Insulin-like system and growth regulation in the Pacific oyster Crassostrea gigas: hrIGF-1 effect on protein synthesis of mantle edge cells and expression of an homologous insulin receptor-related receptor.

The involvement of molecules belonging to the insulin/IGF family in regulation of growth has been investigated in the Pacific oyster Crassostrea gigas. In vitro biological effects of human recombinant IGF-1 (hrIGF-1) on mantle edge cells, involved in oyster shell and soft body growth, were studied over an annual cycle. In mantle edge cells hrIGF-1 stimulates protein synthesis of 56+/-5.1% over basal for 10(-10) M in September with in addition a clear dose-effect corresponding to the highest shell growth period, and 57.5+/-3.45% over basal for 10(-11) M in March and 51+/-5.4% over basal for 10(-10) M in April corresponding to the period of mantle growth. These insulin-like effects were associated with the expression of a recently identified C. gigas insulin receptor-related receptor (CIR) in mantle edge cells as demonstrated by RT-PCR. Moreover, in situ hybridisation (ISH) confirmed this expression at the level of the inner and outer epithelia involved in mantle growth and shell formation.

Cloning, characterization, and comparative activity of turbot IGF-I and IGF-II.

IGF peptides belong to a complex system that is known to play a major role in the control of growth and development in mammals. Even if studies performed in nonmammalian species tend to demonstrate an important function of these molecules, use of heterologous ligands, especially in fish, partly limit our knowledge of the physiological role(s) of IGFs. We report in this study the cloning, production, and characterization in an evolved fish, the turbot Psetta maxima, of mature IGF-I and IGF-II. The deduced 68-amino-acid IGF-I and 70-amino-acid IGF-II show 75% and 74% sequence identity with their mammalian counterparts, respectively, confirming the high sequence homology observed in other species. The development of a simple and efficient system for the production and purification of both IGF-I and IGF-II in Escherichia coli was used to investigate the in vitro regulation of GH release in the turbot. Our results demonstrated for the first time in a Euteleost species that both peptides specifically inhibited GH release. Both hormones were equally potent in inhibiting GH release from dispersed pituitary cells, with maximal inhibitory effects of 92% and 91% at 1 nM doses after 12 days of culture, respectively. The biologically active recombinant turbot IGFs that we obtained will allow us to further investigate potential and perhaps the specific role(s) of these hormones in turbot as, in contrast with mammals, growth in fish is potentially continued during "adult" life.

Fish growth hormone receptor: molecular characterization of two membrane-anchored forms.

A RT-PCR approach was used to clone and sequence the full-length growth hormone receptor (GHR) of a teleost fish, the turbot (Scophthalmus maximus). Total liver RNA was amplified by RT-PCR with degenerate primers designed in extracellular and cytoplasmic regions, and a single DNA fragment of 1100 bp was obtained. The entire coding region was obtained by 5' and 3' RACE assays, and comprises an open-reading frame of 633 amino acids. This sequence shows the characteristic motifs of the class I cytokine receptor superfamily, and its amino acid identity with mammalian, avian, reptilian and amphibian GHRs is 32-36%. The 3' RACE also revealed the occurrence of an alternate messenger encoding a membrane-anchored truncated receptor, which could facilitate the production of GH-binding protein in fish species. This report represents the first data on fish GHR sequence, and it provides evidence for the conservation of this receptor throughout vertebrate evolution.

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2.

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.

Expression pattern of insulin receptor mRNA during Xenopus laevis embryogenesis.

We have identified a member of the insulin receptor (InsR)/insulin-like growth factor-1 receptor (IGF-1R) family. The Xenopus insulin receptor (Xe-InsR) is present as a maternal 6.6 kb transcript. Northern blot analysis reveals the presence of this transcript until the mid-blastula transition (MBT), when levels decrease. At neurulation, two distinct transcripts of 6.6 and 7.7 kb are detected, both of which persist throughout embryogenesis. In situ hybridization analysis shows that InsR expression is restricted to regions of ectodermal and mesodermal origin, notably the encephalon, otic vesicles, optic vesicles, gills, somites and the pronephros.

Insulin and insulin-like growth factor-1 receptors in an evoluted fish, the turbot: cDNA cloning and mRNA expression.

