On the Mechanism of OvulationStudies on Rabbit Ovarian Follicles with Special Reference

to the Overlying Surface Epithelium

A K A D E M I S K A V H A N D L I N G

SOM MED VEDERBÖRLIGT TILLSTÅND

AV REKTORSÄMBETET VID UMEÅ UNIVERSITET

FÖR AVLÄGGANDE AV MEDICINE DOKTORSEXAMEN

KOMMER ATT FRAMLÄGGAS FÖR OFFENTLIG GRANSKNING I

SJUKSKÖTERSKESKOLANS AULA

LÖRDAGEN DEN 22 MAJ 1976 KL. 9.00

av

STEFAN CAJANDERMed. kand.

U M E Â 1976

UMEA UNIVERSITY MEDICAL DISSERTATIONSNew Series No. 23

From the Institute of Pathology, University of Umeå, Umeå, Sweden

On the Mechanism of OvulationStudies on Rabbit Ovarian Follicles with Special Reference

to the Overlying Surface Epithelium

BY

STEFAN CAJANDER

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This thesis is based on the following reports which will be referred to by their Roman numerals:

I. Bjersing, L. and Cajander, S.: Ovulation and the mechanism of follicle rupture. I. Light microscopic changes in rabbit ova­rian follicles prior to induced ovulation. Cell Tiss. Res. 149, 287-300, 1974.

II. Bjersing, L. and Cajander, S.: Ovulation and the mechanism of follicle rupture. II. Scanning electron microscopy of rabbit germinal epithelium prior to induced ovulation. Cell Tiss. Res. 149, 301-312, 1974.

III. Bjersing, L. and Cajander, S.: Ovulation and the mechanism offollicle rupture. III. Transmission electron microscopy of rab­bit germinal epithelium prior to induced ovulation. Cell Tiss. Res. 149, 313-327, I974.

IV. Cajander, S. and Bjersing, L.: Fine structural demonstration of acid phosphatase in rabbit germinal epithelium prior to induced ovulation. Cell Tiss. Res. 164, 279-289, 1975.

V. Cajander, S.: Structural alterations of rabbit ovarian follicles after mating with special reference to the overlying surface epithelium. Submitted to Cell Tiss. Res.

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CONTENTS

INTRODUCTION .................................................. 5

MATERIAL AND METHODS........................................... 6An i ma 1 s .................................................... 6Light microscopy (LM) and transmission electron micro­scopy (TEM) ................................................ 7Scanning electron microscopy (SEM) ....................... 7Ultrahistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

OBSERVATIONS .................................................. 9Light microscopical observations .......................... 9Scanning electron microscopical observations ............. 10Transmission electron microscopical observations ........ 11Observations on the activity of acid phosphatase at theultrastructural level ..................................... 12

DISCUSSION .................................................... 13

SUMMARY AND CONCLUSIONS .... 17

ACKNOWLEDGEMENTS ..... 19

REFERENCES 20

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INTRODUCTION

One of the fundamental events în reproduction is ovulation with release of the ovum from the ovary. The intraovarian mechanism of ovu­lation has been subject to numerous investigations (for reviews, see e.g. Blandau, 1967; Rondell, 1970; Lipner, 1973; Espey, 197*0. For a long time a major question was whether follicle rupture is preceded by an increased intrafol1ieu 1ar pressure or whether the follicle wall is decomposed by enzymatic digestion prior to rupture without an increase in mechanical pressure.

