Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados / Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.

Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol.

The development of the corpus luteum (CL) is accompanied by very active angiogenesis. We hypothesize that during this process endothelial cells (EC) are under the control of several angiogenic factors and steroids. The aim of this study was to examine the expression of the angiogenic growth factor systems - fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) - in EC derived from the bovine CL. Endothelial cells were cultured in serum-free medium and treated for 24 h with different concentrations of oestradiol (range from 10(-13) to 10(-5) mol/l), VEGF or FGF-2 (1, 10 and 100 ng/ml, respectively) and compared with untreated controls. Cells were harvested, total RNA extracted and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Treatment with oestradiol or FGF-2 stimulated the expression of FGF-2, but VEGF treatment showed no effect on the FGF-2 expression. FGF-2 or VEGF treatment resulted in an up-regulation of the FGF receptor (FGFR) mRNA. However, no FGF-1 expression was detected in EC. For the VEGF system, treatment with FGF-2, VEGF or oestradiol did not affect VEGF expression. However, the presence of FGF-2 in the medium up-regulated the expression of both VEGF receptors (VEGFR-1 and VEGFR-2), whereas oestradiol or VEGF treatment showed no effect on the expression of these receptors. Our results reveal that functional angiogenic growth factor systems were expressed in vitro in bovine EC derived from the CL. This suggests that the angiogenic FGF and VEGF system members were regulated by FGF or VEGF, but not by oestradiol-17beta.

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.

Immunocontraception of white-tailed deer with GnRH vaccine.

PROBLEM: Reduction of excess numbers of white-tailed deer (Odocoileus virginianus) is an example of a potential use for immunocontraception as a means of wildlife population management. METHOD OF STUDY: A 4 year multifaceted study was conducted to determine the long term effects of gonadotropin releasing hormone (GnRH) contraceptive vaccine on the fertility and behavior of female and male white-tailed deer. Deer were monitored for breeding behavior, hormone levels, pregnancy, fawning and GnRH specific antibody levels. RESULTS: Treatment lead to reduced fawning rates, altered estrus behavior, reduced concentrations of progesterone, contraception and failure to maintain pregnancy following conception. GnRH immunized does bred to untreated bucks had an 88% reduction in fawning caused by either immunocontraception or immunocontragestion. The vaccine effect is reversible, directly related to the antibody titer. Infertility lasted up to two years without boosting. GnRH immunized bucks demonstrated no interest in sexual activity when paired with control females. Depending on the immunization schedule, antlers either dropped early or remained in velvet. CONCLUSIONS: The results of this study demonstrate that GnRH vaccine is effective in inducing a reversible infertility in white-tailed deer, the infertility lasting up to two years without boosting.

Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis.

Lipocalin-type prostaglandin D synthase in semen has been associated with male fertility, although this relationship is not well defined. To gain insight into potential mechanisms, the objective of the present study was to immunocytochemically localize lipocalin-type prostaglandin D synthase within the testis, efferent ducts, and 4 segments of mouse epididymis. In the testis, immunoperoxidase staining was localized within the Sertoli cells only at stages VI-VIII of the spermatogenic cycle, which is just prior to spermiation. Intense staining was also evident throughout the interstitial tissue, including Leydig cells. The entire epithelium of the efferent ducts, including ciliated and nonciliated cells, was immunoreactive. A distinct pattern of immunostaining for lipocalin-type prostaglandin D synthase was observed in different regions of epididymis, suggesting a possible role in sperm maturation. Staining for lipocalin-type prostaglandin D synthase was strikingly absent in the initial segment. In caput epididymidis, staining was evident throughout the cell cytoplasm of principal cells with some cells more intensely stained than adjacent ones. In the corpus region, overall staining intensity decreased and appeared to be concentrated in the apical region of principal cells, but some cells were completely unreactive. Reaction product in the cauda region was heavily concentrated on microvilli and within the epididymal lumen. In all epididymal regions, expression of lipocalin-type prostaglandin D synthase was specific to epithelial principal cells; no immunoreactivity was apparent in other cell types. The specific localization of lipocalin-type prostaglandin D synthase within the testicular interstitial tissue, Sertoli cells, and principal cells of caput epididymidis strongly suggests that this protein plays an integral role in both the development and maturation of sperm.

