Análisis sobre prácticas de farmacología con animales de laboratorio: una mirada desde los estudiantes de la Universidad Nacional de San Luis, Argentina

Introduction: The subject Pharmacology in the Pharmacy program is theoretical and practical, lasts a semester, and is taught in the fourth year. Practical development involves six practical laboratory sessions (tpl) and four workshops. Three tpl are for in vivo animal experiments, two for computer programs and one for in vitro techniques, allowing students to evaluate the effect of drugs through experimentation, oriented towards biological and biomedical research, and to recognize pharmacological efficacy for the treatment of diseases. Our objective was to conduct a survey to obtain a guidance instrument that allows to increase teaching quality and to know perceptions and preferences about the practices concerned. Method: A cross-sectional, descriptive study was carried out and concluded in the first four-month period of the academic year 2015. Results: 90.48% reported that animal practices were useful for training, and two-thirds considered it necessary to implement alternatives to the use of animals in their education and training. 85.71% preferred in vivo experiments, 9.52% in vitro observations and 4.76% computer programs. 90.48% responded that they would work with laboratory animals again. Conclusions: We believe that it is appropriate to work with animals in this subject; it is important to consider this practical experience in pharmacology. Students expressed their interest in knowing and interpreting the effects and mechanisms of action of drugs through in vivo observations or organs manipulated by them.Introducción: la asignatura Farmacología en Farmacia es de carácter teórico-práctico, dura un cuatrimestre y se imparte en el cuarto año. El desarrollo práctico involucra seis trabajos prácticos de laboratorio (tpl) y cuatro talleres. En tres tpl se realiza experimentación in vivo con animales, en otros dos se trabaja con programas computacionales, y en el restante técnicas in vitro, permitiendo al estudiante valorar el efecto de los fármacos mediante la experimentación, orientado a la investigación biológica y biomédica, además de reconocer la eficacia farmacológica para el tratamiento de enfermedades. Nuestro objetivo fue promover una encuesta para obtener un instrumento orientativo que permita aumentar la calidad de la enseñanza y conocer las percepciones y preferencias sobre las prácticas en cuestión. Metodología: se realizó un estudio transversal y descriptivo, concluido el primer cuatrimestre académico del ciclo lectivo 2015. Resultados: el 90,48 % aludió que las prácticas con animales resultaron útiles para su formación, y las dos terceras partes consideró necesario implementar alternativas al uso de animales en aspectos relacionados a su educación y formación. El 85,71 % prefirió las observaciones in vivo; 9,52 % las in vitro y 4,76% los programas computacionales. 90,48 % respondió que volvería a trabajar con animales de laboratorio. Conclusiones: creemos que es pertinente trabajar con animales en esta asignatura, es importante pensar en esta experiencia práctica en la farmacología. Los estudiantes manifiestan su interés por conocer e interpretar los efectos y mecanismos de acción de los fármacos mediante observaciones in vivo o de órganos manipulados por ellos mismos

Progesterone from the Cumulus Cells Is the Sperm Chemoattractant Secreted by the Rabbit Oocyte Cumulus Complex

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species

Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa

By means of a videomicroscopy system and a computer image analysis, we performed chemotaxis assays to detect true chemotaxis in human spermatozoa, in parallel to immunohistochemistry detection of progesterone inside the cumulus cells. Progesterone indeed chemotactically guides mammalian spermatozoa at very low hormone concentrations, and the cumulus oophorus could be a potential place for sperm chemotaxis mediated by progesterone in vivo.Fil: Teves, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Barbano, María Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Guidobaldi, Héctor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Sánchez, Raúl. Universidad de La Frontera; ChileFil: Miska, Werner. Justus Liebig University; AlemaniaFil: Giojalas, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; Argentin

Versatile action of picomolar gradients of progesterone on different sperm subpopulations.

High step concentrations of progesterone may stimulate various sperm physiological processes, such as priming and the acrosome reaction. However, approaching the egg, spermatozoa face increasing concentrations of the hormone, as it is secreted by the cumulus cells and then passively diffuses along the cumulus matrix and beyond. In this context, several questions arise: are spermatozoa sensitive to the steroid gradients as they undergo priming and the acrosome reaction? If so, what are the functional gradual concentrations of progesterone? Do spermatozoa in different physiological states respond differentially to steroid gradients? To answer these questions, spermatozoa were confronted with progesterone gradients generated by different hormone concentrations (1 pM to 100 µM). Brief exposure to a 10 pM progesterone gradient stimulated priming for the acrosome reaction in one sperm subpopulation, and simultaneously induced the acrosome reaction in a different sperm subpopulation. This effect was not observed in non-capacitated cells or when progesterone was homogeneously distributed. The results suggest a versatile role of the gradual distribution of very low doses of progesterone, which selectively stimulate the priming and the acrosome reaction in different sperm subpopulations

Picomolar gradients of progesterone generated in the chemotaxis chamber stimulate either priming or the acrosome reaction in different sperm subpopulations.

