the ovary. We also obtained new DM-domain homologous sequences in fish, and their analysis suggest that at least four different genes bearing 'DM-domain' (DMRT genes) exist in fish just as in all vertebrate genomes.

Structural organization and regulation of the gene for the androgen-dependent glutathione peroxidase-like protein specific to the mouse epididymis.

Genomic clones containing the gene for the glutathione peroxidase-like androgen-regulated murine epididymal protein of 24 kilodaltons (arMEP24) were isolated. A 9-kilobase DNA fragment was sequenced and found to contain the entire coding region of the gene, which is divided into five exons. The exact sizes and boundaries of the exon blocks were deduced by comparison with the cDNA sequence. One major and four weak transcription initiation sites in the epididymis were localized by primer extension. The promoter of the gene does not contain a conventional TATA box immediately up-stream of the start site; rather, the sequence TATCA occurs at residue -35. Two CAAT boxes in opposite orientation and two putative binding sites for the transcription factor Sp1 were identified up-stream of the TATA-like box. To localize the cis-acting sequences responsible for androgen regulation of expression, fragments of the arMEP24 gene promoter region were cloned in front of the luciferase (LUC) reporter gene and cotransfected with an androgen receptor expression vector into CV-1 cells in a transient assay. LUC activities of CV-1 cells grown in the presence of various concentrations of 5 alpha-dihydrotestosterone were compared to LUC activities of untreated controls. The DNA fragment containing up to 200 nucleotides up-stream from the major transcription start site was sufficient for the full promoter activity, but not for the responsiveness to androgen induction. Depending on the 5 alpha-dihydrotestosterone concentration, a 2- to 4-fold induction of LUC activity was found if a -1797 to -167 arMEP24 gene fragment was used linked to the reporter gene driven by either the homologous promoter or the heterologous thymidine kinase promoter. Two or three copies of the imperfect palindromic sequence TGTTGAgagAGAACA, found at position -896 to -882 in the gene and resembling the consensus steroid hormone-responsive element, are able to confer androgen regulation to the thymidine kinase promoter independently of their orientation. These findings support evidence that transcriptional regulation of the arMEP24 gene occurs via the sequence TGTTGAgagAGAACA. Homologies found in the sequence up-stream of the promoter with several putative binding sites for erythroid-specific trans-acting regulatory proteins are discussed. Finally, the arMEP24 gene is located by in situ hybridization in the [A2-A4] region of mouse chromosome 13.

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation.

Using transgenic mice, we have recently shown that 5 kb of the 5'-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3'-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization analyses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different region-specific gene expression and regulation in the epididymis.

Characterization of an androgen response element within the promoter of the epididymis-specific murine glutathione peroxidase 5 gene.

We have shown in earlier studies, using a mouse model, that the expression of the glutathione peroxidase 5 protein (GPX5) is restricted to the epididymis and that the accumulation of its corresponding mRNA is hormonally, spatially and temporally regulated throughout postnatal development. We report here, using run-on assays, transient expression experiments as well as gel-shift and footprinting analyses on the findings that at least part of the androgenic control of the GPX5 expression is exerted at the transcriptional level via an androgen response element localized in the distal promoter region of the GPX5 gene. The gpx5 androgen response element (ARE) is found to be consistent with the consensus palindromic steroid-receptor target sequence 5'-AGWACWnnnTGTYCT-3' but exhibits a quite weak conservation in the left half site. The data presented here further expand the diversity of sequence able to confer androgen responsiveness.

Spontaneously immortalized epithelial cells from mouse caput epididymidis.

We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

Expression of cytochrome P450(11beta) (11beta-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss.

Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450(11beta) (11beta-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450(11beta) cDNA (rt11betaH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450(11beta) sequence (AF179894) and also to a Japanese eel P450(11beta) (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450(11beta) expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450(11beta) gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450(11beta) gene expression is a key factor for the testicular differentiation in rainbow trout.

GnRH and GnRH receptors in metazoa: a historical, comparative, and evolutive perspective.

