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Biomedical subjects

M Loir

Publications and source records attributed to M Loir.

At least 37 records · Page 2Linked to original sources
PubMed1990

Trout steroidogenic testicular cells in primary culture. I. Changes in free and conjugated androgen and progestagen secretions: effects of gonadotropin, serum, and lipoproteins.

Isolated trout steroidogenic testicular cells were cultured for 10-15 days, either mixed with other round cells or after enrichment in interstitial cells. Free and conjugated progestagen and androgen secretions were assayed using specific radioimmunoassays (RIA). Free progesterone, 17 alpha-hydroxyprogesterone (17 alpha-OH-P), 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha,20 beta-OH-P), androstenedione, testosterone (T), and 11-ketotestosterone (11KT) were produced by testicular cells prepared from testes in spermatogenesis and mature testes. Discrete amounts of dehydroepiandrosterone (DHA) and of estradiol were secreted by mixed testicular cells prepared from mature testes, but no estradiol was detected in interstitial cell media. Conjugated androgens were produced by interstitial cells. While the production of progestagens by cells from spermatogenetic and mature testes either remained constant or increased throughout culture duration, those of free and conjugated androgens progressively decreased to low values whatever the components added to the medium. When salmon gonadotropin (s-GtH) was present permanently, androgen (free and conjugated) and progestagen secretions were stimulated for 3 to 4 days. When GtH was present discontinuously (1 day in every 3 days), the sensitivity of the cells was maintained for at least 7 days. While the GtH-stimulated/basal ratio was high for androgens, it was rather low for 17 alpha 20 beta-OH-P as compared to the values obtained with testis fragments. Trout serum (5%) stimulated the secretion of free and conjugated T and 11KT when testes were mature, but not when they were in spermatogenesis, while it stimulated 17 alpha 20 beta-OH-P secretion at the two stages. Total trout lipoproteins (125-500 micrograms/ml) stimulated 17 alpha 20 beta-OH-P secretion by cells from spermatogenetic testes, but not 11KT secretion.

17-alpha-Hydroxyprogesterone↗
PubMed1990

Trout steroidogenic testicular cells in primary culture. II. Steroidogenic activity of interstitial cells, Sertoli cells, and spermatozoa.

Somatic cells (interstitial cells and Sertoli cells) were prepared either as single cells or in clusters, from spermatogenic and mature trout testes, according to Loir (1988), and cultured for 10-14 days. Sertoli cells are 3 beta-HSD negative when prepared from testes resuming spermatogenesis and from mature testes, but they are 3 beta-HSD positive in spermatogenic testes. Progesterone, 17 alpha-hydroxyprogesterone (17 alpha-OH-P), and free androgens are secreted by interstitial cells, 11-ketotestosterone (11KT) being the predominating steroid produced immediately after seeding. These cells also produce high levels of glucuronated androgens. At least in mature spermiating testes they do not secrete estradiol. After isolation, interstitial cells would lose most of their ability to secrete 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha 20 beta-OH-P) but they would recover it later. Testicular spermatozoa, which convert 17 alpha-OH-P independently of s-GtH, constitute a second source of this progestagen. In addition, our results suggest that Sertoli cells could be able to secrete 17 alpha-OH-P and also progesterone. A possible participation of the intralobular production of the former progestagen to the local regulation of germ cell maturation is evoked.

17-alpha-Hydroxyprogesterone↗
PubMed1989

Trout Sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study.

In order to characterize trout Sertoli cells and germ cells obtained after testis dissociation and cell separation, we have studied their morphology, ultrastructure, survival, and ability to express differentiated activities in primary cultures. After dissociation, the fine structure of Sertoli cells does not differ from that observed in situ and only minor changes are shown for at least 13 days. Until they are flattened in a monolayer, they keep the ability to retain germ cells on their surface. When flattened, some of them are able to divide. At the opposite of meiotic germ cells, spermatogonia can develop independently of Sertoli cells. They are able to proliferate during at least 10 days. Spermatocytes and spermatids are obtained as single cells and multinucleated giant cells (symplasts). In the absence of somatic cells, their maximal viability is approximately 5 days, whereas spermatocytes adhering to Sertoli cells can survive at least 10-12 days, provided trout lipoproteins are present. Spermatocytes are able to differentiate to spermatids, although this process is impaired for some cells. The adhesion of spermatogonia and spermatocytes to Sertoli cells is specific, mediated by desmosome-like junctions and favored by lipoproteins. These data are compared to what is known in mammals and in amphibians.

