A first complete catalog of highly expressed genes in eight chicken tissues reveals uncharacterized gene families specific for the chicken testis.

Based on next-generation sequencing, we established a repertoire of differentially overexpressed genes (DoEGs) in eight adult chicken tissues: the testis, brain, lung, liver, kidney, muscle, heart, and intestine. With 4,499 DoEGs, the testis had the highest number and proportion of DoEGs compared with the seven somatic tissues. The testis DoEG set included the highest proportion of long noncoding RNAs (lncRNAs; 1,851, representing 32% of the lncRNA genes in the whole genome) and the highest proportion of protein-coding genes (2,648, representing 14.7% of the protein-coding genes in the whole genome). The main significantly enriched Gene Ontology terms related to the protein-coding genes were "reproductive process," "tubulin binding," and "microtubule cytoskeleton." Using real-time quantitative reverse transcription-polymerase chain reaction, we confirmed the overexpression of genes that encode proteins already described in chicken sperm [such as calcium binding tyrosine phosphorylation regulated (CABYR), spermatogenesis associated 18 (SPATA18), and CDK5 regulatory subunit associated protein (CDK5RAP2)] but whose testis origin had not been previously confirmed. Moreover, we demonstrated the overexpression of vertebrate orthologs of testis genes not yet described in the adult chicken testis [such as NIMA related kinase 2 (NEK2), adenylate kinase 7 (AK7), and CCNE2]. Using clustering according to primary sequence homology, we found that 1,737 of the 2,648 (67%) testis protein-coding genes were unique genes. This proportion was significantly higher than the somatic tissues except muscle. We clustered the other 911 testis protein-coding genes into 495 families, from which 47 had all paralogs overexpressed in the testis. Among these 47 testis-specific families, eight contained uncharacterized duplicated paralogs without orthologs in other metazoans except birds: these families are thus specific for chickens/birds.NEW & NOTEWORTHY Comparative next-generation sequencing analysis of eight chicken tissues showed that the testis has highest proportion of long noncoding RNA and protein-coding genes of the whole genome. We identified new genes in the chicken testis, including orthologs of known mammalian testicular genes. We also identified 47 gene families in which all the members were overexpressed, if not exclusive, in the testis. Eight families, organized in duplication clusters, were unknown, without orthologs in metazoans except birds, and are thus specific for chickens/birds.

An evolutionary approach to recover genes predominantly expressed in the testes of the zebrafish, chicken and mouse.

BACKGROUND: Previously, we have demonstrated that genes involved in ovarian function are highly conserved throughout evolution. In this study, we aimed to document the conservation of genes involved in spermatogenesis from flies to vertebrates and their expression profiles in vertebrates. RESULTS: We retrieved 379 Drosophila melanogaster genes that are functionally involved in male reproduction according to their mutant phenotypes and listed their vertebrate orthologs. 83% of the fly genes have at least one vertebrate ortholog for a total of 625 mouse orthologs. This conservation percentage is almost twice as high as the 42% rate for the whole fly genome and is similar to that previously found for genes preferentially expressed in ovaries. Of the 625 mouse orthologs, we selected 68 mouse genes of interest, 42 of which exhibited a predominant relative expression in testes and 26 were their paralogs. These 68 mouse genes exhibited 144 and 60 orthologs in chicken and zebrafish, respectively, gathered in 28 groups of paralogs. Almost two thirds of the chicken orthologs and half of the zebrafish orthologs exhibited a relative expression ≥50% in testis. Finally, our focus on functional in silico data demonstrated that most of these genes were involved in the germ cell process, primarily in structure elaboration/maintenance and in acid nucleic metabolism. CONCLUSION: Our work confirms that the genes involved in germ cell development are highly conserved across evolution in vertebrates and invertebrates and display a high rate of conservation of preferential testicular expression among vertebrates. Among the genes highlighted in this study, three mouse genes (Lrrc46, Pabpc6 and Pkd2l1) have not previously been described in the testes, neither their zebrafish nor chicken orthologs. The phylogenetic approach developed in this study finally allows considering new testicular genes for further fundamental studies in vertebrates, including model species (mouse and zebrafish).

New insights into the evolution, hormonal regulation, and spatiotemporal expression profiles of genes involved in the Gfra1/Gdnf and Kit/Kitlg regulatory pathways in rainbow trout testis.

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20ß progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation.

Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss).

Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a, cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reverse-transcriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens up-regulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion.

Gdnf-Gfra1 pathway is expressed in a spermatogenetic-dependent manner and is regulated by Fsh in a fish testis.

