Abstract: 【Objective】 During the ovine antral follicle development, the multilayer cumulus cells closely surround the oocyte to form a special structure named cumulus-oocyte complex (COC), existing in the follicular cavity. The ovine COC will be discharged tube from the mature follicle into the fallopian to be fertilized. The existing technologies for protein identification in COC choose the ovarian tissue containing follicles to be sliced up or to immunostain the whole COC after the COC isolated from the follicle. However, there are significant defects when using the two methods to detect protein expression in ovine COC from the antral follicle. In the present study, the conventional paraffin section technology was improved so as to meet the demand of ovine COC protein identification. 【Method】 The COCs were obtained from the antral follicles by aspiration, and the special structure containing multiple ovine COCs was created by paraffin embedding. Together with the healthy ovarian tissue, the target protein expression, urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), were identified in the COCs structure and the immunostaining effects and procedures were compared between the modified and conventional paraffin immunohistochemical technologies. 【Result】 The samples of ovine COCs and ovaries were obtained by paraffin embedding. The single COC sheets distributed in glass slides and the thin ovarian layer (5µm) was formed following sectioning, plastering and dewaxing. The indirect immunostaining results showed that, ? The target protein expression was consistent in the two types of COCs, which revealed that uPAR was expressed in both bovine cumulus and oocyte, while uPA was only expressed in cumulus. ? In the COC section, a well-preserved bovine COC structure with well-defined layers and easily identified protein location in either the oocyte or cumulus was observed. In the ovarian section, the follicle remained intact, and the clear protein localization in oocyte could be observed; however, cumulus layers and its protein localization were unclear. ?A comparison of the staining procedures between the both methods described herein showed that ovine COCs paraffin-sectioning technique provided a more simplified process and significantly shorter handling for fixation, dehydration, transparent processing, waxing and dewaxing. For example, fixation was shortened from 24 h to 2 h; dehydration was converted from the granual steps of 10 h to the two-step of 1 h; transparent processing was shortened from 30 mins to 10mins; waxing and dewaxing were simplified from 6 h to 1 h, and from the granual steps of 25 mins to the two-step of 10 mins, respectively. 【Conclusion】 The modified COCs paraffin sectioning technique showed a better immunostaining effect and featured the higher generation rate for COCs, the clearer outline of oocyte and cumulus cells, and the more simplified operation process. Based on the complete COCs structure, this new technology effectively detected the target protein expression in ovine COCs, with great methodological significance to reveal the developing mechanism of ovine follicles.

Key words: sheep, cumulus-oocyte complexes-like tissue, ovarian tissue, paraffin section, protein expression