studies of the fine structure of ovarian interstitial tissue

Z. Zellforsch. 113, 133--156 (1971) �9 by Springer-Verlag 1971

Studies of the Fine Structure of Ovarian Interstitial Tissue 5. Effects of G o n a d o t r o p i n s on the Thecal G land of the Domest ic Fowl

EgIx DAnL

Department of Anatomy, Dental Faculty, University of Oslo, Norway

Received September 24, 1970

Summary. The fine structure of ovarian steroid-producing cells of the thecal gland as seen after administration of gonadotropins to the domestic fowl is described. Chorionic gonadotropin and pregnant mare serum gonadotropin were given as intramuscular injections for varying periods (I0-28 days). The main cytoplasmic changes of the steroid-producing cells were a marked decrease in the number and the size of the lipid droplets, increase in the density, number and size of the mitochondria, increase in the amount of endoplasmic reticulum, increase in the size of the Golgi apparatus and in the number of the dense bodies. Furthermore, there was also an increase in the size of the nucleus and the nucleolus. The nuclear bodies increased in number and this organelle also appeared to develope in size. Its internal structure changed with increased fibrillary material and the presence of small vesicles, similar to coated vesicles. An increase in the number of pinocytic vesicles was observed at the surface of the steroid-producing cell. Cilia were occasionally observed. Alterations were also found in the enclosing cells and the theca interna cells, indicating a transformation in these cells toward the morphology of steroid-producing cells. These alterations have never been demonstrated in ovarian steroid-producing cells of the thecal gland before, and are consistent with hypertrophic changes observed in steroid-producing cells in other organs. The findings are discussed in relation to the function of the thecal gland and the theca interna. The study substantiates the view that the thecal gland must have an endocrine function and might be the main target of the gonadotropins in the theca interna.

Key-Words: Ovary--Interstitial tissue--Thecal gland Fowl--Influence of Gonadotro- pins.

Al though the invo lvement of gonadotropins in the regulat ion of ovar ian func- t ion has long been recognized, our knowledge of the act ion of the various pitui- t a ry hormones on the ovary is far from complete (Young, 1961 ; Zuckerman, 1962). Especially little is known about the effects of gonadotropins on the fine structure of the steroid-producing theca in te rna cells in mature animals.

In ters t i t ia l ovar ian cells of juvenile rats and rabbi ts are the only ones t ha t have been examined u l t ras t rue tura l ly after admin is t ra t ion of p regnant mare serum gonadotropin (PMS) and h u m a n chorionic gonadotropin (HCG) (Merker, and Diaz- Encinas, 1969). Fur thermore , Be l t e rmann and Stegner (1968), have invest igated ovaries of newborn mice after admin is t ra t ion of gonadotropins isolated from h u m a n p i tu i t a ry gland (HHG). However, the fine s t ructure of the thecal gland in mature animals, as seen after admin is t ra t ion of gonadotropins, has not been studied.

Previous reports have revealed tha t the ovary of the domestic fowl possesses a steroid-producing gland, the thecal gland (Dahl, 1970b, c, d), which is morphologically almost identical with the adrenal cortex in the same an imal (Kjaerheim,

134 E. Dahl:

1968b) . T h e p u r p o s e of t h e p r e s e n t i n v e s t i g a t i o n was to s t u d y t h e e f fec t of exo-

g e n o u s g o n a d o t r o p i n s o n t h e f ine s t r u c t u r e of t h e t h e c a i n t e r n a w i t h spec ia l

r e f e r e n c e to t h e s t e r o i d - p r o d u c i n g cells in t h e t h e c a l g l a n d .

M a t e r i a l a n d M e t h o d s

Thir ty White Leghorn hens, 18-24 months old, with an average body weight of 1867 g, were used. The animals were housed in individual cages in a well-ventilated, constant climate room (17 ~ C, controlled illumination, light on 7 a.m. and off 7 p.m., relative humidi ty 60%). The diet consisted of commercial chicken fodder, cabbage, sand grits and water ad lib. The main consti tuents of the fodder were proteins (17-19%), fats (2-4%), calcium (1.1-1.2%) phosphorus (0.7%) and sodium chloride (0.5%). The hens were kept for at least 10 days in the animal house in order to get adapted to the environment before the experiment started.

The gonadotropic preparations 1 used were: 1) A purified preparat ion of pregnant mare serum gonadotropin (Antex, LSwen, Copenhagen, Denmark). This will be referred to as PMSG. 2) A purified preparat ion of human chorionie gonadotropin (Physex, LSwen, Copenhagen, Denmark). This will be referred to as HCG.

Of the 30 animals employed in this experiment, 9 served as controls. Since previous experiments with steroids (Dahl, 1970e) had been carried out over a period of 28 days with daily injections, the same regime was followed in the initial phases of the present study. However, since the animals did not tolerate the doses as well as expected, the injections were ad- ministered as follows:

Three hens received 600 I.U. PMSG as an intramuscular injection 18 h before sacrifice.

Three hens received 600 I.U. PMSG as intramuscular injections every second day over a 10 days period (5 injections).

Five hens received 600 I .U. PMSG as daily intramuscular injections for 22 days, and then every second day until the 28th day, total ing 25 injections.

Three hens received 600 I.U. HCG as intramuscular injections every second day over a 1O-days period (5 injections).

Five hens received 600 I.U. HCG as daily intramuscular injections for 22 days, and then every second day until the 28th day, totaling 25 injections.

In addition, two hens were given 600 I.U. PMSG every second day, followed by 600 I.U. HCG every second day, total ing five injections over a l0 days period.

All the animals were sacrificed 24 h after the last injection. Fixat ion was performed as an intracardial perfusion of dextran under nembutal anesthesia (Nembutal sodium Abbot 5 % ), followed by 1.7 % glutaraldehyde in 0.1 M phosphate buffer a t pH 7.3. The perfusion lasted for a minimum of 10 min. Details of the perfusion technique have previously been reported by Kjaerheim (1969a). Following perfusion, the ovary was excised and, while kept in a drop of fixative, cut into th in slices under the dissecting microscope. Samples from follicles of different sizes were then fixed separately in glutaraldehyde for an additional period of 2 h a t 4 ~ C. Subsequently, the tissue blocks were rinsed for 10 min in 0.15 M phosphate buffer at pH 7.3 and fixed in 1% osmium tetroxide at 4~ for 2 h (Millonig, 1961). The blocks were rapidly dehydrated in a graded series of acetone and embedded in Vestopal W (Ryter and Kellenberger, 1958). Ul t ra th in sections were cut on an LKB Ultrotome and t reated with uranyl acetate (3%) for 30 rain, followed by lead citrate (Reynolds, 1963) for 5 rain to increase contrast. The sections were examined in a Siemens Elmiskop I a electron microscope, equipped with 50 micron pla t inum objective apertures. The accelerating voltage was 80 kv. From the same blocks, sections one micron thick were cut for light microscopy and stained for 30 sec on a heated stage with 0.1% toluidine blue adjusted to pH 8.5 with M/15 N%HPO 4.

1 The gonadotropins (Antex and Physex) were a gift from LEO, L~vens kemiske Fabrik, Kobenhavn, Denmark, obtained through the Norwegian representative, Lovens kemiske Fabriks Handelaktieselskap, Oslo. I should like to express my grat i tude for the donation.

Effects of Gonadotropins on the Thecal Gland (Fowl) 135

Results

General

Control Animals

In the domestic fowl only the left ovary normally reached a functional state. The cortex of the ovary possessed little stromal tissue, but was subjected to wide structural variation according to the reproductive state of the individual. In egg-laying hens there was a hierarchy of developing follicles of different sizes. Each follicle was surrounded by the theca interna, where the thecal gland was found as compact lobules. Several glands were seen throughout the whole circum- ference of the follicle, almost at a regular distance from each other. They were composed of two cell types. The vast majori ty of the cells of the thecal gland had a fine structure corresponding to that of steroid-producing cells of other organs. These cells were therefore considered to be steroid-producing cells. The other type, the so-called enclosing cells, were few in number and always located at the periphery of the gland. They were characterized by their tenuous cytoplasmic extensions, which part ly or completely enclosed the steroid-producing cells and also surrounded the nerve processes essentially in the same manner as tha t of the Schwann cell (Dahl, 1970b, c, d).

