Techniques of female reproductive tissue morphology and viability assessment as tools for enhancing cancer research success

Abstract

The incidence of cancer has been higher in the last few decades. The challenge of developing new studies amongst prevention and treatment for these diseases has motivated numerous research groups through the world. Even so, the knowledge about etiology is still scarce and treatments already established are known to cause several side effects in patients, especially in the gonads. Women submitted to chemotherapy may develop gonad dysfunction and become infertile and exhibit iatrogenic or induced menopause. For these patients, reproductive biotechnologies, like ovarian tissue cryopreservation and transplantation are an interesting source to help resume the gonad function and recover fertility. Thus, the objectives of this internship project are: 1) perform immunohistochemistry and western blotting in gerbil mammary and ovary tissue obtained from the experiments developed during the doctorate at UNESP and; 2) participate in an extra project designed by GYNE/IREC/UCL and gain expertise in techniques like tissue cryopreservation, ultrastructure evaluation with transmission electron microscopy (TEM), X-ray computed tomography, in vitro and in vivo tissue culture with human and bovine ovarian tissue after cryopreservation. For the gerbil mammary gland and ovarian tissue already obtained at UNESP, double immunohistochemistry for some markers not available in our institution and already developed at IREC, such as Cx43 and KI67 will be developed. We also aim to perform the western blotting technique in the gerbil mammary gland tissue for detection of the proteins EZH2 and Esr-1. In addition to that doctorate project complementation, it will be developed an experiment to establish an ovarian tissue vitrification protocol. Firstly, a test will be performed at bovine ovaries, which will be warmed, in vitro cultured and transplanted to nude mice. As second part of the experiment, human ovarian biopsies will be obtained from patients to compare the new vitrification protocol to a slow freezing method. Afterwards, the warmed or thawed tissue strips will be xenotransplanted to nude mice for 6 months. Evaluation of the xenografted ovarian tissue will include X-Ray computed tomography in order to certify the cryoprotectant perfusion before vitrification; immunohistochemistry for proliferation (KI67), apoptosis (TUNEL and active caspase-3), vascularization (CD-34) and vessel maturation (a-ASMA) antigen detection; and TEM for membrane integrity and lysosome evaluation. The development of all techniques proposed here must contribute to the improvement of our laboratory abilities and consequently the quality and visibility of publications. It will also enable the establishment of a new research field in our group, such as female reproductive tract morphology.