BINOVULAR FOLLICLES IN THE ADULT HUMAN OVARY

Transcription

1 FERTILITY AND STERILITY Copyright c 1978 The American Fertility Society Vol. 29, No.3. March 1978 Printed in U.S.A. BINOVULAR FOLLICLES IN THE ADULT HUMAN OVARY LUCIENNE PAPADAKI, PH.D.* Department of Gynaecological Pathology, The Bland-Sutton Institute of Pathology, The Middlesex Hospital Medical School, London WIP 7LD, England The human ovary may contain binovular or polyovular follicles at birth, but they are unusual later in life. Binovular follicles were found in bilateral ovarian biopsies from a patient with primary amenorrhea who had been treated with exogenous gonadotropins. The fine structural morphology of these follicles has shown that both oocytes were in the resting meiotic prophase, that their respective vitelline bodies faced each other across the intervening plasma membranes, and that the adjacent oocytes made mutual contacts similar to those found between granulosa cells and oocytes. From the appearance of the multivesicular bodies in nearby stromal fibroblasts it is suggested that these follicles had formed by fusion of two adjacent primordial follicles by a mechanism similar to the depolymerization of the ground substance in the apex of the Graafian follicle that leads to ovulation. Polyovular follicles and polynuclear oocytes have been reported in the ovaries of most mammalian species; they are usually rare except in the opossum, where they may be found in astonishing numbers In man they have been observed sporadically since the end of the last century3.5 (for early references see Hartman1). Binovular follicles are more frequent than ones with larger numbers of ova. 2 At birth, the human ovary may show numerous binovular and polyovular follicles which disappear shortly thereafter. 6 7 Such follicles are probably persistent remains of pairs or groups of germ cells from the sex cords that have failed to become separated into uniovular follicles. In the adult the presence of polyovular follicles and multinuclear oocytes has been linked with both estrogenic influence 8 9 and gonadotropin stimulation During the course of a continuing study of women who are being investigated for amenorrhea and infertility,l1 bilateral ovarian biopsies revealed the presence of binovular follicles in one patient. It was, therefore, of considerable interest to examine the fine structure of these follicles in the hope that the ultrastructural Received September 16, 1977; accepted October 6, *Reprint requests: Lucienne Papadaki, Ph.D., The School of Pathology, The Middlesex Hospital Medical School, Riding House Street, London WIP 7LD, England. morphology might provide some evidence for the mode of origin of these unusual structures. MATERIALS AND METHODS Laparoscopic, bilateral ovarian biopsies were taken from a patient aged 27 with primary amenorrhea. She had previously been treated with several courses of human menopausal gonadotropin (Pergonal; Searle Laboratories, High Wycombe, Bucks., England) containing follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in a 1: 1 ratio. Thin strips of ovarian cortex from the biopsies were fixed both for electron microscopy, in either 0.1 M cacodylate-buffered 2% glutaraldehyde (ph 7.4) or one-half strength Karnovsky fixative,12 and for light microscopy. After 2 t03 hours' fixation for electron microscopy, 0.5-mm cubes containing follicles were obtained by microdissection of the tissue strips. They were then postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded ethanol-water mixtures, and embedded in Epon 812. Thin sections were cut using an LKB Ultrotome, stained with uranyl acetate and lead citrate, and examined in either a Siemens Elmiscop I or a Philips 200 electron microscope. 342

