In previous studies, evidence has been obtained for functional specialization of cells in different regions of the membrana granulosa of Graafian follicles. To examine this phenomenon further, a quantitative cytochemical technique was applied to cryostat sections of rat ovary to measure the relative permeability of lysosomal membranes in cells of the peripheral region, the paraantral region, and the cumulus of the membrana granulosa in the ovulable type of follicle. Measurements were made on the 4 days of the estrous cycle in cells in these three regions. The preservation of the ultrastructure of the granulosa cells in the cryostat sections was first established by electron microscopy. Lysosomal membrane permeability was evaluated by incubating serial sections of ovaries for various periods of time with leucyl-β-naphthylamidase, a substrate of naphthylamidase, a lysosomal enzyme. The reaction was measured at each time point with a scanning and integrating microdensitometer. The time required for the lysosomal membrane to become fully permeable, an index of the original state of the permeability of the lysosomal membrane, was indicated by a sudden increase in the rate of enzyme activity. On each of the 4 days of the estrous cycle, the time of onset of increased enzyme activity (the inflection point) occurred first in the granulosa cells closest to the basal lamina (peripheral granulosa cells), next in the paraantral cells, and last in the cells of the cumulus (e.g. in proestrous, 35, 45, and 55 min, respectively). In all three regions, lysosomal membrane permeability increased on successive days of the cycle, i.e. with the growth of the ovulable type of follicle, being least in estrus and greatest in proestrus. Thus, lysosomes were most fragile in the region of the membrana granulosa thought to be involved in steroidogenesis and at a time in the cycle when steroid hormone production by Graafian follicles reaches its maximum. These data provide additional evidence of functional heterogeneity of the membrana granulosa and reinforce previous suggestions of the involvement of lysosomes in steroid hormone production.
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