Morphological Interaction of Porcine Oocyte and Cumulus Cells Study on in vitro Oocyte Maturation Using Electron Microscopy

Morphological interaction of porcine cumulus-oocyte
complexes (pCOCs) was investigated on in vitro condition using
electron microscope (SEM and TEM). The totals of 1,923 oocytes
were round in shape, surrounded by Zona pellucida with layer of
cumulus cells ranging between 59.29-202.14 μm in size. They were
classified into intact-, multi-, partial cumulus cell layer oocyte, and
completely denuded oocyte, at the percentage composition of 22.80%
32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as
intact- and multi cumulus cell layer oocytes were further culturing at
37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h
in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2
mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 μg/mL porcine
follicle-stimulating hormone, 1 μg/mL LH, 1μg/mL estradiol with
ethanol, and 50 μg/mL gentamycin sulfate. On electron microscope
study, cumulus cells were found to stick their processes to secrete
substance from the sac-shape end into Zona pellucida of the oocyte
and also communicated with the neighboring cells through their
microvilli on the beginning of incubation period. It is believed that
the cumulus cells communicate with the oocyte by inserting the
microvilli through this gap and embedded in the oocyte cytoplasm
before secreting substance, through the sac-shape end of the
microvilli, to inhibit primary oocyte development at the prophase I.
Morphological changes of the complexes were observed after
culturing for 24-44 h. One hundred percentages of the cumulus layers
were expanded and cumulus cells were peeling off from the oocyte
surface. In addition, the round-shape cumulus cells transformed
themselves into either an elongate shape or a columnar shape, and no
communication between cumulus neighboring cells. After 44 h of
incubation time, diameter of oocytes surrounded by cumulus cells
was larger than 0 h incubation. The effect of hormones in culture
medium is exerted by their receptors present in porcine oocyte. It is
likely that all morphological changes of the complexes after hormone
treatment were to allow maturation of the oocyte. This study
demonstrated that the association of hormones in M199 could
promote porcine follicle activation in 44 h in vitro condition. This
culture system should be useful for studying the regulation of early
follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell
technology.

• M. Areekijseree
• W. Pongsawat
• M. Pumipaiboon
• C. Thepsithar
• S. Sengsai
• T. Chuen-Im

• Cumulus cells
• electron microscopy (SEM and TEM)
• in vitro
• porcine oocyte.

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