What makes the spermatogonial stem cells (SSCs) self-renew or differentiate to produce spermatozoa is barely understood, in particular in nonmammalian species. Our research explores possible regulations of the SSC niche in teleost, locally by paracrine factors and peripherally by hormonal regulation. In the present study, we focus on the Gdnf-Gfra1 pathway that plays a major role in the regulation of SSC self-renewal in mammals. We describe a complex evolution of the genes encoding for Gdnf and Gfra1 proteins in trout with the emergence of three gdnf and two gfra1 paralogs. Using quantitative PCR measurements in isolated testicular cell populations, the gdnfb paralog was found expressed in A-spermatogonia and probably in another testicular cell type. In contrast, the transcript of gfra1a, the Gdnf receptor, was preferentially expressed in a population of undifferentiated A-spermatogonia (und A-Spg) separated by centrifugal elutriation. These und A-Spg also demonstrated high stemness potential in transplantation studies and preferentially expressed nanos2, a putative SSC marker in trout (Bellaiche et al., Biol Reprod 2014; 90:79). Flow cytometer experiments demonstrate that only a subfraction of und A-Spg express Gfra1. In trout, spermatogenesis develops along a strict annual cycle, and gdnfb and its receptor were expressed in a spermatogenetic activity-dependent manner. In particular, a dramatic increase of the gdnfb transcript coincided with the progressive cessation of rapid spermatogonial proliferation and of meiosis toward the end of the reproductive cycle. Together these results suggest that, in trout, Gdnfb is involved in the repression of und A-Spg differentiation. Fsh is an endocrine regulator of SSCs self-renewal through the up-regulation of Gdnf in rodents. We demonstrate that in trout, in vitro Fsh treatment stimulated the expression of the gfra1a1 but not of its ligand, gdnfb. Fsh treatment also stimulated the proliferation of und A-Spg cocultured with testicular somatic cells. Based on those results, the Gfra1-positive cells could correspond to the putative SSCs in rainbow trout, and we propose that the balance between SSC self-renewal and differentiation during the trout spermatogenetic cycle is under paracrine regulation by Gdnfb, which represses, and under peripheral regulation by Fsh via the control of gfra1a1 expression.

Spermatogonial stem cell quest: nanos2, marker of a subpopulation of undifferentiated A spermatogonia in trout testis.

Continuous or cyclic production of spermatozoa throughout life in adult male vertebrates depends on a subpopulation of undifferentiated germ cells acting as spermatogonial stem cells (SSCs). What makes these cells self-renew or differentiate is barely understood, in particular in nonmammalian species, including fish. In the highly seasonal rainbow trout, at the end of the annual spermatogenetic cycle, tubules of the spawning testis contain only spermatozoa, with the exception of scarce undifferentiated spermatogonia that remain on the tubular wall and that will support the next round of spermatogenesis. Taking advantage of this model, we identified putative SSCs in fish testis using morphological, molecular, and functional approaches. In all stages, large spermatogonia with ultrastructural characteristics of germinal stem cells were found, isolated or in doublet. Trout homologues of SSC and/or immature progenitor markers in mammals-nanos2 and nanos3, pou2, plzf, and piwil2-were preferentially expressed in the prepubertal testis and in the undifferentiated A spermatogonia populations purified by centrifugal elutriation. This expression profile strongly suggests that these genes are functionally conserved between fish and mammals. Moreover, transplantation into embryonic recipients of the undifferentiated spermatogonial cells demonstrated their high "stemness" efficiency in terms of migration into gonads and the ability to give functional gametes. Interestingly, we show that nanos2 expression was restricted to a subpopulation of undifferentiated spermatogonia (less than 20%) present as isolated cells or in doublet in the juvenile and in the maturing trout testis. In contrast, nanos2 transcript was detected in all the undifferentiated spermatogonia remaining in the spawning testis. Plzf was also immunodetected in A-Spg from spawning testis, reinforcing the idea that these cells are stem cells. From those results, we hypothesize that the subset of undifferentiated A spermatogonia expressing nanos2 transcript are putative SSC in trout.

A promoter fragment of the sycp1 gene is sufficient to drive transgene expression in male and female meiotic germ cells in zebrafish.

The synaptonemal complex protein 1 (Sycp1) is required for the formation of crossovers that occurs during the meiotic prophase. The tissue and cell-specific expression pattern of the Sycp1 protein have been studied in mammals and fish, but data on the corresponding transcript remain scarce. In this report, we described for the first time in zebrafish the tissue- and cell-specific expression pattern of the sycp1 gene. In ovary, the expression of the sycp1 transcript was restricted to the early primary oocytes. In testis, the sycp1 transcript was observed in primary spermatocytes in agreement with a previous report describing the localization of the Sycp1 protein in those cells. Unexpectedly, sycp1 transcript expression remained high in spermatids. To gain insight on the genomic region responsible for the sycp1 gene expression pattern, we generated four independent Dr_sycp1:eGFP transgenic zebrafish lines carrying the -1482/+338 gene fragment fused to the enhanced green fluorescent protein reporter gene. We demonstrate that this promoter fragment contains the information required for the cell-specific expression of the endogenous sycp1 gene in males and in females. However, the GFP protein and its associated fluorescence persist in spermatozoa and maturing oocytes. The Dr_sycp1:eGFP zebrafish lines have the potential to be valuable models to trace meiosis onset in zebrafish and constitute the first transgenic lines expressing the GFP reporter protein only in the male meiotic and postmeiotic cells in fish.

TGFß inhibition and mesenchymal to epithelial transition initiation by Xenopus egg extract: first steps towards early reprogramming in fish somatic cell.

Xenopus egg extract is a powerful material to modify cultured cells fate and to induce cellular reprogramming in mammals. In this study, the response of goldfish fin cells to in vitro exposure to Xenopus egg extract, and subsequent culture, was studied using a cDNA microarray approach, gene ontology and KEGG pathways analyses, and qPCR validation. We observed that several actors of the TGFß and Wnt/ß-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove cultured fin cells towards a mesenchymal-epithelial transition. This indicates that Xenopus egg extract treatment relieved some barriers of somatic reprogramming in fish cells. However, the lack of re-expression of pou2 and nanog pluripotency markers, the absence of DNA methylation remodeling of their promoter region, and the strong decrease in de novo lipid biosynthesis metabolism, indicate that reprogramming was only partial. The observed changes may render these treated cells more suitable for studies on in vivo reprogramming after somatic cell nuclear transfer.

Spermatozoa methylome and its sensitivity to water temperature in a teleost fish.

Global climate change and heat waves are sources of stress which fish are facing in the wild as well as in aquaculture context. In coping with important environmental variations, they demonstrate a great plasticity and a tendency for acclimation throughout generations. Here, we question whether fish might be prone to transmit epigenetic alterations through their gametes to their offspring, thus driving rapid environmental adaptation. The question of epigenetic inheritance in fish has become of crucial interest in the recent years, when the mammalian model of methylome erasure in germ cells and embryos was found not to be conserved. In this work, by sequencing spermatozoa after bisulfite conversion, we characterized the methylation landscape of the paternal gamete in rainbow trout (in comparison to muscle) before to demonstrate its sensitivity to a 4 °C increased rearing temperature during spermatogenesis. We found that spermatozoa methylome specifically primes housekeeping and developmental genes for activation and might be instrumental to early development. Most of these methylation-free promoters were not affected by temperature, attesting the robustness of the epigenetic programming of early development. However, the increase of temperature triggered the differential methylation of 5359 regions, among which 560 gene promoters control spermiogenesis and lipid metabolism. We therefore report, for the first time in fish, that sperm epigenetic landscape carries marks of parental thermal living conditions, suggesting that DNA methylation might be a molecular basis of intergenerational inheritance.

The proximal promoter region of the zebrafish gsdf gene is sufficient to mimic the spatio-temporal expression pattern of the endogenous gene in Sertoli and granulosa cells.

The gonadal soma-derived factor (GSDF) is a new member of the transforming growth factor beta (TGF-beta) superfamily that regulates the proliferation of the primordial germ cells (PGC) in developing embryos and spermatogonia in juvenile male trout. The gsdf transcripts are expressed in the somatic cells supporting germ cell development. In zebrafish, we show that GSDF is encoded by a single copy gene that generates polymorphic transcripts and proteins. We determined that gsdf gene expression occurs before gonadal differentiation and is restricted to the gonads. Gene expression is maintained in adult granulosa cells and Sertoli cells but decreases in the cells that are in contact with meiotic and postmeiotic germ cells. Using zebrafish transgenic lines, we demonstrate that the 2-kb proximal promoter region of the gsdf gene targets high levels of transgene expression in the Sertoli and granulosa cells, and is sufficient to mimic the temporal expression pattern of the endogenous gsdf gene from 16 days postfertilization onward. We identified within the first 500 bp evolutionarily conserved DNA motifs that may be involved in Sertoli and granulosa cell-specific expression. However, the 2-kb proximal promoter region failed to drive efficient expression of the transgene in the gonads in four transgenic medaka lines. We propose that the proximal promoter region can be used to target candidate gene deregulation in zebrafish granulosa and Sertoli cells. Furthermore, the green fluorescent protein-expressing zebrafish lines produced in the present study are new valuable models for cell lineage tracing during sex differentiation and gametogenesis.

