BIOLOGY OF REPRODUCTION 14, 502-506 (1976). The Distribution of Collagenous Connective Tissue in Rat Ovarian Follicles LAWRENCE L ESPEY Reproductive Physiology Laboratory, Trinity University, San Antonio, Texas 78284 ABSTRACT The layers of thecah tissue in the ovarian follicles of mammals normally contain collagen. During the past year, it has been reported that the follicle wall of the rat uterus is an exception. The present report demonstrates that the rat follicle does contain collagen in abundance. This thecal collagen is very conspicuous when the tissue has been stained with phosphotungstic acid. However, collagen is not obvious in ultrathin sections that have been stained with the lead solutions which are commonly used in electron microscopy. These results maintain the current hypothesis that the mechanism of ovulation requires the disruption of collagenous connective tissue in the follicle wall. INTRODUCTION It was recently reported by Parr (1974) that no collagen could be detected in follicle walls at any time in the rat. This observation is in conflict with a recent description of the ultrastructure of rat ovarian follicles by Rhodin (1974). From this data Parr (1975) has concluded that a collagenase would not be expected to mediate ovulation [in the rat]. This is a critical deduction, because it is probable that the basic mechanism of ovulation in most (if not all) mammals is similar; yet, if collagenolysis is not involved in the rupture of rat follicles, then there is reason to doubt the extensive evidence that follicular collagen is decomposed during ovulation in other species (Espey, 1974). Furthermore. Parr s results imply that the term theca externa is a misnomer in the rat, because, by definition, thecal tissue should contain fibrous collagen. This report reveals that mature ovarian follicles of the rat do indeed possess a significant amount of collagen, and that this connective tissue can be consistently demonstrated by using a standard procedure for staining collagen. MATERIALS AND METHODS Nine mature Sprague-Dawley rats were administered pentobarbital ip on the day of proestrus and the whole ovaries were fixed in 2-4 percent glutaraldehyde adjusted to ph 7.2 with 0.1 M cacodylate buffer at 37#{176}C.After 1-2 h the ovaries were transferred to a petri dish which contained only the buffer. A total of 51 large, antral follicles were removed and postfixed for 30 mm with 1 percent 0504 in 0.1 M cacodylate buffer. Subsequently, the tissue was prepared for microscopy as previously described (Espey, 1971), except for modification in the staining procedure. The sections were stained with various combinations of Reynolds lead citrate, 5-7 percent uranyl acetate, and 1 percent phosphotungstic acid according to the staining procedures described by Hayat (1972). RESULTS In all follicles, at low magnification the thecal layers did not appear to contain collagen when the ultrathin sections were stained only with lead citrate (15-60 mm), sequentially with uranyl acetate (45 mm) and lead citrate (15 mm), or sequentially with lead citrate (15 mm) and phosphotungstic acid (15 mm) (Fig. 1). However, abundant collagen was consistently obvious if serial sections from the same follicles were stained only with phosphotungstic acid (5-15 mm) (Fig. 2). At high magnification the collagen is barely discernible following lead citrate staining (Fig. 3), yet very conspicuous when treated with phosphotungstic acid (Fig. 4). DISCUSSION The results show that the rat ovarian follicle Accepted January 15, 1976. Received November 19, 1975. contains an appreciable amount of collagenous connective tissue. Therefore, in order for ovula- 502
COLLAGEN IN RAT OVARIAN FOLLICLES 503 FIG. 1. A cross-sectional view of the thecal tissue between the surface epithehium (se) and theca interna (ti) at the apex of a mature follicle. Note the apparent lack of collagen fibers in the tunics albuginea (ta) and theca externa (te) in this section which has been stained with Reynolds lead citrate. A capillary (cap) appears in the region of the theca interns (7,750X). tion to occur, this layer of fibrous tissue must be degraded. The precise chemical changes which lead to the disruption of the follicle wall are not yet clear. In recent years there has been confusion and question as to whether ovarian follicles possess a true collagenase, i.e., a specific enzyme which hydrolyzes the triple helix of individual collagen fibrils in vitro (Harper and Gross, 1970; Espey, 1974; Parr, 1975). However, it may be that too much emphasis has been placed on the necessity of demonstrating the presence of a so-called true collagenase in ovulatory tissue. As Gross (1953) pointed out more than two decades ago, alterations in thecal tissue might be the result of changes in the collagen fibrils, the mucopolysaccharide matrix which cements these fibrils into