About 10 years ago a new era began. The intrafol1icuiar pressure was recorded in rat and rabbit preovulatory follicles by manometric tech­niques, and it was found that the pressure did not increase (Blandau and Rumery, 1963; Espey and Lipner, 1963; Rondell, 196*+). This was confirmed in subsequent studies on several other species (Lipner, 1973) implying that it is a universal phenomenon. The fact that the increase in size of the Graafian follicle, as ovulation approached, could not be attributed to an increase in intrafol1icular pressure indicated that changes must take place within the wall of the follicle. As a consequence, much in­terest was concentrated upon enzymatic action on the follicle wall and on alterations of the physical properties of the apex before rupture. It has been concluded that the extensibility of the follicle wall is in­creased several times prior to rupture when compared to unstimulated fol­licles (Espey, 1967 a; Rondell, 196*+). Furthermore, proteolytic enzymes injected into unstimulated follicles or applied on the external surface caused decrease of the tensile strength and also induced follicle rup­ture (Espey and Lipner, 1965; Nakayo et al. 9 1973; Espey, 197*+). Lately, it has been more generally accepted that enzymes are essential for fol­licle rupture. However, in spite of intense studies, including several morphological investigations, the intraovarian mechanism of ovulation is still in many respects unknown, e.g. it has not been clarified which is the "ovulatory enzyme" or where it comes from (of. Espey, 197*+).

Then, it appeared essential to study morphological and histochemi- cal changes in preovulatory follicles and surrounding tissues at accura­te times before ovulation. As experimental animal the rabbit was found suitable because it is a reflex ovulator and ovulates at a regular inter­val after an ovulatory stimulus, e.g. mating or intravenous injection of

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human chorionic gonadotrophin (HOG). Furthermore, preovulatory levels of several biologically important substances, e.g. hormonal steroids and prostaglandins, have recently been studied in rabbit ovaries and follic­les. Morphological and biochemical changes in the preovulatory follicles may thus be correlated.

The principal aims of the present investigation may be summarized as follows :

1. To study light microscopical alterations of preovulatory rabbit ovarian follicles and surrounding tissues at different times after an intravenous injection of HCG.

2. To use electron microscopic techniques for a more detailed study of apical wall alterations - found by light microscopy - which might be important for the disintegration and eventual rupture of the follicle wa 11.

3. To combine transmission electron microscopy and histochemistry in order to reveal whether the wall changes found could be related in any way to proteolytic enzymes.

k. To establish whether the preovulatory alterations found after HCG stimulation are the same after natural ovulatory induction, i.e. mating.

MATERIAL AND METHODS

An i ma 1s

Young female virgin rabbits weighing about 3 kg were used in these studies. They were individually caged, fed on standard rabbit pellets and had free access to water. Ovulation was induced either by intraveno­us injection of 25 î.u. HCG (I - IV) or by mating (V). Care was taken to select apparently oestrous animals for the experiment; the vulva should be dark red and moisted with a thin, serous secretion. Another criterion of oestrus, is the acceptance of the buck (Adams, 1972) which, of course, was fulfilled when ovulation was induced by mating (V). Non- -stimulated rabbits and rabbits taken at various intervals after ovula­tory induction were anesthetized with pentobarbitone sodium (Nembutal or Mebumal) given intravenously. After laparatomy by midline incision, the ovaries were ectomized. Special care was taken to minimize manipulation of the vessels in the ligomentum latum \ if the vessels were compressed

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close to the time of follicle rupture there was a risk of inducing pre­mature follicle rupture. After oophorectomy the ovaries were immediately fixed by immersion at 4°C. To facilitate rapid penetration of the fixa­tive to the interior part of the tissue, 1 mm thick slices were general­ly cut from the follicles.

Light microscopy (LM) and transmission electron microscopy (TEM)

Part of the material for LM (i) was fixed in Bouin*s solution, de­hydrated, embedded in paraffin, sectioned (approx. k ym thick sections) and stained with hematoxy1 in-eosin or the van Gieson stain.

Other ovaries for studies by LM and by TEM (I, III, IV, V) were fixed for 12-2A hr in 3-^ % glutara1dehyde in 0.13 M sodium cacodylate or (IV, V) in 2 % glutara1dehyde in 0.1 M sodium cacodylate buffer with 0.1 M sucrose (Brunk and Ericsson, 1972). Postfixation in 1 % osmium tetroxide in 0.15 M sodium cacodylate was generally performed. After rap id dehydration, the follicular tissue was embedded in Epon according to Luft (I960. About 1 ym thick sections from the whole circumference of the follicles were stained with 0.1 % alkaline toluidine blue and exam­ined by light microscopy.