The insulin receptor (IR) and the structurally related insulin-like growth factor type 1 receptor (IGF-1R) belong to the tyrosine kinase (TK) receptor family. In this study, we have carried out the molecular characterization of both receptors from turbot (Psetta maxima), an evoluted teleost flatfish. The cDNA encoding the precursors of IGF-1R and the nearly entire IR (only the first 16 amino acids of the alpha subunit are missing when compared to the published human sequence) were cloned from an embryonic cDNA library. The deduced polypeptides contain all the topological features characterizing the insulin/IGF-1 receptor family. They are highly conserved compared to their mammalian counterparts, particularly within domains involved in the catalytic activity and in the transduction pathways. Nevertheless, some differences in the primary sequences, especially in the carboxy-terminal domain of the precursors, may affect the functions fulfilled by these receptors. As in mammals, the long IGF-1R 5'-untranslated sequence contains open reading frames and potential Sp1 binding sites. Northern blot analyses have revealed a major IR transcript of 11 kb, which is approximately the size of IGF-1R transcript (Eliès, G., Groigno, L., Wolff, J., Boeuf, G., Boujard, D., 1996. Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species. Mol. Cell. Endocrinol. 124, 131-140.). If IR and IGF-1R transcripts are detected by RT-PCR at all developmental stages and in all tissues examined, in situ hybridization studies have shown that these mRNA can be visualized as ubiquitous signals only in young larvae, whereas IGF-1R and IR expression appears weaker during later development and in adult tissues.

Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes.

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 micron < diameter < 1000 micron) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mos synthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively active raf mRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras --> Raf --> MAPKK --> MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the "Mos synthesis machinery." The present results show that incompetence of Xenopus stage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mos synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.

[In vitro oocyte growth and maturation: a mouse example]. / Croissance et maturation ovocytaire in vitro: l'exemple de la souris.

During the last 20 years, numerous studies have been performed to improve culture conditions in order to allow in vitro growth and meiotic maturation of oocytes isolated from primordial to antral follicles. The main objective of these studies is to define optimal culture conditions which will allow to increase the recovery rate of oocytes that can be successfully fertilized. This paper reviews the more recent advances obtained when studying mouse oocyte development in vitro.

Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species.

The insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase which plays essential role in the regulation of growth and development. In this study, we have cloned cDNAs encoding the tyrosine kinase domain of the IGF-1R from two species of fish. The turbot and trout nucleotide sequences share 82% identity. Moreover, the deduced polypeptides are also highly conserved (> 90% identity) compared with the IGF-1R sequences described in other vertebrates, particularly within domains involved in the catalytic activity and in the transduction pathway. Northern blot analyses have revealed a unique 13-kb mRNA transcript. Using an RT-PCR approach, we have also shown that the polyadenylation status seems to vary according to the developmental stage in turbot: polyadenylated in oocytes and in the first larval stages, the mRNA becomes undetectable in the polyadenylated fraction in later stages or in adult somatic tissues. These results suggest that IGF-1R mRNA undergoes complex post-transcriptional regulation.

Insulin-like growth factor I receptor messenger expression during oogenesis in Xenopus laevis.

To study Xenopus insulin-like growth factor I (IGF-I) receptor messenger expression during oogenesis, we isolated a complementary DNA (cDNA) corresponding to the beta-subunit of the receptor. There is a high degree of conservation between the deduced polypeptide and the three mammalian sequences previously described for the IGF-I receptor (75% homology) even though it is lower than the homology among mammals themselves (95% homology). IGF-I receptor messenger RNAs were specifically detected by reverse transcription-PCR in oocytes, embryos, and adult liver and muscle. By in situ hybridization, these messenger RNAs could be visualized only in oocytes. Quantification showed that they accumulated from the previtellogenic stage until early vitellogenesis. No specific labeling could be detected in oocytes after stage IV of vitellogenesis. Thus, the IGF-I receptor messenger stock does not seem to increase further beyond this point or may even decrease. The long 3'-untranslated sequence (1.8 kilobases) included in the cDNA presents no homology with those of mammalian receptor cDNAs and could be longer, as no polyadenylated tail is observed. Some motifs corresponding to sequence described as cytoplasmic polyadenylation element or that have been described in unstable messengers could be observed. Moreover, a deadenylation of this RNA occurs in the postvitellogenic stage. These results suggest that translation occurred very early during oogenesis. Therefore, IGF-I receptor could play a role early on, during oocyte growth, in addition to its involvement in the maturation process.