Expression of the lipocalin-type prostaglandin D synthase gene in the reproductive tracts of Holstein bulls.

The aim of this study was to localize expression of the prostaglandin D synthase gene in the reproductive tracts of Holstein bulls using northern blotting and in situ hybridization. For northern blotting, a digoxigenin-labelled prostaglandin D synthase cDNA probe was used to probe blots containing RNA isolated from the testes, epididymides, vas deferens, ampullae, seminal vesicles, prostate and bulbourethral glands of bulls. The digoxigenin-labelled cDNA for the bovine homologue of prostaglandin D synthase hybridized to a single band (approximately 0.9 kb) to RNA samples from the caput, corpus and cauda epididymides, as well as RNA samples from the vas deferens and the ampulla. The probe also detected a single band in testis samples, although the transcript size was slightly larger (approximately 1.0 kb) than the transcript found in the other tissues. The highest expression of prostaglandin D synthase was observed in the testes and caput epididymides. Prostaglandin D synthase transcripts were not found in the seminal vesicles or the prostate or bulbourethral glands using northern blotting. For in situ hybridization, antisense and sense riboprobes were synthesized and used to hybridize to cryosections obtained from the reproductive tissues of bulls. In situ hybridization of bull testes showed that prostaglandin D synthase transcripts were present within the germ cells in the adluminal compartment of the seminiferous tubules containing round and elongated spermatids, indicating that expression varied with stage of development of the seminiferous tubules. Prostaglandin D synthase expression was observed in the epithelial cells of the epididymides with greatest expression occurring in the caput epididymidis. Some expression was also observed in the epithelial cells of the vas deferens and a few cells of some lobules in the prostate and bulbourethral glands. Expression of the prostaglandin D synthase gene was not detected in ampullae or seminal vesicles by in situ hybridization.

Immunocontraception of white-tailed deer using native and recombinant zona pellucida vaccines.

We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and three recombinant rabbit zona pellucida vaccines (RC55, RC75a and a combination of RC55, RC75a and RC75b) as an initial phase of developing a recombinant immunocontraceptive vaccine to control reproduction in overpopulated herds of white-tailed deer (Odocoileus virginianus). Forty captive white-tailed does were divided into five groups (one sham and four treated), of eight each and injected with a 500microg prime dose of vaccine. Each prime dose was followed by a 300microg booster dose at 3-7 weeks post prime. The frequency and number of months of observed breeding were higher in PZP immunized does than in sham controls. Although the antibody titers of the three recombinant groups were 1000 or less, as compared with the PZP group with titers often over 128,000, the fawning rates of the two recombinants were significantly lower than that of the control group. The combined antigen group did not have a significantly lower fawning rate.

Long-term effects of PZP immunization on reproduction in white-tailed deer.

A 6-year study was conducted to determine the long-term effects of porcine zona pellucida (PZP) vaccine on the immune and hormonal responses, and reproduction of the white-tailed deer. The first 2 years of active immunization resulted in an 89% reduction in fawning. Vaccination with PZP produced reversible infertility lasting 1-4 years. Infertility was directly related to immune titers to PZP. Doe fertility was restored when the antibody titer dropped to minimal levels, but following re-immunization, infertility was reestablished. Reduction in fawning throughout the 6-year study was 76%. It was also observed that immune responses among deer were variable, especially in the first year of treatment. Variability was also observed among deer for the duration of infertility following the initial vaccination.

Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma.

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.

Effect of bovine ampullary and isthmic oviductal fluid on motility, acrosome reaction and fertility of bull spermatozoa.

Motility, acrosome reaction and oocyte fertilizing ability were assessed for bull spermatozoa after incubation in regional (isthmic or ampullary), bovine oviductal fluid, pooled by stage of the oestrous cycle. Oviductal fluids collected daily from isthmic and ampullary cannulae implanted in the same oviduct were divided into pools, representing two oestrous cycle stages, based on daily serum progesterone concentrations. Ejaculated bull spermatozoa were incubated for 0-6 h in each type of oviductal fluid. Incubation in isthmic oviductal fluid collected during the nonluteal stage, including oestrus and ovulation, decreased overall sperm motility (from 71.7% motile spermatozoa to 34.0%) and both path (78 microns s-1 versus 86-89 microns s-1) and progressive (74 microns s-1 versus 83-85 microns s-1) velocities of spermatozoa motion. Spermatozoa incubated in isthmic, non-luteal oviductal fluid had a higher rate and extent of sperm acrosome reaction (213% of control versus 136-161% of control by 2 h incubation) compared with spermatozoa incubated in other oviductal fluid types. However, incubation in nonluteal ampullary fluid increased the number of spermatozoa, which were both acrosome reacted and live, and able to fertilize bovine ova (88.7% fertilized versus 75-81%). Glycosaminoglycan concentrations were similar among types of oviductal fluid (0.77-0.88 mg ml-1). These findings indicate that oviductal fluid differentially affects sperm function, depending on the oviduct region and the stage of the oestrous cycle at which the fluid was obtained.

