Improvement of 2D-PAGE resolution of human, porcine and equine follicular fluid by means of hexapeptide ligand library.

The major challenge of follicular fluid proteomic analysis is the presence of high-abundance proteins that originate from plasma. These proteins can prevent the detection of lower abundant ones, produced locally by follicle cells and that may have important roles in follicular activity. In this study, the novel technology called hexapeptide ligand library was evaluated to enrich the low-abundance proteins in follicular fluid of human (HFF), porcine (PFF) and equine (EFF) prior 2D-PAGE. Our results showed that the new strategy enabled detection of many new protein spots, increased resolution and highly improved the intensity of low-abundance proteins by 2D-PAGE.

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.

Human epididymal secretome and proteome.

The proteins that are neosynthesized and secreted in the different regions of the human epididymis were determined by in vitro biosynthesis of epididymal tubules, and the luminal proteins were collected by microperfusion of each tubule. The preparations were analyzed by two-dimensional gel electrophoresis and the proteins were identified by mass spectrometry. Some of the major proteins identified corresponded to serum compounds such as albumin, transferrin and alpha-1-antitrypsin. The other proteins identified included lactotransferrin, clusterin, PEBP, NCP2/CTP/HE1, HE3, Crisp, actin, calmodulin, E12, PGDS, l-lactate dehydrogenase, malate dehydrogenase, carbonic anhydrase, triose phosphate isomerase, glutamyltransferase, glutathione S-transferase P, thioredoxin peroxidase, superoxide dismutase, cathepsin D and cystatin. Epididymal activity is highly regionalized in most species. However, in this study in humans, there were only minor changes in the major proteins secreted. It is suggested that this specificity might be related to the difference between species in the location of the epididymis where sperm become fertile.

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.

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0). Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH. These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid.

The epididymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymis. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA library of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the cDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit cDNA, similarly to the results previously obtained for cDNAs encoding the epididymis-specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20 microM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosomal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantly retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol.

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures [corrected].

Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C.

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

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.

Studies of the androgen binding protein in the rete testis fluid of the ram and its relation to sexual season.

An androgen binding protein (ABP) with an electrophoretic mobility (Rf) of 0.56 is present in the rete testis fluid of adult rams. Its steroid specificity was found to be in the following order: 5alpha-DHT, testosterone, oestradiol-17 beta, dehydroepiandrosterone 5beta-DHT, androstenedione, cyproterone, cyproterone acetate, cortisol and progesterone. The characteristics of the ABP are similar to those found for the ABP of the testis and the epididymis of the rat and the rabbit. The concentration of ABP, determined by the dextran-coated charcoal method and sometimes confirmed by the steady-state polyacrylamide gel electrophoresis method, was significantly higher in the breeding season than in the non-breeding season (4.40 +/- 0.98 X 10(-9) M vs. 2.60 +/- 0.62 X 10(-9) M; P less than 0.037). The affinity constant of the ABP was independent of the season (2.45 +/- 0.21 X 10(9) M-1 vs. 2.66 +/- 0.1 X 10(9) M-1; NS). In addition, ABP was positively correlated with 5alpha-DHT (r = 0.506; P less than 0.0009), testosterone (r = 0.445; P less than 0.0003), total protein (r = 0.329; P less than 0.02) and spermatozoa (r = 0.406; P less than 0.006) in the RTF and with blood plasma testosterone (r = 0.584; P less than 0.0001). Furthermore, testosterone and 5alpha-DHT in RTF were positively correlated (r = 0.582; P less than 0.0001). These androgens were also correlated with plasma testosterone (r = 0.262, P less than 0.052 for testosterone in RTF; r = 0.341, P less than 0.018 for 5 alpha-DHT). Total proteins and spermatozoa were found to be positively correlated in the RTF (r = 0.789; P less than 0.0001).

Post-testicular sperm environment and fertility.

When mammalian spermatozoa exit the testis, they show a highly specialized morphology; however, they are not yet able to carry out their task: to fertilize an oocyte. This property, that includes the acquisition of motility and the ability to recognize and to fuse with the oocyte investments, is gained only after a transit through the epididymis during which the spermatozoa from the testis travel to the vas deferens. The exact molecular mechanisms that turn these cells into fertile gametes still remain mysterious, but surface-modifying events occurring in response to the external media are key steps in this process. Our laboratory has established cartographies of secreted (secretomes) and present proteins (proteomes) in the epididymal fluid of different mammals and have shown the regionalized variations in these fluid proteins along the epididymis. We have found that the main secreted proteins are common in different species and that enzymatic activities, capable of controlling the sperm surface changes, are present in the fluid. Our studies also indicate that the epididymal fluid is more complex than previously thought; it contains both soluble and particulate compartments such as exosome-like vesicles (epididymosomes) and certainly specific glycolipid-protein micelles. Understanding how these different compartments interplay to modify sperm components during their transit will be a necessary step if one wants to control and to ameliorate sperm quality and to obtain valuable fertility markers helpful to establish a male fertility based genetic selection.

The role of an investigator in healthcare protection.

The author discusses three types of investigations the healthcare security officer may be concerned with--employee theft, homicide and violent crimes, and fraud. He examines the role of the investigator as puzzle solver and the steps that need to be taken to put all the pieces of the puzzle in place.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE: Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.

[In vivo imaging of ram spermatozoa in the ewe genital tract using fibered confocal microscopy]. / Visualisation in vivo des spermatozoïdes de bélier dans le tractus génital de brebis par microscopie confocale fibrée.

Sperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit.