THE DEVELOPMENT OF RABBIT OVARIAN FOLLICLES FOLLOWING COPULATION*

Transcription

1 FERTILITY AND STERILITY Copyright" 1975 The American Fertility Society Vol. 26, No. 3, March 1975 Printed in U.SA. THE DEVELOPMENT OF RABBIT OVARIAN FOLLICLES FOLLOWING COPULATION* DAVID D. CHERNEY, PH.D, LffiERATO J. A. DIDIO, M.D., PHD, AND PIETRO MO'ITA, M.D.t Department of Anatomy, Medical College of Ohio, Toledo, Ohio The morphologic changes within the ovarian follicle leading up to ovulation are well established. 1-3 Nonetheless, some of the interrelated processes that enable release of the ovum from the mammalian ovary are still under investigation.4-6 This comprehensive light and electron microscopic study of the morphologic changes leading up to ovulation was undertaken in order to correlate the structural changes occurring with other parameters involved in ovum release which have been reported in the literature. MATERIALS AND METHODS Fourteen young, virgin, New Zealand White rabbits were divided into six groups of at least two each. One group was not mated (control); the other five were mated and the ovaries removed 2, 4, 6, 8, or 10 hours after copulation. The tenhour group contained four rabbits so that both preovulatory and postovulatory follicles might be obtained. Ovaries were removed by laparotomy from Nembutal-anesthetized animals and placed in cold 3% buffered glutaraldehyde (ph 7.4) for two hours. The follicles were excised by microdissection, postfixed Received April 4, *Supported in part by the Medical College of Ohio, General Research Support Fund, grant 5-S01- RR tvisiting professor, Fulbright scholarship. Permanent address: Department of Normal Human Anatomy, University of Rome, Italy. for two hours in buffered 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in an Araldite Epon mixture. 6 Sections (2 IJ-) were stained with 1% toluidine blue for light microscopy. An LKB Ultrotome and a Porter-Blum Sorvall MT2 ultramicrotome were used to trim blocks for thin sectioning. Sections were mounted on copper grids and stained with uranyl acetate and lead citrate for study with Philips 300 and a Hitachi HUllE-1 electron microscope. RESULTS Controls. In the unstimulated follicle, the ovum within the antrum was attached to the granulosa layer by column(s) of cells (Fig. 1). The cells of the granulosa layer generally appeared to be uniformly distributed and lay adjacent to the richly vascularized theca interna. The theca externa at the apex was composed of dense connective tissue as it became continuous with the tunica albuginea. The surface epithelium appeared as a single cell layer following the contour of the epithelium and underlying connective tissue. The evaginations of the epithelium and underlying connective tissue seen are a normal occurrence and are referred to as ovarian villi. 7 Higher magnification of the apex of the follicle in Figure 1 shows that the granulosa cells were radially arranged with the long axis of each cell perpendicular to the 257

2 258 CHERNEY ET AL March 1975 FIG. 1. Light micrograph of a rabbit ovarian follicle before copulation. The ovum (0) within the antrum (A) is attached to the granulosa layer (G) by columns of cells. The surface epithelium is evaginated at some places (arrows), forming ovarian villi (x 38). basal lamina (Fig. 2). The theca interna contained light-staining cells similar in appearance to granulosa cells (Figs. 1 and 2). The connective tissue fibers of the theca externa and tunica albuginea were arranged in whorls and were parallel to the surface epithelium. The nuclei of the surface epithelial cells were at the apex. With the electron microscope, the granulosa cells appeared elongated with lobulated nuclei (Fig. 3). A few ribosomes were attached to the moderately dilated endoplasmic reticulum, but most were in clusters within the cytoplasm. The granulosa cells were separated from the theca interna by a thin basal lamina. Large gaps between the granulosa cells were virtually nonexistent. A capillary within the theca interna was surrounded by cells and collagen fibrils. Superficial epithelal cells contained large, lobulated nuclei and were joined by tight junctional complexes (Fig. 4). Where these junctions did not appear, the cells were separated by small spaces containing t,