The material embedded in Epon as described above was also used for TEM. Furthermore, the material studied in publications I and III (by LM and TEM, respectively) was identical. Thin sections were generally taken from the apical region of the follicles. They were cut on an LKB-ultro- tome, mounted on uncoated copper grids and stained with saturated uranyl acetate, followed by lead citrate.

Scanning electron microscopy (SEM)

Whole ovaries were fixed in 2 or 3 % glutaraldehyde buffered in 0.1 M sodium cacodylate with 0.1 M sucrose or in 0.15 M cacodylate at k°C. The ovaries were fixed for 1 to 7 days. Then the large follicles with adjacent ovarian tissue were dissected out and dehydrated. Two de­hydration techniques were used, viz. freeze-drying (il) and critical- -point-drying (V). The material submitted to freeze-drying was rinsed in four changes of glass redistilled water for 2Q-AQ min. and then plunged into isopentan, cooled by liquid nitrogen. Special care was taken to leave a minimal amount of free water on the epithelial surface to minimize freeze artifacts {of. Bergström, 1971). The material was

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then quickly transferred to a vacuum subìimator (Edwards-Pearse Speedi- vac Tissue-Dryer) and dried at -6Q°C for 2k hr. The material submitted to cri tical-point-drying was dehydrated in a graded series of ethanol (70 % 9 80 % 9 90 %, 95 % and 100 % 9 each step lasting 10 min.) and then brought to amylacetate (ethanol: amylacetate, 7:3, 4:6, 3:7, 1:9, and to 100 % amylacetate, each step lasting 10 min.). The material was then cri t i ca 1-point-dried from CO^ in a Polaron E 3000 apparatus.

The follicle specimens were subsequently mounted on aluminium prep­aration holders with silver conductive paint and afterwards vacuum-coated with gold in an Edwards Vacuum-Coat Unit M k. The specimens were rotated and tilted by means of a home-made motor driven apparatus to allow an even coating from all angles (Horstedt and Stenling, 1976).

Alternative dehydration methods with subsequent air-drying were al­so tested (II). These methods, however, caused considerable gross dis­tortion, even if the fine structure was often well preserved.

The material was examined and photographed in a Cambridge Scanning Electron Microscope S k.

Ultrah i stocherni stry

The material was fixed in 2 % glutaraldehyde in 0.1 M sodium caco- dylate buffer pH 7-2, with 0.1 M sucrose at 4°C for 2k hr. Thereafter, the material was rinsed in the same buffer with sucrose. Part of the ma­terial (IV) was rapidly frozen in isopentan chilled by liquid nitrogen. Cryostat sections, kO ym thick, were incubated for 60 min. at 37°C in Gomori's acid phosphatase medium with 0.22 M sucrose, essentially accord­ing to Brunk and Ericsson (1972). Another part of the material was in­cubated as 1 mm thick slices without previous freezing. The follicle ma­terial in report V was cut in 40 ym thick sections by vibratome (Oxford)technique prior to incubation in Gomori's acid phosphatase medium. Sec­tions incubated in medium without substrate and sections treated for 1 min. in boiling water before incubation were used as controls. After in­cubation the material was rinsed in 0.1 M sodium cacodylate buffer with0.1 M sucrose for 2 min. with three changes of the buffer. Some of thesections were postfixed for 1 hr at 4°C in 1 % osmium tetroxide. Afterrapid dehydration the blocks were embedded in Epon, according to Luft (1961). Thick sections of the Epon embedded material were stained with 1 % alkaline toluidine blue and examined by light microscopy. Thin se- tions taken from the apical part of the follicles were generally stained

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with saturated uranyl acetate and lead citrate. Some sections, however, were studied in the electron microscope without previous staining.