<p>Percentage of spermatozoa primed (A) or acrosome-reacted (B) after a 15 min exposure to gradients of different progesterone concentrations (1 pM to 100 µM). Percentage of spermatozoa-primed (C) or acrosome-reacted (D), after a 15 min exposure to a homogeneous distribution of different progesterone concentrations (1 pM to 100 µM). Data are shown as means ± s.e.m. calculated from 7 independent experiments, counting 200–600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Inset: Percentage of acrosome-reacted spermatozoa after a 30 min exposure to a homogeneous 10 µM progesterone concentration. Data are shown as means ± s.e.m. calculated from 3 independent experiments, counting 200-600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Percentage of spermatozoa-primed (E) or acrosome-reacted (F) in capacitated (+cap) or non-capacitated (-cap) populations, after a 15 min exposure to a gradient of 10 pM progesterone. Data are shown as means ± s.e.m. calculated from 4 independent experiments, counting 200–600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Percentage of spermatozoa-primed (G) or acrosome-reacted (H) in the presence or absence of progesterone, located in different longitudinal areas of the connection between wells (A, close to the progesterone well; B, in the middle; and C, close to the culture medium well).</p

Chambers for generating progesterone gradients.

<p>Both chambers consist of two wells (W1 and W2) with a connection (C) between them, where progesterone (P) is loaded in W2 and diffuses to W1 (containing culture medium; M) through the connection, forming a one-dimensional concentration gradient. The difference between systems lies in the way spermatozoa (S) are exposed to the gradient. A, in the CH chamber the sperm population is stuck to a coverslip (co) which is positioned upside down at the connection containing the progesterone gradient; therefore, the cells are exposed to the progesterone gradient from a fixed position. B, in the SSA device, spermatozoa are loaded in W1, facing the progesterone gradient while swimming across the connection between wells. C and D, theoretical representation of the rate of change of progesterone (ΔP) over time in the CH and SSA chambers, respectively. ΔP was calculated according to the formula [P]_t+1 - [P]_t, where [P] is the concentration of progesterone and <i>t</i> is the time. C, ΔP shown in cells positioned at different arbitrary distance (x) from the progesterone-containing well (the closest position being 0.15 mm); insets: i, represents a magnification of ΔP values during the first 60 s of the experiment, in cells located at different distances from the progesterone source; ii, represents a magnification of ΔP for the same cells shown in i, but from 500 s until the end of the experiment. D, ΔP is shown in 3 theoretical cells swimming at different linear speeds (20, 30 and 40 µm/s) getting into the connection between wells at different times (300 s up to 1200 s).</p

Picomolar gradients of progesterone generated in the chemotaxis chamber stimulate either priming or the acrosome reaction in different sperm subpopulations.

<p>Percentage of spermatozoa primed (A) or acrosome-reacted (B) after a 15 min exposure to gradients of different progesterone concentrations (1 pM to 100 µM). Percentage of spermatozoa-primed (C) or acrosome-reacted (D), after a 15 min exposure to a homogeneous distribution of different progesterone concentrations (1 pM to 100 µM). Data are shown as means ± s.e.m. calculated from 7 independent experiments, counting 200–600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Inset: Percentage of acrosome-reacted spermatozoa after a 30 min exposure to a homogeneous 10 µM progesterone concentration. Data are shown as means ± s.e.m. calculated from 3 independent experiments, counting 200-600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Percentage of spermatozoa-primed (E) or acrosome-reacted (F) in capacitated (+cap) or non-capacitated (-cap) populations, after a 15 min exposure to a gradient of 10 pM progesterone. Data are shown as means ± s.e.m. calculated from 4 independent experiments, counting 200–600 cells per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Percentage of spermatozoa-primed (G) or acrosome-reacted (H) in the presence or absence of progesterone, located in different longitudinal areas of the connection between wells (A, close to the progesterone well; B, in the middle; and C, close to the culture medium well).</p

Picomolar gradients of progesterone stimulate singular intracellular calcium changes in spermatozoa.

<p>A, cells exposed to a gradient of 10; Aii, comparison of the curves showing mean values of the subpopulation of oscillating sperm with calcium values higher than the mean population value (red) and the mean of the rest of the cells (blue); Aiii, absolute progesterone concentration in the middle of the CH chamber along time where the starting point corresponds to 2 min due to the delay between CH sealing and start of recording. ^a, significant differences vs. the blue curve (<i>p</i><0.0001). B, cells exposed to a step application of 10 pM progesterone. Bi, calcium variations in several single cells along time. Representative images of several sperm cells exposed either to a gradient or a step 10 pM progesterone (Aiv and Bii, respectively). In Ai and Bi, data are shown as calcium variation expressed as relative fluorescence intensity (%) of individual cells of one representative experiment of five, analyzing 50 cells per treatment and experiment.</p

Picomolar gradients of progesterone generated in the SSA chamber stimulate either priming or the acrosome reaction in different sperm subpopulations.

<p>Percentage of spermatozoa primed (A) or acrosome-reacted (B) after a 20 min exposure to a gradient of 10 pM progesterone. Data are shown as means ± s.e.m. calculated from 5 independent experiments, counting 200–600 cells per well per treatment. ^a, significant differences vs. without progesterone (<i>p</i><0.05). Percentage of spermatozoa-primed (C) or acrosome-reacted (D) exposed to 0–10 pM or 10–20 pM of progesterone gradients. Data are shown as means ± s.e.m. calculated from 5 independent experiments, counting 200–600 cells per well per treatment. ^a, significant differences vs. the other treatments (<i>p</i><0.05). E, theoretical progesterone gradient formation over time and the connection between wells, when spermatozoa are exposed to 0–10 pM or 10–20 pM gradient of progesterone. In each set of curves, the lower represents time = 300 s, the middle = 600 s, and the upper = 900 s. The inset (i) represents the ΔP in the middle of the connection between both wells, for a 0–10 pM (blue dash line) and 10–20 pM (red dashed line) gradients.</p