For the purpose of description, the end of the cells facing the basal lamina and the surrounding tissue will be named " the basal port ion", and the opposite pole will be designated " the apical por t ion" of the cell.

The cytoplasm of the steroid cells of the thecal gland was characterized by a large number of lipid droplets and tubular mitochondria. The Golgi apparatus was always present within the juxtnuclear region. The smooth-surfaced endoplasmic reticutum occurred in a relatively larger amount than in the adrenal cortex (Kjaerheim, 1968b), while the rough-surfaced endoplasmic reticulum was almost completely absent. The cytoplasmic organelles revealed a pronounced polarity in their distribution. The majori ty of the lipid droplets, the mitochondria and the smooth endoplasmic reticulum were found in the basal cytoplasm, whereas the Golgi complex, dense bodies, a t tachment devices, and rough endoplasmic reticulum were observed apically. The nucleus contained one or two nucleoli and, occasionally, nuclear bodies. The enclosing cells were morphologically quite different from the steroid-producing cells. Normally, they did not contain any lipid droplets, the mitochondria were smaller, the endoplasmic reticulum was of the granular type and the nucleus was elongated, rich in chromatin, but usually without a distinct nucleolus. The enclosing cell had a close relationship to the basal lamina. The steroid-producing cells had membraneous contact with autonomic nerve fibers (Dahl, 1970d).

Experimental Animals

Injections of the gonadotropins had a rather marked effect on the hens. However, there were differences, both as to the hormone used and the extent of the experiment. Generally, PMSG seemed to have a more pronounced effect, and the following changes were observed: PMSG: No definite effect could be demonstrated after a single injection. After 48 h the comb had increased slightly in size and after 4 days it was significantly larger. The appetite and plumage increased. However, the hens ceased laying eggs already after 3-5 days. After about 15 days

Figs. 1 and 2

E. Dahl: Effects of Gonadotropins on the Thecal Gland (Fowl) 137

their appeti te decreased, and from the 22nd day they had to get injections every second day since their physical condition had become gradually reduced. They were only in a moderate physical condition at the end of the experimental period.

Examinat ions of the ovary did not reveal any macroscopic alterations after 24 h. After 10 days the ovary contained numerous follicles, about 11/.~ cm in diameter, and there also seemed to be an additional increase in the number of small follicles, however , the hierarchy which normally exists in egg-laying hens had disappeared. After 28 days there were m a n y large follicles of about 4 cm in diameter, most of them with a black-green content, and the whole ovary was enormously enlarged.

HCG: Injections of HCG seemed to leave the hens almost unaffected for the first 10 days except for a slight increase in weight. However, later on they seemed to gain weight steadily, their comb increased and they were egg-laying during the whole period. They were in good condition at sacrifice. The ovary was probably larger than normal, there was a normal hierarchy of the follicles, and no degenerated follicles were found. Light microscopic examinat ion of the sections stained with tolnidine blue showed tha t the greatest deviation from normal was found in the PMSG-trea ted animals, especially those which were t reated for 28 days, with the occurrence of round nuclei with large nucleolus, depletion of lipid and, probably, an enlargement of the whole gland. The polarit iy of the cells was not altered, bu t there seemed to be an enlargement of the apical par t of the cytoplasm. Also by electron microscopy, changes were most pronounced in the long-term PMSG- t rea ted animals.

Changes within the Organelles The Steroid-Producing Cells

Lipid Droplets. The most striking change in m a n y of the steroid-producing cells as seen after st imulation with gonadotropins, was the depletion of lipid compared with the control animals (Figs. 1 and 2). No definite al terat ion could be demonst ra ted after 18 h in the PMSG-t rea ted animals. After 10 days the lipid droplets were smaller than in the normal material and significantly reduced in number. After 28 days cells could be seen, in which the cytoplasm was almost completely devoid of lipid (Fig. 2). Wi th regard to the HCG-t rea ted animals, no

Fig. 1. Survey electron micrograph of the normal thecal gland of the domestic fowl. The gland is surrounded by a well-defined membrane (arrows). The major part of the gland is formed by the steroid-producing cells, which are characterized by lipid droplets (L), dense bodies (Db) and a spherical nucleus (N) with nucleoli (Nu). The lipid droplets are located mainly in the basal parts of the cell, i.e. the end adjacent to the connective tissue (CT) or the capillaries (Ca), whereas the dense bodies and the Golgi apparatus (G) are located in the opposite region of the cell. The enclosing cells (EC) are seen at the periphery of the gland. Capillaries and nerve fibres (Ne) are always seen adjacent to the gland. Mitochondrion (M).