OBSERVATIONS

The ovaries excised from non-stimulated rabbits were small and pale with a slightly wrinkled surface. Four to five large and protruding fol­licles were seen on each ovary. After HCG-stimulation or mating, the ova­ries became progressively more hyperemic, and distended, bloodfilled ves­sels were particularly evident around the large follicles. The closer to the time of follicle rupture the more swollen were the ovaries and the ovarian surface smoothed out. At the later stages prior to rupture, a pale area, usually called the stigma, was seen at the follicle apex.

Light microscopical observations (I, V)

The observations were mainly based on studies of the Epon embedded material, since this was superior when studying cellular and subcellular structures. In sections from mature ovarian follicles of non-stimulated rabbits, the surface or germinal epithelium covering the follicles con­sisted of closely apposed, low to cuboidal cells seated on a basement membrane. The cytoplasm harboured occasional small, dark granules and/or some light vacuolar spaces. The surface cells between follicles were higher and closer to each other than those over the follicles.

The surface epithelium over follicles 1/3 “ 1/2 hr after HCG stimu­lation showed essentially the same characteristics as described for non- -stimulated follicles.

At A hr after HCG treatment or mating, the epithelial cells at the follicle apex displayed distinct changes. They had enlarged markedly in size and contained an increased number of dark, cytoplasmic granules.From A hr and onwards the apical epithelial cells showed an increasing amount of intracellular granules. Maximal accumulation of granules was seen at 8 hr after HCG treatment, somewhat later after mating. At this stage, the surface epithelial alterations were more wide-spread than earlier and equally prominent alterations were found in the lateral regions of the follicle dome. However, no increase of granules was found in the surface epithelium between preovulatory follicles. Vacuolar spaces in the epithelial cytoplasm were comparatively few at A hr but more nu­merous and generally larger at 8 hr.

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In the time interval between maximal epithelial accumulation of granules and follicle rupture, the surface cells flattened and the granules decreased in amount. Just prior to rupture the epithelial cells were thin and even missing in small apical areas.

A series of structural alterations were also seen in the follicle wall below the surface epithelium. At 8 hr after ovulatory induction, but sometimes earlier, fibroblasts of the tunica albuginea began to dis­sociate at the top of the follicle and an interstitial oedema appeared. Later, the dissociation of the fibroblasts and other cell components in­creased and spread towards the centre of the follicle as well as later­ally.

As early as k hr after ovulatory stimulation, a prominent oedema was seen in the theca interna region at the follicle apex. The theca in­terna cells were haphazardly distributed in the oedematous area, either in small groups or apparently isolated. Closer to the time of follicle rupture, the oedema was about the same or even less than at A hr.

The vessels encircling the follicle were dilated and contained more blood corpuscles after ovulatory stimulation than before. This was par­ticularly evident prior to rupture. However, at this stage the apical vessels were compressed and practically devoid of blood cells.

The granulosa cells, all around the follicle antrum, dissociated in the preovulatory stage. Obvious cell dispersion was found at the fol­licle apex as early as A hr after HCG or mating. Later, a more pro­nounced cell dissociation was seen, especially in the apical region, where granulosa cells even could be missing in small areas.

Close to the time of follicle rupture, granulocytes were found in the follicle wall and also in the follicle antrum.

The alterations after ovulatory induction followed the general time schedule described above. However, moderate variations between rabbits were found, more after mating than after HCG treatment (V). In addi­tion, some of the mated rabbits displayed no response at all and their ovaries remained small and pale.

Scanning electron microscopical observations (II, V)

The ovarian surface was easy to study, both in survey view and in considerable detail, by SEM. The surface epithelium over and between follicles was scrutinized and compared. Cells situated far from follic­les had the same general appearance both before and after ovulatory

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stimulation and were similar to most follicular surface cells in non- -stimulated animals. The epithelial cells showed a convex peritoneal surface, generally covered by microvilli of varying size and number.Small pits, probably invaginations of the surface epithelium, were seen both over and between most follicles (V). Surface papillomas were also frequent.