A nonsteroidal follicular factor is involved in maturation process of Xenopus laevis oocytes.

Xenopus laevis postvitellogenic ovarian follicles release into conditioned medium nonsteroidal factor(s) that potentiate progesterone-induced oocyte maturation. The molecular weight of this factor is between 1000 and 10,000 Da. The potentiating activity is suppressed when follicles are incubated with cyanoketone to inhibit steroidogenesis, suggesting that the secretion of the potentiating factor can be modulated in vitro. These results indicate that the steroid-independent pathway in vitro, previously reported in oocytes exposed to insulin or IGF-1 to induce meiotic resumption, may be of physiological importance.

Localization of the estradiol receptor mRNA in the forebrain of the rainbow trout.

In situ hybridization was used to localize the cells that express the estradiol receptor gene (ER) in the forebrain (hypothalamus, preoptic area, telencephalon) of the rainbow trout (Oncorhynchus mykiss). Both sense and anti-sense [35S]UTP-labeled single-stranded RNA probes were generated from the estradiol binding domain of the ER cDNA. The sense probe was used to evaluate the background of the hybridization reaction. In the forebrain, specific signal appeared in three areas: the posterior hypothalamus, the preoptic area, and the ventral telencephalon. Our localization correlates with [3H]estradiol binding studies in other teleost species. In the pituitary, we observed a weak signal when compared to the signal observed in the forebrain (about ten grains/cell in the pituitary against 35 grains/cell in the posterior hypothalamus). A significant difference was also observed between the intensity of labeling per cell when different forebrain nuclei were compared. We provide here evidence for a tissue-specific regulation of the ER mRNA levels in the trout hypothalamo-pituitary axis.

Patterns of protein synthesis during Xenopus oocyte maturation differ according to the type of stimulation.

We examined the qualitative patterns of protein synthesis in fully grown prophase-blocked oocytes of Xenopus laevis and after meiosis reinitiation accompanying maturation of the oocytes. Newly synthesized proteins labelled with [35S]methionine were run on isoelectric focusing gels and further separated in the second dimension on SDS-polyacrylamide slab gels. Three types of maturation inducer were compared: progesterone, considered as the natural inducer of Xenopus oocyte maturation, hCG (human chorionic gonadotropin) and insulin. Three polypeptides with apparent molecular masses of 37 kDa (pI 4.7-4.8), 78 kDa (pI 4.7) and 138 kDa (pI 4.6-4.7) were found to be always synthesized in all three types of stimulation, while the synthesis of a fourth one (molecular mass 116 kDa, pI 4.7) was arrested during oocyte maturation. Moreover, when the follicular cells surrounding the oocytes were part of the stimulating pathway, which is the case during hCG-induced maturation, an additional polypeptide was synthesized by the oocytes (molecular mass 106 kDa, pI 6.0-6.2). This polypeptide was not synthesized during progesterone- or insulin-induced oocyte maturation, two types of stimulation which do not require the presence of the follicular cells. The biological significance of the hCG-induced polypeptide, not necessary for oocyte maturation, is discussed. On the other hand, the four other modifications in protein synthesis taking place during all three types of maturation-inducing stimulation appear to be necessary for oocyte maturation, since oocytes which failed to mature in response to stimulation always missed one or several of these four polypeptides.

The dynamics of the steroidogenic response of perifused Xenopus ovarian explants to gonadotropins.

Steroid release before and during maturation was studied in Xenopus ovarian follicles under various conditions of stimulation of LH in a perifusion system. Acute stimulation by 15 micrograms of LH induces a 10-fold increase in the androgen (testosterone and androstenedione) level which reaches a maximum 4 hr later and then slowly decreases until the 25th hour. Repeated stimulations every 2 or 4 hr are followed by the same androgen increase during the first 8 or 10 hr and then by a slow decrease in the secretion despite new LH injections. A significant increase in progesterone secretion is seen only after at least two stimulations (8 hr). Estradiol secretion slowly increases to a moderate level during the first 5 hr and then remains stable whatever the stimulation. During continuous stimulation (LH 0.5 microgram/ml) androgen levels reach an initial maximum after 4 hr and then fluctuate with a 2-hr period. Addition of theophyllin to the medium enhances these fluctuations. After 12 hr when the progesterone has increased, androgen secretion diminishes to reach a basal level without fluctuations. Germinal vesicle breakdown occurs only in follicles that have been appropriately stimulated to secrete androgens and progesterone during the required time.

