An ultrastructural morphometric analysis of in vitro inhibition of luteinization of mature porcine ovarian granulosa cells by fluid from immature porcine follicles
Alexander, J. Steven
MetadataShow full item record
A comparison was made of the ultrastructure of preovulatory porcine granulosa cells after two days of culture in media which were either inhibitory or permissive of luteinization. Granulosa cells were collected from mature antral (6-12 mm) follicles. Follicular fluids were collected from immature and mature antral follicles. Granulosa cells were cultured in Medium 199 supplemented with either 50% fluid from immature porcine antral follicles (inhibitory of luteinization), fluid from mature antral follicles (permissive of luteinization) or adult female porcine serum (permissive of luteinization). Four x 10? cells were inoculated in 0.5 ml of medium in Falcon multiwell plates on carbon-coated round glass coverslips for 48 hours at 37 °C. Cells were fixed in 3% glutaraldehyde in 0.15 M phosphate buffer (pH 7.4) at 37 °C and allowed to reach room temperature during 45 minutes of fixation. Following buffer rinses, specimens were post- fixed in 1% OsO? in phosphate buffer for 30 minutes. Cells were dehydrated in a graded series of ethanol and embedded in a Epon-Araldite resin mixture while attached to the coverslips. Thin sections parallel to the plane of the coverslip were cut with glass knives, stained with uranyl acetate and lead citrate, and examined in a Hitachi HS-9 electron microscope. Some cultures were processed for scanning electron microscopy and were examined in a JEOLCO electron microscope. Cellular fine structure was examined quantitatively at the ultrastructural level through use of two stereo- logical techniques. Organelle volumes, in terms of percent cell cytoplasm, were determined by applying a point lattice grid to electron micrographs of 17,500 magnification. Membrane surface areas were determined using a line-intersect method on micrographs of 35,000 magnification. Statistical analysis was by single factor ANOV and Student-Newman-Keuls test. Granulosa cells cultured in fluid from immature follicles contained significantly less lipid bodies, mitochondria, lysosomal-like bodies, surface areas of outer and inner mitochondrial membranes, and agranular endoplasmic reticulum membranes than cells cultured in luteinization permissive media. Cells cultured in fluid from immature follicles also contained smaller and fewer whorled configurations of AER. Cells cultured in fluid from immature follicles and serum contained the same amount of GER membranes, which was significantly more than the GER content of cells cultured in fluid from mature follicles. No differences in Golgi complex cisternae surface areas between culture groups were observed. Scanning electron microscopic observations indicated a variety of surface features, such as microvilli and blebs, but no differences in cell shapes or surface features between culture groups were noted. Junctional complexes were classified as abutment and annular types. Portions of single cilium were occasionally observed in cell indentations. Analysis of all data indicated that granulosa cells cultured in fluid from immature follicles were less differentiated after 48 hours in culture in the direction of luteal cells than similar cells cultured in fluid from mature follicles or adult female porcine serum. Fluid from mature follicles was found to be the most permissive of morphological luteinization of porcine granulosa cells in vitro.