After HCG or mating, the cells covering the follicles showed a gen­eral increase in size and the microvilli successively decreased in length and number. These changes were first observed over the apex of the fol­licles and some of them were obvious 6 hr after HCG. In later stages the contours of intracel1ular rounded bodies could be visualized at the fol­licle apex; this was particularly evident in the freeze-dried specimens. Just prior to rupture fragments of the surface epithelium were often missing at the top of the follicle.

Transmission electron microscopical observations (ill/ IV and V)

The epithelial cells covering follicles of non-stimulated rabbits showed a convex peritoneal surface with numerous microvilli. Regular and sometimes wide intercellular spaces, apparently filled with liquid, seem­ed to be separated from the peritoneal cavity by intercellular junctions. The cells were standing on a basement membrane and, especially on the lower surface of the cells, there were many small pinocytotic vesicles. Below the basement membrane there was a moderately thin zone of electron lucent, amorphous substance. The nuclei of the surface epithelium cells usually showed deep indentations with moderate amounts of chromatin at the periphery. Small, round, electron dense cytoplasmic bodies surround­ed by a distinct membrane were seen in moderate or small amounts. The cytoplasm contained relatively few mitochondria and rather small amounts of rough endoplasmic reticulum which occasionally seemed to be in direct continuity with the agranular endoplasmic reticulum. Golgi complexes were predominantly found in the supranuclear region (the region between the nucleus and the peritoneal cell surface).

The first alterations of the surface epithelium were noted at the top of the follicle apex k hr after ovulatory stimulation. The cells were considerably larger and many of them contained large, round elec­tron dense bodies. The cytoplasm appeared sometimes darker and the nu­clear chromatin more margina ted. Some of the cells harboured several

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dense hodîes and their peritoneal surface was smoothly rounded with only a few microvilli. The dense bodies in these cells occurred both below and above the nucleus. Apparent fusions between dense bodies were com­monly seen.

At 6 and 8 hr after HCG or mating, the changes in the apical epi­thelial cells had progressed and many of the cells contained a lot of large, dense bodies. Some bodies had a less electron dense content (semi- dense bodies), often with a fine fibrillar matrix, and there were also light bodies or vacuoles in the cytoplasm. The vacuoles were usually found in the intranuclear region. It was commonly seen that dense bodies protruded into each other or into semidense bodies or into large vacu­oles. Degenerating or dead cells were occasiona1ly found at the apical region.

Between 9 and IQ hr after mating the surface cells flattened and the dense bodies decreased in amount parallel with an increase of semi­dense bodies and vacuoles. Some of the vacuoles contained fragments of dense, semidense or membranous material. In favourable sections the large vacuoles in the infranuclear region showed communications between each other and also openings towards the basement membrane. Immediately before follicle rupture the surface cells at the apex were extremely at­tenuated, often with signs of degeneration, and there were only a few granules left. Occasionally, parts of the surface epithelium were mis­sing at the stigmal area.

The surface epithelium at some distance from the follicle showed only small changes or no changes at all.

Observations on the activity of acid phosphatase at the ultrastructura1 level (IV and V)

Before HCG treatment or mating, the cells covering Graafian follic­les showed acid phosphatase reaction product in Golgi cisternae as well as in some small cytoplasmic vesicles. Small dense bodies, generally seen below the nucleus, were also enzyme positive. Sections from control incubations showed no precipitation.

At k hr after ovulatory induction, the apical surface cells had en­larged and contained more and larger dense bodies. Several of these bodi­es showed acid phosphatase activity, but some of them were enzyme nega­tive. Reaction product was occasionally also found in the cytoplasm just outside the dense bodies, both at this time and later (IV).

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At 6 and 8 hr after HCG or mating, the same distribution of enzyme activity was recorded. Closer to the time of fol 1icle rupture the sur­face epithelial cells were attenuated and thin. They contained only a few large dense bodies but the enzyme activity showed the same pattern as earlier, i.e. concentrated deposition in the Golgi complexes and a weaker reaction in some of the large, dense bodies.