Electrophoretic characteristics of nuclear proteins from human spermatozoa.

A comparative electrophoretic analysis of nuclear proteins was investigated in ejaculated human semen. The results confirm the biochemical heterogeneity of nucleoproteins in sperm with normal routine parameters and demonstrate the same heterogeneity in semen with defective routine parameters: nucleoproteins comprise histones, intermediate proteins, and protamines in the two groups. Individual qualitative and quantitative differences are observed within and between the two groups. The results allow a better knowledge of nuclear characteristics of ejaculated human spermatozoa but it does not permit the establishment of a relationship between biochemical heterogeneity of sperm nucleus and decreased fertility.

Human spermatozoal nuclear maturity in normozoospermia and asthenozoospermia.

Ejaculated human spermatozoa were studied to assess their nuclear maturity. After SDS or SDS-EDTA treatment, asthenozoospermic semen had a lower resistance to decondensation than normozoospermic semen and contained more stained immature nuclei after aniline blue staining. It showed a higher uptake of ethidium bromide, specific for DNA. There was no difference in the binding of 14C iodoacetamide in the two groups. Therefore, asthenozoospermic semen could be characterized by its relative nuclear immaturity.

Effect of duration of abstinence on maturity of human spermatozoa nucleus.

Human sperm are a heterogeneous population, particularly with respect to their morphology, motility, and degree of nuclear maturity. The characteristics of human sperm and the degree of nuclear condensation with variable sexual abstinence times (long, 7 days; short, 12 h) have been studied. Long abstinence led to an increase in the number of sperm and a decrease in their motility, but their morphology remained unchanged. The DNA-protein complex demonstrated by ethidium bromide uptake was unchanged, but there was a significant increase in nuclear stability upon treatment with SDS. The duration of abstinence hardly affected the degree of nuclear condensation or stability of human sperm. The heterogeneity observed is essentially of testicular origin.

PIP: Spermatogenesis is a constant process; however, periods of abstinence do affect the number of sperm stored in the tail of the epididymis and the vas deferens as well as the percentage of stable nuclei. Volunteers either abstained for a short period of 1 ejaculation/day or a longer period of 1 ejaculation/7 days. Longer periods of abstinence showed an increase in sperm volume, concentration, and total count, as well as decreased sperm motility. Sperm morphology, however, showed no change. Longer abstinence also produces an increase in the percentage of stable nuclei, as shown when 50 mcl sperm was combined in solutions of 0.5 ml 1% sodium dodecyl sulphate and the same solution with 0.5 ml 1% ethidium bromide. Finally, shorter periods of abstinence were not shown to increase homogeneity of sperm populations, the composition of which is determined by testicular origin.

The dynamics of the steroidogenic response of perfused Xenopus testis explants to gonadotropins.

The dynamics of androgen production by perfused Xenopus testis explants has been investigated under various gonadotropic stimulations. Androgens were measured in perfusate by radioimmunoassay. The behavior of hormones in the perfusion system was assessed. Pieces of testis were capable of producing androgens after more than 20 hr in perifusion. Amplitude and duration of the response to a gonadotropic stimulation were the same 3 and 10 hr after the beginning of the experiment. After stimulation by pituitary extract, hCG, or carp GTH, there was a rapid tenfold increase in androgen secretion with a maximum after 1.5 hr and then a slow decrease to a basal line level. A typical log dose-response curve was obtained with hCG and cGTH. After 6 hr continuous stimulation by pituitary extract, steroid secretion increased and reached a plateau 4 hr later. The decrease occurred only after the end of stimulation. The same amount of pituitary extract injected into the column at the rate of 4 min stimulations every hr gave the same type of response. As far as is known, the present data are the first on perifusion systems on the testes of lower vertebrates.

Regional distribution of steroids in the dog epididymis.

Concentrations of delta 4 androstenedione, testosterone and dihydrotestosterone were measured in the post-nuclear supernatant of the dog epididymis. In addition, they were measured in the efferent ducts and the testis. From the efferent ducts to the corpus there was a significant decrease in testosterone and delta 4 androstenedione concentrations. On the contrary, the concentration of dihydrotestosterone increased from the efferent ducts to the corpus where this steroid predominated. The marked differences in steroid concentration along the epididymis of the dog suggested regional differences in androgen metabolism.