2 Vol. 29, No.3 BINOVULAR FOLLICLES 343.,.. FIG. 1. Photomicrograph of the ovarian cortex showing a binovular follicle (arrow) in a field containing primordial and early primary follicles (hematoxylin and eosin, x 180). RESULTS The laparoscopic biopsies measured approximately 0.4 x 1.0 x 0.3 cm. Light microscopic examination of serial sections revealed a basic histologic structure common to many patients with amenorrhea, in which there appears to be an abundance of primordial and primary follicles but no evidence of maturation beyond the secondary follicular stage and no recent luteal formation.ll Remarkable in these bilateral ovarian biopsies, however, was the incidence of approximately 1 in 20 of binovular primordial or primary follicles. In these follicles two oocytes were lying in direct contact with each other (Fig. 1). Other than this, there were no nuclear or cytoplasmic features which distinguished these oocytes from those in the remaining population of uniovular follicles. All of the binovular follicles examined in the electron microscope were primordial and were encompassed by a flattened layer of follicular epithelial cells (Fig. 2). The two oocytes within each follicle were somewhat flattened along their apposing surfaces, with the result that, in midcross-section, their respective nuclei were nearer to the plasma membrane along the flattened side than elsewhere (Fig. 2). The oocyte nuclei were spheroidal and the nuclear membrane profiles were smooth with prominent nuclear pores. The nuclear chromatin was mainly dispersed with occasional clumps of heterochromatin near the periphery. The nucleoli had a reticular structure typical of that seen in the dictyotene stage or resting prophase of meiosis (Fig. 2). The two oocytes within each binovular follicle were so polarized that their crescent-shaped vitelline bodies faced each other across the intervening plasma membranes, resulting in a mirrorimage orientation (Figs. 2 and 3). These bodies were composed of compound aggregates, portions of annulate lamellae, coarse fibers, and mitochondria. The mitochondria were frequently distorted, an artifact that has been assoicated with the tonicity of the glutaraldehyde fixativey Unfortunately, the binovular follicles in the tissue processed for electron microscopy were fixed in glutaraldehyde and not in the more hypertonic half-strength formaldehyde-glutaraldehyde mixture of Karnovsky.12 The close apposition of cell membranes of the adjacent germ cells in the binovular follicles was similar to that found between oocytes and granulosa cells. There were interdigitating cytoplasmic projections along the plasma membrane interfaces, and coated vesicles occurred either adjacent to or in continuity with the oocyte membranes (Fig. 4, a and b). This fine structural relationship is well known and, when present between oocytes and granulosa cells, is indicative of the influential role of the latter in the nutrition and maturation of the germ cells. 13, 14 It is probable, therefore, that the two oocytes in the binovular follicles are mutually interdependent. The pregranulosa cells surrounding the binovular follicles were attached to each other by typical desmosomes (Fig. 4b). They also made direct contact with the oocytes either immediately at the cell body or through cytoplasmic projections from their surfaces (Figs. 4b and 5a). The latter junctional complexes were desmosomelike and were identical with the pregranulosa

3 344 PAPADAKI cell-oocyte attachments found in uniovular follicles, which Hertig and Adams 13 and Zamboni 15 have referred to as desmosomes. Although these complexes are more commonly associated with microtubules and small projections of endoplasmic reticulum, especially on the oocyte side, than with the microfilaments or tonofilaments characteristic of the common spot desmosome, they probably March 1978 perform the same function. Since they resemble more closely the early stages in desmosome formation, it may be preferable to refer to them merely as rudimentary or developing desmosomes. More important, perhaps, was the occurrence in the binovular follicles of similar rudimentary desmosomes along the closely apposed oocyte membranes of the adjacent germ cells (Fig. 5b ), which FIG. 2. Part of a binovular follicle surrounded by pregranulosa cells (G). The two nuclei (N) are in close proximity to the apposing oocyte plasma membranes, and the nucleolus ( nuc) is reticular in structure. Compound aggregates ( ca), part of the annulate lamellae ( al), mitochondria ( m), and coarse fibers (cf) can be seen, all of which are constituents of the vitelline bodies (x5,600).

4 Vol. 29, No.3 BINOVULAR FOLIJCLES 345 FIG. 3. Mirror-image orientation of the two vitelline bodies in a binovular follicle. Note the compound aggregates (ca), mitochondria (m), and coarse fibers (cf) adjacent to the oocyte-oocyte interface. Their respective nuclei (N) are also nearby (x8,500). suggests that there is mutual support between the two oocytes. In other areas of the ovarian cortex, uniovular follicles were positioned very close to each other where there were often only one or two slender fibroblasts, together with a few collagen and reticular fibers, in the intervening space (Fig. Sa). Sometimes a follicle was found in which the flattened layer of pregranulosa cells was very attenuated or even incomplete. In such instances the oocyte plasma membrane abutted directly on the follicular basal lamina and was thereby exposed to the underlying stroma (Fig. 6b). The stroma surrounding both the binovular and uniovular follicles consisted mainly of fibroblasts and intervening collagen. Within the peripheral cytoplasm of a number of fibroblasts were unusual multivesticular structures (Fig. 7a). The vesicles within these structures sometimes appeared tubular, while others were more rounded; both contained diffuse material of moderate electron density. There was no particular organelle with which they could be associated. Rarely did these multivesicular structures protrude from the cell, but sometimes the cytoplasmic ground substance around them had become dissociated (Fig. 7a). There were also groups of vesicles occurring beneath the plasma membrane of other fibroblasts (Fig. 7b). These clusters of microvesicles are probably precursors of the multi vesicular bodies. Similar multivesicular structures found in thecal fibroblasts at the time of ovulation are thought to play an important role in the decomposition of the follicle prior to rupture. 16 DISCUSSION There are several possibilities that should be examined when considering the origin of these binovular follicles. Binovular and polyovular follicles are not uncommon in the human infant. 7 When they do occur they are probably remnants of the syncytial groups of cells in the sex cords which have failed to separate and become individually encapsulated following the mitotic phase of oogenesis. If such follicles were examined

