Pepsin diffusion in dairy gels depends on casein concentration and microstructure.

Fundamental knowledge of gastric digestion had only focused on acid diffusion from the gastric fluid, but no data are available for pepsin diffusion. Using fluorescence recovery after photobleaching technique, diffusion coefficients D of fluorescein isothiocyanate (FITC)-pepsin were measured in rennet gels across a range of casein concentrations allowing to form networks of protein aggregates with different structures. To investigate the microstructural parameters of native gels, electron microscopy image analysis were performed and qualitatively related to diffusion behavior of FITC-pepsin in these dairy gels. This study is the first report on quantification of pepsin diffusion in dairy product. Pepsin diffusion in rennet gels depends on casein concentration and microstructure. Models of polymer science can be used to assess D in dairy gel. Such data should be confronted with pepsin activity in acidic environment, and will be very useful as input parameters in mathematical models of food degradation in the human stomach.

Sex differentiation in an all-female (XX) rainbow trout population with a genetically governed masculinization phenotype.

Sex determination is known to be male heterogametic in the rainbow trout, Oncorhynchus mykiss; however, scattered observations that deviate from this rather strict genetic control have been reported. Here, we provide a detailed morphological and histological characterization of the gonadal differentiation and development (from 43 days postfertilization to 11 months of age) in an all-female (XX) population with a genetically governed masculinization phenotype. In comparison with control males and females, the gonadal differentiation in these animals was characterized by many perturbations, including significantly fewer germ cells. This decrease in germ cells was confirmed by the significantly decreased expression of 2 germ cell maker genes (vasa and sycp3) in the masculinized XX populations as compared with the control females and control males. Although only a proportion of the total adult population was partially or fully masculinized, this early differentiating phenotype affected nearly all the sampled animals. This suggests that the adult masculinization phenotype is the consequence of an early functional imbalance in ovarian differentiation in the entire population. We hypothesize that the lower number of germ cells that we observed in this population could be one cause of their masculinization.

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.

Trout gill cells in primary culture on solid and permeable supports.

Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.

In situ hybridisation of a large repertoire of muscle-specific transcripts in fish larvae: the new superficial slow-twitch fibres exhibit characteristics of fast-twitch differentiation.

Much of the present information on muscle differentiation in fish concerns the early embryonic stages. To learn more about the maturation and the diversification of the fish myotomal fibres in later stages of ontogeny, we investigated, by means of in situ hybridisation, the developmental expression of a large repertoire of muscle-specific genes in trout larvae from hatching to yolk resorption. At hatching, transcripts for fast and slow muscle protein isoforms, namely myosins, tropomyosins, troponins and myosin binding protein C were present in the deep fast and the superficial slow areas of the myotome, respectively. During myotome expansion that follows hatching, the expression of fast isoforms became progressively confined to the borders of the fast muscle mass, whereas, in contrast, slow muscle isoform transcripts were uniformly expressed in all the slow fibres. Transcripts for several enzymes involved in oxidative metabolism such as citrate synthase, cytochrome oxidase component IV and succinate dehydrogenase, were present throughout the whole myotome of hatching embryos but in later stages became concentrated in slow fibre as well as in lateral fast fibres. Surprisingly, the slow fibres that are added externally to the single superficial layer of the embryonic (original) slow muscle fibres expressed not only slow twitch muscle isoforms but also, transiently, a subset of fast twitch muscle isoforms including MyLC1, MyLC3, MyHC and myosin binding protein C. Taken together these observations show that the growth of the myotome of the fish larvae is associated with complex patterns of muscular gene expression and demonstrate the unexpected presence of fast muscle isoform-expressing fibres in the most superficial part of the slow muscle.

Muscle fiber differentiation in fish embryos as shown by in situ hybridization of a large repertoire of muscle-specific transcripts.