DISCUSSION

The rabbit is a reflex ovulator and ovulates fairly regularly 10-12 hr after HCG treatment or mating (Adams, 1972). In the present work the rabbit follicle wall was studied by morphological and ultrahistochemical means at different times after ovulatory induction. The preovulatory al­terations found in the wall were essentially the same after HCG as after mating (V).

Already by light microscopy, alterations, not previously described, were found in the surface or germinal epithelium covering preovulatory follicles. After ovulatory induction, the surface epithelial cells at the follicle apex enlarged concomitant with a rise in the amount of cyto­plasmic granules. At 4 hr after HCG or mating, a marked increase of gran­ules was observed at the follicle apex before any signs of disintegra­tion of the connective tissue elements in the tunica albuginea. Maximal accumulation of granules was found at 8 hr. Then, they gradually dis­appeared parallel with a disintegration of the underlying tunica albu­ginea. This sequence of events suggests that the disintegration of the rabbit follicle apex starts in the outermost region of the follicle, as described in the preovulatory follicle of mice (Byskov, 1969). From a logical point of view, it appears necessary that synthesis and/or release of any enzyme(s) active in the process of wall disintegration should pre­cede signs of wall dissolution. The increase in surface epithelial gran­ules was seen several hours before the wall disintegration which appear­ed about 8 hr (of. Espey, 1967 b) after ovulatory induction. However, at this stage of the investigation, it was not known at all if the granules contained any substance which could participate in this process.

Since so much effort had been spent in the search for the origin of the "ovulatory enzyme" (for review, see Rondell, 1970; Lipner, 1973; Espey, 197*0, it appeared correct to investigate the surface epithelium over preovulatory follicles in more detail. Therefore, further studies

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by scanning electron microscopy, transmission electron microscopy, and ultrahistochemistry were performed.

The scanning electron microscope appeared to be a suitable instru­ment to start with, since it allows rapid screening of large areas both in survey view and in consîderable detaî1. Distinct changes of the sur­face epithelial cells covering preovulatory follicles were observed. The cells increased in size and microvilli decreased in amount approaching the time of follicle rupture, as also described by other investigators (Motta et al., 1971; Nilsson and Munshi, 1973; Motta and Van Blerkom, 1975). Furthermore, the outlines of intracellular spherical bodies, prob­ably corresponding to the granules seen by light microscopy, could be visualized. The granules were more protruding in the freeze-dried mate­rial (II) than in the eritical-point-dried material (v) which indicates a difference in the preservation of tissue between the two drying tech­niques (V).

Transmission electron microscopical studies (ill, V) showed that the cytoplasmic granules were electron dense and surrounded by membrane. Furthermore, in material incubated for demonstration of acid phosphatase at the ultrastructural level (IV, V), a positive enzyme reaction was found in many of the granules. Thus, these granules may, both from struc­tural and histochemica1 standpoints, be regarded as lysosomes (IV). The observation that there was a marked increase in lysosomal granules in the apical surface ep î thel i urn before follicle rupture is most interest­ing as regards the ovulatory mechanism, since, lately, it has been more generally accepted that lysosomes may contribute to extracellular de­gradation processes both in pathological and physiological situations (Reynolds, 1969; Vaes, 1969; Lucht, 1974; Anderson and Fejerskov, 1975). In the surface epithelium at 8 hr and later, the granules showed signs of release in the direction of the underlying tunica albuginea (V; Ca- jander and Bjersing, 1976), which suggests that material from the gran­ules may help to break down the follicle apex.

