Carlos Eduardo A. Souza

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-β-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-β-glycoprotein, apolipoprotein A-1, β 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.

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams ☆

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

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.

Reproductive Development of Santa Ines Rams During the First Year of Life: Body and Testis Growth, Testosterone Concentrations, Sperm Parameters, Age at Puberty and Seminal Plasma Proteins

We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two-dimensional SDS-PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 +/- 0.7 to 54.3 +/- 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non-significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 +/- 0.05 to 1.14 +/- 0.24 x 10(9) cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 +/- 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 +/- 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands.

Early prepubertal testis criteria, seminiferous epithelium and hormone concentrations as related to testicular development in beef bulls

The present study was conducted to evaluate testis size, spermatogenesis and hormone concentrations before and when peripheral testosterone reached 1 ng/ml as related to further gonad development of beef bulls (n=28). Blood samples were taken weekly starting at 10 weeks (wk) and when testosterone reached 1 ng/ml (AGE1), the left testis was surgically excised. From AGE1 until 54 wk, blood samples were collected to follow basal and GnRH-stimulated hormone profiles. At 54 wk, the second testis was removed. Testosterone reached 1 ng/ml at 20±0.6 wk and, at this developmental state, the seminiferous tubules occupied 57±1.1% of the testis parenchyma. At this phase, 79.3±1.4% of tubule sections had no germ cells and only 2.4±0.3% of the remaining tubules had spermatocytes as the most advanced germ cell type. Also at AGE1, testis size was correlated with the number of Sertoli cells per testis (r=0.67; P<0.05), but not (P>0.05) with the percentage of tubules with germ cells. There was a consistent increase in body weight and testis size throughout the study showing that hemicastration did not impair the development of the bulls. At 54 wk, seminiferous tubules represented 76±0.7% of the testis parenchyma and 72.3±1.7% of tubule sections were found with either round or elongated spermatids. Quantitative criteria of spermatogenesis in the second testis (excised at 54 wk) were not correlated (P>0.05) with the percentage of seminiferous tubules with germ cells in the first testis (excised at AGE1). As determined by regression analysis, testis diameter measured between 30 and 44 wk (AVTD) was associated with AGE1 and testis diameter averaged at 12 wk and AGE1 (R(2)=0.77; P<0.01). Also, AVTD was related to AGE1, testis diameter at 12 wk and concentrations of 17β-estradiol (estradiol; basal+GnRH-stimulated) averaged between 10 wk and AGE1 (R(2)=0.79; P<0.01). Yearling testis weight, in turn, was linked to AGE1 and testis weight at AGE1 (R(2)=0.49, P<0.01). In conclusion, early detection of 1 ng of testosterone/ml, larger testis size and greater estradiol before and at that developmental period positively relate to future testis attributes. When testosterone reached 1 ng/ml, the seminiferous tubules had Sertoli cells, spermatogonia and a few spermatocytes and events occurring before and at that phase are potential markers of testis growth and sperm-producing capacity of sires.

The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles

The effects of varying concentrations of EGF were evaluated in terms of in vitro follicular development and the mRNA expression levels of EGF, EGF-R, FSH-R and P450 aromatase. After 6 days, the addition of 50 ng/mL of EGF to the culture medium increased the antrum formation rates in comparison to cultured control and after 18 days of culture produced oocytes with higher rates of meiosis resumption when compared to the other treatments (P<0.05). The daily follicular growth rates in presence of EGF (50 or 100) were increased in comparison to the cultured control (P<0.05). Treatment with EGF 50 stimulated the expression of EGF mRNA but reduced EGF-R mRNA expression and estradiol secretion as compared to the cultured control (P<0.05). After 18 days of culture, the mRNA levels for FSH-R and P450 aromatase were greater than those of the non-cultured controls (P<0.05). In conclusion, the effects of EGF treatment on the mRNA levels for EGF, EGF-R, FSH-R, and P450 aromatase varied according to the stage of follicle development.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 μg mL⁻¹ insulin, 5.5 μg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Proteome of Soybean Seed Exudates Contains Plant Defense-Related Proteins Active against the Root-Knot Nematode Meloidogyne incognita

Several studies have described the effects of seed exudates against microorganisms, but only few of them have investigated the proteins that have defensive activity particularly against nematode parasites. This study focused on the proteins released in the exudates of soybean seeds and evaluated their nematicidal properties against Meloidogyne incognita. A proteomic approach indicated the existence of 63 exuded proteins, including β-1,3-glucanase, chitinase, lectin, trypsin inhibitor, and lipoxygenase, all of which are related to plant defense. The presence of some of these proteins was confirmed by their in vitro activity. The soybean exudates were able to reduce the hatching of nematode eggs and to cause 100% mortality of second-stage juveniles (J2). The pretreatment of J2 with these exudates resulted in a 90% reduction of the gall number in tobacco plants. These findings suggest that the exuded proteins are directly involved in plant defense against soil pathogens, including nematodes, during seed germination.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production

OBJECTIVE Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.

Proteomics changes during the incompatible interaction between cowpea and Colletotrichum gloeosporioides (Penz.) Penz and Sacc.

Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.