3 Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 259 / FIG. 2. Photomicrograph of the apex of an unstimulated ovarian follicle. The granulosa cells (G) are radially arranged and appear to stain similarly to cells of the theca interna (T). Nuclei of the surface epithelial cells are located at the apex (arrow) (x 83). " proteinoid material. The superficial epithelium rested upon a thin basal lamina that separated it from fibroblast-type cells and compact bundles of collagen fibrils of the theca externa and tunica albuginea. Rabbits two hours after copulation. The follicular changes two hours after copulation were minimal compared to controls. An increase in the width of the intercellular spaces between granulosa cells lining the follicular antrum was apparent. Another noticeable difference was a slight edema in the area directly under the surface epithelium. Rabbits four hours after copulation. At this stage of development, the increase in width of the intercellular spaces surrounding the granulosa cells was quite apparent (Fig. 5). The edema (noted at two hours) was very pronounced around the cells and vessels of the theca intema. In addition, both the cells of the theca intema and of the granulosa layer contained more vacuoles. Most of the surface epithelial cells at the follicular apex increased in size as a result of extensive fluid-filled vacuoles. There were no breaks in the walls of blood vessels at this stage; however, some degree of hyperemia was observed. With the electron microscope the clear spaces within the granulosa cells appeared to be fluid-filled dilations of cellu-. lar structures (Fig. 6). In the theca intema, the fluid causing the edema precipitated as a homogeneous proteinoid material. Where it surrounds vessels and cells, it disrupted the local relationships

4 260 CHERNEY ET AL March FIG. 3. Apex of a rabbit ovarian follicle before copulation. The granulosa cells have lobulated nuclei (N) and are separated from the theca intema by a thin basal lamina (arrow) (X 4,470). of the cells and capillaries within the theca interna. Rabbits six hours after copulation. The granulosa cells were widely separated by follicular fluid-filled spaces and many of the basal cells were attached to the region of the basal lamina by long cytoplasmic processes (Figs. 7 and 8). In the follicular wall of the apex, the light microscope revealed edema more prevalent than that observed in previous stages or in other subapical perifollicular areas (Fig. 7). Electron micrographs identified the edema as large fluid-filled vacuoles within and between the superficial epithelial cells (Figs. 8 and 9). The collection of fluid beneath the superficial epithelium resulted in the dissociation of the closely knit fibrils and fibroblast cells (Fig. 9). Rabbits eight hours after copulation. An increase in the accumulation of fluid was noted at this stage. The superficial epithelium appeared to have been pushed upward like a blister as a result of the accumulation of subepithelial fluid (Fig. 10). The cells of the superficial epithelium still maintained continuity; they were attached to each other by tight junctions. Concurrently, the fluid-filled spaces between the connective tissue elements of the tunica albuginea and the theca externa seemed to have increased. Rabbits ten hours after copulation. At this time, follicles in different stages of development could be identified. The...

5 Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 261 FIG. 4. Electron micrograph of the apex of a rabbit ovarian follicle before copulation. The cells of the superficial epithelium contain large, lobulated nuclei (N) and are separated from the theca externa by a thin basal lamina (arrow). Note the density of the cells and collagen bundles within the theca externa. (X 3,400). range was estimated to be from onehalf hour before ovulation (Fig. 11), just ready to rupture (Fig. 12), to about onehalf hour after ovulation (Fig. 13). These light micrographs show that, before ovulation, hyperemia can be observed as a hemostasis of dark-staining red blood cells within the vessels of the theca interna at the apex of the ovarian follicle (Fig. 11). As the apical wall became edemic, the extracellular fluid dissociated the dense elements of the wall and the apex became thinner as a result of the intrafollicular pressure. Finally, the blood vessels, which were weakened and unable to stretch any further, ruptured, releasing their contents into the antrum (Fig. 12). When the cohesive forces in the other cellular layers of the apex became less than the physical forces within the follicle, ovulation occurred, releasing the follicular fluid containing small amounts of blood and granulosa cells into the peritoneal cavity (Fig. 13).

6 262 CHERNEY ET AL March 1975 ;o. FIG. 5. Photomicrograph of the apex of a rabbit ovarian follicle four hours after copulation. Note the spaces surrounding the granulosa cells (G). Extracellular fluid or edema (E) can be observed around the cells and vessels of the theca interna. Many vacuoles can also be seen within the cells of the superficial epithelium (arrow) (x 600). \(

7 Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 263 FIG. 6. Electron micrograph of the apex of a rabbit ovarian follicle four hours after copulation. Note the fluid-filled dilations within the granulosa cells (G) and the edemic fluid separating the theca interna (T) from the basal lamina (arrow) (x 6,840)..

8 264 CHERNEY ET AL March 1975 FIG. 7. Light micrograph of an ovarian follicle six hours after copulation. Fluid-filled areas of the apex (arrows) will presumably be the place of rupture. The granulosa cells are not as close together as they were in earlier stages (x 83).

9 Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 265 FIG. 8. Apex of a follicle six hours after copulation. Granulosa cells (G) are attached to the basal lamina by long cytoplasmic strands. Edema extends around!''.lll!' (T) and vessels (V) of the theca interna (x 1,350)..

10 266 CHERNEY ET AL March 1975 FIG. 9. Electron micrograph of the apex of a follicle six hours after copulation. Note the decrease in the density and dissociation of the connective tissue under the superficial epithelium (arrow) as the result of edema (x 3,600).

11 .. Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 267 FIG. 10. Photomicrograph of a follicular apex eight hours after copulation. The superficial epithelium (arrow) has been pushed upward by the fluid accumulation beneath it. Note the dissociation of connective tissue of the theca externa, of the theca interna (T), and of the granulosa cells (G) (x 545). DISCUSSION The rabbit ovary is excellent for investigations on ovulation because at any one time in t~sexually mature, captive rabbit, follicles can be found in various stages of development. Those mature follicles may ovulate about ten hours after copulation. Mature follicles in the mammalian ovary are characterized by a fairly large antrum filled with fluid thought to arise from granulosa cells 8 or from plasma After copulation, the coital stimulus reaches the median eminence in a matter of seconds and luteinizing hormone-releasing factor (LH-RF) reaches the an-

12 CHERNEY ET AL March 1975 FIG. 11. Photomicrograph of a follicle about one-half hour before ovulation. Hemostasis occurs within blood vessels at the apex (small arrows) and is recognized as the dark-staining blood cells. The area of expected rupture is thin and has lost its denseness as a result of edema (large arrow) (x 41). FIG. 12. Photomicrograph of a follicle just prior to rupture. The ovum (0) is free within the antrum along with the contents of ruptured blood vessels (arrows). The apex appears as a single limiting boundary (x 70).

13 Vol. 26, No.3 RABBIT FOLLICULAR DEVELOPMENT 269 FIG. 13. Photomicrograph of a follicle about one-half hour postovulation. Vascular congestion and hemorrhages are evident (arrows) (x 65). terior pituitary and secretion oflh begins within minutes.u LH remains in the circulation for about two hours, but retains its potency as an ovarian stimulus for an additional four hours. 12 This hormone initiates the morphologic changes within the mature follicle leading up to ovulation. Our study indicates that the initial morphologic change within the stimulated preovulatory follicle is the development of edema at the apex of the follicle. This event coincides with the increase of estrogen (three hours postcoitus) and the site of production (theca cells). 13 The influx of fluid resulting in edema is probably a transudate from the blood since estrogen is known to increase the permeability of the blood-liquor barrier The hemostasis within the apical perifollicular capillaries is also thought to be a consequence of locally secreted estrogen, 18 since it was not observed in adjacent immature follicles. Both the edema and hyperemia are thought to result from locally produced vasoactive amines following estrogen initiation As fluid retention increases within the apex of the follicle, the elements of the connective tissue are increasingly separated by fluid in the interstitial spaces. Enzyme-related, fibrinolytic activity within the apex has been suggested Furthermore, in the endometrium of rats in late estrus, fibrinolytic activity in blood vessels has been observed during high

