Biomedical subjects

J L Dacheux

Publications and source records attributed to J L Dacheux.

At least 19 recordsLinked to original sources

PubMed1995

Direct evidence for the elevated synthesis and secretion of procathepsin L in the distal caput epididymis of boar.

The proteins which are secreted from the restricted part of the epididymis are suggested to sustain sperm maturation. In porcine species, as the potential abilities of sperm for movement and fertilization greatly increase in the corpus epididymis, the secretions in both the caput and corpus epididymis seem to be very important for the sperm maturation. In this study, we have directed our attention to the 40 kDa protein which is detected in the fluid of the distal caput epididymis of boar. It was purified from the porcine cauda epididymal fluid and its cDNA was cloned from the cDNA library of the distal caput epididymis. According to the deduced amino acid sequence, the 40 kDa protein has been identified as procathepsin L. Northern blot analysis showed that the procathepsin L mRNA was most abundant in the distal caput epididymis among the tissues as examined. Consistent with the distribution of the procathepsin L mRNA in the epididymis, the activity of procathepsin L was absent in the fluid of the proximal and mid caput epididymis and first appeared in the distal caput epididymal fluid, whose contents gradually decreased with the passage through the epididymis. These results first appeared in the first distal caput epididymis expresses very high levels of procathepsin L and unusually secretes it into the luminal fluid instead of targeting it to lysosomes. It has been also found that the mRNA of PDGF, which is known to enhance cathepsin L expression in the culture cells, is very high in the mid caput epididymis, which just precedes the site of procathepsin L secretion. This result indicates that PDGF directly regulates the locally restricted expression and secretion of procathepsin L in the epididymis, which is one of the possible mechanisms involved in the functional differentiation in the epididymis.

Amino Acid Sequence↗

PubMed1995

Increased zona-binding ability after incubation of spermatozoa with proteins extracted from spermatozoa of fertile semen.

The involvement of proteins extracted from spermatozoa of fertile semen in sperm-zona binding was examined under hemizona assay conditions. One droplet of a suspension of spermatozoa was exposed to sperm proteins and then tested for zona binding, while a parallel semen suspension droplet incubated with culture medium served as a control. The reliability of the test was increased by relating the number of spermatozoa bound to each inseminated hemizona to the surface area of the hemizona and expressed as the binding index. For spermatozoa incubated with extracted proteins, the binding index was greater than (P = 0.001) that of controls (125.2 +/- 45.1 versus 63.6 +/- 29.2, respectively). As a first control, two other protein sources (fetal calf serum and human follicular fluid) were tested in the hemizona assay. No significant differences were found in zona binding for other protein-exposed spermatozoa compared with controls. As a second and reverse control, exposure of one hemizona to sperm proteins before insemination with untreated spermatozoa induced a marked decrease (P = 0.0003) in sperm binding, compared with that of the matched hemizona not exposed to sperm proteins (control) (3.4 +/- 1.4 versus 74.5 +/- 6.8, respectively). Taken together, these findings confirm the involvement of extracted sperm proteins in sperm-zona interactions. Therefore, in the cases in which fertilization in vitro fails because of a lack of sperm-zona binding, incubation of deficient spermatozoa with proteins extracted from spermatozoa of fertile ejaculates should restore their ability to interact with the oocyte and, thus, should enhance the prognosis for in vitro fertilization.

Culture Media, Conditioned↗

PubMed1994

Characterisation of boar sperm dynein heavy chains by UV-vanadate dependent photocleavage.

Intact and Triton X-100 demembranated boar spermatozoa possess two main heavy chains with molecular masses (M(r)) of 430 and 460 kDa. These heavy chains were photo-cleaved within the axoneme under V1 conditions and produced two main fragments at 245 kDa and 185 kDa. Two minor fragments at 170 and 90 kDa were also obtained. In the presence of low Mg2+ (1 mM) a supplementary fragment of 200 kDa was also observed. The heavier chain was cleaved in the absence of ATP to give the 245 kDa fragment. The boar axonemal heavy chains cannot be directly extracted by high salt treatment of the demembranated sperm in presence of high level of protease inhibitors (HPI) but were extracted when the solution contained low protease inhibitor (LPI) concentrations. Electron microscopy showed that high salt treatment in presence of LPI extracted the outer arm mainly from the principal piece of the flagellum and less from the intermediate piece. Fractionation of the LPI high salt by chromatography or sucrose gradient allowed the obtention of a particle with ATPase activity, a size of 1.2 MDa and a sedimentation coefficient of about 20S. The particle was composed of two heavy chains of M(r) 320 and 340 kDa. These heavy chains can be photo-cleaved under V1 conditions and in absence of Mg2+. The sucrose gradient 20S fractions contained also two chains at 110 and 87 kDa which could be either intermediate chains or proteolytic fragments of the heavy chains. A chain at 63 kDa was also associated with the 20S fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation.