5 346 PAPADAKI March 1978 FIG. 4. Similarity of plasma membrane interfaces between (a) the two oocytes (0) and (b) a pre granulosa cell (G) and an oocyte (0). In both there is interdigitation of cytoplasmic projections (p) and coated vesicles ( cv) in continuity with the oolemma. Figure 4b also shows a junctional contact (j) between the pregranulosa cell and oocyte as well as a typical desmosomal attachment (d) between two pregranulosa cells (a, x 25,000; b, x 24,200). in the electron microscope, one might expect to find intercellular bridges between the two oocytes. Intercellular bridges are generally believed to be formed during the mitotic stages of oocyte development, resulting from incomplete cytokinesis and characterized by continuity of the cytoplasm of the conjoined cells. 17 It appears that these intercellular bridges synchronize oogenesis/7 with subsequent separation of individual oocytes probably be the pregranulosa cells, during follicluar formation. 18 Thus, if the binovular follicles observed in the adult ovaries in the cur- FIG. 5. Junctional complexes between (a) a pregranulosa cell and an oocyte and (b) two oocytes in a binovular follicle. Associated with these rudimentary desmosomes are both microtubules (arrow) and elements of the endoplasmic reticulum ( er) (a, x25,500; b, x55,900).

6 Vol. 29, No.3 BINOVULAR FOLLICLES 347 rent study were surviving pairs of oocytes which had separated as a pair from the syncytial groups of germ cells, presumably their intercellular bridges would also remain. However, after an extensive search, not even the remnants of bridges could be found. Fifty years ago Hartman 1 suggested that polyovular follicles may result from division of multinucleate ova. In neither ovarian biopsy, however, were any binucleate ova seen from which the binovular follicles could have arisen. Some form of parthenogenetic cleavage may have occurred. That is, the primary oocyte may have divided equally instead of unequally during the first meiotic division, giving rise to two secondary oocytes. Maturation of follicular oocytes up to a parthenogenetic cleavage in vitro is not unknown and has been attributed to the extreme sensitivity of the oocytes to a nonspecific environment rather than stimulation by steroid hormones or gonadotropins. 19 Indeed, parthenogenetic embryos do occur in mammals. 20 Presumably this would happen only after the primary oocyte had reached the Graafian follicle stage when meiosis is resumed. All of the binovular follicles in question, however, were in the primordial or early primary stage of development. Thus, parthenogenesis does not appear to be im- FIG. 6. a, The close proximity of two primordial follicles. Only two slender fibroblasts (F) and a few bundles of collagen separate the pregranulosa cells (G). The two oocytes (0) are also apparenl b, Part of a primordial follicle. The surrounding layer of pre granulosa cells (G) is discontinuous and the oocyte (0) rests directly on the follicular basal lamina (bl), Cytoplasmic projections from a pregranulosa cell are protruding into the ooplasm (arrow) (a, x20,400; b, x29,700).

7 348 PAPADAKI March 1978 FIG. 7. a, Part of a cortical stromal fibroblast. A multivesicular body (arrow) appears in the pripheral cytoplasm around which the cytoplasmic ground substance has become electron-lucent and dissociated. b, Microvesicles grouped beneath the plasma membrane of a stromal fibroblast representing a stage in the formation ofthe multivesicular structures (a, x31,000; b, x 27,000). plica ted in any way in the formation of these follicles. Mitotic division of a primordial follicle seems extremely unlikely, as the germ cells will all have entered the resting meiotic prophase at the time of birth or soon after.21 There they remain until antrum formation takes place, when condensation of the nucleolar material is one of the earliest observable changes as the oocyte proceeds toward meiotic metaphase.22 One would, therefore, have to envisage the most unlikely occurrence of a reversion to a mitotic phase. Finally, it seems most probable that, in the adult, binovular follicles are formed by fusion of two nearby follicles. Very often two primordial follicles can be observed lying in close proximity with very little intervening collagen or connective tissue cells. Indeed, in some primordial follicles the single layer of flattened pregranulosa cells may be very attenuated or even incomplete, with the result that a portion of the oocyte plasma membrane may abut directly against its surrounding basal lamina. This may in turn lie against that of an adjacent follicle, an observation not unknown in the adult human ovary as well as in other primates.1s On the other hand, with regard to polynuclear oocytes, existing evidence favors the view that they arise by fusion of two or more adjacent mononuclear germ cells Underlying the events which could lead to follicular fusion are a number of possible hormonal influences. Estrogen stimulation has long been implicated in the formation of polyovular follicles. Greater numbers of multiple follicles are seen during the follicular phase than during the luteal phase of the ovarian cycle in the squirrel monkey.8 Moreover, after prolonged treatment of certain primates with diethylstilbestrol, up to 30% of the primordial follicles are found to be polyovular.9 Harrison8 also observed that, in the goat, multiovular follicles were mainly associated with estrus and with "a certain period in early pregnancy," occasions which could be associated just as easily with high gonadotropin levels as with estrogenic stimulation. In general, treatment with oral contraceptive steroid hormones inhibits follicular development; mainly primordial and early primary follicles are found with the occasional antral follicle. 23 Of all of the ovarian tissue that has been examined from women taking these estrogen-progestogen compounds,. no binovular follicles or binuclear oocytes have been reported. However, it has been suggested that estrogen at the preovulatory peak can change the physical structure of the connective tissue, thereby assist-