Cholesterol, phospholipid and phospholipase activity of ampullary and isthmic fluid from the bovine oviduct.

Cholesterol and phospholipid concentrations and phospholipase activity were measured in fluid from cannulae collected from the bovine oviductal isthmus and ampulla at different stages of the oestrous cycle. The cholesterol concentration and cholesterol normalized by protein were significantly (P = 0.03) greater in isthmic oviductal fluid (224.3 +/- 42.7 micrograms ml-1 over all stages) than in ampullary oviductal fluid (164.5 +/- 11.3 micrograms ml-1), and maximal concentrations (284.5 +/- 25.5 micrograms ml-1) were found during the luteal stage (serum progesterone concentration > or = 1.5 ng ml-1). The concentrations of the phospholipids sphingomyelin and lysophosphatidylcholine increased at different stages of the cycle and in different regions. In the ampulla, the concentration of sphingomyelin was significantly (P < 0.05) greater in oviductal fluid collected during the luteal phase (12.1 +/- 2.7% of total phospholipids) than in fluid collected near oestrus and ovulation (7.5 +/- 1.5% and 6.9 +/- 1%, respectively). The concentration of lysophosphatidylcholine was greater (P < 0.01) in ampullary (19.2 +/- 1.6% of total phospholipids) than in isthmic oviductal fluid (9.9 +/- 1.1%) collected near ovulation. The ratio of cholesterol to total phospholipid was highest in oviductal fluid collected from the isthmus during all stages (2.3 micrograms ml-1:% total phospholipid), while the minimal ratio was found in ampullary fluid collected near ovulation (1.5). Phospholipase activity was higher (P = 0.03) in isthmic oviductal fluid (20.4 +/- 3.2% product formed) than in ampullary oviductal fluid (14.6 +/- 1.4%); the lowest activity (12.6 +/- 1.7% product formed) was in fluid collected during the phase of the oestrous cycle immediately before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cannulation of the bovine ampullary and isthmic oviduct.

Procedures were developed in the bovine for cannulating the ampulla and isthmus regions of the same oviduct. A total of 21 dual cannulation procedures were performed with an average patency of 103 +/- 16 days for the ampulla and 73 +/- 14 days for the isthmus. The major cause of catheter failure was avulsion. Daily fluid flow around estrus for the ampulla averaged 1.04 mL, but only 0.50 mL for the isthmus. Volumes were about half the estrus values during the luteal phase. Catheter materials used in the study were evaluated to determine their ion diffusion properties. These studies indicated that diffusion of 24Na was negligible.

Libido, hormone concentrations in blood plasma and semen characteristics in Holstein bulls.

Relationships among bull libido, serum hormone concentrations and semen characteristics were studied using 18 Holstein bulls that were 4 to 5 yr old. The hormones studied included testosterone, estradiol (E2), prolactin (PRL), LH and cortisol. Two ejaculates were collected three times per week from each bull during a 5-wk trial. During the last week of the trial, on a day semen was not collected, blood was collected from indwelling catheters every 15 min for 6 h to determine the hormonal profiles of each bull. On the following day, blood was sampled every 10 min before and after the time of semen collection. Libido factors were quantified, and semen volumes and sperm concentrations were recorded. The libido factors included reaction time to first service, latency time between the first and second semen collections, and duration of time the bull mounted the teaser prior to the first (TM1) and second (TM2) semen collection. Average reaction and latency times were correlated (r = .524; P = .026), as were TM1 and TM2 (r = .597; P = .015). Latency times were correlated with average TM2 (r = .669; P = .003). Average PRL concentrations were correlated with average latency times (r = .467; P = .05). Low libido bulls tended to have higher E2:testosterone ratios than did high libido bulls. Both PRL and cortisol concentrations increased at semen collection.

