Stereotactic radiosurgery to surgical cavity post resection of brain metastases: Local recurrence and overall survival rates. A single-centre experience.

INTRODUCTION: We reviewed local control (LC) and overall survival (OS) post intracranial SRS to cavity post resection of brain metastases at one institution, and factors affecting LC. METHODS: A retrospective review was conducted of adjuvant SRS at one institution from 2013 to 2016. Patient records, treatment plans and diagnostic images were reviewed. Local failure was MRI defined. Categorical variables were analysed using chi-square and Fisher's exact tests. Continuous variables were analysed using Mann-Whitney tests. The Kaplan-Meier method was used to estimate survival times and the log-rank test was used to compare differences in survival. RESULTS: Forty-seven patients with 48 cavities were treated with SRS post operatively. LC rate was 69%, and the distant intracranial failure rate was 47% for entirety of the follow-up period. The 12-month freedom from local recurrence (FFLR) was 77% (63-91%). Median OS (95% CI) was 22.7 (14.6-30.8) months. Patients with a single metastasis had longer FFLR (30.1 vs 14.4 months; P = 0.014). Median interval from surgery to SRS was 6.3 weeks. Patients with interval >7 weeks had increased local recurrence (LR) (62%) than <7 weeks (37%), P = 0.025. Patients with a margin < 2 mm were more likely to experience LR (48%) than those with margin equal to 2 mm (20%); this approached statistical significance (P = 0.063). The median follow-up for all patients was 15.4 months (2-41). CONCLUSIONS: We determined LC and OS post adjuvant SRS at our institution. Based on the findings of this retrospective review SRS should be given promptly post operatively with a 2 mm PTV margin.

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.

Physiology and endocrinology symposium: evidence that oviduct secretions influence sperm function: a retrospective view for livestock.

The mammalian oviduct has long been recognized as an organ essential for successful reproduction. Bovine, ovine, porcine, and equine animal models have offered clear advantages for oviduct study related to gamete physiology, fertilization, and early embryonic development. Livestock species are amenable to surgical alteration of the reproductive tract, estrous cycle manipulation, gamete cryopreservation, and AI, as well as in vitro fertilization and embryo production. Although most reproductive technology developed for livestock was intended to benefit production animal agriculture, these techniques are a treasure trove of tools for researchers to better understand how the oviduct influences gamete function. Oviduct secretions obtained from in vitro tissue cultures or via indwelling oviduct catheters have been used for analyses to define the protein, lipid, carbohydrate, enzyme, and electrolyte compositions of the secretions during the estrous cycle or in response to hormone treatment. Oviduct secretions or components purified from them have also been used in in vitro assays to assess their ability to bind to sperm, influence sperm viability, motility, sperm capacitation, the acrosome reaction, sperm-egg binding, and egg penetration, as well as subsequent embryonic development. Compelling data have emerged which show that the composition of secretions differs during the estrous cycle and that their composition differs whether they originate from the ampullary or isthmic regions of the oviduct. These differences in composition are functionally relevant and associated with different responses by sperm. Evidence indicatess that oviduct-specific glycoproteins, glycosaminoglycans, carbohydrates, norepinepherine, catecholamines, heat-shock protein, and osteopontin are components of the oviductal milieu that have the capacity to modulate sperm function. Future research on the livestock oviduct will likely define the role that oviduct secretions have in modulating sperm function and how these modifications ultimately affect fertilization and embryo development.

Ion, protein, phospholipid and energy substrate content of oviduct fluid during the oestrous cycle of buffalo (Bubalus bubalis).

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (µm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (µmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.

Influence of osteopontin in bovine uterine tube fluid on sperm binding and fertilization in RCA-1 lectin-treated oocytes.

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 microl of fertilization medium (FM); (ii) 250 microl of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 microl of FM with 250 microl of NLAUTF and 4 microl of RCA-1 lectin; (iv) 250 microl of FM with 250 microl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 microl of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 microg heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Effect of osteopontin (OPN) on in vitro embryo development in cattle.

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 microg/mL OPN. There were greater percentages (P<0.01) of cleavage and compact morulae-blastocysts (79.7 and 43.3%, respectively) with 10 microg/mL OPN than in the control group (without OPN; 71.2 and 32.1%, respectively). Furthermore, percentages of advanced blastocysts were greater in the group receiving 40 microg/mL OPN versus control (56.4% vs. 42.0%, P<0.05). Capacitation was assessed by the ability of sperm to undergo the acrosome reaction after incubation with lysophosphatidylcholine. Semen from three bulls was incubated for 2h in either TALP medium alone (control) or with TALP medium containing 0.01 mM heparin, or with TALP medium containing 10 or 20 microg/mL OPN. Incubation with 10 and 20 microg/mL OPN produced more (P<0.01) capacitated sperm (14.4 and 13.6%, respectively) than the untreated control group (8.3%), but both untreated sperm and those treated with OPN had significantly fewer capacitated sperm than those treated with 0.01 mM of heparin (30.5%). In conclusion, OPN improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.