Animals↗
PubMed1988

Trout Sertoli and Leydig cells: isolation, separation, and culture.

Trout testes at various stages of maturation were dissociated by perfusion at 12 degrees C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: 1) the testis cell suspension was separated by sedimentation at unit gravity into "isolated cell" and "cell cluster" populations; 2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10-15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3 beta-HSD positive and produced progesterone and 17 alpha, 20 beta-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.

Animals↗
PubMed1988

Immunocytochemical localization of protamine in the boar testis.

Protamine was specifically demonstrated in spermatids and spermatozoa of the boar by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera, and three different direct or indirect labelling techniques. The two isomers of the protamine could not be labelled separately. The protamine is present in the cytoplasm of elongating spermatids and it enters the nuclei throughout the elongation process after possible storage in the cytoplasm or in the nuclear envelope of spermatids, or both. These findings differ from previous observations in other species.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1986

High mobility group proteins in ram spermatids.

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.

Amino Acids↗
PubMed1986

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies.

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.

Androgen-Binding Protein↗
PubMed1985

Interactions of nuclear proteins with DNA, during sperm differentiation in the ram.

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nucleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous "knobby fibers" that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30-200 A region.

Animals↗
PubMed1985

Isolation and characterization of the ram spermatidal nuclear proteins P1, 3 and T.

Ram spermatidal proteins P1, 3 and T were isolated from non-round spermatid nuclei and characterized by amino acid analysis. Spermatidal proteins are small arginine- and cysteine-rich basic proteins. Proteins P1 and T are unusually rich in serine and the histidine content of P1 is particularly high. The NaCl molarities required to dissociate these proteins from the spermatid nuclei were determined. These proteins are present only during the reorganization of the spermatid chromatin.

Amino Acids↗
PubMed1984

Primary structure of the ram (Ovis aries) protamine.

The amino acid sequence of the protamine isolated from mature sperm nuclei of the ram (Ovis aries) has been established from automated sequence analysis of the S-carboxymethylated protamine. Ram and bull protamines differ only by two point changes and the deletion in bull protamine of the tripeptide Cys39-Arg-Arg41. In mammalian protamines the central region (residues 13-36) consisting mainly of arginine clusters appears to be conserved whereas the N-terminal and C-terminal regions are more variable.

Amino Acid Sequence↗
PubMed1984

Purification and characterization of ubiquitin from mammalian testis.

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.

Amino Acid Sequence↗
PubMed1984

Structural function of the basic nuclear proteins in ram spermatids.

The function of the cysteine-containing spermatidal proteins and of protamine in the packaging and stabilization of chromatin during ram spermiogenesis was investigated. Extractions of the histones and spermatidal proteins from the nonround spermatid nuclei decreases the nuclear stability (sonication resistance), decondenses the chromatin, and reduces the diameter of the largest chromatin threads (100-200 A vs. 380 A in the control nuclei). Extractions by acid, salt, or heparin have no effect on the protamine-containing electron-opaque chromatin. In contrast, treatment by dithiothreitol alone decondenses all the nonround spermatid nuclei at a rate which decreases with the maturation state of the nuclei. The electron-opaque chromatin is then resolved in 35-A-thick filaments. Experimentally induced fluctuations of the level of SS bonding appear to influence the chromatin stabilization and ultrastructure in most of the nonround spermatid nuclei. These data evidence that noncovalent interactions play a main structural role at the beginning of chromatin reorganization, and SS bonding between spermatidal proteins and then between protamine molecules increases progressively and becomes mainly responsible for the chromatin stabilization in the protamine-containing nuclei.

Animals↗
PubMed1983

Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis.

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.

Animals↗
PubMed1982

An electrophoretic investigation of mammalian spermatid-specific nuclear proteins.

Using standardized methods for protein extraction and analysis, the testes of rams, bulls, goats, boars, stallions, rats, cats, hedgehogs, European mink and ferrets were examined for basic spermatid nucleoproteins by electrophoresis. The results suggest that differences exist in the total number of these proteins as well as in the number and amount of the cross-linked cystein-containing proteins. These differences appear to be more family-specific than species-specific.

Animals↗