Expresión y actividad de las caspasas iniciadoras (-2, -8 y -9) y la principal caspasa efectora (-3) en la regresión del cuerpo lúteo del mono rhesus y la rata

El cuerpo lúteo (CL) es una glándula endocrina transciente que surge, luego de la ovulación, de la luteinización de las células de granulosa y tecales del folículo ovárico. Las células luteales son responsables de producir progesterona para permitir el comienzo y el mantenimiento del embarazo. En caso de no producirse la fecundación, el CL se degenera en un proceso denominado luteólisis, decayendo los niveles de progesterona. La formación y la regresión del CL requieren de una extensa reestructuración del tejido, cuyos mecanismos moleculares aún permanecen en gran parte desconocidos. La apoptosis o muerte celular programada ha sido relacionada con la regresión o luteólisis del CL en distintas especies. Sin embargo existen controversias acerca de la participación de este proceso en el CL. Los miembros de la familia de las caspasas son cisteín-proteasas que se encuentran involucradas tanto en los eventos tempranos como en los tardíos de la apoptosis, jugando un papel central en dicho proceso. La expresión de algunas caspasas ha sido descripta en la luteólisis, con resultados contradictorios de acuerdo a los distintos modelos estudiados. La hipótesis de esta tesis fue que cambios en la expresión y/o actividad de las caspasas están involucrados en la regresión o luteólisis del CL. Por lo antes expuesto, el objetivo de esta tesis fue evaluar los cambios de expresión y/o actividad de diferentes caspasas durante la formación y regresión del CL utilizando distintos modelos. Obteniéndose los siguientes resultados y conclusiones: Varios miembros de la familia de las caspasas (-2, -8, -9 y -3) se encuentran expresados y activos en el CL durante la fase lutea del ciclo menstrual natural del mono rhesus. Asimismo, se observó un aumento transciente en la actividad de las tres caspasas iniciadoras y la principal caspasa efectora en MLCL (donde los niveles de progesterona aún son elevados). La regulación de las caspasas en el CL de primates sería a nivel de la traducción o de la activación enzimática y no a nivel de la transcripción. En cuanto a los resultados obtenidos del modelo de remoción y reemplazo hormonal, éstos sugieren que las gonadotrofinas y esteroides locales, estarían regulando principalmente la activación de la cascada de las caspasas a nivel de las iniciadoras (-2, -8 y -9). Durante la formación y regresión del CL en el ciclo estral y la preñez en la rata, se observó una expresión y/o actividad dinámica en las caspasas iniciadoras (-2, -8 y -9) y la principal efectora (-3). La actividad de las caspasas-2, -3 y -9 aumentó en el CL del ciclo anterior en E durante la luteólisis funcional, seguido de un aumento en el número de células apoptóticas y disminución en el peso en el CL del ciclo anterior en DII. Asimismo, se observó un aumento transciente en la actividad de las tres caspasas iniciadoras y la principal caspasa efectora en el CL de preñez en los días 19 y/o 21 de gestación. Este aumento fue previo o coincidente con la disminución en los niveles luteales de progesterona. Por otro lado, se observó un aumento significativo en el número de células apoptóticas e incremento en la actividad de la 20α-HSD luego del parto. Por lo tanto, un camino apoptótico independiente de las caspasas podría estar involucrado en la regresión estructural del CL de la preñez luego del parto en la rata. El tratamiento in vivo con PGF-2α a ratas en el día 14 de preñez, disminuyó los niveles luteales de progesterona a las 24 horas post inyección y esta disminución podría ser la responsable del aumento significativo en la actividad de las cuatro caspasas evaluadas a las 36 horas post tratamiento. Mientras que, no fue capaz de producir cambios notorios en el porcentaje de células apoptóticas en el CL de la preñez a los distintos tiempos post inyección evaluados. En conclusión, la activación temprana de las caspasas en el ciclo mentrual del mono rhesus, y en el ciclo estral y la preñez de la rata, sugieren que estas proteasas están involucradas en la etapa temprana de la regresión del CL. Mientras que, la administración exógena con PGF-2α no necesariamente mimetiza la luteólisis fisiológica, ya que la caída en los niveles de progesterona precede la activación de las caspasas. Además, de acuerdo a los resultados obtenidos en los distintos modelos estudiados, el control en la expresión de las caspasas puede diferir respecto al de su actividad. Dado que la actividad de las tres caspasas