Skeletal muscles are composed of different fiber types, largely defined by differential expression of protein isoforms involved in myofibrillogenesis or metabolism. To learn more about the gene activations that underlie the differentiation and the diversification of embryonic fish myotomal fibers, we investigated the developmental expression of 25 muscle genes in trout embryos by in situ hybridization of muscle-specific transcripts. The earliest event of muscle differentiation, at approximately the 25-somite stage, was the expression of a variety of muscle-specific genes, including slow-twitch and fast-twitch muscle isoforms. The activation of these muscle genes started in the deep somitic domain, where the slow muscle precursors (the adaxial cells) were initially located, and progressively spread laterally throughout the width of the myotome. This mediolateral progression of gene expression was coordinated with the lateral migration of slow adaxial cells, which specifically expressed the slow myosin light chain 1 and the SLIM1/FHL1 genes. Subsequently, the fast and slow skeletal muscle isoforms precociously expressed in the course of the mediolateral wave of muscle gene activation became down-regulated in the superficial slow fibers and the deep fast fibers, respectively. Finally, several muscle-specific genes, including troponins, a slow myosin-binding protein C, tropomodulins, and parvalbumin started their transcription only in late embryos. Taken together, these findings show in fish embryos that a common myogenic program is triggered in a mediolateral progression in all muscle cells. The acquisition of the slow phenotype involves the additional activation of several slow-specific genes in migrating adaxial muscle cells. These events are followed by sequential gene activations and repressions in fast and slow muscle cells.

Expression patterns of collagen I (alpha1) encoding gene and muscle-specific genes reveal that the lateral domain of the fish somite forms a connective tissue surrounding the myotome.

Somites are repeated, epithelial structures that are derived from the unsegmented paraxial mesoderm located lateral to the notochord. In higher vertebrates, somites differentiate into a sclerotome that subsequently forms the vertebrae and the ribs and into a dermomyotome that gives rise to a myotome, from which arises the skeletal muscle, and to a dermatome, from which arises the dermis. Fish somites have been shown to produce a sclerotome and a myotome, but very little is known regarding their participation in the formation of connective tissues, especially at the junction between the epidermis and the myotome. To investigate the formation of connective tissues in fish somites, we have examined the expression pattern of the collagen I (alpha1) chain. As somitogenesis proceeds rostrocaudally, collagen I (alpha1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (alpha1) chain appeared in a distinct epithelial-like monolayer situated at the periphery of the developing somite facing the surface epidermis. This epithelial monolayer of somitic cells that covered the superficial slow muscle cells, did not express the myogenic transcriptional regulator myogenin and was devoid of contractile filament. As the somite increased in size, these collagen-expressing epithelial cells flattened, forming a thin cellular layer underlying the epidermis and recovering the lateral surface of the myotome. In conclusion, the lateral domain of the fish somite forms a distinct epithelial cell layer sharing many characteristics with amniote dermatome.

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.

Red and white muscle development in the trout (Oncorhynchus mykiss) as shown by in situ hybridisation of fast and slow myosin heavy chain transcripts.

The axial muscle of most teleost species consists of a deep bulk of fast-contracting white fibres and a superficial strip of slow-contracting red fibres. To investigate the embryological development of fast and slow muscle in trout embryos, we carried out single and double in situ hybridisation with fast and slow myosin heavy chain (MyHC)-isoform-specific riboprobes. This showed that the slow-MyHC-positive cells originate in a region of the somite close to the notochord. As the somite matures in a rostrocaudal progression, the slow-MyHC-positive cells appear to migrate radially away from the notochord to the lateral surface of the myotome, where they form the superficial strip of slow muscle. Surprisingly, the expression pattern of the fast MyHC showed that the differentiation of fast muscle commences in the medial domain of the somite before the differentiation and migration of the slow muscle precursors. Later, as the differentiation of fast muscle progressively spreads from the inside to the outside of the myotome, slow-MyHC-expressing cells become visible medially. Our observations that the initial differentiation of fast muscle takes place in proximity to axial structures and occurs before the differentiation and migration of slow muscle progenitors are not in accord with the pattern of muscle formation in teleosts previously described in the zebrafish Danio rerio, which is often used as the model organism in fishes.

Expression of the prolactin receptor (tiPRL-R) gene in tilapia Oreochromis niloticus: tissue distribution and cellular localization in osmoregulatory organs.