Spermatogenesis in fish.

Spermatogenesis is a developmental process during which a small number of diploid spermatogonial stem cells produce a large number of highly differentiated spermatozoa carrying a haploid, recombined genome. We characterise morphologically the different germ cell stages with particular attention for the spermatogonial generations, including the stem cells and their specific capacity to colonise a recipient's testis after transplantation. We propose a nomenclature for fish germ cells to improve the comparability among different teleost fish but also to higher vertebrates. Survival and development of germ cells depends on their continuous and close contact to Sertoli cells, and we review their multiple roles in the cystic mode of spermatogenesis seen in fish. We then discuss gene expression patterns associated with testis maturation. The endocrine system of vertebrates has evolved as master control system over spermatogenesis. In fish, both pituitary gonadotropins LH and FSH stimulate gonadal sex steroid hormone production directly by activating Leydig cells. Information is reviewed on the effects of progestin, androgens, and estrogens on global testicular gene expression patterns (microarray analysis), and on the molecular mechanisms by which steroids regulate specific candidate genes (identified by subtractive hybridization approaches) during early stages of testis maturation. Moreover, progestin and androgen effects on spermiation and milt hydration are discussed. Sex steroids mainly act via receptors expressed by Sertoli cells. One type of response is that Sertoli cells change growth factor expression, which subsequently modulates germ cell proliferation/differentiation via mechanisms yet to be characterised. Finally, we review data on germ cell autonomous processes, mainly derived from loss-of-function mutant fish lines, before identifying a number of focus areas for future research activities.

Expression profiling of rainbow trout testis development identifies evolutionary conserved genes involved in spermatogenesis.

BACKGROUND: Spermatogenesis is a late developmental process that involves a coordinated expression program in germ cells and a permanent communication between the testicular somatic cells and the germ-line. Current knowledge regarding molecular factors driving male germ cell proliferation and differentiation in vertebrates is still limited and mainly based on existing data from rodents and human. Fish with a marked reproductive cycle and a germ cell development in synchronous cysts have proven to be choice models to study precise stages of the spermatogenetic development and the germ cell-somatic cell communication network. In this study we used 9K cDNA microarrays to investigate the expression profiles underlying testis maturation during the male reproductive cycle of the trout, Oncorhynchus mykiss. RESULTS: Using total testis samples at various developmental stages and isolated spermatogonia, spermatocytes and spermatids, 3379 differentially expressed trout cDNAs were identified and their gene activation or repression patterns throughout the reproductive cycle were reported. We also performed a tissue-profiling analysis and highlighted many genes for which expression signals were restricted to the testes or gonads from both sexes. The search for orthologous genes in genome-sequenced fish species and the use of their mammalian orthologs allowed us to provide accurate annotations for trout cDNAs. The analysis of the GeneOntology terms therefore validated and broadened our interpretation of expression clusters by highlighting enriched functions that are consistent with known sequential events during male gametogenesis. Furthermore, we compared expression profiles of trout and mouse orthologs and identified a complement of genes for which expression during spermatogenesis was maintained throughout evolution. CONCLUSION: A comprehensive study of gene expression and associated functions during testis maturation and germ cell differentiation in the rainbow trout is presented. The study identifies new pathways involved during spermatogonia self-renewal or rapid proliferation, meiosis and gamete differentiation, in fish and potentially in all vertebrates. It also provides the necessary basis to further investigate the hormonal and molecular networks that trigger puberty and annual testicular recrudescence in seasonally breeding species.

The nanos1 gene was duplicated in early Vertebrates and the two paralogs show different gonadal expression profiles in a shark.

Nanos are RNA-binding proteins playing crucial roles in germ cell development and maintenance. Based on phylogenetic and synteny analyses, this study reveals that nanos1 gene has undergone multiple duplications and gene copies losses in Vertebrates. Chondrichthyan species display two nanos1 genes (named nanos1A/1B), which were both retrieved in some Osteichthyes at basal positions in Sarcopterygii and Actinopterygii lineages. In contrast, Teleosts have lost nanos1A but duplicated nanos1B leading to the emergence of two ohnologs (nanos1Ba/1Bb), whereas Tetrapods have lost nanos1B gene. The two successive nanos gene duplications may result from the second and third whole genome duplication events at the basis of Vertebrates and Teleosts respectively. The expression profiles of nanos1A and nanos1B paralogs were characterized in the dogfish, Scyliorhinus canicula. Nanos1A was strongly expressed in brain and also localized in all germ cell types in the polarized testis. In contrast, nanos1B was detected in testis with the highest expression in the germinative zone. In addition, Nanos1B protein was predominantly located in the nuclei of male germinal cells. In the ovary, both paralogs were detected in germinal and somatic cells. Our study opens new perspectives concerning the complex evolution of nanos1 paralogs and their potential distinct roles in Vertebrates gonads.

