Vol.29-Issue 3 - 2005

Seasonal variations in the heterologous binding of viscacha spermatozoa. 
A scanning electron microscope study
CLAUDIA AGUILERA MERLO, ESTELA MUÑOZ, SUSANA DOMINGUEZ, MABEL FÓSCOLO*, 
LUIS SCARDAPANE, AND JUAN CARLOS DE ROSAS*
Cátedra de Histología y Embriología, Facultad de Química, Bioquímica y Farmacia, 
Universidad Nacional de San Luis, 5700 San Luis, Argentina.
*Instituto de Histología y Embriología (IHEM), Facultad de Ciencias Médicas, 
Universidad Nacional de Cuyo, CONICET, 5500 Mendoza, Argentina.

Key words: viscacha (Lagostomus maximus maximus), spermatozoa, heterologous binding, seasonal variations.

ABSTRACT: Seasonal changes in the reproductive activity of the adult male viscacha (Lagostomus maximus maximus) were investigated during the annual reproductive cycle. Assays of heterologous in vitro binding between compatible gametes were used to evaluate the ability of viscacha spermatozoa to achieve primary binding during its annual reproductive cycle. Sperm were collected by mincing cauda epididymis in HECM-3 medium and the sperm concentration and motility were evaluated. Cumulus-free and zona-free oocytes obtained from superovulated hamsters were inseminated in vitro with capacitated sperm suspensions, incubated at 37ºC, 5% CO2 for 3 h, and then processed for studies by scanning electronic microscopy. Statistical analysis was used to compare the quantitative differences. The number of spermatozoa significantly decreases during the regression period, while sperm motility was progressive speed in both periods. During the active period elevated sperm binding to cumulus-free and zona-free oocytes was observed, while the binding during the regression period decreased drastically. In both periods, oocyte microvilli covered sperm heads and tails. These results suggest that the ability of viscacha spermatozoa to participate in gamete recognition is profoundly affected. This would likely be related to different functional stages of the spermatozoa and their epididymal microenvironment during the annual reproductive cycle of viscacha.

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Construction and application of a yeast expression system for thymosin α1
CHEN FENG*, CHEN XIANG-MING**, CHEN ZHI*, JIANG HAN-LIANG*, PAN XIAO-PING*,
HU ZHONG-RONG*, LIU RONG-HUA* AND CHEN XIAO-MING*
* Institute of Infectious Diseases, 1st Affiliated Hospital, Medical College of Zhejiang University, Key Lab. of Infectious
Diseases, Ministry of Public Health, Hangzhou, 310003, CHINA.
** Department of Anesthesiology, 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, 310009,
CHINA.

Key words: thymosin α1, yeast expression system, biological activity, orally administered.

ABSTRACT: We want to construct a yeast expression system for thymosin α1 (Tα1) to make the orally administered Tα1 preparation possible. The whole Tα1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Tα1 in recombinant coincided with the original one reported in Genbank. When pYES2-Tα1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Tα1 expression. Western blot was performed to identify the quality of the expressed Tα1. Dried yeast containing pYEST2-Tα1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Tα1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Tα1 and synthesized Tα1 peptide can significantly increase the CD8+ level (22.74±1.09 and 18.77±4.72 vs 7.49±2.14, p<0.01), while both of them had little effect on the CD4+ lymphocytes (61.86±6.94 and 65.91±4.78 vs 57.93±10.40, p>0.05). We concluded that a high effective yeast expression system for Tα1 was constructed successfully and the Tα1 protein expressed by this system can improve CD8+ level in immune inhibited mice.

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Extracellular breakdown of collagen by mice decidual cells.
A cytochemical and ultrastructural study
SIMA GODOSEVICIUS KATZ
Department of Histology, School of Medicine, Federal University of São Paulo. São Paulo, SP, BRAZIL.

Keywords: decidua, acid phosphatase, fasting, electron microscopy, mice.

ABSTRACT: The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates
(disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.

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Ubisch bodies and pollen ontogeny in Oxalis articulata Savigny
SONIA ROSENFELDT AND BEATRIZ G. GALATI
Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales. Universidad de Buenos
Aires. Buenos Aires, Argentina.

