The porcine oocyte is encapsulated by cumulus cells forming the cumulus-oocyte complex (COC), which is essential for successful in vitro fertilization (IVF). This article details the isolation and maturation of COCs from porcine ovarian follicles, emphasizing the importance of intercellular interactions in maintaining oocyte viability.
The porcine oocyte is surrounded by a compact multi-layered cluster of cumulus cells forming the cumulus-oocyte complex or COC. The innermost cumulus cells extend the processes through the zona pellucida of the oocyte. These processes develop both gap and intermediate junctions, which help in the intercellular transfer of molecules between the oocyte and surrounding cumulus cells. These bi-directional interactions maintain the viability of COCs which is a crucial determinant of successful in vitro fertilization or IVF.
To isolate COCs, submerge a few porcine ovarian follicles in a Petri dish containing the maturation media. This media contains hormones necessary for in-vitro oocyte maturation. Dissect ovarian follicles to release COCs and follicular fluid into the Petri dish. This fluid supplements the maturation media with additional nutrients and hormones essential for oocyte competence.
Aspirate the released follicular content and transfer it to another Petri dish. Visualize the plate under a microscope to identify undamaged COCs that exhibit normal morphology consisting of round oocytes with moderately granulated cytoplasm and an intact polar body surrounded by cumulus cells.
Isolate a single COC and transfer it to the maturation media-containing central well of an IVF plate. Store the plate at an optimum temperature until further use.
To isolate the COCs from medium-sized follicles, transfer two to three ovaries to a sterile 10-cm Petri dish filled with HM. Gently cut the surface of the protruding ovarian follicles with a sterile surgical # 15 blade, which will cause the follicular fluid, and the COCs to flow out into the Petri dish. Aspirate the follicular contents with a 28-gauge needle attached to a disposable syringe, and transfer it into another Petri dish.
To prepare the IVF Petri dishes, add 1 milliliter of HM to the central wells, and place three to four drops of HM in the outer rings. Then, use a polycarbonate micropipette to move the undamaged COCs to drops of HM in the outer rings, and briefly rinse them three to four times. When finished, individually transfer them into the central well. Store the IVF plates in the incubator.
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