As pointed out above, not all granules in the surface epithelium showed acid phosphatase activity. What these granules represent is un­known. Anderson and Yatvin (1970) suggested that the germinal epithelium in the frog ovary might synthesize collagenase after an ovulatory stimu­lus. Lazarus et al. (1968) showed that granulocytic leukocytes contained "active" collagenase in vivo. Since then, active collagenase has been identified in other cells, e.g. the Kupffer cells of the liver (Fujiwara

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et al., 1973). Of interest is that collagenase production in many in­stances is associated with epithelium and not with deep connective tis­sue elements (Riley and Peacock, 1967; Eisen, 1969; Brown and Weller,1370; of. Cajander and Bjersing, 1976). Whether some granules in the germinal epithelium contain collagenase, as the specific granules in polymorphonuclear leukocytes do (Robertson et al.f 1972), is still un­known. In this context it may be of significance that "exudates" col­lected over preovulatory follicles showed about 3 times higher collagen- ase-like activity than "exudates" col 1ected over follicles from non- -mated rabbits (Rondell, 1970). This enzyme could, of course, originate from cell components deep in the follicle wall, e.g. the granulosa or theca interna cells. Parallel estimation of enzyme activity in the fol­licular fluid, however, showed relatively low activity and it was almost the same in mated and unmated rabbits (Rondell, 1970). Another indica­tion of the possible significance of the surface or germinal epithelium is the observation that the ovaries of the mature mare (Hammond and Wod- zicki, 19^1; Bergin and Shipley, 1968) and of the rodent Galea mustel- oides (Weir and Rowlands, 197*0 have a circumscribed area called the "ovulation fossa" which is the only part of the ovarian surface covered by surface or germinal epithelium. The follicles, on the other hand, are distributed throughout the ovaries but burst only in this fossa.

Other cells may, however, very well contribute degradati ve enzymes essential for the ovulatory process, i.e. the granulosa'cel Is*, the theca interna cells and the fibroblasts (of. Rondell, 1970; Espey, 1971 a; Motta, et al.9 I97I). But morphological evidence for an increase or release of such enzyme(s) has, so far, not been presented. The "mult ivesicular bodi­es" in fibroblasts, which appear in increased number before follicle rup­ture, have been thoroughly investigated by Espey (1971 a, b). It is pos­sible that they are relevant and contribute to non-1ysosoma1 (Espey,197*0 proteolytic enzymes. On the other hand, the "multivesicular bodies" may also be arti factual due to OsO^-fixation, as pointed out by Espey himself (Espey, 1971 a).

The possibility that the increase of lysosomes in the surface epi­thelium may be partly a degenerative phenomenon, as seen in several tis­sues during involution (Ericsson, 1969), should also be considered. In such instances, the activity of lysosomal enzymes appears to be in­creased (of. Ericsson, 1969). In the later stages, just prior to follic­le rupture, degenerating epithelial cells are common at the apex (Byskov,

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1969; Motta et al., 1971; Nilsson and Munshi, 1973; I). The increase of epithelial lysosomes, however, was found as early as 4 hr after mating. Furthermore, ovulation will not occur in the absence of oxygen and many metabolic inhibitors as well as inhibitors of protein synthesis will prevent it (Rondell and Wright, 1957, 1958; Pool and Lipner, 1966; Yat- vin and Anderson, 1967; Rondell, 1970; Lipner and Greep, 1971). There­fore, it is more likely to be an active,rather than a passive, degenera­tive process.

Much remains to be done before the complex process of ovulation is solved. Several intraovarian factors in addition to degradative enzymes are probably involved in the mechanism of follicle rupture, e.g. the capillary vessels surrounding the membrana granulosa, smooth muscle cells and the nerves in the follicle wall (cf. Lipner, 1973; Bahr et al., 1974; Bjersing and Cajander, 1974 a, b, 1975; Owman et al., 1976; III,IV, V).

One important question that remains to be solved is: What is the basic mechanism that induces the increase and disappearance of the sur­face epithelial lysosomes? In papers III - V, it was suggested that sex steroids, secreted in large amounts early in the preovulatory phase (YoungLai, 1972), may stimulate the increase in the lysosomal pool. Fur­thermore, it was suggested that the lysosomal membranes might be labil- ized by the high levels of prostaglandins found in the follicle during the late stages of the ovulatory process (LeMaire et al., 1973). This hypothesis will be tested in future studies.

