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We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

The present study was designed to determine whether rat pre-antral follicles can grow under in-vitro conditions. Emphasis is on whether follicular interaction is involved in in-vitro follicle culture, and furthermore its role in follicular development has been assessed. Pre-antral follicles were isolated mechanically from 10-day old rat ovaries. They were divided into small (50 microm < diameter < 100 microm) and large (120 microm < diameter < 200 microm) pre-antral follicles and cultured individually or in groups for 6 days in medium with or without fetal calf serum (FCS). Based on morphological criteria, large pre-antral follicles cultured in groups in serum-free medium had significantly higher survival rates than those cultured individually. In the presence of FCS, no significant difference was detected with respect to the survival. However, the large pre-antral follicles cultured in groups had a significantly greater increase in diameter than those cultured individually. Furthermore, follicles cultured in groups in FCS-containing medium exhibited significantly more follicular cell proliferation than those in serum-free medium, based on DNA measurement. The present culture system (with or without FCS) proved to be insufficient for small pre-antral follicles to stimulate growth comparable to that of large pre-antral follicles. The transmission electron microscopical (TEM) study revealed the ultrastructural differences between follicles cultured in FCS-containing and serum-free media. Taken together, the results suggest that interfollicular factors are involved in follicle development in vitro, which especially at the early folliculogenesis stage plays a positive role in terms of follicular growth as well as survival. The present culture model allows further investigation of factors that regulate early folliculogenesis., (Copyright 2000 Wiley-Liss, Inc.)

Porcine follicular maturation begins by recruitment from a continually proliferating pool of small antral follicles; those receiving the appropriate stimulus differentiate rapidly through a series of structural and functional changes. Such ovarian activity can be induced in prepubertal gilts with a single injection of equine chorionic gonadotropin (eCG). Average follicular diameter in eCG treated females increased from approximately 2 mm before stimulation to 3.5 mm by 24 hr after injection, with subsequent growth to ovulatory size (8 or 9 mm) by 96 hr. Both theca and granulosa layers increased in thickness and complexity, and a prominent capillary bed evolved immediately outside the basement membrane separating the two layers. Cytoplasmic organelles associated with increased metabolic activity and steroidogenesis proliferated within the first 24 hr. Progressive changes included increasing amounts of lipid and rough and smooth endoplasmic reticulum, with the latter occurring in vesicular or lamellar forms and as lipid-associated whorls. Bizarre mitochondrial forms also appeared, often associated with lipids. The amount and proportion of rough and smooth endoplasmic reticulum shifted dramatically as follicles matured. By 24 hr, rough endoplasmic reticulum in thecal cells increased from 4.2 to 7% of cell volume, while the amount in granulosa cells increased from less than 3.5% to more than 10%; the quantity remained relatively constant in the theca but declined to prestimulation values in the granulosa layer. Rough endoplasmic reticulum predominated over smooth in the first 24 hr following stimulation but the proportions were then reversed, so that more than 10% of both layers was composed of smooth endoplasmic reticulum by the time ovulation was imminent. Some follicles had or were in the process of ovulating by 96 hr. Their walls were collapsed into prominent folds with the two cell types beginning to mix. Slight undulations and some regions of discontinuity were observed in basement membranes of large unovulated follicles at this time. In specimens collected at 96 hr poststimulation and processed for retention of lipid, lipid-like material was noticeable in the extracellular matrix surrounding cells that contained organelle configurations suggestive of steroidogenesis.

Corpora lutea were obtained from ten pregnant rhesus monkeys during implantation, and the ultrastructure of granulosa and theca lutein cells was characterized. Specimens were individually staged with regard to the extent of implantation and the relationship to the rise in circulating progesterone and estrogen which is characteristic of early pregnancy. Structural changes characteristic of granulosa lutein cells occurring during implantation included: change in form of endoplasmic reticulum from predominantly agranular tubules to predominantly granular cisternae; reduction in size and number of lipid droplets; increase in area occupied by the Golgi and increase in length of the cisternae of the Golgi complex; development of numerous microvillus-lined intracellular spaces; increase in numbers of membrane-bound dense bodies including peroxisomelike bodies, multivesicular bodies within lobopodia, and other lysosomelike bodies; and alterations of mitochondrial cristae. These changes were suggestive of the production of a secretory protein, rapid utilization of existing steroid precursor reserves for the production of progesterone, and a reduction in capability for steroid precursor accumulation and processing by granulosa lutein cells. Structural changes characteristic of theca lutein cells occurring during implantation included an increase in size and number of lipid droplets, an increase in agranular endoplasmic reticulum, and an increase in area occupied by the Golgi complex. These changes were suggestive of an increased capability for steroid precursor accumulation and processing, perhaps for estrogen production, by the theca lutein cells.

The autonomic innervation of the interstitial gland of the rat ovary was studied on days 4, 6, 10, 14 and 18 of pregnancy with the acetylcholinesterase procedure, the Falck-Hillarp technique and electron microscopy after 5-hydroxydopamine treatment. Acetylcholinesterase-positive nerves were present as perivascular plexuses at all stages studied. Adrenergic nerves were present in the interstitial gland in all stages studied. The number and intensity of interstitial fluorescent adrenergic nerves increased as pregnancy progressed. Measurement of norepinephrine with the fluorometric procedure showed a highly significant (p less than 0.05) increase in the neurotransmitter in the ovary on days 14 and 18 as compared to day 4. Fine-structural studies after administration of the false transmitter, 5-hydroxydopamine, showed that the innervation of the steroidogenic cells of the interstitial gland is adrenergic.