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum.

Animals↗

PubMed1992

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.

Age Factors↗

PubMed1991

Water insoluble fraction of egg yolk maintains porcine sperm motility by activating adenylate cyclase.

The decrease in motility of porcine cauda epididymal sperm was less than that of caput epididymal sperm in the medium containing bicarbonate. This may be due to the difference of sensitivity of adenylate cyclase to bicarbonate between mature and immature sperm; activation of mature sperm enzyme by bicarbonate was higher than that of immature sperm. Nondialysable fraction of egg yolk prevented the decrease in motility of immature sperm in the presence of bicarbonate, but it was not effective for the motility of mature sperm under the same condition, because only bicarbonate is sufficient for the maintenance of its motility. In the absence of bicarbonate, both mature and immature sperm required egg yolk to maintain motility. The favorable effect of egg yolk on the motility is ascribed to the enhancement of intracellular cAMP level. Partial fractionation of egg yolk showed that water-insoluble lipoprotein fraction contains factor(s) which activates adenylate cyclase in sperm plasma membrane. This is the first report in which high molecular weight activator of the sperm enzyme was demonstrated.

Adenylyl Cyclases↗

PubMed1991

Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.

Animals↗

PubMed1989

Androgenic control of antagglutinin secretion in the boar epididymal epithelium. An immunocytochemical study.

Antagglutinin, a specific protein synthesized by the boar epididymis, was secreted by the principal cells of the initial segment, the caput and the corpus, but was not detectable in the caudal cells. Castration completely abolished the synthesis and secretion of antagglutinin in all epididymal cells. Androgen replacement suggests that the epithelial cells from different segments have differential regulatory mechanisms. The proximal zone appeared refractory to exogenous testosterone; the median zone was a typical androgen-dependent region; and the caudal cells, where an unusual secretion of antagglutinin was detected, revealed still a different reaction pattern. It is postulated that these latter cells depend not solely on androgen but also or exclusively on other factors. Our results, which demonstrate a primary role of the Golgi complex in the secretory process in the epididymal cells, also suggest that the apical smooth endoplasmic reticulum may be implicated in the intracellular transport of glycoproteins to the cell surface.

Androgens↗

PubMed1989

Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis.

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.

Animals↗

PubMed1989

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar.

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.

Animals↗

PubMed1988

Immunocytochemical localization of antagglutinin in the boar epididymis.

Antagglutinin, a specific protein synthesized by the boar epididymis, was localized by the biotin-streptavidin method in all the principal cells of the caput and corpus epididymidis as well as in the lumen of this organ. Intracellular staining, which was first detected in the initial segment, appeared stronger in the distal caput and in the corpus but diminished and disappeared in the caudal epididymal cells. In all the principal cells, a consistent reaction product was localized in the large Golgi complex. Only slight and diffuse immunoreactive material was detected in the cytoplasm, except in the middle caput where the heterogeneous reactive granules appeared to be intracellular sites of degradation of this protein. In the lumen, the intensity of reaction increased from the caput to the cauda. Antagglutinin appeared strongly associated with the luminal surfaces, especially around and between the stereocilia. However, the spermatozoa also exhibited a distinct pattern of immunostaining. The results are discussed in relation to protein secretion in the epididymis and to the role of antagglutinin in the gamete-interaction process.

Animals↗

PubMed1988

[Differential regulation of antagglutinin secretion in pig epididymis: an immunocytochemistry study].

Antagglutinin, a specific protein synthesized by the boar epididymis, was localized in all the principal cells of the initial segment, of the caput and of the corpus but was not detectable in the caudal cells. Castration completely abolished the synthesis and secretion of antagglutin in all the epididymal cells. Androgen treatment led to (1) the restoration of antagglutinin secretion in the caput (middle and distal) and in the corpus, (2) the failure to restore the secretion in the initial segment and in the proximal caput and (3) the emergence of a new secretion of antagglutinin by the caudal cells. These results suggest that (1) antagglutinin is an androgen-dependent protein and (2) the epithelial cells from different segments have differential regulatory mechanisms.

Androgens↗

PubMed1988

[Maturation of ram spermatozoa. Preliminary study of the flagellar movemnent characteristic of the transitory forms of the epididymal corpus].

