Trinucleate uterine epithelial cells as evidence for White-tail Deer trophoblast binucleate cell migration and as markers of placental binucleate cell dynamics in a variety of wild ruminants.

INTRODUCTION: The unicellular trophoblast epithelium of all ruminants so far investigated contains 15-20% binucleate cells with numerous secretory granules. Electron microscope (EM) studies of the domesticated cow, ewe, goat and deer species have established that these BNC migrate out of the trophoblast epithelium to fuse with the apposed maternal uterine epithelial cells or derivative to form fetomaternal tissue throughout pregnancy. However there is one careful EM study of the trophoblast of a wild ruminant, the White-tail deer, which found the usual number of BNC but no evidence of any migration or fusion. Since there are up to 200 species of wild ruminants, it was important to establish whether there really are two possible scenarios for BNC function. MATERIALS AND METHODS: This paper reports a light microscope (LM) immunocytochemical study of cell dynamics in ruminant placentas using 1-2 mµ deresinated sections. RESULTS: The results clearly demonstrate that the White-tail deer and all of the other 15 (see Table 1) randomly selected wild ruminants show the same BNC migration and fusion pattern. DISCUSSION: These results suggest that this remarkable cellular behaviour is fundamental to the ruminant evolutionary success.

Analysis and characterisation of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionisation mass spectrometry imaging.

In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.

Proteomics of cauda epididymal fluid from mature Holstein bulls.

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-ß-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-ß-glycoprotein, apolipoprotein A-1, ß actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.

The single-shot GnRH immunocontraceptive vaccine (GonaCon) in white-tailed deer: comparison of several GnRH preparations.

PROBLEM: An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations. METHOD OF STUDY: Two GnRH conjugates, GonaCon (GnRH-KLH) and GonaCon-B (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments. RESULTS: The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine. CONCLUSION: One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer. GonaCon-B provided a longer lasting contraceptive effect.

Binding patterns of bovine seminal plasma proteins A1/A2, 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid.

Previous studies from our laboratory have reported empirical associations between bovine seminal plasma protein(s) (BSP) A1/A2 and 30 kDa and osteopontin (OPN) in accessory sex gland fluid and bull fertility. These BSP and OPN are believed to bind to sperm at ejaculation and to remain bound until sperm reach the oviduct. The objective of the present study was to evaluate the topographical distribution of BSP A1/A2, 30 kDa and OPN binding on: (1) bovine ejaculated sperm; (2) ejaculated sperm incubated with isthmic oviductal fluid (ODF); (3) ejaculated sperm+isthmic ODF incubated in ampullary ODF. From each of these media, aliquots of sperm for BSP and OPN were processed for immunocytochemistry and analysis by laser scanning confocal microscopy. Isthmic and ampullary ODF was collected from indwelling catheters and used as pools from three cows in the non-luteal phase of the estrous cycle. Anti-BSP A1/A2 was detected bound to the midpiece, post-equatorial and equatorial segments and acrosome of sperm after ejaculation and after incubation with isthmic and ampullary ODF. The BSP A1/A2 fluorescence was more concentrated on the midpiece and increased as acrosome-intact sperm came in contact with ODF. As compared with acrosome-intact sperm, non-intact acrosome intact sperm had 39 and 68% reductions of acrosome fluorescence and 36% and 90% increases of post-equatorial fluorescence after contact with isthmic and ampullary ODF (P<0.05). Anti-BSP 30 kDa was more intense on the midpiece than on post-equatorial, equatorial and acrosome regions of sperm after ejaculation and contact with ODF. However, equatorial fluorescence was 141% and 89% more intense and acrosome stainning was 80% and 76% less (P<0.05) in non-intact acrosome sperm than in acrosome intact cells, during all ODF incubations. Anti-OPN was identified on the acrosome of ejaculated sperm, but with less fluorescence (P<0.05) on the post-equatorial segment and midpiece. Incubation of sperm with isthmic ODF increased fluorescence on post-equatorial segment (P<0.05). There were 72% and 78% reductions (P<0.05) of acrosome fluorescence and intensification (P<0.05) in equatorial fluorescence in non-intact acrosome sperm as compared with acrosome intact cells incubated with isthmic and ampullary ODF. In summary, interactions of BSP A1/A2 and 30 kDa and osteopontin with the sperm membrane undergo modifications dictated by the oviductal fluid. The BSP are thought to modulate cholesterol and phospholipid movement from the sperm membrane and help sperm binding to the oviductal epithelium. Furthermore, our model suggests that OPN participates in sperm-oocyte interaction, affecting fertilization and early embryonic development.

Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization.

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane.

Proteins of the accessory sex glands associated with the oocyte-penetrating capacity of cauda epididymal sperm from holstein bulls of documented fertility.

We previously reported that accessory sex gland fluid (AGF) from high fertility (HF) bulls influenced the oocyte-penetrating capacity of cauda epididymal sperm from low fertility (LF) bulls, based on in vitro fertilization (IVF) assays. The present study determined if AGF proteins were associated with these effects. Nineteen IVF assays with 12 bulls were grouped as follows. Group I (n = 8): assays where sperm from LF bulls exposed to AGF from HF bulls had greater oocyte penetration than exposed to homologous AGF. Group II (n = 7): sperm from LF bulls to AGF from HF bulls versus homologous AGF showed no significant differences. Group III (n = 4): sperm from LF bulls treated with homologous AGF had greater fertility than sperm treated with AGF from HF bulls. Sire fertility was based on nonreturn rates (NNR) and AGF collected by artificial vagina from bulls with cannulated vasa deferentia. Two-dimensional SDS-PAGE maps of AGF were analyzed by PDQuest and proteins identified by tandem mass spectrometry and Western blots. Differences in spot intensity between AGF of HF and LF bulls were compared across groups of IVF assays (P < 0.05). The expression of BSP A1/A2 and A3, BSP 30 kDa, clusterin, albumin, phospholipase A(2) (PLA(2)), and osteopontin was greater in the AGF of HF bulls in Group I as compared to Groups II and III. Conversely, there was less nucleobindin in the AGF of HF bulls in Group I than in Groups II and III. This is the first report of nucleobindin (58 kDa/pI 5.6) in male reproductive fluids, using both immunoblots and mass spectrometry. Thus, the effect of AGF from HF bulls on epididymal sperm is likely the result of specific proteins expressed in the AGF.

A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls.

The expression of proteins in accessory sex gland fluid (AGF) of proven, high use mature Holstein bulls was evaluated. Thirty-seven bulls with documented fertility based on their non-return rates were studied. AGF was obtained by artificial vagina after bulls were surgically equipped with cannulae in the vasa deferentia. Samples of AGF were evaluated by two-dimensional SDS-PAGE, gels stained with Coomassie blue and polypeptide maps analyzed by PDQuest software. A master gel generated by the software representing the best pattern of spots in the AGF polypeptide maps was used as a reference for protein identification. Proteins were identified by Western blots and capillary liquid chromatography-nanoelectrospray ionization tandem-mass spectrometry (CapLC-MS/MS). The product ion spectra were processed using Protein Lynx Global Server 2.1 prior to database search with both PLGS and MASCOT (Matrix Science) software. The entire NCBI database was considered for mass fingerprint matching. An average of 52+/-5 spots was detected in the AGF 2D gels, which corresponded to proteins potentially involved in capacitation (bovine seminal plasma protein-BSP-A1/A2 and A3, BSP 30 kDa, albumin); sperm membrane protection, prevention of oxidative stress, complement-mediated sperm destruction and anti-microbial activity (albumin, clusterin, acidic seminal fluid protein--aSFP, 5'-nucleotidase--5'-NT, phospholipase A2--PLA2); acrosome reaction and sperm-oocyte interaction (PLA2, osteopontin); interaction with the extracellular matrix (tissue inhibitor of metalloproteinase 2, clusterin) and sperm motility (aSFP, spermadhesin Z13, 5'-NT). The 20 spots distinguished in all gels were matched to proteins associated with these functions. Proteins identified by tandem mass spectrometry as ecto-ADP-ribosyltransferase 5 and nucleobindin, never described before in the accessory sex gland secretions, were also detected. In summary, we identified a diverse range of components in the accessory sex gland fluid of a select group of Holstein bulls with documented fertility. Known characteristics of these proteins suggest that they play important roles in sperm physiology after ejaculation.

Modifications of surface carbohydrates on bovine spermatozoa mediated by oviductal fluid: a flow cytometric study using lectins.

The objective of the present study was to characterize and quantify changes in exposed saccharide residues of bovine sperm during capacitation in oviductal fluid (ODF) using flow cytometry (FC). Bovine sperm were incubated with 0% or 50% non-luteal ODF for 30 min or 3.5 h. After incubation, sperm were labelled with 11 fluorescein isothiocyanate-labelled lectins and evaluated for lectin binding with FC. Furthermore, inhibiting sugars were used to determine specificity of lectin binding to oligosaccharides on the sperm surface. After 30 min incubation, there was a 91% decrease in fluorescence intensity of labelled sperm incubated in WGA, a 76% decline for Con A, 75% decline for BS-I and a 36% decline for DBA. These differences remained approximately the same over the 3.5-h incubation. Interestingly, although there was no reduction in UEA-I binding at 30 min, a significant reduction (23%) was observed at 3.5 h. Con A fluorescence was mostly inhibited with either alpha-d-glucose or alpha-d-mannose (86% and 90% respectively). BS-I fluorescence was reduced after prior incubation of the control samples with N-acetyl-galactosamine and galactose by 74% and 80% respectively. After prior incubation with N-acetyl-galactosamine DBA fluorescence reduced by 18% in the control samples. With UEA-I no fluorescence reduction was observed after prior incubation with l-fucose. We have demonstrated that capacitation of bovine sperm in ODF is accompanied by a quantitative reduction in individual lectin binding sites. These modifications may be crucial to the subsequent signalling events involved with sperm-zona binding, zona penetration or interaction with the oolema.