The expression of the prolactin receptor (PRL-R) gene has been investigated in various tissues of tilapia (Oreochromis niloticus) reared in fresh or brackish water. Using a cDNA probe spanning the extracellular domain of the tilapia PRL-R and Northern blot analysis, the presence of tilapia PRL-R mRNA has been confirmed in the osmoregulatory organs and has been detected in other tissues, including the skin, the brain, the reproductive organs, and the two major hematopoietic organs (spleen and head kidney), as well as circulating lymphocytes. These findings suggest a conservation of the physiological processes regulated by prolactin throughout the vertebrates, including immunity and central nervous activity. A non-radioactive in situ hybridization procedure has allowed us to detect the expression of the tilapia PRL-R in the branchial chloride cells and the intestinal mucosal layer of fresh water animals, confirming the direct control exerted by prolactin on the water and ionic exchanges in tilapia. In all the tissues examined one unique PRL-R transcript has been detected with a similar size (3.2 kb) whatever the salinity conditions. Thus, the transcriptional expression of the tilapia PRL-R strongly differs from the complex RNA pattern reported for the higher vertebrates PRL-R and provides an additional argument for the existence of a single PRL-R for both prolactin isoforms in this fish species.

The interstitial cells of the trout testis (Oncorhynchus mykiss): ultrastructural characterization and changes throughout the reproductive cycle.

The major non-vascular cell types present in the interstitial compartment in trout testes have been ultrastructurally characterized and cell changes in the course of the two first reproductive cycles have been studied. Three major cell types are always present fibroblasts, myoid cells and Leydig cells. Their structure varies with the maturational stage of the gonad. Fibroblasts are centrally located in the interstitial areas. Numerous typical myoid cells are always present near the basal lamina. However, at the end of a cycle some of them display degenerative changes, then disappear. At the beginning of the next cycle, fibroblasts appear to differentiate to new myoid cells. Leydig cells present before the start of the first spermatogenesis are replaced by new ones which probably also arise from fibroblasts. These cells progressively differentiate during spermatogenesis, so that most of the Leydig cells present during the spermiation phase are fully-differentiated steroidogenic cells. At the end of a cycle, a certain number of the Leydig cells disappear and are replaced at the beginning of the next cycle by new ones most likely derived from fibroblasts. The remaining Leydig cells dedifferentiate to cells which may redifferentiate during gonadal activity. Macrophages are present mostly at the end of a cycle and participate at this time in the removal of dead interstitial cells. In conclusion, at least some fibroblast-like cells are precursor cells, able to replace myoid cells and Leydig cells which disappear between two consecutive reproductive cycles.

Evidence of GnRH receptors in cultured pituitary cells of the winter flounder (Pseudopleuronectes americanus W.).

For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.

Ovarian early postovulatory development and oestrogen production in rainbow trout (Salmo gairdneri R.) from a spring-spawning strain.

Ovarian early postovulatory development and oestrogen production were studied in rainbow trout from a spring-spawning strain, 5 to 50 days postovulation (PO). Early postovulatory development was assessed by the ovarian development index, corresponding to the relative volume occupied in the ovary by oocytes in an advanced stage of differentiation (containing cortical alveoli). This index was shown to be related to the maximal oocyte diameter and to the time since ovulation, as well as to metabolic criteria. In contrast, gonadosomatic index was not correlated with any of these, confirming its unreliability for assessing the gonadal development, at least at this stage. Size of follicles and presence of vitellus (lipoprotein) in the oocytes of several animals indicate that ovarian development was more advanced in our females than in other studies. It is suggested that young follicles start developing before ovulation of the preceding batch of oocytes. Oestradiol (E2) plasma levels were low but detectable, and showed, in some females, peak values (max 5.5 ng/ml) within the first month PO. Besides, in vitro metabolism was studied using two isotopes of androstenedione as precursors. No sharp discrepancies were observed between the two types of incubation. Conjugation of the precursor or its metabolites was high but decreased with time postovulation. This suggests that it proceeds from postovulatory follicles. E2 production potential was demonstrated from the shortest time postovulation. Moreover, it increased with ovarian development, suggesting that E2 is synthesized in developing follicles.