iniciadoras evaluadas aumentan significativamente en el CL del ciclo menstrual del mono, de la preñez de rata y post administración de PGF-2α; ambos caminos apoptóticos (camino mitocondrial y camino de receptores de muerte) se encontrarían involucrados.The corpus luteum (CL) is a transient endocrine gland that it is formed after ovulation by the luteinization of the granulosa and theca cells of the follicle. Luteal cells are responsible for secreting progesterone, which permits the initiation and maintenance of pregnancy. If pregnancy does not occur, the CL regresses by a process called luteolysis and progesterone levels decline. The formation and regression of the CL involves extensive tissue remodeling, and most of the molecular mechanism are unknown. Apoptosis, or programmed cell death, is associated with luteolysis or regression of the CL in many species. However, there is some controversy about the involvement of apoptosis during luteolysis. Members of the caspase family are cysteine proteases involved in either the early or the final apoptotic events, playing a key role in this process. Information regarding the expression of different caspases during the regression of the CL has been described, with contradictory results depending on the models studied. The hypothesis of this thesis was that changes in expression and/or activity of the caspases are involved in the regression of the CL. Therefore, the aim of this thesis was to evaluate changes in expression and/or activity of different caspases during formation and regression of the CL with different models. The following results and conclusions were obtained. Several members of the caspase (-2, -3, -8, and -9) family are expressed and active in the rhesus monkey CL throughout the luteal phase of the natural menstrual cycle. Also, a transient rise in initiator and effector caspase activity at mid-late luteal phase was observed (when the progesterone levels are still high). Regulation of the caspases in the primate CL was not at the transcriptional level, but rather at the level of translation and/or enzyme activation. Regarding hormone ablation/replacement treatment, the results suggest that gonadotropic hormones and local steroids act primarily to regulate initiator (-2, -8, and -9) caspase activity rather than the main effector caspase-3, in the primate CL. Dynamic expression of the initiator caspases (-2, -8 and -9) and the main effector caspase-3 was observed during the formation and regression of the CL in the estrous cycle and pregnancy of the rat. The activity for caspase-2,-3 and -9 increased in the old CL at estrus during the functional regression, followed by an increase in the number of apoptotic cells and a decrease in the CL weight in the old CL at diestrus II. Moreover, a transient increase in initiator and effector caspase activity in the CL on days 19 and 21 of pregnancy was observed. This increase was prior to or coincident with the decrease in luteal progesterone content. Also, the number of apoptotic cells and activity of 20α-HSD increased after parturition. Therefore, a caspase-independent apoptotic mechanism may be involved in the structural regression of the rat CL after parturition. In vivo PGF2α treatment on day 14 of pregnancy decreased luteal progesterone content by 24 hours post injection and this drop may stimulate the activation of the four caspases studied at 36 hours post PGF-2α administration. However, PGF-2α treatment failed to produce any significant change in the number of apoptotic cells in the CL of pregnancy at the selected times post injection. In conclusion, the early activation of the caspases in CL during the menstrual cycle of the rhesus monkey, and the estrous cycle and pregnancy of the rat, suggests that these proteases are involved in the early stages of the luteal regression. In addition, exogenous administration of PGF-2α to rats does not necessarily mimic physiological luteolysis since the drop of luteal progesterone content precedes caspase activation. Also, according the results from the different experimental models, the control of caspase protein may differ from the activity of these proteases. Since a significant increase of the three initiator caspases studied was found in the CL during the menstrual cycle of the rhesus monkey, in the CL of pregnancy and post PGF-2α administration in the rat, both apoptotic pathways (the extrinsic death receptor and the intrinsic mitochondrial pathways) appear involved in luteolysis.Fil: Peluffo, Marina Cinthia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum

Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy (days 7, 17, 19, and 21 of gestation), postpartum (days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2α. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2α injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2α. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2α may regulate members of the caspase family.Fil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Stouffer, Richard L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Direct role of the C-C motif chemokine receptor 2/monocyte chemoattractant protein 1 system in the feline cumulus oocyte complex

Studies were designed to a) evaluate the mRNA expression of the C-C motif chemokine receptor 2 (CCR2) and its chemokine ligands, as well as genes related to periovulatory events, within the cumulus oocyte complex (COC) and follicle wall after a luteinizing hormone (LH) stimulus in cultured feline antral follicles; b) assess the immunolocalization of CCR2 and its main ligand (monocyte chemoattractant protein 1, MCP1) within the feline COC; and c) examine the direct effects of exogenous recombinant MCP1 on mRNA expression of the CCR2 receptor and MCP1 as well as key periovulatory genes in the COC, using a feline COC culture system. Both culture systems were developed by our laboratory and exhibit physiological response to gonadotropin stimuli. In summary, this study demonstrated mRNA expression of CCR2 receptor and its assessed ligands (MCP1, MCP2, MCP3 and MCP4) within the feline COC and follicle antral wall, and a significant increase in CCR2 mRNA by LH within the COC. Also, CCR2 and MCP1 immunoreactivity was observed in the oocyte and cumulus cells of the feline COC. Remarkably, this is the first report, in any species, describing a direct effect of the recombinant MCP1 in the CCR2/MCP1 system within the COC, by increasing the mRNA levels of key genes involved in the ovulatory cascade, as well as its own receptor CCR2. Together, these data suggest that CCR2 receptor signaling in the COC may regulate events critical for promoting cumulus oocyte expansion and/or oocyte maturation.Fil: Rojo, Julieta Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Jaworski, Juan Pablo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentin

Expression of caspase-2, -3, -8 and -9 proteins and enzyme activity in the corpus luteum of the rat at different stages during the natural estrous cycle

Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since caspases play a central role in apoptosis, we studied several initiators (-2, -8, and -9) and the main effector (-3) caspase in the CL during the estrous cycle of the rat. Two different populations of CL (old and new) were identified on ovaries at estrus and diestrus II (DII). Diminished (P < 0.05) luteal progesterone content and P450scc levels suggested that functional luteolysis occurred between the new CL at DII and old CL at estrus, whereas the decline (P < 0.05) in luteal weight indicated that structural regression was occurring between old CL at estrus to DII. Immunostaining for caspase-2 in luteal and endothelial cells appeared to increase as the luteal phase progressed, peaking at DII in the old CL. However, caspase-8 and -9 immunostaining showed little change with a slight increase at estrus in the old population. Notably, caspase-3 staining appeared to peak at DII in the new CL. Enzyme activity of caspase-9 increased (P < 0.05) in the new CL at DII, followed by that of caspase-2 and -3 in old CL at estrus. Caspase-8 activity did not change at any stage. The number of apoptotic cells increased at DII in the old CL. These results suggest an important role for this protease family during early events of luteolysis in the rat estrous cycleFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Bussmann, L. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Stouffer, Richard L.. Oregon Health & Science University; Estados UnidosFil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Felis catus as a model to study follicle biology in vitro

Purpose The current study was designed to evaluate the responseof individual intact antral follicles from adult femaledomestic cats to a luteinizing hormone (LH) stimulus in vitroby assessing cumulus-oocyte expansion (C-OE) and steroidproduction.Methods C-OE and steroid levels (estradiol [E2] and progesterone[P4]) obtained from individual antral feline follicles(n=366 follicles; n=56 cats) were analyzed after 12 or 24 hof culture in the presence or absence of LH (low [3.4 ng/ml] orhigh [100 ng/ml]).Results At the end of the culture, the highest percentage ofexpanded cumulus-oocyte complexes (COCs) was observedin the LH groups at 12 or 24 h in comparison to their controls(p<0.001). There was a significant increase in expandedCOCs when comparing LH concentrations (high vs. low) at12 or 24 h. Higher levels of both E2 and P4 were observed inthe media from antral follicles after 12 and 24 h of culture inthe presence of LH (both concentration, p<0.05). There wasno association between hormone levels and follicle diameter;high variability was observed in the steroid levels produced byantral follicles within all treatment groups.Conclusions These data indicate, for the first time, that felineantral follicles (0.5-2 mm) from different stages of the naturalestrous cycle can be cultured and will respond to an LH stimulus,based on an increase in steroid levels as well as C-OEafter 12 or 24 h in culture.Fil: Rojo, Julieta Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Linari, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Musse, Mariana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentin

Stromal-derived factor 1 directly promotes genes expressed within the ovulatory cascade in feline cumulus oocyte complexes

Purpose We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. Methods Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. Results MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. Conclusions These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.Fil: Rojo, Julieta Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Linari, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Young, Kelly A.. California State University; Estados UnidosFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentin

Oocyte maturation and in vitro hormone production in small antral follicles (SAFs) isolated from rhesus monkeys

PURPOSE: The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro. METHODS: Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5 mm; n = 5 monkeys) was evaluated after 34 h of culture in the absence (control) or presence of hCG. RESULTS: Of the total cohort (n = 91), 49 % of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1 mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes. CONCLUSIONS: These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.Fil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina. Oregon National Primate Research Center; Estados UnidosFil: Hennebold, Jon D.. Oregon National Primate Research Center; Estados UnidosFil: Stouffer, Richard L.. Oregon National Primate Research Center; Estados UnidosFil: Zelinski, Mary B.. Oregon National Primate Research Center; Estados Unido