Proteins of the cauda epididymal fluid associated with fertility of mature dairy bulls.

We evaluated the relationships between proteins in cauda epididymis fluid (CEF) and fertility scores of dairy bulls. Fertility was expressed as the percentage point deviation (PD) of bull nonreturn rate from the average fertility of all bulls at an artificial insemination center. The number of services for each bull ranged from 1074 to 52 820, and PD values ranged from +7.7% to -6.6%. CEF from 20 bulls was obtained from vasa deferentia cannulae and was separated from sperm by centrifugation immediately after collection. Samples were evaluated by 2-dimensional (2-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis gels stained with Coomassie blue, and polypeptide maps were analyzed by PDQuest software. Protein quantities, defined as the total integrated optical density of the spots, were compared between groups of high-fertility sires (n = 12; PD >or= 0) and low-fertility sires (n = 8; PD < 0) and were also used as independent variables in regression analysis. Proteins were identified by capillary liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry. An average of 118 spots was detected in 2-D maps of the CEF, but we were unable to distinguish any protein that was expressed only in high-fertility or in low-fertility bulls. However, the amount of alpha-L-fucosidase 2 and cathepsin D was 2.3- and 2.4-fold greater (P < .05) in high-fertility than in low-fertility bulls, respectively. Conversely, the intensities of 3 isoforms (24-27 kd; pl 6.3-5.8) of prostaglandin D-synthase (PGDS) were from 3.2- to 2.2-fold greater in low-fertility sires (P < .05). An empirical regression model established that a significant proportion (R(2) = 0.72; P < .0001) of the variation in fertility scores (PD values) was explained by the intensities of cathepsin D and 1 isoform of PGDS (24 kd; pl 6.3). Thus, multiple proteins present in the CEF are potential biomarkers of fertility in high-use, mature Holstein bulls.

Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: a proteomic approach.

We evaluated the expression of proteins in the accessory sex gland fluid (AGF) and their relationships with fertility indexes of dairy bulls. Fertility was normalized as the percentage point deviation of their nonreturn rates (PD) from the average fertility of all bulls from a given artificial insemination center. Services associated with each sire ranged from 269 to 77 321 and PD values from +7.7% to -18.1%. AGF, from 37 bulls, was obtained with an artificial vagina after cannulation of the vasa deferentia. Proteins from AGF were separated by 2-dimensional SDS-PAGE followed by staining with Coomassie blue and analysis of polypeptide maps using PDQuest software. Bulls were divided in groups based on PD values and the optical density of spots in the AGF gels used as independent variables to predict bull fertility. Proteins were identified by capillary liquid chromatography nanoelectrospray ionization tandem mass spectrometry (CapLC-MS/MS). An average of 52 +/- 5 spots was detected in the AGF gels, but there were no spots unique to groups of either high- (PD > or = 0) or low- (PD < 0) fertility sires. The former were neither less nor more homogeneous than the latter based on correlations of all matched spots between pairs of AGF maps. However, high fertility of dairy bulls was significantly associated with lower expression of 14-kDa spermadhesin Z13 isoforms and higher amounts of 55-kDa osteopontin and 58-kDa phospholipase A2 (PLA2) isoforms. The average intensity of 5 spots identified as BSP 30 kDa in the AGF gels had a quadratic association with fertility indexes (R2 = .18; P = .03). PD values of bulls were related (R2 = .56) to the quantity of spermadhesin, osteopontin, and BSP 30 kDa in the AGF polypeptide maps. Bull fertility was also determined by another equation (R2 = .53) with spermadhesin, BSP 30 kDa, and PLA2 as independent variables. We conclude that interactions among several proteins in accessory sex gland fluid explain a significant proportion of the variation in fertility scores of mature dairy sires.