Key word: pollen, ubisch bodies, ontogeny, ultrastructure, Oxalis

ABSTRACT: The correlation between the ontogeny of Ubisch bodies and pollen development in Oxalis articulata was studied with Transmission Electron Microscopy (TEM). The ultrastructural changes observed during the different stages of development in the tapetal cells are related to Ubisch bodies, sporopollenin and pollen-kitt formation. The pro-orbicules have the appearance of lipid globuli and their formation is related to the endoplasmic reticulum of rough type (ERr). The lipid globules or pro-orbicules disappear in the mature Ubisch bodies, and the places that they occupied remain free of contents or with pollen-kitt.

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Seed weight variation of wyoming sagebrush in Northern Nevada
CARLOS A. BUSSO* AND BARRY L. PERRYMAN**
* Departamento de Agronomia-CERZOS (CONICET), Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
** Department of Animal Biotechnology, College of Agriculture, Biotechnology and Natural Resources, Mail Stop 202,
University of Nevada, Reno, Nevada, USA.

Keywords: Artemisia tridentata ssp. wyomingensis, climate effects, rangeland restoration, seed size.

ABSTRACT: Seed size is a crucial plant trait that may potentially affect not only immediate seedling success but also the subsequent generation. We examined variation in seed weight of Wyoming sagebrush (Artemisia tridentata ssp. wyomingensis Beetle and Young), an excellent candidate species for rangeland restoration. The working hypothesis was that a major fraction of spatial and temporal variability in seed size (weight) of Wyoming sagebrush could be explained by variations in mean monthly temperatures and precipitation. Seed collection was conducted at Battle Mountain and Eden Valley sites in northern Nevada, USA, during November of 2002 and 2003. Frequency distributions of seed weight varied from leptokurtic to platykurtic, and from symmetry to skewness to the right for both sites and years. Mean seed weight varied by a factor of 1.4 between locations and years. Mean seed weight was greater (P<0.05) in 2003 than in 2002 at both sites. This can partially be attributed to 55% greater precipitation in 2003 than 2002, since mean monthly temperatures were similar (P>0.05) in all study situations. Simple linear regression showed that monthly precipitation (March to November) explained 85% of the total variation in mean seed weight ( P=0.079). Since the relationship between mean monthly temperature (June-November) and mean seed weight was not significant (r2=0.00, P=0.431), this emphasizes the importance of precipitation as an important determinant of mean seed weight. Our results suggest that the precipitation regime to which the mother plant is exposed can have a significant effect on sizes of seeds produced. Hence, seasonal changes in water availability would tend to alter size distributions of produced offspring.

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Cyclic nucleotide phosphodiesterase inhibition increases tyrosine
phosphorylation and hyper motility in normal and pathological human spermatozoa
ROBERTO YUNES, PEDRO FERNÁNDEZ, GUSTAVO F. DONCEL, AND ANÍBAL A. ACOSTA
The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology,
Eastern Virginia Medical School, Norfolk, VA. USA.

Key words: PDE inhibition, phosphotyrosine proteins, sperm hyperactivation, sperm motility, assisted reproduction.

ABSTRACT: Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples
incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.

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Microsporogenesis in tetraploid accessions of Brachiaria
nigropedata (Ficalho & Hiern) Stapf (Gramineae)
KARINA SAYURI UTSUNOMIYA*, MARIA SUELY PAGLIARINI* AND CACILDA BORGES DO VALLE**
* Department of Cell Biology and Genetics, State University of Maringá,
Av. Colombo 5790, 87020-900, Maringá, Paraná, Brazil.
** Embrapa Beef Cattle, P. O. Box 154, 79002-970 Campo Grande, Mato Grosso do Sul, Brazil.

Key words: Brachiaria nigropedata, chromosome number, meiosis, polyploidy, apomixis, grasses.