Oviductal fluid proteins associated with the bovine zona pellucida and the effect on in vitro sperm-egg binding, fertilization and embryo development.

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding.

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development.

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 microg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 microg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 microg/mL OPN (bull 2=85+/-4% and bull 5=78+/-4%) than without OPN (bull 2=75+/-4% and bull 5=69+/-4%). Those bulls also had increase in cleavage and embryo development (bull 2=85+/-3%, 41+/-1.9%; bull 5=76+/-2%, 37+/-1.8%) compared with control (bull 2=75+/-3%, 30+/-2%; bull 5=68+/-2%, 29+/-2%). Incubating matured oocytes in 10 microg/mL OPN (87+/-3%) and 100 microg/mL OPN (88+/-3%) significantly increased fertilization than control (73+/-3%). OPN also improve cleavage, and embryo development in treatments with 10 microg/mL OPN (82.7+/-1.3%; 31.7+/-1.4%) and 100 microg/mL OPN (85.8+/-1.3%; 33.8+/-1.5%) when compared with control (74.1+/-1.3%; 24.2+/-1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.

Influence of arginine-glycine-aspartic acid (RGD), integrins (alphaV and alpha5) and osteopontin on bovine sperm-egg binding, and fertilization in vitro.

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<0.05) fertilization compared to the in vitro-fertilized control. These data provided evidence that integrin ligands existed on bovine oocytes and spermatozoa that contained RGD recognition sequences, and that antibody to OPN, a protein that contains that RGD sequence, was capable of reducing sperm-egg binding and fertilization in vitro.

Immunocontraception of Florida feral swine with a single-dose GnRH vaccine.

PROBLEM: Methods to limit fertility of feral swine are needed to reduce transmission of diseases and agricultural and ecosystem damage. Method of Study We evaluated a single-shot GnRH immunocontraceptive vaccine in both male and female feral swine for its effect on fertility and functional status of the reproductive tissues. Captive feral pigs were randomly assigned to receive 1000 or 2000 microg GnRH-KLH vaccine treatments or no treatment. RESULTS: After 36 weeks, none of the 2000-microg-treated females and only 20% of the 1000-microg-treated females were pregnant. This corresponded to reduced serum progesterone, regressed tissues within the reproductive tract and lack of evidence for follicular development leading to ovulation. Males were less responsive to the vaccine than females, but more responsive to the lower dose of the vaccine than the higher dose. CONCLUSIONS: The single-shot GnRH vaccine is effective in controlling fertility of female feral swine and may be useful for population reduction.

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine.

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.

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.

Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs.

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES-1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles.

Expression of haptoglobin mRNA in the liver and oviduct during the oestrous cycle of cows (Bos taurus).

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events of mammalian reproduction. The objective of this study was to determine whether haptoglobin mRNA was expressed in the bovine ovary and oviduct, and to evaluate whether expression of haptoglobin mRNA in reproductive tissues and liver was associated with a specific phase of the bovine oestrous cycle. Oestrus was synchronized in Holstein cows by prostaglandin injection and tissues were collected during the luteal and peri-oestrous stages of the oestrous cycle. Total RNA was isolated and reverse-transcription polymerase chain reaction (RT-PCR) was performed using primers designed against regions of similarity in human, rat and mouse haptoglobin sequences. Haptoglobin mRNA expression was detected in oviductal cells and liver, during both stages of the oestrous cycle, but not in ovarian follicular cells. The 302 bp PCR product was determined to share 82-83% identity with reported primate haptoglobin sequences. Analysis by Northern blotting revealed 1.2 and 1.4 kb haptoglobin mRNA transcripts in the oviduct and liver, and indicated that hepatic haptoglobin mRNA expression was elevated above basal levels in a greater proportion of peri-oestrous cows (4/4) than luteal cows (1/5). Haptoglobin cDNA was cloned and in vitro transcribed to generate probes for in situ hybridization. Haptoglobin mRNA was detected in the liver, but not in the ovary or oviduct. We conclude that haptoglobin mRNA expression in the bovine liver is up-regulated during the peri-oestrous phase of the oestrous cycle, and that the bovine oviduct expresses a low level of haptoglobin mRNA constitutively. This temporal pattern of haptoglobin mRNA expression would expose reproductive tissues to elevated concentrations of serum haptoglobin during the peri-oestrous stage, and suggests that haptoglobin may be important in reproductive events occurring during this time period.

