Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines

Abstract

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens

“…Cyclic AMP is an important regulator of growth and muscle tone (relaxing agent), and dynamic changes in cAMP play an active role on oocyte maturation [ 5 ], fertilization, and embryo implantation, development, growth, and differentiation [ 21 , 45 ]. Exposure of embryos to the adenylyl cyclase stimulator forskolin or the PDE IV inhibitor rolipram prevented embryo development [ 24 ]. Notably, exposure of mice to caffeine, a PDE inhibitor at high concentrations, during the implantation window impaired embryo transport, embryonic development and uterine receptivity, leading to abnormal implantation and pregnancy loss [ 23 ].…”

“…The pathophysiological significance of our finding that extracellular cAMP levels are induced by natural E2 and EE as much as by PDE inhibitors can only be speculated. The fact that various PDE inhibitors and cAMP inducers inhibit early embryo development [ 21 , 22 , 23 , 24 , 25 , 26 , 52 ] suggests that abnormal increases in cAMP may be deleterious. Indeed, cAMP has been shown to inhibit growth [ 7 , 8 ]; moreover, FT cells can convert cAMP to adenosine, which may result in activation of cAMP in distant cells, including the early embryo, via A 2B receptors [ 38 ].…”

“…Interestingly, treatment of embryos with agents that increase cAMP levels, for example AC activators or PDE inhibitors, induces deleterious effects on embryo maturation suggesting that cAMP can influence embryo growth and fertility [ 21 , 22 ]. Drugs known to elevate cAMP by inhibiting PDEs have been shown to impair oviductal embryo transport, embryo development and uterine receptivity [ 23 , 24 , 25 , 26 ]. Whether these effects are a result of cAMP produced within the FT or due to direct actions on the embryo is unclear.…”

Evaluación del YM976 como alternativo a las hormonas gonadotrópicas en la maduración in vitro de oocitos bovinos y su competencia para el desarrollo embrionario

“…A su vez, los niveles de AMPc son regulados por las enzimas fosfodiesterasas (PDE) que hidrolizan el AMPc en AMP, con una expresión especifica de compartimiento en la unidad folicular, donde la PDE tipo 3 (PDE-3) se expresa en el oocito y la PDE tipo 4 en las células del cúmulo (López et al, 2008;Gupta et al, 2020). Así entonces, la utilización del YM976 debería prevenir la degradación de AMPc en oocitos bovinos, resultando en su acumulación en cúmulos y oocitos, manteniendo estables las uniones gap, retardando la reanudación de la meiosis (Santana et al, 2019). Esta prolongación de las uniones gap durante la maduración in vitro en presencia de inhibidores de PDE-4 podría permitir el paso de metabolitos, iones, nucleótidos y aminoácidos, que mejoran la maduración citoplasmática del oocito, aproximándose a una sincronización entre la maduración citoplasmática y nuclear (Luciano et al, 2011).…”

“…Concentraciones bajas de YM976 (1 y 10nM) favorecieron el desarrollo de los embriones hasta la etapa de blastocisto, con porcentajes similares a los obtenidos en los oocitos madurados en presencia de gonadotropinas. Esto es concordante con el uso de Rolipram (López et al, 2008, Santana et al, 2019 y Burolactona I (Marques et al, 2011) como inhibidores de PDE en el medio de cultivo. Estas diferencias en el porcentaje de blastocistos con YM100, a pesar de que el porcentaje de maduración nuclear y de clivaje fueron similares a los resultados con YM1, sugieren que el parámetro del porcentaje de blastocisto es un criterio de evaluación de la competencia y calidad del oocito.…”