8 Vol. 29, No.3 BINOVULAR FOLLICLES 349 ing dissociation of collagen at ovulation.24 Nevertheless, neither estradiol nor progesterone, separately or in combination, has any effect on the lysis of reconstituted collagen in vitro.25 In a patient with primary amenorrhea, however, whose ovaries contain no maturing follicles, it seems unlikely that these hormones are implicated. The suggestion has also been made that the presence of polynuclear oocytes in the human adult ovary may be linked with high gonadotropin levels.5 Such abnormalities have been ascribed more specifically to fluctuations in the ratio of FSH to LH, FSH permitting development of multinucleate ova while LH favors polyovular follicles. lo Furthermore, Jones4 has found binovular follicles in operative specimens after treatment of patients with gonadotropins. It could be, therefore, that under particular circumstances stimulation by exogenous gonadotropins may actually induce fusion of adjacent follicles. In infertile women whose ovaries show little evidence of follicular maturation, the use of exogenous gonadotropins more readily accounts for the stimulus for follicular fusion. The same process which allows the mature ovum to be released l6 may be invoked to explain the mechanism of follicular fusion. In the apex of a Graafian follicle shortly before ovulation, there is dispersion of collagen fibrils and ground substance. 16 After the midcycle gonadotropin peak a collagenolytic enzyme is elaborated from the multivesicular structures present in cytoplasmic processes of the thecal fibroblasts. 16 Thus, the following sequence of events may have occurred in the present case. The high levels of exogenous gonadotropins persistently administered to this patient could have resulted in the release of hydrolytic and proteolytic enzymes from multi vesicular bodies in the cytoplasmic processes of the interfollicular stromal fibroblasts. This would lead to degradation of connective tissue and de polymerization of the ground substance between closely adjacent follicles. The consequent reduction in tensile strength of the connective tissue environment could induce fusion of the follicles, followed by dispersion or suppression of the active enzyme. Certainly after ovulation further collagenolysis is suppressed. 16 Follicular fusion being the exception rather than the rule after gonadotropin stimulation may be explained by the presence of a naturally occurring lytic enzyme inhibitor which prevents collagenolysis from extending beyond the site of ovulation.25 Fusion may, however, be triggered when exogenous gonadotropins are administered in amounts that create abnormally high levels within the ovary. Recently, Woodruff and his colleagues26 have presented new evidence suggesting that the prevalence of binovular follicles and/or binuclear oocytes in the human ovary is greater than was previously thought. Although all of the patients in their study had menstruated, many had infertility problems, and it would be interesting to know whether any had been treated with exogenous gonadotropins prior to oophorectomy. Acknowledgments. Grateful thanks are due to Dr. J. O. W. Beilby for helpful criticism, to Dr. R. M. Hicks for providing laboratory facilities, and to Mr. A. L. E. Barron for his skilled photographic assistance. REFERENCES 1. Hartman CG: Polynuclear ova and polyovular follicles in the opossum and other mammals, with special reference to the problem of fecundity. Am J Anat 37:1, Brambell FWR: Ovarian changes. In Marshall's Physiology of Reproduction, Vol 1, Pt 1, Third Edition, Edited by AS Parkes. London, Longmans, Green and Co Ltd, 1956, P Pankratz DS: Some observations on the Graafian follicles in an adult human ovary. Anat Rec 71:211, Jones GS: Induction of ovulation. Annu Rev Med 19:351, Kennedy JF, Donahue RP: Binucleate human oocytes from large follicles. Lancet 1:754, Bacsich P: Multinuclear ova and multiovular follicles in the young human ovary and their probable endocrinological significance. J Endocrinol 6:i, Curtis EM: Normal ovarian histology in infancy and childhood. Obstet Gynecol 19:444, Harrison RJ: The changes occurring in the ovary of the goat during the estrous cycle and in early pregnancy. J Anat 82:21, Graham CE, Bradley CF: Polyovular follicles in squirrel monkeys after prolonged diethylstilboestrol treatment. J Reprod Fertil 27:181, Collins DC, Kent HA Jr: Polynuclear ova and polyovular follicles in the ovaries of young guinea pigs. Anat Rec 148:115, Steele SJ, Beilby JOW, Papadaki L: Visualization and biopsy of the ovary in the investigation of amenorrhea. Obstet Gynecol 36:899, Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell BioI 27:137 A, Hertig AT, Adams EC: Studies on the human oocyte and its follicle. 1. Ultrastructural and histochemical observations on the primordial follicle stage. J Cell BioI 34: 647, Gondos B: Granulosa cell-germ cell relationship in the developing rabbit ovary. J Embryol Exp Morphol 23: 419, 1970

9 350 PAPADAKI 15. Zamboni L: Fine morphology of the follicle wall and follicle cell-oocyte association. BioI Reprod 10:125, Espey LL: Ovarian proteolytic enzymes and ovulation. BioI Reprod 10:216, Gondos B, Zamboni L: Ovarian development: the functional importance of germ cell interconnections. Fertil Steril 20:176, Stegner H-E, Wartenberg H: Elektronenmikroskopische Untersuchungen an Eizellen des Menschen in verschiedenen Stadien der Oogenese. Arch Gynaekol 199:151, Nicosia SV: Luteinization of rabbit preovulatory granulosa cells cultured in vitro in presence offollicular oocytes. 1. Growth characteristics and progestin biosynthesis. Fertil Steril 23:791, Beatty RA: Parthenogenesis in vertebrates. In Fertilization. Comparative Morphology, Biochemistry and Immunology, Edited by CB Merz, A Monroy. London, Academic Press, 1967, p 413 March Baker TG: A quantitative and cytological study of germ cells in human ovaries. Proc R Soc Lond [BioI] 158:417, Baca M, Zamboni L: The fine structure of human follicular oocytes. J Ultrastruct Res 19:351, Maqueo M, Rice-Wray E, Calderon JJ, Goldzieher JW: Ovarian morphology after prolonged use of steroid contraceptive agents. Contraception 5:177, Yang S-L, Okamura H, Beer AE: The effect of estrogen on collagen synthesis at the site of a skin autograft. Am J Obstet Gynecol 116:694, Espey LL, Coons PJ: Factors which influence ovulatory degradation of rabbit ovarian follicles. BioI Reprod 14:233, Sherrer CW, Gerson B, Woodruff JD: The incidence and significance of polynuclear follicles. Am J Obstet Gynecol 128:6,1977