Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P < 0.05) after 7 days of culture in all treatments compared to the fresh control. A significantreduction in the percentage of primordial follicles and an increase (P < 0.05) in the growing ones were observed in all treatments compared to fresh control. Furthermore, both the control medium and 1 ng/mL EGF promoted an increase (P < 0.05) in follicular activation compared to other EGF treatments. The PCNA-positive cells in the EGF treatment were higher (P < 0.05) than in fresh control and α-MEM+. Pretreatment of ovarian tissue with PI3K inhibitor significantly inhibited (P < 0.05) α-MEM+-stimulated primordial follicle activation, but had no effect on EGF-stimulated activation (P > 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.(AU)

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.(AU)

Modelo experimental estável de aneurisma sacular em artéria carótida de suínos utilizando veia jugular interna / Stable experimental model of carotid artery saccular aneurysm in swine using the internal jugular vein

OBJETIVO: Desenvolver um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna. MÉTODOS: Em 12 suínos sadios, com peso variando entre 25 e 50kg, cinco machos e sete fêmeas, foi confeccionado aneurisma na artéria carótida comum direita. Após arteriotomia elíptica, foi realizada anastomose terminolateral com coto distal de veia jugular interna. O volume do aneurisma era calculado de maneira que o valor não excedesse em 27 vezes o valor da área da arteriotomia. Após seis dias, era realizada angiografia e análise microscópica do aneurisma para avaliar perviedade e trombose parcial ou total. RESULTADOS: Houve ganho de peso significante dos suínos no intervalo de tempo entre a confecção do aneurisma e a angiografia (p = 0,04). Foi observada perviedade aneurismática em dez suínos (83%). Ocorreram infecções de feridas operatórias em dois animais (16,6%), ambas com início de aparecimento em três dias após a confecção do aneurisma. Análise histológica dos aneurismas mostrou trombos ocluindo parcialmente a luz em nove suínos (75%). Nesses animais, observou-se que, em média, 9% da luz aneurismática estava preenchida por trombos. CONCLUSÃO: Pôde ser desenvolvido um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna.

OBJECTIVE: To develop an experimental model of stable saccular aneurysm in carotid of pigs using the internal jugular vein. METHODS: In 12 healthy pigs, weighing between 25 and 50kg, five males and seven females, we made a right common carotid artery aneurysm. After elliptical arteriotomy, we carried out a terminolateral anastomosis with the distal stump of the internal jugular vein. Aneurysm volume was calculated so that the value did not exceed 27 times the area of the arteriotomy. After six days angiography and microscopic examination were performed to assess patency of the aneurysm and the presence of total or partial thrombosis. RESULTS: There was a significant weight gain of pigs in the time interval between the manufacture of the aneurysm and angiography (p = 0.04). Aneurysmal patency was observed in ten pigs (83%). Operative wound infections occurred in two animals (16.6%), both with early onset, three days after the making of the aneurysm. Histological analysis showed aneurysm thrombus partially occluding the light in nine pigs (75%). In these animals, it was observed that on average 9% of the aneurysmal diameter was filled with thrombi. CONCLUSION: It was possible to develop a stable experimental model of saccular aneurysms in pig carotid artery by use of the internal jugular vein.