ABSTRACT: The genus Brachiaria (Trin.) Griseb. has achieved considerable importance to cattle production systems, as a result of the good production and adaptation of a few cultivars to poor and acid soils of the Brazilian savannas. Many of its species and accessions are polyploid and apomictic, which limits direct hybridization. To assist the breeding program, cytogenetic characterization has been undertaken on the accessions of Brachiaria collection at the Embrapa Beef Cattle Research Center. In this study, chromosome number and meiotic behavior are reported for the Brachiaria nigropedata (Ficalho & Hiern) Stapf collection. The 20 available accessions are tetraploid (2n = 4x = 36). Chromosomes paired preferentially as bivalents, but quadrivalents were found in high frequencies in some cells. Meiotic behavior was, in general, irregular, and varied among accessions. Most accessions presented more than 20% of abnormal tetrads. The most common meiotic abnormalities were those related to irregular chromosome segregation due to polyploidy, leading to
micronuclei formation in the tetrad stage. A low frequency of other meiotic abnormalities such as the absence of cytokinesis, chromosome stickiness, cell fusion, anaphase bridges, and chromosome transfer among microsporocytes were also recorded in some accessions. Limitations of these accessions for use in hybridization programs are discussed.

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TNFα increases in vitro migration of human HPV18-positive
SW756 cervical carcinoma cells
K. HIDALGO1, I. G. ROJAS2, A. B. PENISSI3, AND M. I. RUDOLPH1
1Faculty of Biological Sciences and 2College of Dentistry, Universidad de Concepción, Concepción, Chile.
3IHEM-CONICET, Faculty of Medical Sciences, Universidad Nacional de Cuyo, Mendoza, Argentina.

Key words: TNFα, migration, carcinoma, SW756, wound healing assay.

ABSTRACT: TNFα has been associated with both, tumor survival and apoptosis. This cytokine is also involved in promoting cell migration during wound healing and tumorigenesis. SW756 is a HPV18-positive cervical carcinoma cell line, which has been used to study different mechanisms of cervical cancer progression. An in vitro assay of scratch wound healing onto monolayers of SW756 cells was used to assess the effect of TNFα on cell migration into a wound space. It was found that SW756 cells have the ability to migrate, but not proliferate in response to scratch wounding in a serum-free medium supplemented with TNFα. RT-PCR analysis showed that SW756 cells express TNFα mRNA when incubated in medium with and without serum. Wound closure and migration rate of SW756 cells were significantly increased in the presence of serum-free media supplemented with TNFα (10 ng/mL) as compared to serum-free media, and media supplemented with either anti-TNFα antibody or both TNFα and anti-TNFα antibody (p<0.05). The results showed a stimulatory effect of TNFα on the migration of SW756 cervical carcinoma cells, suggesting a novel and important role for TNFα in cervical cancer progression.

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Brief Note

Plant regeneration after long term callus culture in clones of
Asparagus officinalis L.*
A. C. PONTAROLI AND E. L. CAMADRO
Estación Experimental Agropecuaria (EEA) Balcarce, Instituto Nacional de Tecnología Agropecuaria (INTA) and Facultad
de Ciencias Agrarias (FCA), Universidad Nacional de Mar del Plata (UNMdP); C. C. 276, (7620) Balcarce, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina. Current Address: Dept.of Genetics,
University of Georgia, 30602 Athens, Georgia, U.S.A.

Key words: callus, shoot primordia induction, rooting, medium x genotype interactions.

ABSTRACT: Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg.l- 1 kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg.l- 1 2,4-D and 1 mg.l-1 kinetin. Shoot primordia (SP) induction from >18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone ‘265’ on MS medium with 5 mg.l-1 2iP, 1 mg.l-1 IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone ‘265’ on MS medium with 0.1 mg.l-1 kinetin, 0.1 mg.l-1 NAA, 1.32 mg.l-1 ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Abbreviations: 2,4-D: 2,4-dichlorophenoxyacetic acid; 2iP: 2-isopentenyladenine; BA: 6-benzylaminopurine; FFW: final fresh weight; IAA: indoleacetic acid; IFW: initial fresh weight; MS: Murashige and Skoog (1962); NAA: 1-naphtaleneacetic acid; PGRs: plant growth regulators; PVP: polyvinylpyrrolidone; SP: shoot primordia.

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