The Study of Estrous Cycle Phases Female Rats With the Use of N-Acetylcysteine in Ovarian Graft

Ovarian graft may be the target of the biochemical effects of oxidative stress caused at the time of transplantation. In order to evaluate the effect of N-acetylcysteine on the ovarian graft, regarding the estrous cycle preservation , 50 female and virgin EPM-1 Wistar rats, weighing up to 250g, originating from CEDEME of UNIFESP, were kept in adequate sanitary conditions. , receiving their own food and water. Daily vaginal smears were performed to identify the estrous phase for 8 days. The animals were randomly distributed into 05 groups: 1st Group (GTx), saline was administered subcutaneously, 2nd (NAC 150mgKg), 3rd (NAC 300mg / Kg), 4th (NAC 600mg / Kg) and 5th (NAC 1200mg / Kg) , that were administered NAC subcutaneously on the abdominal face, 60 minutes before left unilateral ovarian transplantation in retroperitoneum and contralateral oophorectomy for purposes of histomorphological analysis, with colpocytological evaluation. Euthanasia was performed by means of anesthetic lethal dose in half of the animals on the 4th postoperative day, with a single vaginal smear collection and euthanasia on the rest of the animals, between the 14th and 16th days, after the material was collected in order to define the estrus phase. It was evaluated in the graft that the animals exhibited in all groups return of estrous cycle in the later phase of the post-transplant, with better definition of regular cycle in the highest dosages of N-acetylcysteine. N-acetylcysteine induced the return of the estrous cycle in the rats’ ovarian graft , mainly in the highest dosage, proving its effectiveness in revascularization of the tissue after ischemia and reperfusion. Keywords: Acetylcysteine. Reperfusion. Histocompatibility Antigens. Menstrual Cycle. ResumoO enxerto ovariano pode ser alvo dos efeitos bioquímicos do stress oxidativo causado no momento do transplante. Com o objetivo de avaliar o efeito da N-acetilcisteína no enxerto ovariano, quanto à preservação do ciclo estral, foram utilizados 50 ratos EPM-1 Wistar, fêmeas e virgens, pesando até 250g, originários do CEDEME da UNIFESP, mantidos em adequadas condições sanitárias, recebendo ração própria e água. Realizados esfregaços vaginais diários para identificação da fase estral durante 08 dias. Os animais foram distribuídos aleatoriamente em 05 grupos: 1º Grupo (GTx), administrada solução salina via subcutânea, 2º (NAC 150mgKg), 3º (NAC 300mg/Kg), 4º (NAC 600mg/Kg) e 5º (NAC 1200mg/Kg), aos quais foi administrada NAC por via subcutânea em face abdominal, 60 minutos antes do transplante unilateral esquerdo do ovário em retroperitônio e à ooforectomia contra-lateral para fins de análise histomorfológica, com avaliação colpocitológica. A eutanásia foi realizada por meio da dose letal do anestésico em metade dos animais no 4º dia de pós-operatório, realizado única coleta de esfregaço vaginal e a eutanásia no restante dos animais, entre o 14 º e 16º dia, após a coleta do material para definição da fase estro. Foi avaliado no enxerto que os animais apresentaram em todos os grupos retorno de ciclo estral na fase mais tardia do pós-transplante, com melhor definição de ciclo regular nas dosagens mais elevadas de N-acetilcisteína. A N-acetilcisteína induziu o retorno do ciclo estral no enxerto ovariano de ratas, principalmente na maior dosagem comprovando sua eficácia na revascularização do tecido após isquemia e reperfusão. Palavras-chave: Acetilcisteína. Reperfusão. Transplante. Ciclo Ovariano.

Pretreatment with melatonin improves ovarian tissue cryopreservation for transplantation.

Background: Melatonin has anti-inflammatory and antioxidative actions at the mitochondrial level. This indole may protect the ovarian graft during the cryopreservation process. Therefore, our study aimed to determine whether melatonin pretreatment improves rat ovarian graft quality. Methods: Twenty female rats were allocated to two study groups of ten animals each: 1) control group: ovaries cryopreserved using the standard protocol; and 2) melatonin group: ovaries cryopreserved in a medium with melatonin. Following 24-h freezing, whole ovaries underwent autologous and avascular transplants with retroperitoneal placement. After postoperative (PO) day 15, daily vaginal smears were obtained for estrous cycle characterization. Between PO days 30 and 35, animals were euthanized and ovarian grafts were recovered for histological and immunohistochemical (Ki-67, cleaved caspase-3, TUNEL, von Willebrand factor, estrogen, and progesterone receptors) analysis. The ovaries of the three remaining rats from each group were studied immediately after thawing to assess cryopreservation effects. The ANOVA and the Tukey tests were used, and the rejection level of the null hypothesis was set at 0.05 or 5% (p<0.05).Results: Melatonin promoted faster restart of the estrous cycle and increased the expression of mature follicles, collagen type I, von Willebrand factor, Ki-67, and cleaved caspase-3 on corpora lutea and estrogen receptors in the ovaries. There was a reduction in apoptosis by TUNEL on follicles, corpora lutea, and collagen type III. Conclusion: Melatonin may promote the quality of ovarian grafts through the evaluated parameters. Reproductive function enhancement could be further studied.