María Jesús García-López⁽¹⁾, María Jesús Ramírez-Expósito⁽²⁾, José Manuel Martínez-Martos⁽³⁾, María Dolores Mayas-Torres⁽⁴⁾, Isabel Prieto-Gómez⁽⁵⁾, Garbiñe Arechaga-Maza⁽⁶⁾, Manuel Ramírez-Sánchez^{(7)
(1)(3)(4)(5)(6)(7)}Unit of Physiology. University of Jaén - Jaén. Spain
⁽²⁾Unit of Physiology. University of Jaen - Jaén. Spain

	[Biochemistry]	[Endocrinology]	[Neuroscience]	[Physiology]	[Reproduction Sciences]

MATERIAL & METHODS

In this work, forty male mice Balb-C mice were used (30.575 g body weight). The animal were randomly divided into 5 groups of 8 mice each one. All the animals had free access to fed and water and were housed at a constant temperature of 25 ºC with lights on from 7:00 am to 7:00 pm. Four groups of mice were orchiectomized and the fifth group was used as controls. Fifteen days after gonadectomy, three of this orchiectomized group were injected with a testosterone solution (2 mg/ml), in an increasig doses during ten days. The concentration administred to the groups were 1, 2 and 3 mg of testosterone respectively (E1, E2 and E3). The fourth group orchiectomized (ORX) and the control were only injected with 100 µl of sesame oil, used as vehicle. After that, all animals were sacrificed. The animals were anaesthetized by an intraperitoneal injection of equithesin. Blood samples were obtained from the left cardiac ventricle and hypophysis, hypothalamus, frontal cortex and adrenal glands were also obtained. Blood samples were measured the same day and the tissues were frozen to -80ºC until its measure.

Tissue samples were homogenized in 10 volumes of 10mM HCl-Tris buffer (pH 7.4) and ultracentrifuged at 100 000 g for30 min (4 ºC) to obtain the soluble rraction. The resulting supernatants were used to measure soluble enzymatic activity (Sol) and protein content, assayed in triplicate. To solubilize membrane proteins, the pellets were rehomogenized in HCl-Tris buffer (pH 7.4) plus 1% Triton X-100. After centrifugation (100 000g, 30 min, 4 ºC) the supernatants were used to measure membrane-bound activity (M-B) and proteins, also in triplicate. To ensure complete recorvery of activity, the detergent was removed from the medium by adding adsorbent polymeric Biobeads SM-2 (100 mg/ml) and shaking the samples for 2 h at 4 ºC.

GluAP activity was measured in a fluorimetric assay using Glutamyl- -naphthylamide (GluNNap), in accordance with the method of Tobe et al., (1980), with modifications. Asp AP activity was measured in a fluorimetric assay too, using Aspartyl- -naphthylamide following the method of Cheung and Cushman (1971). The amount of -naphthylamine released as a result of the enzimatic activity was measured fluorimetrically at an emission wavelength of 412 nm and an excitation wavelength of 345 nm. Proteins were quantified in triplicate by the method of Bradford (1976), using BSA as a standar. Specific Sol an M-B aminopeptidase activities were expressed as pmol of pGluNNap hydrolyzed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content. We used one-way analysis of variance (ANOVA) to analyze differences between groups. Post-hoc comparisons were made using the paired Student´s t test; P values below 0.05 were considered significant.

Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [IMAGES] [DISCUSSION] [REFERENCES] [Discussion Board]

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	[Biochemistry]	[Endocrinology]	[Neuroscience]	[Physiology]	[Reproduction Sciences]

María Jesús García-López, María Jesús Ramírez-Expósito, José Manuel Martínez-Martos, María Dolores Mayas-Torres, Isabel Prieto-Gómez, Garbiñe Arechaga-Maza, Manuel Ramírez-Sánchez
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Last update: 15/01/00