C-C motif chemokine receptor 2 as a novel intermediate in the ovulatory cascade

Expression of immune function genes within follicle cells has been reported in ovaries from many species. Recent work from our laboratory showed a direct effect of the monocyte chemoattractant protein 1/C-C motif chemokine receptor 2 system within the feline cumulus oocyte complex, by increasing the mRNA levels of key genes involved in the ovulatory cascade in vitro. Studies were designed to evaluate if C-C motif chemokine receptor 2 acts as a novel mediator of the ovulatory cascade in vitro. Therefore, feline cumulus oocyte complexes were cultured in the presence or absence of a highly selective C-C motif chemokine receptor 2 antagonist together with known inducers of cumulus-oocyte expansion and/or oocyte maturation to assess mRNA expression of key genes related to periovulatory events in other species as well as oocyte maturation. Also, the effects of recombinant monocyte chemoattractant protein 1 on spontaneous or gonadotrophin-induced oocyte maturation were assessed. This is an in vitro system using isolated cumulus oocyte complexes from feline ovaries. The present study reveals the modulation of several key ovulatory genes by a highly selective C-C motif chemokine receptor 2 antagonist. However, this antagonist was not enough to block the oocyte maturation induced by gonadotropins or amphiregulin. Nonetheless, recombinant monocyte chemoattractant protein 1 had a significant effect on spontaneous oocyte maturation, increasing the percentage of metaphase II stage oocytes in comparison to the control. This is the first study in any species to establish C-C motif chemokine receptor 2 as a mediator of some actions of the mid-cycle gonadotrophin surge.Fil: Jaworski, Juan Pablo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; ArgentinaFil: Urrutia, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Dascal, Eduardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Jaita, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentin

Effect of gonadotropin-releasing hormone agonist and antagonist on proliferation and apoptosis of human luteinized granulosa cells

The GnRH agonist leuprolide acetate (LA) inhibited DNA synthesis in epidermal growth factor-stimulated human granulosa luteinized cell cultures. This effect was blocked by the prior addition of a GnRH antagonist antide (ANT), and this compound per se was able to produce a stimulatory effect of DNA synthesis on basal conditions. Leuprolide acetate produced an increase in the percentage of apoptotic cells, and when these two factors were co-incubated, ANT blocked the apoptotic effect produced by LA.Fil: Vitale, Alejandra Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Abramovich, Dalhia Nurit. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Peluffo, Marina Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Meresman, Gabriela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Local effects of the sphingosine 1-phosphate on prostaglandin F2 alpha -induced luteolysis in the pregnant rat

Since the regression of the corpus luteum (CL) occurs via a tightly controlled apoptotic process, studies were designed to determine if local administration of the antiapoptotic agent sphingosine 1-phosphate (S1P) effectively blocks the luteolytic action of prostaglandin F-2alpha (PGF-2alpha). On day 19 of pregnancy, 2 hr before systemic PGF-2alpha administration, rats were injected intrabursa with either S1P or vehicle (control). The activity of four caspases, which contribute to the initial (caspase-2, -8, and -9) and final (caspase-3) events in apoptosis was measured in pooled CL from four individual ovaries at 0 and 4 hr after PGF-2alpha injection. The expression of the phosphorylated form of AKT (pAKT) and tumor necrosis factor-alpha (TNF-alpha) was analyzed by ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 4 and 36 hr after PGF-2alpha injection. The activity of caspase-2, -3, and -8 was significantly greater by 4 hr after PGF-2alpha, but not caspase-9 activity. In contrast, expression of pAKT and TNF-alpha decreased significantly. Administration of S1P suppressed (P < 0.05) these effects, decreasing caspase activities and increasing pAKT and TNF-alpha expression. The administration of S1P also significantly decreased the percentage of luteal apoptotic cells induced by PGF-2alpha. PGF-2alpha treatment increased the prevalence of luteal cells with advanced signs of apoptosis (i.e., multiple nuclear fragments, chromatin condensation, or apoptotic bodies). S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P blocks the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3, and -8 activities and increasing AKT phosphorylation and TNF-alpha expression.Fil: Hernandez, Silvia Fátima. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Peluffo, Marina Cinthia. Oregon Health and Science University; CanadáFil: Bas, Diana Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Stouffer, Richard L.. Oregon Health and Science University; CanadáFil: Tesone, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin