As a basis for the interpretation of the experimental findings to be presented, it may be well to review the histological changes which occur in the development, differentiation, and maturation of the loose or areolar connective tissue from mesenchyme.Mesenchyme, from which all the connective tissues are derived, is composed of undifferentiated mesenchymal cells separated by amorphous intercellular substance with only a hint of a fibrillar appearance, which has been accentuated by fixation. In the umbilical cord, the intercellular substance is of a soft jelly-like consistency, and only a few tiny fibrils have appeared.As the mesenchymal cells differentiate into fibroblasts within such a matrix, fine fibers, ensheathed by a somewhat modified amorphous substance, first appear. Some of these are later cemented together into bundles as collagen fibers.FIGURE 1 is a phase-contrast photomicrograph of a fresh preparation of adult-mouse subcutaneous tissue lightly stained with toluidine blue. Here are fully clifferentiated cells such as the fibroblast and several mast cells. The intercellular substances, both amorphous and fibrous, i.e., collagen fibers of various sizes, reticular fibers, and elastic fibers (just out of focus), have been derived from the optically homogeneous amorphous substance of the mesenchyme.Material and Methods. The following experiments were undertaken in an attempt to determine the relation of connective tissue cells to the intercellular substances and the specific effect on both of the spreading factor of testicular extracts.Testicular extracts were prepared in the laboratory and compared with extracts kindly supplied by the Schering Corporation. No attempts were made to determine the potency of the extract other than the rate and degree of spread of India ink injected with the extract subcutaneously. Though the results varied in degree, they were similar in kind in all the series. 0.1 cm3 of testicular extract was injected subcutaneously under the shaved skin of each animal of the series. Tissues were removed from a control mouse, from an injected mouse within 5 minutes after the injection, and subsequently from each succeeding mouse in the series at daily intervals following the day of injection. In each case, fresh tissue studies were made by the "bulh" method and by dried spreads of the connective tissue stained metachromatically with toluidine blue. Pieces of skin were removed from the site of injection, fixed in formol-saline, embedded in paraffin, sectioned, and stained with a variety of stains.The two routine stains most useful for the fixed tissue were found to be hematoxylin and eosin for tissue relationships and a neutral dye of pina-
983Others become coated to form elastic fibers.Several series of mice and rats were used.