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.

Bovine oviductal cannulations.

Oviductal fluid was collected from 23 dairy cows following cannulation of the oviduct in 38 procedures utilizing silastic catheters. Standing laparotomy in the caudal abdomen enabled exteriorization of the ipsilateral ovary, oviduct, and uterine horn tip. A simple plastic device inserted through the ostium facilitated placement of an indwelling catheter. The exteriorized catheter connected to a collection device attached to the flank that provided ready access for harvesting fluids. Typical fluid volumes collected ranged from 0.1 to 3 mL per day depending on the estrus cycle stage. Patency varied from 2 to 156 days with an average duration of 68.2 days. Fluid flow ceased most commonly when a catheter was avulsed from the surgical site (58%) or became occluded with fibrin (14%). Slight modification of the stanchion housing has increased the longevity of the preparations.

Effects of long- and short-term vasectomy on structural and functional parameters of the rat.

The effects of vasectomy were examined by comparing various parameters from sham operated and vasectomized rats that had undergone surgery at 90 days of age and were killed at 190 or 390 days of age. Significant alterations in the vasectomized rats from sham rats included: testicular and epididymal hypertrophy, formation of pathologic vas deferens granulomas, decreased total serum protein, lowered alpha-globulin levels as shown by serum electrophoresis, and increased sperm agglutinin antibody titers. For vasectomized rats, the differential white blood cell count showed increased numbers of neutrophils and large lymphocytes and decreased numbers of small lymphocytes and basophils. Both the number and extent of many vasectomy-induced alterations were greater in long-term vasectomized than in short-term vasectomized rats.

Isolation of epithelial cells from the corpus epididymidis and analysis for glycerylphosphorylcholine, sialic acid, and protein.

Enzymatically dispersed cells from the rat corpus epididymidis were separated by unit gravity sedimentation and centrifugal elutriation. Based on differential cell counts, principal cells isolated by centrifugal elutriation were 55% pure, while basal cells and fibroblasts were 88% and 61% pure, respectively. Compared to unit gravity separation, purity was an average of 7% higher in principal and basal cell fractions obtained by elutriation. Viability was greater than 87% by either method, but yields following elutriation averaged 23% higher for basal cells, 78% higher for principal cells, and 290% for fibroblasts than corresponding yields following unit gravity separation. Analyses for epididymal secretory products indicated that cellular content (mumoles/10(9) cells) of glycerylphosphorylcholine (GPC), sialic acid, and protein was greater in principal cells than other cell types (p less than .05). When expressed on the basis of cellular volume, concentrations of GPC and protein also were significantly greater in basal and principal cells than fibroblasts (p less than .05). Caput sperm contained significantly more GPC than either corpus or cauda sperm (p less than .005). Protein concentrations in caput and corpus sperm were similar, but concentrations were lower in cauda sperm (p less than .005). Levels of sialic acid did not vary among sperm from different epididymal regions. It was concluded that principal cells from the corpus epididymidis contain major quantities of three epididymal secretory products and that regional differences in the function of the epithelium are present.

Isolation of principal and basal cells from the epithelium of the hamster caput epididymidis by unit gravity sedimentation.

PIP: A technique for the isolation of principal and basal cells from the epithelium of the hamster caput epididymides by unit gravity sedimentati on is described. The technique enzymatically disaggregates cells comprising the caput epididymides, and the resulting mixture of disperse d cells is separated by sedimentation in a shallow bovine serum albumin gradient at unit gravity into populations of spermatozoa, erythrocytes and several nucleated types. The separated somatic cell types and the homogeneity of each population were identified by light and electron microscopy. The purest fractions of the 6 populations, from smallest to largest, contained an average of 84% erythrocytes, 76% basal cells, 82% fibroblasts and intraepithelial lymphocytes, 68% small principal cells and 34% smooth muscle cells or 58% large principal cells. Cell viability following sedimentation was excellent, as concluded from elect ron micrographs revealing adenosine triphosphate content and fine struct ure. This technique should enable critical analyses of epididymal function in isolated epithelial cells.^ieng