Maurice Loir

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin‐like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF‐I cDNA, we confirmed that IGF‐I transcription occurs in trout testis. Testicular IGF‐I mRNA abundance may be increased by long‐term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH‐II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF‐I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 × 1010 M1; Bmax = 10 to 20 fmol/107 cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF‐I > IGF‐II ⋙ insulin) and the molecular size of its α‐subunit labelled with 125HGF‐I (Mr125 ‐ 140 kDa). 125HGF‐II also bound to the type 1 receptor whereas IGF‐II/mannose 6 phosphate receptors could not be detected.

Spermatogonia of rainbow trout: II. in vitro study of the influence of pituitary hormones, growth factors and steroids on mitotic activity.

At the present time, in spite of recent advances, knowledge about the factors regulating germ cell proliferation in the teleost testis is limited. This study was designed to investigate, in vitro, the ability of various hormones, growth factors, and steroids to influence the proliferation of trout spermatogonia (Go) present in mixed cultures of somatic and germ cells prepared from testes, either prespermatogenetic or spermatogenetic. The tested molecules were usually present for the duration of culture (4.5 days) and 3H‐thymidine (3H‐Tdr) for the last day in culture. In our cell culture conditions, homologous gonadotropin I (tGTH‐I) and growth hormone (tGH) moderately stimulated 3H‐Tdr incorporation by Go, with ED50 equal to 5.5 ± 3.0 and 1.8 ± 0.4 ng/ml respectively. Insulin growth factor I (rhIGF‐I) and fibroblast growth factor (rhFGF‐2) stimulated 3H‐Tdr incorporation by Go from spermatogenetic testes only, with ED50 equal to 16.2 ± 9.3 and 2.4 ± 0.3 ng/ml respectively. The effects of the most efficient concentrations of rhIGF‐I combined with those of either tGTH‐I or tGH were additive. Seventy to one hundred μM suramin stimulated 3H‐Tdr incorporation by Go from testes at all maturation stages and this effect was additive with that of tGTH‐I. We assume that this effect of suramin could result from the inhibition of an unidentified antimitogenic factor. No effect was observed with homologous prolactin, human epidermal growth factor, activin A and B, transforming growth factor‐β1, testosterone, 11‐ketotestosterone, 17β‐estradiol, pregnenolone, 11β‐hydroxyprogesterone, and 22‐hydroxycholesterol. In conclusion, our in vitro results suggest that GTH‐I, GH, IGF‐I, and FGF‐2, are potent in situ modulators of the proliferative activity of trout Go at the time of induction, speeding up, then slowing down spermatogenesis, through direct or indirect additive and/or antagonistic influences. Mol. Reprod. Dev. 53:434–442, 1999.

Transformation of ram spermatid chromatin

Abstract In order to investigate the sequence of changes in nuclear basic proteins throughout ram spermiogenesis, we have used different techniques to obtain populations of spermatid nuclei in specific stages of maturation. Sedimentation of testis cells at 1 gravity followed by treatment with Triton X-100 resulted in one population of round spermatid nuclei (steps 1–a), one of non-round spermatid nuclei (steps 8b-15), and one of elongated spermatid nuclei (steps 12–15). Populations of non-round spermatid nuclei were obtained by treatment with EDTA (steps 9–15), by sonication (steps 12–15) and digestion by DNase (steps 13–15). Nuclear proteins, extracted either directly with dilute acid or following a reducing treatment with 2-mercaptoethanol were characterized by polyacrylamide gel electrophoresis. The most striking alterations in protein composition occur during the elongation phase (steps 8–12). The five histones are displaced from chromatin at the same rate. When they are freed of histones (step 12), the nuclei start to accumulate the sperm-specific protein (BNSP) which is then partly extractable by dilute acid without a thiol that is required for its extraction from more mature nuclei. This stepwise replacement process is accompanied by a reduction of the basic protein amount bound to DNA. As soon as histones begin to disappear, eight spermatidal protein fractions are present in the nuclei until the BNSP synthesis reaches its maximum rate. Except for one, they all contain cysteine and are partially intermolecularly cross-linked in the chromatin. After in vivo and in vitro labelling experiments, they are synthesized in elongating spermatids (steps 8–11). None are degradation products of histones. Correlations of the times of onset of EDTA, sonication and DNase resistances with changes in the basic nuclear proteins point out that stabilization and condensation of spermatid chromatin is promoted through a progressive increase in disulfide bridges.

