Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus(R) e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.

Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.Fil: Losinno, Antonella Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Morales, Alfonsina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Fernández, Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lopez, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Oral misoprostol does not hasten oviductal transport of day-5 horse embryos

In horses, prostaglandin E2 (PGE2) is produced by embryos around Day 5 post-ovulation; PGE2 functions directly at the oviduct promoting embryo transport into the uterus. Non-surgical collection of horse embryos for cryopreservation is recommended at Day 6.5–7 post-ovulation. It was proposed that misoprostol administered orally will hasten oviductal transport of horse embryos. In Experiment 1 (n = 15) there was comparison of time of embryo recovery (Day 6 and 6.5 post-ovulation) from mares administered misoprostol (Day 5 and 5.5) orally to that of untreated mares. On Day 6, embryo collections were attempted; if no embryo was collected, there was a second attempt on Day 6.5. In Experiment 2, (n = 16) misoprostol treatment was initiated on Day 4.5; there was the first embryo collection attempt on Day 5.5, followed by Day 6 and 6.5 if no embryo was collected. Blood samples were collected at 12 h intervals on Day 4.5 or 5, to Day 6.5. In Experiment 1, on days 6 and 6.5, respectively, there was collection of seven and one of a total of eight embryos detected at the time of collection per group (P = 1). In Experiment 2, 12 embryos were collected during 15 cycles with there being a total of three, two, and one collected from mares of both groups on Day 5.5, 6, and 6.5 post-ovulation, respectively (P = 1). Serum progesterone concentrations were not different (P ≥ 0.05). In conclusion, misoprostol, when administered orally, does not hasten oviductal transport of horse embryos.Fil: Checura, Celina M.. Clemson University; Estados UnidosFil: Momont, Harry W.. University of Wisconsin; Estados UnidosFil: Castañeira, Catalina. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; ArgentinaFil: Flores Bragulat, Ana Paula. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Losinno, Luis. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; Argentin

Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.Fil: Gambini, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Duque Rodriguez, Matteo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Rodriguez, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Briski, Olinda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Flores Bragulat, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Demergassi, Natalia. Fundación Temaikén; ArgentinaFil: Losinno, Luis. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria; ArgentinaFil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; Argentin

Wilsher cervical forceps for artificial insemination technique in jennies

The pregnancy rate after artificial insemination (AI) with fresh semen between jennies and mares seems to be similar.Fil: Flores Bragulat, Ana Paula. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Carolina Natalia. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; ArgentinaFil: Castañeira, Catalina. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; ArgentinaFil: Losinno, Luis. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; Argentin

Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule

The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.Fil: Losinno, Antonella Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Sorrivas, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Ezquer, Eduardo Marcelo. Universidad del Desarrollo; ChileFil: Ezquer, Fernando. Universidad del Desarrollo; ChileFil: Lopez, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Morales, Alfonsina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Beta actin: its implication in the seminiferous tubule

Junctional devices in Sertoli cells play a key role in maturation and differentiation of germ cells. However, the cellular and subcellular organization of these specializations are still not totally understood. By combining light and electron microscopic techniques using â-actin immunolabeling and prosaposin and glutaredoxin antibodies to label Sertoli cytoplasm, we observed the structural (by confocal microscopy) and fine structural (by electron microscopy) organization of tight and adherent junctions, which are the morphological substrate of the blood testis barrier (BTB). The association to the beta actin also characterizes ectoplasmic specializations (ES) found at two different level of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubular bulbar complexes (TBC), a known component of apical ectoplasmic specializations. These different approaches also allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for â-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the â-actin network, the junctional complexes of the BTB and the ectoplasmic specializations were detected at different stages of the seminiferous cycle. â-actin immunolabeling and prosaposin and glutaredoxin antibodies to label Sertoli cytoplasm, we observed the structural (by confocal microscopy) and fine structural (by electron microscopy) organization of tight and adherent junctions, which are the morphological substrate of the blood testis barrier (BTB). The association to the beta actin also characterizes ectoplasmic specializations (ES) found at two different level of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubular bulbar complexes (TBC), a known component of apical ectoplasmic specializations. These different approaches also allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for â-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the â-actin network, the junctional complexes of the BTB and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.Fil: Losinno, Antonella Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lopez, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Capani, Francisco. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto Alberto C. Taquini de Investigaciones En Medicina Traslacional. - Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiologicas "prof. Dr. Alberto C. Taquini". Instituto Alberto C. Taquini de Investigaciones En Medicina Traslacional.; ArgentinaFil: Foscolo, Mabel Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ibañez, Jorge Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Cavicchia, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Equine spermatozoa at optimum physiological state are selected by chemotaxis toward progesterone

The success of assisted reproduction techniques depends inpart on sperm quality, which influences not only fertilization butalso embryo development and implantation. In our laboratory, wedesigned the Sperm Selection Assay (SSA) based on chemotaxistowards progesterone, which selects human sperm at optimumphysiological state (capacitated, with low levels of DNA fragmentation and reactive oxygen species). The aim of this study was todefine the experimental conditions to apply the SSA in unsexedand sexed equine sperm samples. Cryopreserved sperm samples ofthree stallions were conventionally thawed, removing the seminalplasma and cryoprotectant by a modified swim up procedure.Spermatozoa were incubated in BWW media with or without capacitating conditions (25 mM NaHCO3 and 0.3% BSA), at 38.5C atan atmosphere of 5% CO2 on air, for 45 minutes. The SSA deviceconsists of two wells connected by a tube. Well 1 (W1) was filledwith the sperm suspension and well 2 (W2) with the attractantsolution, which diffused along the connecting tube as a gradient.The percentage of sperm accumulation in W2 was determined asthe difference between with and without attractant. Firstly, weestablished the capacitation conditions in equine sperm samplesby inducing the the acrosome reaction (AR) with A23187 calciumionophore, and by the protein tyrosine phosphorylation pattern(PY). The level of capacitated spermatozoa was significantlyincreased at 45 minutes of incubation vs non-capacitated control. Next, we defined the experimental conditions to set up theSSA with frozen-thawed, unsexed and sexed equine spermatozoa, determining the percentage of accumulated spermatozoa inW2 under several dose response conditions and timing: byplacing 2 million sperm per ml in W1 (162% and 192%,respectively), 10 pM progesterone in W2 as the attractant solution (132% and 172%, respectively), and running the SSA for10 min (92% and 182%, respectively). We next verifiedwhether the sperm selection in the SSA was indeed mediated bychemotaxis. Thus, sperm accumulation in W2 was only observedwhen capacitated spermatozoa were loaded in W1 and progesterone was displayed as an ascending gradient (102%). Thequality of selected spermatozoa in W2 containing progesteronewas better than that of spermatozoa without being selected bythe SSA where a significant higher level of capacitated spermatozoa (PY) and lower level of DNA fragmentation (evaluated bythe ?Halo sperm test?), for sexed and unsexed samples, wereobserved. In conclusion, equine spermatozoa are selected bychemotaxis towards progesterone are at the optimum functionalstate, at a similar extent in sexed and unsexed samples. Theresults have potential application to improve current equinereproductive biotechnologiesFil: Dominguez, Esteban Mauricio. 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: Moreno, Ayelen. 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: Flores Bragulat, Ana Paula. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ramírez Castex, Hernan. Centro de Reproducción Equina Bioteq; ChileFil: Losinno, Luis. Universidad Nacional de Rio Cuarto. Facultad de Agronomia y Veterinaria. Departamento de Producción Animal. Laboratorio de Reproducción Equina; ArgentinaFil: 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