Epididymis-specific lipocalin promoters.

Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 (Lcn5 or mE-RABP) and Lipocalin 8 (Lcn8 or mEP17) are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis (Lcn5 to the distal caput and Lcn8 to the initial segment), indicating that these promoter fragments contain important cis-regulatory element(s) for epididymis-specific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 (Foxa2) interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function.

High-resolution 3D imaging of whole organ after clearing: taking a new look at the zebrafish testis.

Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.

Epididymis-specific promoter-driven gene targeting: a transcription factor which regulates epididymis-specific gene expression.

Mammalian spermatozoa undergo several modification and finally acquire the ability to fertilize during epididymal transit. One of the distinct features of the epididymis is that it displays a highly regionalized pattern of gene expression. This tissue-, region-, and cell-specific pattern of gene expression is critical for the maintenance of a fully functional epididymis. One would hypothesize that disrupting this process provides an ideal approach to male contraception, since it would not interfere with testicular endocrine output or sperm production. To achieve this purpose, we studied a cluster of epididymis-specific lipocalin genes for understanding the specific mechanisms involved in the control of gene expression in the epididymis. We have identified six epididymis-specific lipocalin genes that are differently regulated and regionalized in the epididymis. Lipocalin 5 [Lcn5 or epididymal retinoic acid-binding protein (E-RABP)] is a member of this epididymis-specific lipocalin gene cluster, which binds hydrophobic molecules such as retinoic acid. We have previously shown that the 5kb promoter fragment of the Lcn5 gene confers both androgen-dependent regulation and epididymis-specific gene expression in transgenic mice whereas 0.6 kb promoter fragment does not. To further narrow down the important cis-regulatory elements that regulate gene expression in the epididymis, we studied the Lcn5 promoter in both transgenic mice and immortalized epididymal cells. We have found that 1.8kb promoter fragment of the Lcn5 gene was sufficient for tissue- and region-specific expression in transgenic mice, and that a transcription factor Forkhead box A2 (Foxa2) interacts with the androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 and 1.3 kb. Our finding provides a framework for further analysis of the epididymal lipocalin gene regulation and modulated control of epididymis-specific expression.

The 5'-flanking region of the murine epididymal protein of 17 kilodaltons gene targets transgene expression in the epididymis.

A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.

Identification, immunolocalization, regulation, and postnatal development of the lipocalin EP17 (epididymal protein of 17 kilodaltons) in the mouse and rat epididymis.

Several lipocalins are present in the mouse epididymis and are thought to play a role in sperm maturation by transporting lipophilic molecules. We have previously reported that two lipocalin genes, mERABP (mouse epididymal retinoic acid binding protein), and mEP17 (mouse epididymal protein of 17 kDa), derived from an ancestral gene, are specifically expressed in the epididymis. In the present study, a polyclonal antibody was raised against a recombinant protein to investigate the presence and the regulation of mEP17. mEP17 was detected in the supranuclear region of the principal cells of the initial segment, the clear cells of the caput epididymidis, and the lumen of the mid/distal caput but not of the distal epididymis. Initial segment and caput tissue extracts were subjected to HPLC separation. After electrophoresis of the immunoreactive mEP17-enriched fractions, the immunoreactive band was analyzed by mass spectrometry to identified mEP17 unambiguously. After two-dimensional electrophoresis, mEP17 appeared as a train of five 22-kDa spots with a range of pI (isoelectric point) from 5.8-6.7. N-glycanase digestion gave rise to a single spot of 17 kDa and pI 6, the predicted mass and pI. During ontogeny, mEP17 was detected as early as 3 wk of age and increased afterward. After bilateral orchiectomy, mEP17 disappeared 2 d after surgery and was not restored after testosterone replacement. After unilateral orchiectomy, mEP17 levels decreased only in the orchiectomized side. After cryptorchidism or busulfan treatment, mEP17 levels were either greatly diminished or not detected. This suggests that mEP17 is dependent on testicular factor(s) that may have a germ cell origin. Altogether, our data demonstrate that mEP17 spatial expression, regulation, and fate are different from that of the highly related mouse epididymal retinoic acid binding protein. This suggests that these two related proteins exhibit distinct functions in the mouse epididymis.