A cytochemical study of nuclear changes in boar, bull, goat, mouse, rat, and stallion spermatids.

En bloc staining with alcoholic phosphotungstic acid for lysine detection was applied to the testis of boar, bull, goat, mouse, rat, and stallion. Known changes in spermatid nucleoproteins were reflected in modification of the stainability of the nuclei over five successive periods of spermiogenesis. Species-specific differences occur in the way the chromatin is condensed, the presence and development of a peripheral layer of dense chromatin, the way lysine-rich nucleoproteins are lost from nuclei, and the staining abilities of nuclei of old spermatids. The observed staining pattern adds information about the precise timing of nucleoprotein changes during spermiogenesis and also about the heterogeneous distribution of nucleoproteins in nuclei of spermatids during several steps of differentiation.

Proteins of seminal fluid and spermatozoa in the trout (Oncorhynchus mykiss): partial characterization and variations.

The protein composition of seminal fluid, blood serum, sperm plasma membrane and flagellum of rainbow trout were analysed by SDS-polyacrylamide gel electrophoresis. Immunological identity between proteins of the 2 fluids and sperm components was studied using crossed immunoelectrophoresis, rocket immunoelectrophoresis and immunoblotting. Results indicate that many seminal proteins are antigenically-related to serum proteins, proteins of sperm origin are present in seminal fluid in varying amounts, depending on the animals and sampling time, and several serum-like seminal proteins are bound to spermatozoa.Lipoproteins were isolated from seminal fluid (mean level: 33 μg/ml) and characterized. They were identified as being HDL-like lipoproteins. A possible physiological role is proposed for these seminal lipoproteins.

Nuclear reorganization in ram spermatids.

Nuclear changes as a function of ram spermatid differentiation have been studied using electron microscopy, cytochemistry, and treatment of isolated nuclei with chemical and mechanical agents. In round nuclei, chromatin consists of intertwined knobby fibers about 210 and 120 A in diameter. In flattening nuclei, these fibers progressively change into smooth filaments at least 25–33 A thick. This change is interpreted as reflecting somatic histone removal known to occur in these nuclei (M. Loir and M. Lanneau, Exp. Cell. Res. 115 , 231, 1978). The packaging of chromatin is carried out by aggregation of the smooth filaments into large (about 300 A) contorted threads which finally coalesce to form a homogeneous mass. In flattened nuclei a small fraction of chromatin (basal knobs) does not undergo complete packaging and retains both cytochemical properties and resistance to disruption similar to those of chromatin in flattening nuclei. Correlations of changes in nuclear morphology, ultrastructure, and resistance to chemical and physical degradation with changes in nucleoproteins suggest that the cystine-containing spermatid-specific proteins could play an important role in chromatin-structure reorganization, shaping and increasing stabilization of the nucleus. The sperm-specific protein promotes terminal packaging and stabilization of chromatin. Other nuclear components considered in this study are: the RNP-containing structures, the nuclear posterior space, and the redundant nuclear envelope; the last two structures are possibly related to the transfer of proteins leaving the chromatin for the cytoplasm.

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis.

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO - Igepal(R) 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine (3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations > or =10 micromol/l. The presence of IGF-I (10-100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25-50 micromol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1-100 micromol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 micromol/l), NP2EO and NP (30 micromol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1-100 nmol/l 17beta-estradiol had no effect in our in vitro system, Triton(R) X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.

Isolation and characterization of two protamines St1 and St2 from stallion spermatozoa, and amino-acid sequence of the major protamine St1.

Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.

Spermatogonia of rainbow trout: I. Morphological characterization, mitotic activity, and survival in primary cultures of testicular cells.

Prerequisites of developing in vitro studies for a better understanding of the control mechanisms underlying the proliferation and differentiation of spermatogonia (Go) in the teleost testis are: (1) to be able to identify the different types of Go; (2) to maintain in culture the structural relationships occurring in situ between the various testicular cell types, as intact as possible; and (3) to know how the Go survive and proliferate in culture for several days. After very gentle homogenization of trout testes treated with collagenase, a cell suspension containing mainly spermatocysts (one or several Sertoli cells enclosing one Go or a clone of germ cells) and clusters of myoid cells and Leydig cells was seeded in culture onto a laminin plus fibronectin coating. After 4.5–6 days in culture, then staining with May‐Grünwald and Giemsa reagents, the determination of the nuclear and cellular size of the various Go and of the number of Go present in clones has enabled the identification of two types of large Go, in pairs or alone (Go A) and six successive types of smaller Go (Go B). Cell viability determination by staining with Rhodamine 123/propidium iodide (PI)/Hoechst 33342 and with FITC‐Annexin V/PI indicated that after 5–7 days in culture, all the somatic cells and most of the Go were viable. Only some of the Go, mainly among the most differentiated ones, underwent apoptosis, as it was the case for a number of spermatocytes and spermatids increasing with the time in culture. Brdu labelling and 3H‐Thymidine (3H‐Tdr) incorporation indicated that the proliferative activity of Go was at a maximum after 4.5 days in culture and that the response to at least two molecules (QAYL‐IGF‐I and GTH‐I) remained unchanged between 3 and 6 days. As only very scarce somatic cells from immature/spermatogenetic testes synthesized DNA up to 6 days in culture, the measurement of 3H‐Tdr incorporation by cells from such testes reliably reflected synthesis of DNA by only the Go (and eventually also by primary spermatocytes when they are present). In conclusion, this study provides information allowing a detailed analysis of the events related with the mitotic phase of spermatogenesis in the trout and it establishes that primary cultures of testicular cells carried out in the reported conditions represent a useful tool to develop an analysis of the mechanisms participating in the control of this phase. Mol. Reprod. Dev. 53:422–433, 1999.

Sexuality and gonadal cycle of the common dentex (Dentex dentex) in intensive culture

Abstract The common dentex ( Dentex dentex ) is a highly valued table fish in the Mediterranean region. In culture conditions, this sparid fish has a high specific growth rate and spawns spontaneously. However, the proportion of females that spawn spontaneously and the timing of their spawning are highly unpredictable. In addition, knowledge of the sexual cycles of this species is very limited. The main objective of this study was to provide basic knowledge on the reproductive biology of the common dentex. Study of male and female gametogenesis was performed using four groups of 0 + -, 1 + -, 2 + - and 3 + -year-old fish born in captivity. These groups were sampled either every 1 or 2 months, for either 1 or 2 consecutive years. Histological analysis of the gonads from a total of 448 individuals enabled the definition of five maturity stages for male and five for female fish. Sexual differentiation occurred between 5 and 12 months of age. Among the fish studied, neither bisexual gonads nor any other indication of sexual inversion was found in fish up to 4 years of age, suggesting that common dentex is a gonochoristic fish. In Crete, Greece, spawning took place between the end of March and May. Males and females older than 1 year (1 + ) matured almost simultaneously. All the 2-year-old males produced milt. In our sampling conditions, the highest percentages of females observed undergoing maturation during their second, third and fourth years were 67%, 100% and 100%, respectively. Sexually mature females were detected at the minimum standard length of 21 cm. After the spawning period, and until the following January, all the females were in previtellogenesis and in some males, spermatogenetic activity resumed gradually. In February, under increasing photoperiod, cortical alveoli appeared in growing oocytes and the development of spermatogenesis greatly increased. Between February and April, vitellogenesis occurred in females and the gonadosomatic index (GSI) increased from 0.2–1% to 3–6% in both sexes. The fecundity estimates indicated that common dentex display a spawning strategy similar to many temperate marine teleosts. It is a multiple spawner, exhibiting asynchronous oocyte development (unknown annual fecundity). Batch relative fecundity ranged from 32,000 to 393,000 eggs/kg body weight, and a positive linear relationship between batch fecundity and body size was found.