504 ESPEY FIG. 2. A serial section of the same follicle illustrated in Fig. 1, however the tissue has been stained with phosphotungstic acid instead of lead citrate. Note the conspicuous collagen fibers which are distributed in the extracellular spaces among the thecal fibroblasts (7,750X). bundles (fibers), or both. Therefore, it seems that a variety of enzymic or nonenzymic agents potentially could weaken this kind of tissue (Espey, 1974; Espey and Coons, 1976). Because of this possibility, a highly specific in vitro assay might not be adequate to identify the agent(s) responsible for the dynamic changes which occur in vivo in the collagenous wall of an ovulatory follicle. Regardless of the nature of the active substances, there is evidence (Espey, 1974) that follicular collagen becomes flaccid and distensible during the FIG. 3. Even at high magnification it is difficult to detect the collagen (c) which is distributed between the fibroblasts (f) if the tissue is stained with lead citrate (40,000X). FIG. 4. In contrast to Fig. 3 the collagen is obvious in tissue stained with phosphotungstic acid (40,000X).
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506 ES PE V process of ovulation, and, in a functional sense, these changes are collagenolytic. Therefore, more progress might be made towards understanding the mechanism(s) of the physiological remodelling of mammalian collagen in situ if more attention were given to the evaluation of false collagenases or thecalases, i.e., less specific enzymes which may be capable of loosening thecal tissue, but not necessarily capable of satisfying the highly specific definition of a true collagenase. In ovulation, such enzymes might include plasmin (personal communication with W. H. Beers, The Rockefeller University, N.Y.; Parr, 1975), or other types of fibrinolytic agents (Espey, 1974; Espey and Coons, 1976). In any event, the present data continue to support the theory that mammalian ovulation requires the decomposition of collagenous connective tissue. ACKNOWLEDGMENTS This work was carried out with the reliable assistance of Patrice J. Coons. I am thankful to Dr. Jon M. R. Rawson for his constructive criticism of the manuscript and to Dr. Andrew C. Cowles for his generous donation of the electron microscope and other facilities essential to the conduct of this study. This research was supported by NIH Grant HD-06371. REFERENCES Espey, L. L. (1971). Multivesicular structures in proliferating fibroblasts of rabbit ovarian follicles during ovulation. J. Cell Biol. 48, 437-442. Espey, L L. (1974). Ovarian proteolytic enzymes and ovulation. Biol. Reprod. 10, 216-235. Espey, L. L. and Coons, P. J. (1976). Factors which influence ovulatory degradation of rabbit ovarian follicles. Biol. Reprod. 14, 233-245. Gross, J. (1953). Evaluation of structural and chemical changes in connective tissue. N. V. Acad. Sci. 56, 674-683. Harper, E. and Gross, J. (1970). Separation of collagenase and peptidase activities of tadpole tissues in culture. Biochim. Biophys. Acts 198, 286-292. Hayat, M. A. (1972). Basic Electron Microscopy Techniques. Van Nostrand Reinhold Company, New York. pp. 75-88. Parr, E. L. (1974). Histological examination of the rat ovarian follicle wall prior to ovulation. Biol. Reprod. 11,483-503. Parr, E. L. (1975). Rupture of ovarian follicles at ovulation. J. Reprod. Fert. Suppl. 22, 1-22. Rhodin, J. A. C. (1974). Histology: A Text and Atlas. Oxford University Press, New York. p. 713. RECOMMENDED REVIEWS Espey, L L. (1974). Ovarian proteolytic enzymes and ovulation. Biol. Reprod. 10;216-235. Espey, L L and Coons, P. J. (1976). Factors which influence ovulatory degradation of rabbit ovarian follicles. Biol. Reprod. 14, 233-245.