Biomedical subjects

C Finaz

Publications and source records attributed to C Finaz.

At least 19 recordsLinked to original sources

PubMed2001

Cloning and sequencing of SOB3, a human gene coding for a sperm protein homologous to an antimicrobial protein and potentially involved in zona pellucida binding.

We have previously characterized an 18-19 kDa cationic protein, SOB3, that was detected in the epididymis and localized within the acrosome and on the neck region of human spermatozoa. We suggested that it is involved in secondary sperm binding to the zona pellucida. The present study describes its purification to homogeneity by preparative electrophoresis and non-equilibrium pH gradient electrophoresis. Degenerate primers deduced from microsequencing were used to amplify a specific fragment from human epididymal RNA by reverse transcription-polymerase chain reaction (RT-PCR). This 164 bp fragment was extended by 5' and 3'-RACE to obtain the 548 bp full length cDNA. The open reading frame encodes a 170 amino acid protein. SOB3 is a single copy gene. It is 98% identical to prepro-FALL39 and 100% identical to CAP18, two human genes which were initially identified by screening a human bone marrow (lambda)gt11 library, and which encode an antimicrobial protein. Northern blots of human tissues revealed a 1 kb transcript in corpus and cauda epididymis only, while RT-PCR showed presence of the mRNA in the three epididymal regions and also in round spermatids. The above results suggest that SOB3 has two roles in sperm protection and fertilization, depending on its dual origin and final sperm localization.

Amino Acid Sequence↗

PubMed2000

Adhesion proteins expressed on human gamete surfaces and egg activation.

The initiation and propagation of a Ca2+ signal through the egg seems to be the pivotal event in triggering of meiosis resumption. Over the past decade evidence has accumulated suggesting that sperm contact is essential for this phenomenon to occur in most physiological groups. Given their ability to transduce signals, adhesive proteins which are involved in various binding mechanisms such as cell migration, lymphocyte activation, phagocytosis and virus fusion may play a similar role in fertilization. They have been the subject of serious investigation in non-human mammals and some emerging data indicate that they are active in humans as well. Our goal is to review the presence of such molecules on human gametes and their relevant physiological role, i.e., integrins and their ligands, selectins, IgG Fc receptors and leucocyte differentiation markers. We will discuss how they might trigger egg activation through signaling pathways in light of their identified functions in other adhesion systems. The putative participation of specific human sperm proteins will also be evaluated.

A Kinase Anchor Proteins↗

PubMed1999

Cloning and characterization of SOB1, a new testis-specific cDNA encoding a human sperm protein probably involved in oocyte recognition.

A human sperm-oocyte binding protein, SOB1, was purified by two dimensional gel electrophoresis and sequenced. This protein was selected because it was recognized by a monoclonal antibody that inhibited the binding of human sperm to zona-free hamster oocytes. The sequences of the tryptic peptides were used to design degenerate primers. These were used to amplify a specific fragment from human testis cDNA by the polymerase chain reaction. This 1233 bp fragment was extended in 3' and 5' by RACE to obtain the 3 kb full length SOB1 cDNA. Sequence analysis indicated that the deduced open reading frame encodes a 853 amino acid protein, with a molecular mass of 94. 7 kDa. This is a new testis-specific cDNA. It is 27, 32.8 and 34.4% homologous to three sperm proteins, HI, Fsc1 and AKAP82 respectively. A single 3kb transcript was demonstrated only in the testis by northern blot analysis. It is a single copy gene, well conserved among mammals and located on human chromosome 12 at band p13.

A Kinase Anchor Proteins↗

PubMed1999

Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients.

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Adult↗

PubMed1999

Flow cytometric method to isolate round spermatids from mouse testis.

The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting. Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density. A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids. Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was obtained with a single cell using nested primers. The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia. Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.

Acrosome↗

PubMed1998

SOB3, a human sperm protein involved in zona pellucida binding: physiological and biochemical analysis, purification.

LB5 antibody was selected from a monoclonal antibody (mAb) library directed against human sperm proteins. LB5 mAb detected the corresponding protein SOB3 in the neck region and the flagellum of most live ejaculated sperm while it labelled, in addition, the acrosome of about 10-20% of spermatozoa. The percentage of LB5 acrosome-stained sperm was significantly correlated with the percentages of either spontaneous or A23187-induced acrosome-reacted sperm. While SOB3 could not be detected in the testis, it appeared in spermatozoa from the corpus epididymis segment. LB5 mAb impaired neither sperm motion parameters, acrosomal reaction triggering, nor sperm binding to zona-free hamster oocytes. By contrast, LB5 Fab fragments (200 micrograms/ml) inhibited sperm binding to human zonae pellicidae by 35.7%. If sperm were induced to acrosome react with A23187 prior to LB5 treatment, the inhibitory effect shifted to 59.9%, while no significant effect was observed following A23187 incubation alone. Western blotting of human sperm and cauda epididymis extracts revealed two bands of 18 and 19 kDa. While no cross-reaction was observed with other tested organs, a similar 18-kDa band was revealed in erythocytes and one of 19 kDa in B-lymphocytes. No cross-reactivity could be evidenced in any animal sperm analyzed. SOB3 was first separated in a 17- to 20-kDa preparative electrophoresis fraction and finally purified by isoelectrofocusing according to its pl of 9.8. These results suggest that SOB3 is localized under the outer acrosomal membrane, that it participates in secondary sperm binding to the zona pellucida, and that it shares homologies with the immune system.

Acrosome↗

PubMed1997

Characterization and isolation of SOB2, a human sperm protein with a potential role in oocyte membrane binding.

G12 monoclonal antibody (mAb), one of a library of constructed mAb directed against human sperm proteins, was found by immunoperoxidase staining to label the post-acrosomal and neck regions of fixed human cauda epididymal and ejaculated spermatozoa. Epithelium and fluid of caput epididymis were strongly labelled while there was no staining on testis and efferent ducts. Western lot analysis revealed that G12 antibody reacted with proteins of 17.5, 18 and 19 kDa in human spermatozoa. This pattern seems to be specific for mature human spermatozoa, as it has not been observed either in other human tissues tested, or in spermatozoa from different animals. SOB2, the corresponding protein, was isolated from NP40-extracted human spermatozoa by using preparative electrophoresis, followed by isoelectrofocusing according to its isoelectric point of 6.4 G12 Fab fragments strongly inhibited binding of human spermatozoa to zona-free hamster oocytes (up to 86% inhibition at 200 micrograms/ml). Impairment of binding was dependent on the concentration of purified G12 immunoglobulin (Ig)G1, and significant even at 10 micrograms/ml. There was no inhibitory effect of G12 antibody on sperm motility parameters or triggering of the acrosome reaction and it did not inhibit binding to human zona pellucida. These results indicate that SOB2 is likely to participate in membrane oocyte binding, and my be potential candidate for the development of a contraceptive vaccine.

Animals↗

PubMed1995

FLB1, a human protein of epididymal origin that is involved in the sperm-oocyte recognition process.

CA6 antibody was selected out of a monoclonal antibody library raised against human sperm proteins primarily for its ability to recognize an epididymal antigen and to modify sperm adhesion to zona-free hamster oocytes. In the present study, CA6 was shown to decrease sperm binding to zona-free hamster and human oocytes by 40-92% and 38-48%, respectively. The corresponding protein, which was referred to as FLB1, was found to be secreted by the epididymis and to bind specifically to a human, macaque, and rodent subacrosomal sperm region. Western blotting revealed a molecular mass of 94 kDa in human epididymal extracts and of 100 kDa in human, macaque, mouse, rat, and hamster sperm, suggesting further modifications after its binding to sperm. An equivalent protein was not observed in human liver, ovary, testis, plasma, or epidermis. Two-dimensional electrophoresis showed that FLB1 is formed of two subunits with the same 47-kDa molecular mass and slightly different pI (5.8, 5.9). Microsequencing of the protein revealed a partial homology with human cytokeratins 1 and 10. These results suggest that FLB1 is an epididymis-specific cytokeratin-like protein that is involved in the sperm-oocyte recognition process.

Amino Acid Sequence↗

PubMed1994

Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3 beta-hydroxysteroid dehydrogenase/delta (5-4)-isomerase and 17 alpha-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line.

Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17 alpha-hydroxylase/C17-20 lyase (P-450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3 beta HSD and P-450 17 alpha were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17 alpha and 3 beta HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17 alpha expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0 kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Human sperm proteins from testicular and epididymal origin that participate in fertilization: modulation of sperm binding to zona-free hamster oocytes, using monoclonal antibodies.

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme-linked immunosorbent assay (ELISA). Immunoperoxidase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the beginning to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona-free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation.

Animals↗

PubMed1991

Inhibition of luteinizing hormone-human chorionic gonadotropin binding by retinoids in a Leydig cell line.

Treatment of K9 mouse Leydig cells with 3 x 10(-6) M retinol (R) and retinoic acid (RA) resulted in 75% and 65% reduction of 125I-labeled hCG binding respectively, when assayed at 35 degrees C. This effect was dose-dependent and was first detected 12 h after initiation of treatment: it was maximal at 48 h for RA. R and RA had no significant effect on the rate of internalization and degradation of 125I-hCG as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloracetic acid-soluble label in the medium. When exposed to increasing concentrations of hCG for 24 h, both retinoid-treated and control cells 'down-regulated' their gonadotropin receptors with the same dose-dependent pattern. The kinetics of reappearance of the receptors was similar for retinoid-treated and control cells, but for treated cells the maximal number of receptors reinitiated at 24 h never exceeded 40% of the values observed with control cells. Scatchard plot analysis confirmed a decrease in hCG receptor number from approximately 26,000 to approximately 6400 and approximately 3500 sites per cell after R and RA treatment. Kd values for 125I-hCG binding were 2 x 10(-10) M, 7.3 x 10(-11) M and 6.9 x 10(-11) M for control, R- and RA-treated cells respectively. On the basis of our data it is likely that retinoid-induced reduction in 125I-hCG binding to K9 Leydig cells is due to decreased receptor synthesis.

Animals↗

PubMed1991

Characterization of rat epithelial epididymal cells purified on a discontinuous Percoll gradient.

A method for the purification of epithelial cells from the three anatomical regions of the rat epididymis (corpus, caput and cauda) is described. An enzymic digestion followed by sedimentation of crude cell suspension on discontinuous Percoll gradient yielded quite pure active epithelial cell population as judged by morphological and functional studies. Electron microscopy analysis showed that cells from bands corresponding to densities 1.055 and 1.06 g/ml the gradient preserved a morphology compatible with their epithelial origin and their absorptive and secretory functions. Moreover, they stained positively with anticytokeratin antibody (95-97%) and were negative for antidesmin antibody. They selectively bound L-carnitine through a time-dependent and saturable system and differences in the rate of binding were apparent according to the three anatomical regions of the epididymis.

Animals↗

PubMed1988

Steroidogenesis expression depends on negative control(s): analysis in Leydig X adrenal intraspecific cell hybrids.

Hybrids constructed by fusing mouse Leydig cells with mouse adrenal Y1 cells were able to randomly express all the parental specific traits but for the response to gonadotropin (hCG) and corticotropin (ACTH): three of them, YDYL 14, 17 and 19, metabolized both progesterone and dehydroepiandrosterone into testosterone accounting for 17 alpha-hydroxylase, 17-20-lyase, 17-ketoreductase and 3 beta-hydroxysteroid dehydrogenase activities. Under basal conditions, 17 alpha-hydroxylase and 17-20-lyase activities were high in the three clones as compared to parental Leydig cells, and were no longer stimulated by cAMP in YDYL 17 and 19. The hybrids responded to various hormones such as prostaglandin E2 (PGE2), vasoactive intestinal peptide (VIP) and prolactin (PRL) which are not directly implicated in the expression of steroidogenesis; they generally retained the Y1 morphological response to 8-bromo cAMP. On extended culture, reexpression of ACTH sensitivity occurred in one clone, YDYL 9. This reexpression was correlated with a Robertsonian translocation between mouse chromosomes 2 and 11, while extinction required the presence of an intact mouse chromosome 11.

17-alpha-Hydroxyprogesterone↗

PubMed1987

Construction of a Leydig cell line synthesizing testosterone under gonadotropin stimulation: a complex endocrine function immortalized by cell hybridization.

Hybridization between a mouse Leydig tumor cell line, MA-10, which produces cyclic AMP and progesterone under human chorionic gonadotropin (hCG) stimulation, and freshly isolated mouse Leydig cells gave rise to 54 hybrid clones, one of which, LK17, was capable of hCG-stimulated testosterone production. Subcloning of this hybrid resulted in the emergence of a subclone, K9, whose testosterone production is more than 10 times that of parent clone LK17, after hCG stimulation, with an ED50 of 37 pM. Testosterone synthesis by K9 cells was multiplied by 25 after gonadotropin stimulation, and binding of hCG declined after prolonged exposure to the hormone. These similarities with murine Leydig cells in primary culture make the K9 clone an attractive alternative for physiological studies.

Animals↗

PubMed1986

Systematic shut-off of the hormone receptors in intraspecific adrenal x Leydig cell hybrids.

The mouse Y1 adrenal cell line was fused with mouse Leydig cells in primary culture. The selected hybrids were examined for their response to gonadotropin (hCG) and ACTH. None of them bound specifically [125I]hCG, nor did they augment their cAMP production in response to gonadotropin or ACTH stimulation, whereas their adenylate cyclase remained responsive to forskolin and cholera toxin, thus indicating a repression of hCG receptor synthesis and probably a loss of ACTH receptors, rather than a lesion of the coupling between the hormone receptor complex and the adenylate cyclase. Basal pregnenolone production in 17 hybrids was close to that of Leydig and Y1 cells and was enhanced after 8-bromo adenosine 3',5'-monophosphate (8-Br-cAMP) stimulation in 11 of them. Therefore, the negative control leading to the extinction of both parental functions acts preferentially at the first step of steroidogenesis, i.e., the gene(s) coding for the hormone receptors.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1986

Panel of twenty-five independent man-rodent hybrids for human genetic marker mapping.

To increase the efficiency of the human gene mapping, a panel of 25 man-rodent hybrids was selected out of 200 independent man-rodent hybrids produced in our laboratory since 1969. The hybrid panel was selected in such a fashion as to allow the asignment of a human marker M by respecting the two following complementary criteria: correlation between M and a chromosome and exclusion of the other chromosomes. The panel characteristics allowing the use of these two criteria were presented and discussed. A first series of enzyme markers were analysed to test the validity of the hybrid panel for the human gene mapping. The different possibilities and limits of the hybrid panel were also discussed, especially for the assignment of markers with DNA probes.

Animals↗

PubMed1985

Inhibitory or stimulatory effect of human genes on the expression of adrenal function in human Leydig X Y1 cell hybrids.

In order to investigate the expression and the regulation of steroidogenesis, human Leydig cells were fused with a functional mouse adrenal cell line (Y1). Six independent hybrid clones were analysed for hormone receptors and for cAMP and steroid response to ACTH, hCG, 8Br-cAMP or forskolin. All hybrids had lost hCG receptors and their ability to produce testosterone. With respect to the response of adenylate cyclase to ACTH and/or forskolin, hybrids could be classed into two groups. In the first group, the pattern of response was qualitatively similar to Y1 parental cells; The second group was far less responsive to ACTH than are Y1 cells, and when added together, forskolin and ACTH only had an additive effect. All hybrids responded to ACTH and 8Br-cAMP with an increased production of pregnenolone (P5). The amounts of P5 produced both under basal conditions and following 8Br-cAMP stimulation were significantly higher in three hybrids when compared to Y1 cells. However, the ability of two of these three hybrids to produce 20 alpha-dihydroprogesterone (20 alpha OHP4) was very low. The metabolism of [14C]P5 revealed that in one of these hybrids, there was a loss of 3 beta-hydroxysteroid dehydrogenase/isomerase whereas in the other case, there was a low 20 alpha-hydroxylase activity. The inhibition of cell growth by ACTH was related to the ability of the hormone to stimulate cAMP. Conversely, the inhibitory growth effects of 8Br-cAMP were not always inversely correlated with the ability of this nucleotide to stimulate P5 production. Since hybrids contained two mouse genomes and retained variable human chromosomes, these results suggest that extinction or enhancement of murine genes coding for some of the enzymes involved in steroidogenic response to ACTH was due to the regulation by human genes.

Adrenal Glands↗

PubMed1985

Modulation of cultured mouse Leydig cells adenylate cyclase by forskolin and hCG.

The diterpene, forskolin, stimulated cAMP accumulation about 15-fold over basal levels in purified mouse Leydig cells; however, it remained far less potent than hCG. Simultaneous addition of forskolin and hCG resulted in a striking synergistic stimulation of cAMP production. In contrast, forskolin-enhanced testosterone accumulation was never synergistic with that produced by maximal concentrations of hCG. hCG (3 X 10(-9) M) lowered about 6-fold the ED50 for forskolin-elicited cAMP accumulation and increased the maximal response to forskolin about 16-fold. Conversely, forskolin 10(-6) M) reduced the ED50 for hCG 2-fold but had a much smaller effect (2-3-fold) on maximal response. Moreover, pretreatment with hCG induced only a homologous desensitization of adenylate cyclase, whereas the enzyme became partially resistant to both hCG and forskolin in cells pretreated with forskolin. The homologous hCG-induced desensitization and the partial heterologous one induced by forskolin suggest that more than the catalytic unit of the cyclase is required for the diterpene activation.

Adenylyl Cyclases↗