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SUMMARY AND CONCLUSIONS

To elucidate the intraovarian mechanism of follicle rupture, rab­bit ovarian follicles were studied by morphological and u1trahistochem- ical means before and at different times after an ovulatory stimulus. Special attention was paid to the overlying surface epithelium. The rab­bit was found suitable as an experimental animal because it is a reflex ovulator and ovulates fairly regularly 10-12 hr after mating or HCG stimulation.

1. It was found that ovulation induced by HCG or mating caused essen­tially the same kind of structural and ultrahistochemica 1 alterations of the preovulatory follicle wall.

2. Preovulatory follicles studied by light microscopy showed a series of structural alterations. Of particular interest were the alterations- not previously described - of the surface epithelium covering the fol­licles. Up to 8 hr after ovulatory induction an increase of epithelial granules occurred. Then, prior to rupture, the granules disappeared con­comitant with a disintegration of the underlying connective tissue ele­ments of the tunica albuginea. This sequence of events suggested that the granules in the surface epithelium might contain material essential for disintegration of the apical follicle wall. Therefore, the surface epithelium over preovulatory follicles was studied in more detail.

3. By scanning electron microscopy it was found that the surface epi­thelial cells at the follicle apex enlarged markedly after ovulatory induction parallel with a decrease of microvillous projections. A few hours before expected ovulation, the outlines of intracellular rounded bodies - corresponding to the granules found by light microscopy - were visualized. Just prior to follicle rupture, fragments of the apical epi­thelium were sometimes sloughed off.

k. By transmission electron microscopy it was shown that the granules or rounded bodies seen by light microscopy and SEM, were electron dense and membrane surrounded. Maximal accumulation of these bodies over the follicle dome was found about 8 hr after an ovulatory stimulus. There­after, they gradually disappeared, and, just prior to rupture, the ap­ical epithelial cells were extremely attenuated, sometimes with obvious signs of degeneration and cell death.

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5. Acid phosphatase, the most commonly used ultrahistochemical "mark­er" enzyme for lysosomes, was found to be localized to many of the den­se bodies in the surface epithelium. These bodies can thus be regarded as lysosomes.

6. A hypothesis on the mechanism of ovulation and the role of the ovarian surface epithelium was put forward. It implied that lysosomes, known to contain a broad spectrum of enzymes, e.g. proteolytic enzymes, contribute to the preovulatory apical wall disintegration. Furthermore, it was suggested that the large amount of sex steroids present in the rabbit follicle early in the preovulatory phase may stimulate the lyso­somal growth. It was also suggested that lysosomal membranes may be labilized by the high levels of prostaglandins in the follicle during the late stages in the ovulatory process.

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ACKNOWLEDGEMENTS

I wish to express my sincere and deep gratitude to my teacher and friend, Professor Lars Bjersing, who introduced me into this field of research. His extensive knowledge, inspiring and encour­aging guidance, generous advice and never failing interest, have been invaluable for this investigation.

I am also indebted to:

Professor Sture Falkmer for encouragement and valuable advice throughout the study and for placing excellent facilities at my di spo sa i ,

All my colleagues at the Institute of Pathology for their support and assistance in many ways,

Miss Ingalis Fransson, Miss Kerstin Nilsson, Mrs. Ulla-Britt West­man and Mr. Kjell Öhman for excellent and devoted technical assist­ance,

Mr. Bengt Carfors for expert photographic work and Fil. kand. Per Horstedt for technical assistance,

Mrs. Viola Bernander for skilful and never failing patience in typ­ing the manuscripts,

Mrs. Inga-Gret Bäckman and Mrs. Gerd Gustafsson for support and valu­able secretary assistance,

Fi 1. lic. Robert Elston for linguistic revision of the manuscripts.

This investigation was supported by grants from the Swedish Med­ical Research Council (Projects No. B72-12X-78-07A, B73“ 12X-78-08B, B7^-12X-78-09C and B 75“12X— 78—1OA), The Magn. Bergvall Foundation and from the Medical Faculty of the University of Umeå.

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