Effect of plasmin on movement characteristics of ejaculated bull spermatozoa.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Several observations suggest that the plasminogen activator/plasmin system might also play a role in mammalian fertilization. Movement characteristics of bovine sperm incubated with different concentrations of plasmin were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4h in a modified Tyrode's medium (control) and 0.1, 1, 10 and 100 mU/ml of plasmin. The percentage motile sperm was significantly higher at 0 h for sperm incubated in 1, 10, and 100 mU of plasmin. Relative to sperm incubated in control medium, lateral head displacement (ALH), curvilinear velocity, beat cross frequency, path velocity and straight line velocity (VSL) of sperm treated with 100 mU of plasmin for 0 h were increased. After 2h of incubation, sperm treated with 100 mU of plasmin showed an increase in ALH, but a decrease in VSL, straightness and linearity. The effect of plasmin on most motility parameters appears to be direct since all these parameters were affected at 0 h of incubation. Our results support the notion of hyperactivation of bovine spermatozoa following incubation with different concentrations of plasmin. The present work provides additional information to further characterize motility movement of bovine sperm associated with final preparation for fertilization.

In search of the active PZP epitope in white-tailed deer immunocontraception.

Native porcine zona pellucida (PZP) has been shown to be highly effective as an immunocontraceptive in white-tailed deer. However, the immunogenicity of PZP extracted from pig ovaries may vary from lot to lot and the extract has the potential of containing either viral or pathogenic material. Determination of the immunocontraceptive epitopes of PZP would allow portions of the molecule to be synthesized or inserted into a recombinant system for production of a consistent and safe vaccine. In this study, epitopes of PZP were selected and tested by in vitro binding, immunogenicity in rabbits, immunogenicity and immunocontraception in deer. Sera from PZP immunocontracepted deer were tested on ELISA plates containing immobilized peptides from ZP1 and ZP3alpha. Peptides with which sera from infertile deer reacted (six peptides from ZP1 and six peptides from ZP3alpha) were selected, synthesized and tested for immunogenicity in rabbits. Deer were then immunized with combinations of peptides from either the ZP1 or ZP3alpha groups. ZP3alpha peptides induced high immune titers against native PZP, but did not induce infertility in the deer. Although ZP1 peptides induced lower titers, deer immunized with two ZP1 peptides exhibited multiple estrus events and infertility, typical of that for deer immunized with native PZP vaccine. Competitive inhibition assays using the ZP1 peptides demonstrated that the peptide comprising pins 10-16 was most effective in blocking binding by the serum antibody of native PZP immunized deer. This peptide was used to immunocontracept deer, resulting in a significant reduction in fawning for 1 year.

Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine.

Identification of norepinephrine (NE) within the microenvironment of the bovine oviduct suggests a potential role for catecholamines in the events surrounding fertilization. Previous studies have shown that the catecholamines capacitate and induce the acrosome reaction in spermatozoa from several species. The current project was undertaken to investigate the role of catecholamines in bovine sperm capacitation and the acrosome reaction. Freshly ejaculated bovine spermatozoa were incubated in NE (0-1000 ng/mL) and induced to acrosome-react with lysophosphatidylcholine (LPC). Additionally, spermatozoa capacitated with heparin were incubated with NE (0-1000 ng/mL) to assess its ability to induce the acrosome-reaction in capacitated spermatozoa. Concentrations of NE were chosen on the basis of physiological concentrations previously determined for bovine oviductal fluid. NE at concentrations of 10 and 20 ng/mL capacitated bovine spermatozoa after 2 hours of incubation. Additionally, spermatozoa incubated for 2 hours with heparin were induced to acrosome-react with 10 and 20 ng/mL NE. Interestingly, higher concentrations of NE inhibited both capacitation and the acrosome reaction. Incubating spermatozoa with dopamine or epinephrine did not result in capacitation or the acrosome reaction, suggesting that the action of NE was specific to that catecholamine. The ability of NE to capacitate or induce the acrosome reaction appears to be dependent on the presence of another membrane-destabilizing factor. Although adrenergic receptors have not been identified on spermatozoa from any species, the action of NE on spermatozoa may be a receptor-mediated event. This study suggests a possible function for oviductal catecholamines in sperm preparation prior to fertilization.

Immunohistochemical localization of microsomal PGE synthase-1 and cyclooxygenases in male mouse reproductive organs.

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in the male mouse reproductive organs. Northern blotting revealed that the mPGES-1 mRNA was expressed intensely in the epididymis and weakly in the lung, spleen, skin, kidney, colon, and brain. In the male reproductive tract, the expression of mPGES-1 increased from the testis to the cauda epididymis and was highest in the vas deferens when examined by Northern blotting, RT-PCR, and Western blotting. By immunohistochemistry, mPGES-1 was detected in Leydig cells of the testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In addition, the caput and cauda regions of the epididymis and the vas deferens in this order showed a progressive increase in the expression of COX-1 mRNA and immunoreactivity, whereas COX-2 was dominantly expressed in the vas deferens. COX-1 was localized in epithelial cells of the caput, corpus and cauda epididymis and of the vas deferens, and COX-2 was evident in epithelial cells of the distal cauda epididymis and vas deferens. These results show that mPGES-1 is expressed coordinately with COX-1 and COX-2 and is involved in PGE(2) production in male genital organs.