14 270 CHERNEY ET AL March 1975 levels of estrogen. 22 Prior to ovulation, estrogen production decreased and blood vessels ruptured releasing their contents into the antrum. The rupture is thought to be due to the low levels of estrogen and the high levels of follicle-stimulating hormone (FSH) that occur before ovulation. Clinical studies show a relationship between capillary fragility and low estrogen levels. 18 Human chorionic gonadotropin, which has FSH-like properties, can also induce ovarian hemorrhages.23 SUMMARY The morphologic development of rabbit ovarian follicles was studied by means of light and electron microscopy 0, 2, 4, 6, 8, and 10 hours after copulation. The progressive changes at the apex of the follicle included extensive edema, hyperemia, and rupture of blood vessels, and then rupture of the follicular wall. Ovulation occurred when the cohesive forces in the cellular layers of the follicular apex became less than the physical forces within the follicle. Most of the morphologic changes in the follicle as it approached ovulation could be related to the effects of locally produced estrogens. REFERENCES 1. Walton A, Hammond J: Observations on ovulation in the rabbit. Br J Exp Bioi 6:190, Hisaw FL: Development of the Graafian follicle and ovulation. Physiol Rev 27:95, Blandau RJ: Anatomy of ovulation. In Ovulation. Edited by L. Mastroianni. New York, Harper and Row, 1967, p Rondell P: Follicular processes in ovulation. Fed Proc 29(6):1875, Espey LL: Decomposition of connective tibsue in rabbit ovarian follicles by multivesicular structures of thecal fibroblasts. Endocrinology 88:437, Motta P, Cherney DD, DiDio LJA: Scanning and transmission electron microscopy of the ovarian surface in mammals with special reference to ovulation. J Submicrosc Cytol 3:85, Cherney DD, Motta P, DiDio LJA: Ovarian villi studied with light, scanning and transmission electron microscopy. J Microsc (Oxf) 17:37, Hadek R: Electron microscope study on primary liquor folliculi secretion in the mouse ovary. J Ultrastruct Res 9:445, Manarang-Pangan S, Menge AC: Immunologic studies on human follicular fluid. Fertil Steril 22(6):367' Carvaglios R, Cilotti R: A study of the protein in the follicular fluid of the cow. J Endocrinol 15:273, Frith DA, Hooper KC: The action of some ovulation inhibitors on the rabbit hypothalamus. Acta Endocrinol 66:221, Hilliard J, Hayward JN, Sawyer CH: Postcoital patterns of section of pituitary gonadotropin and ovarian progestin in the rabbit. Endocrinology 75:957, Bjersing L, Hay HF, Kann G, et al: Changes in gonadotropins, ovarian steroids and follicular morphology in sheep at estrus. J Endocrinol 52:465, Packham B, Kiehofer W: The movement of tritium-labeled water in the human ovarian follicle. Am J Obstet Gynecol 78(5):1012, Burr JH, Davies JI: The vascular system of the rabbit ovary and its relationship to ovulation. Anat Rec 111(3):273, Zachariae F: Studies of the mechanism of ovulation. Acta Endocrinol 27:339, Morris B, SaBB MB: The formation of lymph in the ovary. Proc Soc Med 164:557, Rona G: The role of vascular mucopolysaccharides in the hemostatic action of estrogens. Am J Obstet Gynecol 87(4):434, Wurtman RJ: An effect of luteinizing hormone on the fractional perfusion of the rat ovary. Endocrinology 75:927, Szego CM, Gitin ES: Ovarian histamine depletion during acute hyperaemic response to luteinizing hormone. Nature 201:682, Espey LL, Rondell P: Collagenolytic activity in the rabbit and sow Graafian follicle during ovulation. Am J Physiol 214(2):326, Kwaan HC, Albrechtsen OK: Histochemical study of fibrinolytic activity in the rat uterus in normal and hormonally induced estrus. Am J Obstet Gynecol 95(4):468, Loide SS: Ovarian hemorrhages induced in immature mice with human placental gonadotropin. Experientia 28(2):199, 1972,...