The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

Abstract The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-olds lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.(AU)

EFEITOS DO INIBIDOR DA RHO QUINASE (ROCK, Y-27632) SOBRE O ENDOTÉLIO CORNEAL EM CÃES SUBMETIDOS À FACOEMULSIFICAÇÃO

A catarata situa-se entre as afecções oculares mais frequentes em cães. A facoemulsificação é o método que melhor se adéqua à sua terapia e o preferido entre os oftalmologistas. Dentre as complicações pós-operatórias destacam-se lesões endoteliais que podem produzir descompensação permanente da córnea, alterando a sua transparência. Buscou-se avaliar os efeitos do inibidor de ROCK (Y-27632), na proteção endotelial, em cães submetidos à facoemulsificação. Utilizaram-se 20 olhos de 10 pacientes, machos ou fêmeas, com sete a 12 anos, portando catarata senil bilateral. Dois grupos foram concebidos: Olhos controles (Oc) (n=10), receberam solução salina balanceada e Olhos Tratados (Ot) (n=10), receberam intracameralmente, 0,3 mL de Y-27632, na concentração de 100 mol/L, imediatamente após a sutura corneal. O endotélio corneal foi avaliado à microscopia especular de não contato, antes e em diferentes momentos após a facoemulsificação. Avaliaram- se a densidade (células/mm2) e a área celular (m2), a espessura corneal (mm), a hexagonalidade (%) e o coeficiente de variação do tamanho celular (%). Valores de P iguais ou inferiores a 0,05 foram considerados significativos. Com relação à densidade de células endoteliais, ambos os grupos apresentaram perdas, sem diferença estatística. Relativamente à espessura corneal e hexagonalidade, não houve diferença. No Oc, a área celular aumentou significativamente entre os momentos avaliados, o que não foi observado em Ot. Admite-se que a aplicação intracameral de 0,3mL contendo 100mol/L de Y-27632 durante o procedimento de facoemulsificação em cães em condições normais, colabora com a estabilidade das células do endotélio corneal, diminuindo sua perda (7,4%) em comparação com sua não utilização (11,8%).

Cataract is among the most common eye diseases in dogs. Phacoemulsification is the method that best fits its therapy. Among the complications related to this surgery, there are endothelial injuries that can induce permanent corneal decompensation, altering its transparency. The goal of this study was to evaluate the effects of ROCK inhibitor Y-27632 on endothelial protection in dogs undergoing phacoemulsification. Twenty eyes of 10 patients, male or female, aged 7 to 12 years, with bilateral senile cataract were used. Two groups were conceived: Control eyes (Oc) (n = 10), that received balanced saline solution (BSS) and Treated Eyes (Ot) (n = 10), which received intracamerally 0.3 mL of Y-27632 at a concentration of 100 mol. / L, immediately after corneal suture. The corneal endothelium was assessed via non-contact specular microscopy at multiple time points before and after phacoemulsification. Cell density (cells/mm2) and area (m2), corneal thickness (mm), hexagonality (%), and the coefficient of variation of cell size (%) were all assessed. P values equal to or less than 0.05 were considered significant. With respect to the density of endothelial cells, both groups showed losses, without statistical difference. There were no differences in corneal thickness and hexagonality. In Oc, the cellular area increased significantly between the evaluated moments, what was not observed in Ot. It is admitted that the intracameral application of 0.3mL containing 100mol/L of Y-27632 during the phacoemulsification procedure in dogs under normal conditions, collaborates with the stability of the corneal endothelium cells, decreasing their loss (7.4%), compared to their non-use (11.8%).