Mammalian spermatozoa acquire the ability to move forward during epididymal maturation and the corpus occurs as a critical region where all the patterns of movements can be observed: immature, 'erratic' and mature. The absence of coordination of the flagellar movement of the transitory forms provides a difference in the initiation sites and the waves propagation between the two sides of the axonema. The mechanisms that may be required to activate selectively the tubulin-dynein system on the two sides of the flagellum during the initiation of the movement of immature sperm in mammals are unknown.

Animals↗

PubMed1988

[Flagellar motility amd movement of boar spermatozoa during epididymal transit].

The motility of boar spermatozoa during epididymal transit was analysed in vitro using various techniques. From the head to the cauda there was an increase of the percentage of motile and progressive spermatozoa. During maturation there was a progressive reduction of flagellar bend curvature while flagellar beat frequencies increased. A three dimensional pattern of flagellar beating responsible for cell rotation and straight line progression of spermatozoa was observed only in caudal epididymis. The addition of epididymal fluid protein to the media could increase the number of motile cells at the various levels but had no influence on the characteristics of flagellar bending.

Animals↗

PubMed1988

[Acetylcarnitine and spermatozoa: relationship with epididymal maturation and motility in the boar and man ].

The level of carnitine and acetylcarnitine in spermatozoa of boar epididymal origin and of human ejaculates was demonstrated. In the epididymal fluid of boars, the concentration of carnitine (nmol/mg protein) began to increase from 20 in the distal caput to rise progressively to 700 in the distal cauda. By contrast, the carnitine content of spermatozoa only started to increase in the proximal cauda where the concentration of carnitine in the fluid was 200-300 nmol/mg protein, then gradually increased in spermatozoa from more distal sites. The increase in the acetylcarnitine content of spermatozoa paralleled that of the carnitine amount, represented 50% of total carnitine (carnitine + acetylcarnitine) and coincided with the acquisition of progressive motility. In two populations of human seminal spermatozoa selected by migration and characterised by a very large difference in their percentage of progressively motile cells, higher carnitine and acetylcarnitine contents (40%) were found in migrated spermatozoa compared to the residual population. These results suggest that accumulation of carnitine and its metabolite may be an important factor in the acquisition and the maintenance of progressive motility. Measurement of acetylcarnitine content of human seminal spermatozoa could be used as a marker of epididymal maturation.

Acetylcarnitine↗

PubMed1987

Analysis of the flagellar bending waves of ejaculated ram sperm.

The variability of flagellar movement, illustrated by the highly heterogeneous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 X 36 camera system. The multiple-moving-exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.

Animals↗

PubMed1987

The intracellular pathway of antagglutinin secretion in the boar caput epididymidis as revealed by immunogold labelling.

Antagglutinin, a specific protein synthesized by the boar epididymis, was localized by an ultrastructural immunogold-labeling procedure in the principal cells of the three regions of the caput epididymidis, most notably at the sites of synthesis and secretion. The intensity of the reaction was variable in the three epididymal zones. Labeling was of low intensity in the proximal and middle caput, except in the granules of the latter. These granular "storage sites" did not correspond to typical secretory granules but appeared to be intracellular sites of degradation of this protein. In the distal caput, which was devoid of these granules, intense secretory activity for antagglutinin was detected. Few gold particles were localized in the RER profiles but labeling was detected in the Golgi zone, in numerous dense vesicles, in structures distributed between the Golgi zone and the apex of the cell, and in the epididymal lumen. This study has enabled us to visualize immunocytochemically antagglutinin along its intracellular secretory pathway, i.e. at the site of its synthesis, during its passage via the Golgi zone, and its intracellular transport to the lumen.

Animals↗

PubMed1987

The distribution of carnitine and acetylcarnitine in the epididymis and epididymal spermatozoa of the boar.

In the epididymal fluid of boars, the concentration of carnitine (nmol/mg protein) began to increase from 20 in the distal caput, then rose progressively to 700 in the distal cauda. By contrast, the carnitine content of spermatozoa only started to increase in the proximal cauda where the concentration of carnitine in the fluid was 200-300 nmol/mg protein then gradually increased in spermatozoa from more distal sites. The increase in the acetylcarnitine content of spermatozoa paralleled that of the carnitine amount and represented 50% of the total carnitine (carnitine + acetylcarnitine). We conclude that the acetylcarnitine content of epididymal spermatozoa may be used as a marker of maturation.

Acetylcarnitine↗