Domestication is associated with differential expression of pikeperch egg proteins involved in metabolism, immune response and protein folding.

Domestication is a condition in which the breeding, care and feeding of animals are, at least in part, controlled by humans. Information regarding the changes in the protein composition of eggs in response to domestication is very limited. Such data are prerequisite for improvements in the reproduction of domesticated fish. The aim of this study was to examine the impact of domestication on the proteome of pikeperch eggs using two-dimensional differential in-gel electrophoresis. We analysed high-quality eggs from domesticated and wild pikeperch fish to reveal proteins that were presumably only related to the domestication process and not to the quality of eggs. Here, we show that domestication has a profound impact on the protein profile of pikeperch eggs. We identified 66 differentially abundant protein spots, including 27 spots that were more abundant in wild-caught pikeperch eggs and 39 spots that were enriched in eggs collected from domesticated females. Eggs originating from wild-caught females showed higher expression levels of proteins involved in folding, apoptotic process, purine metabolism and immune response, whereas eggs of domesticated females showed higher expression levels of proteins that participated mainly in metabolism. The changes in metabolic proteins in eggs from domesticated females can reflect the adaptation of pikeperch to commercial diets, which have profoundly distinct compositions compared with natural diets. The decrease in the abundance of proteins related to immune response in eggs from the domesticated population suggests that domestication may lead to disturbances in defence mechanisms. In turn, the lower abundance of heat shock proteins in eggs of domesticated fish may indicate their adaptation to stable farming conditions and reduced environmental stressors or their better tolerance of stress from breeding. The proteins identified in this study can increase our knowledge concerning the mechanism of the pikeperch domestication process.

Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17ß on mRNA expression.

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth.

The Nme gene family in fish.

The Nme gene family, also known as Nm23 or NDPK, is a very ancient gene family that can be found in all kingdoms of life. In the late eighties, a gene of the Nme family, NME1, was identified as the first metastatic suppressor gene, resulting in a major interest for this family. Due to the complexity of the family, the need for a unified and evolutionary-supported gene nomenclature was recently stressed by the scientific community. Based on a complete evolutionary history study of the gene family in metazoans and vertebrates, a unified nomenclature was recently proposed and accepted by gene nomenclature consortia. In addition to its well-documented role in tumor metastasis, members of the Nme family are also involved in a wide variety of cellular and physiological processes. Available data in non-mammalian species remain, however, scarce with the noticeable exception of Drosophila in which a major role in development was reported. In fish, very few studies have specifically investigated the role of nme genes. Several transcriptomic and proteomic studies have, however, revealed the expression of nme genes in various fish organs and tissues, in mature oocytes, and during embryonic development. Altogether, interest for the Nme gene family in fish is growing and new functions/roles in fish biology are expected to be discovered in the forthcoming years. Here, we briefly review the current knowledge of the Nme family in fish.

Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis.

In contrast to the classical model describing the synthesis of androgens and estrogens as restricted to somatic cells, a previous study demonstrated that Xenopus laevis oocytes participate in androgen synthesis. The objective of our study was to determine whether Xenopus oocytes are also involved in estrogen synthesis. More precisely, we analyzed aromatase expression by in situ hybridization and RT-QPCR and measured aromatase activity. Aromatase, the enzyme responsible for estrogen synthesis, appears to be expressed and active not only in the follicular cells but also in the vitellogenic oocytes. During late oogenesis, aromatase oocyte expression and activity decreased concomitantly with the trend observed in surrounding follicular layers. In order to investigate the role of estradiol-17ß (E(2)), we studied its effect on oocyte meiotic resumption. It appears that, as in Rana pipiens, E(2) inhibited the follicle-enclosed maturation of Xenopus oocytes, likely through inhibition of LH-induced maturation-inducing steroid synthesis. In addition, E(2) exerted a slight enhancing action on denuded oocyte maturation whose biological significance remains unclear. Together, our results demonstrate that Xenopus oocyte significantly participates in ovarian E(2) synthesis and this may be a common feature of vitellogenic vertebrates.

The NME gene family in zebrafish oogenesis and early development.

After the recent report of the expression of several nme genes in the zebrafish gonads, the present study aimed at further analyzing the expression of nme genes in the ovary with special attention for the nme transcripts that are maternally inherited and could thus participate in the determination of oocyte developmental competence. The expression levels of all groups I and II nme genes were characterized by QPCR in a panel of zebrafish tissues. The nme genes exhibiting an ovarian expression were subsequently monitored throughout oogenesis and early development, and their expression sites characterized using in situ hybridization. Here, we show that nme2b1, nme3, nme4, and nme6 are highly expressed in the ovary and present in the zebrafish oocyte throughout oogenesis. While the four transcripts are maternally inherited, nme3 and nme6 display a typical maternal profile and are detected in the zebrafish early embryo. In contrast to nme3, nme6, abundance exhibits a sharp decrease during early embryogenesis. After zygotic genome activation, we observed an increased expression of nme2b1, nme2b2, nme3, and nme6. The present study provides a comprehensive overview of the expression of nme family members during zebrafish oogenesis and early development. In addition, the maternal origin of two nme transcripts in the early embryo is reported here for the first time in any vertebrate species. Together, our observations suggest an important role of the nme family in oocyte and embryo development in vertebrates.

Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

Cysteine protease inhibitor is specifically expressed in pre- and early-vitellogenic oocytes from the brook trout periovulatory ovary.

A cDNA fragment hybridizing with a transcript abundant in the periovulatory ovary was obtained while performing subtractive cloning on brook trout ovulatory and postovulatory ovarian tissue. Using this fragment as a probe, a 478 bp full-length cDNA was obtained by screening an ovulatory ovarian cDNA library. This cDNA presumably codes for an 88 amino acid protein that is structurally related to a new family of cysteine protease inhibitors characterized by the presence of a type I thyroglobulin motif in the amino acid sequence. Therefore, the protein was tentatively named an oocyte cysteine protease inhibitor (OCPI). On Northern blots, the OCPI cDNA hybridizes with a 0.5 kb transcript present in the ovary during the periovulatory period. The OCPI transcript and protein were localized to the cytoplasm of pre- and early-vitellogenic oocytes. On Northern blots of RNA from other tissues, the OCPI transcript was detected only in the ovary. On Western blots, OCPI was detected in the ovarian tissue at all periovulatory stages tested. The specific localization of both OCPI transcript and protein to pre- and early-vitellogenic oocytes and the structural similarity to protease inhibitors, suggest that OCPI might be involved in the protection of oocytes during the periovulatory period or in the regulation of yolk formation and degradation.

Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary.

Using degenerative primers, partial cDNAs of a TNF (tumor necrosis factor) receptor and two TNF ligands were obtained by PCR of zebrafish and trout cDNAs, or cDNA libraries. These fragments were then used to screen cDNA libraries of appropriate tissues to obtain clones containing full coding sequences. A zebrafish cDNA was obtained that presumably codes for a 438 amino acid ovarian TNF receptor (OTR) that was identified as a death-domain-containing member of the TNF receptor family. On Northern blots, the OTR cDNA hybridized with a 3.4-kb transcript that is abundant in the zebrafish ovary but lightly detected in all other tissues tested. A zebrafish cDNA presumably coding for a 214 amino acid protein with sequence similarity to mammalian TRAIL (TNF-related apoptosis inducing ligand), was also isolated. In addition, a fragment of the brook trout TRAIL homologue was obtained. Finally, a full-length brook trout cDNA, that presumably codes for a 255 amino acid protein with sequence similarity to mammalian TNF-alpha and lymphotoxin-alpha, was isolated. This study is the first report of a death-domain-containing TNF receptor and the first published report of a TNF ligand in fish.

Conservation of death receptor-6 in avian and piscine vertebrates.

One of the most recently identified members of the tumor necrosis factor receptor family, death receptor-6 (DR6), has been shown to mediate apoptosis following overexpression in HeLa cells. The avian and piscine orthologs of DR6 have now been identified, and the deduced amino acid sequence for each demonstrates a high level of conservation compared to the mammalian sequence. Expression of dr6 mRNA occurs widely across tissues of both the mature chicken and brook trout. It is now well-established that ovarian follicular atresia occurs via apoptosis originating within the granulosa cell layer. Accordingly, DR6 expression within the ovary was examined to assess the relationship between stage of follicle development and relative levels of this death receptor. Of particular interest was the finding that elevated levels of dr6 mRNA, as well as the translated protein, are expressed in atretic compared to healthy follicles of the hen ovary, thus providing the first association between DR6 expression and apoptosis, in vivo. We conclude that DR6 is a highly conserved and widely expressed death-domain-containing receptor and may be implicated in regulating follicle atresia within the vertebrate ovary.

An ovarian progastricsin is present in the trout coelomic fluid after ovulation.

An up-regulated cDNA fragment was isolated using a differential display polymerase chain reaction between ovulatory and postovulatory brook trout ovarian tissues. Using this fragment as a probe, a full-length cDNA of 1783 base pairs was obtained from an ovarian cDNA library. The cDNA presumably codes for a 383-amino acid protein with strong sequence similarity to an aspartic protease, progastricsin (EC 3.4.23.3), also known as pepsinogen C. On Northern blots of ovarian tissue, the trout progastricsin cDNA hybridized with a 1.8-kilobase transcript that was strongly up-regulated 4-6 days after ovulation. Of all other tissues tested, a transcript was only detected in the stomach. A recombinant trout progastricsin protein was produced and used to raise an antibody. On Western blots of ovarian tissue, the progastricsin antibody recognized a single 39-kDa protein that was present in the ovary only following ovulation. On Western blots of coelomic fluid, the 39-kDa protein was strongly detected 4-10 days after ovulation. The trout progastricsin was immunocytochemically localized to the granulosa cells of postovulatory follicles, suggesting that it is released from this tissue into the coelomic fluid following ovulation. Progastricsin has been found in the stomach, prostate, seminal vesicle, seminal fluid, and pancreas of vertebrates; however, this is the first report of a progastricsin in an animal ovary.