About 50years after Harris's first demonstration of its existence, GnRH has strongly stimulated the interest and imagination of scientists, resulting in a high number of studies in an increasing number of species. For the endocrinologist, GnRH, via its actions on the synthesis and release of pituitary gonadotrophins, is first an essential hormone for the initiation and maintenance of the reproductive axis, but recent data suggest that GnRH emerged in animals lacking a pituitary. In this context, this review intends to explore the current status of knowledge on GnRH and GnRH receptors in metazoa in order to see if it is possible to draw an evolutive scenario according to which GnRH actions progressively evolved from the control of simple basic functions in early metazoa to an indirect mean of controlling gonadal activity in vertebrates through a sophisticated network of finely tuned neurons developing in a rather fascinating way. This review also intends to provide an evolutive scenario based on the recent advances of whole genome sequencing possibly explaining the number of GnRH and GnRH receptor variants according to the 2R and 3R theories accompanied by gene losses.

Isolation, sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation.

A cDNA clone (Bc329) encoding calmodulin was isolated from a Bryonia cDNA library by screening with cloned Arabidopsis calmodulin cDNA. The cDNA Bc329 was 899 bp full-length clone. The predicted amino acid sequence consists of 149 residues and reveals a high homology with other known plant calmodulins (91 to 99% identity). Genomic southern blot suggests that Bryonia calmodulin is encoded by a single-copy gene. The Bc329 clone was used as a probe to study the expression of calmodulin mRNA after a mechanical stimulus applied on young Bryonia internodes. The steady-state of calmodulin mRNA reached a maximum 30 min after the treatment before it progressively decreased. The role of calcium and calmodulin as second messengers is discussed with regard to environmental changes.

The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5.

This report presents data that suggest that the tissue-restricted polyoma enhancer activator protein (PEA3) of the Ets oncogene family of DNA-binding proteins is a putative modulator of the epididymis-specific glutathione peroxidase 5 gene gpx5. Northern and polymerase chain reactions on reverse-transcribed epididymal RNAs were used to show that the PEA3 factor is spatially and temporally expressed within the mouse epididymis in a manner consistent with gpx5 characteristics of expression. Then, using contransfection experiments carried out in heterologous tissue-culture cells with various deletions of the gpx5 promoter driving a CAT reporter gene, we have shown that the transcriptional activity of the gpx5 promoter is modulated by the presence of the PEA3 protein. Subsequently, we have shown using gel-shift assays that DNA sequences located within the 5' flanking region of the gpx5 gene have the ability to bind specifically to the PEA3 protein. Finally, using Northern assays we present data that suggest that PEA3 mRNA accumulation in the mouse caput epididymidis is controlled by androgens and testicular factors. Altogether, these results strongly suggest that the PEA3 factor might participate in the transcriptional control of the murine epididymis caput-specific gpx5 gene.

Type II iodothyronine deiodinase is preferentially expressed in rainbow trout (Oncorhynchus mykiss) liver and gonads.

It is well admitted that thyroid hormones (TH) play a role in the development of vertebrates. The major secretory product of the thyroid is a pro-hormone, T(4), which is activated in peripheral tissues by outer ring deiodination to T(3). We have isolated from rainbow trout testis, a full length cDNA encoding type II iodothyronine deiodinase (rtD2). The cDNA was 2410 nucleotides long and coded for a polypeptide of 264 amino acids including a selenocysteine residue. The predicted molecular weight of rtD2 was 29.3 kDa and the isoelectric point 8.71. The deduced amino acids sequence showed 80% identity with Fundulus heteroclitus D2 (fhD2) but only 68-69% identity with rat, mouse, and human D2. The 3' UTR contained a putative selenocysteine insertion sequence (SECIS) similar to that described in human cDNA. The rtD2 gene was isolated and the gene structure was similar to that described in human with two exons separated by a large intron. We studied rtD2 gene expression by Northern blot analysis using total RNA extracted from testis, ovary, and other tissues. We found a high expression of a 3 kb transcript in liver and in gonads. A lower expression was also detected in posterior kidney. In testis, rtD2 mRNA expression was dependent on spermatogenic stages: it increased at the onset of spermatogenesis. Our results show that the structural characteristics of the D2 protein and gene have been highly conserved during evolution. The rtD2 mRNA expression in the gonads suggests that rtD2 may be a key factor regulating local supply of active T(3) during rainbow trout gametogenesis.