Evidence for the role of oviduct secretions in sperm function, fertilization and embryo development.

The oviduct is a dynamic organ which facilitates gamete function, fertilization and embryo development. Secretions of the oviduct, recovered by tissue culture or cannulation techniques have been used to define the composition of the oviduct milieu, as well as functions associated with stage of the reproductive cycle or region of the oviduct. Several oviduct proteins have been shown to associate with the gametes and embryos. Ongoing studies are directed at identifying oviduct proteins and determining their function. Oviduct-specific glycoproteins (OSG) have been purified from the oviduct and shown in vitro to have positive affects on sperm capacitation, sperm-ovum binding, ovum penetration and embryo development. Osteopontin, another oviduct secretion, also has been shown to stimulate fertilization and embryo development. The picture emerging is that some components of the oviduct milieu have overlapping functions to collectively provide a failsafe system to ensure fertility in vivo so that success is not dependent on a single component.

Identification and immunohistochemical localization of a haptoglobin-like protein in the tissues and fluids of the bovine (Bos taurus) ovary and oviduct.

The objective of this study was to establish the identity of a 40 kDa bovine oviductal fluid protein as a haptoglobin-like protein and to evaluate the association of the haptoglobin-like protein with ovarian and oviductal tissues and fluids. An oviductal fluid protein band corresponding to a molecular mass of 40 kDa was excised and electroeluted from SDS-PAGE gels. Sequence analysis revealed an N-terminal region sharing 81% identity with the beta-subunit of bovine haptoglobin. The 40 kDa oviductal fluid protein crossreacted on immunoblots with antiserum against rabbit endometrial haptoglobin and with an anti-human haptoglobin polyclonal antibody. Two-dimensional PAGE revealed four protein variants ranging in pI from 7.7 to 8.6, which appeared identical, with respect to molecular weight, number of isoforms and pI, to bovine haptoglobin in acute phase serum. The haptoglobin-like protein was localized using immunohistochemistry to the lumina of blood vessels and to the extracellular matrix of ovarian and oviductal tissues. Immunostaining for the haptoglobin-like protein was also detected in the oviductal lumen, in the mucosa of the ampullary oviduct but not the isthmic oviduct, and in intermittent ampullary epithelial cells. Within the ovary, the haptoglobin-like protein was localized to the avascular granulosa cells and follicular fluid of antral follicles, but not in the theca cells or in preantral follicles of any developmental stage. It was concluded that the haptoglobin-like protein is a normal constituent of bovine ovarian and oviductal tissues and fluids, and it was hypothesized that the haptoglobin-like protein contributes to ovarian follicular development and oviductal function.

Induction of the acrosome reaction in bull spermatozoa with plasmin.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.

Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle.

Osteopontin and integrin alpha(v)beta(3) are known to mediate cell-cell attachment and cell migration. Western blot analysis was used to demonstrate the presence of osteopontin in oviductal fluid collected from ampullar and isthmic regions. Three different osteopontin isoforms of 55 kDa, 48 kDa and 25 kDa were detected in the oviductal fluid. Each isoform was observed during the luteal and non-luteal phases and in both ampullar and isthmic fluids. The 25 kDa osteopontin was the most prevalent isoform in oviductal fluid except in isthmic fluid during the non-luteal phase of the oestrous cycle. RT-PCR was performed with RNA from oviductal cells collected from cows in the post-ovulatory, early to mid-luteal, late luteal or pre-ovulatory stages of the oestrous cycle to reveal the oviduct as a site of osteopontin and integrin synthesis. Only one osteopontin mRNA transcript was detected, and amounts did not vary throughout the oestrous cycle. In contrast, the relative expression of the integrin subtypes alpha(v) and beta(1) during the late luteal phase was lower compared with the other oestrous cycle phases. Integrin beta(3) mRNA content increased significantly from the lowest level during the late luteal phase to the highest level before ovulation. In conclusion, differential presence of osteopontin isoforms and integrins in the bovine oviduct throughout the oestrous cycle indicate that osteopontin-integrin interactions have functional roles in normal oviduct physiology which may potentially influence interactions between the gametes, the embryo, and the epithelium.