A novel osteopontin-like protein is expressed in the trout ovary during ovulation.

Using suppression subtraction hybridization between ovulatory and postovulatory trout ovaries, a down-regulated cDNA was obtained that presumably encodes a novel ovarian protein ('NOP'). NOP mRNA is present in the ovary during ovulation and down-regulated by 48 h postovulation, suggesting an important role for NOP during ovulation. Besides the ovary, NOP is also strongly expressed in the testis and at lower levels in the skin, gills, kidney and gastrointestinal tract. While the overall identity is not high, NOP shares several sequence similarities with mammalian and chicken osteopontins, including the percentage of aspartate, serine and alanine residues and the presence of a cell attachment motif.

A S100 homologue mRNA isolated by differential display PCR is down-regulated in the brook trout (Salvelinus fontinalis) post-ovulatory ovary.

A down-regulated cDNA fragment was isolated using differential display PCR between ovulatory and post-ovulatory brook trout ovarian tissue. Using this cDNA as a probe, a full-length cDNA of 538bp was obtained by screening an ovarian cDNA library. The cDNA isolated presumably encodes for a 101 amino acid protein that is similar in sequence to a group of calcium binding proteins called S100 proteins. Within the S100 family, the differentially expressed trout protein is more similar to S100 A1 and A10 proteins. A comparison of the trout S100 protein with mammalian S100 A1 proteins and a S100 protein previously isolated from the loach suggests that the trout S100 protein could be a new member of the S100 family. On Northern blots of ovarian tissue, the trout S100 cDNA hybridized with a 550bp transcript which appeared to progressively decrease throughout ovulation and was significantly down-regulated by 48h post-ovulation. A transcript was also detected in several other tissues such as the heart, skin, spleen, gills, intestine, stomach and testis. Using in situ hybridization the trout S100 mRNA was strongly detected in the granulosa cells of the ovulated follicle. These results suggest that a member of the S100 protein family may play a role in the trout ovary during the periovulatory period.

Embryonic muscle development in rainbow trout (Oncorhynchus mykiss): a scanning electron microscopy and immunohistological study.

Embryonic muscle development was studied in rainbow trout (Oncorhynchus mykiss) at low and high temperature using scanning electron microscopy (SEM) and immunohistology. Somite development was described starting at stage 16 (Vernier JM. 1969. Ann Embryol Morphogen 4:495-520) for both temperatures, with special interest in their shape and size. Muscle differentiation, associated with somite growth, is characterized by a larger increase in height compared to width and by acquisition of a chevron shape. Thin structures such as striation, sarcomeres, and myofibrils within muscle cells and myotubes were observed starting at the eyed stage (stage 24). Immunohistological analyses showed appearance of embryonic fast myosin at stage 20 in the deep part of the somite. The area where myosin was expressed extended in the somite throughout embryonic development and the presence of myosin was observed in the entire somite at hatching (stage 30). Slow myosin was expressed in a monolayer of superficial cells at the eyed stage and during the entire embryonic development. Then it was expressed in a few layers of cells located in the red muscle area. These results suggest that muscle differentiation, characterized by myosin expression, is engaged at stage 20. Myogenesis starts in the deep part of the somite, near the notochord and progresses laterally to cover the complete somite at hatching when the somite is composed of muscle fibres exhibiting a high degree of maturity. No significant difference was observed in terms of muscular development between low- and high-temperature conditions. J. Exp. Zool. 286:379-389, 2000.

A tumor necrosis factor decoy receptor homologue is up-regulated in the brook trout (Salvelinus fontinalis) ovary at the completion of ovulation.

An up-regulated cDNA fragment was obtained from differential-display polymerase chain reaction of brook trout ovarian tissue stimulated by phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187. Using this cDNA as a probe, a full-length cDNA of 2267 base pairs was obtained by screening a library of PMA/A23187-stimulated ovarian cDNA. The mRNA obtained presumably encodes for a 302-amino acid protein showing similarities with several members of the tumor necrosis factor (TNF) receptor superfamily. The protein contains several cysteine-rich domains characteristic of mammalian TNF receptor members and is most similar to human decoy receptor 3 and osteoprotegerin, two soluble decoy TNF receptors. Consequently, this TNF receptor homologue was tentatively named a trout decoy receptor (TDcR). On Northern blots of ovarian tissue, TDcR hybridized with a 2.2-kilobase transcript that was strongly up-regulated under phorbol ester stimulation. TDcR mRNA was localized in granulosa cells and was detected in the ovary during and after natural ovulation. Its expression was up-regulated at the end of ovulation and progressively down-regulated after 48 h postovulation. Among other trout tissues tested, the transcript was present only in the testis. To our knowledge this is the first description of a member of the TNF receptor family from a lower vertebrate and the first report of a decoy-like TNF receptor in the vertebrate ovary.