Structural organization, chromosomal localization, expression and phylogenetic evaluation of mouse glutathione peroxidase encoding genes.

We have reported earlier the cloning and the chromosomal localization of 2 GPX-encoding sequences expressed differentially within the mouse epididymis, gpx5 and gpx3. Here, we have mapped on the mouse chromosomes the third known murine GPX-encoding gene, the cytosolic GPX or gpx1. We have compared the degree of identity of the 3 GPX proteins, the respective organization of the 3 corresponding single copy genes and, using degenerated oligonucleotides designed in highly conserved domains of the proteins, we have analyzed the expression of GPX-encoding genes in the mouse epididymis as well as in control tissues known to express GPX proteins (the liver for GPX1 and the kidney for GPX3). The 3 genes characterized to date were found expressed in each of the tissues tested but in a highly tissue-restricted manner. Nucleotidic sequences comparisons were carried out on GPX-encoding sequences from various species and were used to draw a dendrogram. Phylogenetic evaluation of the sequence information, as well as the chromosomal localizations, suggest that the GPX genes have evolved by duplication events followed by random insertions from a single ancestral gene.

Stage-dependent and alternative splicing of sGnRH messengers in rainbow trout testis during spermatogenesis.

The gonadotropin releasing hormone (GnRH) has long been considered as a neuropeptide involved in the control of the reproductive cycle. However, the presence of GnRH and its receptors in various tissues, including ovary and testis, suggests a role as autocrine/paracrine factor. In the present study, we report the expression of the sGnRH-1 and sGnRH-2 genes encoding salmon GnRH in rainbow trout testis throughout testicular development and spermatogenesis. We demonstrate that both sGnRH mRNA are expressed prior of sexual differentiation. In adult, northern blot analysis indicates that sGnRH-2 transcripts are expressed in the testis at higher levels than sGnRH-1 messengers. Moreover, we observed that the expression of sGnRH-2, and not sGnRH-1, messengers was stage-dependent. sGnRH-2 mRNA expression decreases at the onset and progressively rebounds at the end of spermatogenesis. In addition, we demonstrate that a complex stage-dependent and differential splicing of the sGnRH-2 messengers occurs throughout spermatogenesis. We isolated five transcripts corresponding to sGnRH-2 messengers. Two of them may encode a novel and shortened GnRH-associated peptide containing 18 residues instead of 46. Our data provide new insight in the putative role of GnRH and GAP peptides as autocrine/paracrine factors of spermatogenesis.

Characterization, regulation of the expression and putative roles of two glutathione peroxidase proteins found in the mouse epididymis.

Two glutathione peroxidase genes (gpx5 and gpx3) were found to be expressed in the mouse epididymis. Gpx5 was shown to be epididymis specific and restricted to the caput epididymidis, while gpx3 was found to be expressed in a wide array of tissues including the caput, corpus and cauda epididymides. Both single copy genes are regulated by androgens as well as being developmentally regulated during postnatal ontogenesis of the epididymis. In this report data collected to date concerning the mechanisms by which these genes are regulated in the mouse epididymis are summarized. The putative roles of these antioxidant enzymes in the sperm maturation process are discussed.

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene.

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa-predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, "CACCC-boxes," NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene.

Characterization of an androgen-specific response region within the 5' flanking region of the murine epididymal retinoic acid binding protein gene.

The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5' flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions -445/-459 and -102/-88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the -543/-88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

Structure and putative function of a murine epididymal retinoic acid-binding protein (mE-RABP).

Vitamin A is required to maintain the epididymal epithelium. In this report, the characterization and putative functions of a murine epididymal retinoic acid-binding protein (mE-RABP) that is secreted into the lumen from the mid-/distal caput epididymidis are discussed. The amino acid sequence analysis of the mE-RABP preprotein shows that mE-RABP is the mouse orthologue of the rat epididymal secretory protein I (ESPI). These proteins belong to the lipocalin superfamily and bind to active retinoids but not to retinol. Therefore, we propose that mE-RABP may function as an extracellular retinoid carrier-protein involved in the paracrine regulation of epididymal function by retinoids.