Golgi apparatus (G). x 7560

Fig. 2. Survey electron micrograph of the thecal gland from a domestic fowl injected with pregnant mare serum gonadotrophin (PMSG) for 28 days. The cells are rich in smooth-surfaced endoplasmic reticulum (SER), which is diffusely distributed. The mitochondria (M) are darker than normal. There is an increase in number of dense bodies (Db). The nuclei (N) are spherical. Note the paucity of the lipid droplets (L) and the hypertrophy of the enclosing cells (EC).

Capillary (Ca). x 7 560

Figs. 3 and 4


significant alteration could be demonstrated after 10 days. Mter 28 days definite changes were noted in the size as well as number of lipid droplets.

As observed in the normal steroid-producing cells (Dahl, 1970c), there was a gradual change in the morphology of the lipid droplets from the basal to the apical area of the cell (Fig. 3). Round, light droplets predominated in the basal area, but in the apical, paranuclear region some of the lipid droplets were partly by parallel myelin-like membranes.

Smooth - Sur/aced Endoplasmic Reticulum. Generally there was a significant increase in the smooth endoplasmic reticulum (Fig. 4). A single dose of the gonadotropins did not seem to cause any ultrastructural alterations when studied 18 h later. Definite changes could be observed after 10 days in the PMSG-treated animals, and after 28 days also in the HCG-treated animals. The smooth endoplasmic reticulum was diffusely dispersed in the cytoplasm with especially large accumulation in the form of "parasomes" (Bjersing, 1967) in the basal part of the cell (Fig. 15). In the long-term treated animals the smooth endoplasmic reticulum was occasionally found separated from the cell membrane by a brim of cytoplasmic matrix with a rather fibrillar appearance relatively free of ribosomes (Fig. 18). I t was striking that the areas exceptionally rich in smooth endoplasmic reticulum were poor in lipid droplets compared with the normal appearance.

Mitochondria. No definite alteration could be traced within the mitochondria after 18 h. After 10 days, changes were striking, and after 28 days they were quite considerable. Several alterations could regularly be found, and they were most pronounced in the PMSG-treated animals. There was a definite increase in number (Fig. 2). The mitochondrial population appeared more variable in size than normally, with gigantism (Figs. 8-9), elongated forms (Figs. 11 and 12) and an increased density of the matrix (Figs. 8-12). The pattern of the predominantly tubular cristae was more complex than normally (Fig. 9). Dense material was regularly present within the matrix (Fig. 10), and mitochondria with a light substance in the matrix could also regularly be encountered. However, the most outstanding phenomenon was the occurrence of vacuoles or blebs at the surface of the mitochondria (Figs. 12, 13). The outer mitochondrial membrane was invariably con- tinuous. No protrusion of intra-mitochondrial material through the membrane was seen, but regularly the blebs or vacuoles could be seen forming membranous structures in continuation with the outer surface of mitochondria (Fig. 13). How- ever, communication with vacuoles outside the mitochondria could not be demonstrated. These vacuoles were membrane-bounded, but did not always seem to be of the same type as the smooth-surfaced endoplasmic reticulum.

Fig. 3. Apical portion of steroid-producing cells after injection of gonadotrophin for 28 days. There is an increase of dense bodies (Db), and the Golgi apparatus (G) is prominent. The whole portion of the cell is hypertrophic. Lipid droplets (L). Nucleus (N). Nuclear body (Nb).

Mitochondrion (M). Microtubules (Mr). • 13500 Fig. 4. This illustration shows steroid-producing cells almost completely depleted of lipid droplets. The mitochondrial population (M) is variable in size with increase in density of the matrix. Note the amount of smooth endoplasmic reticulum (SER), dense bodies (Db) and the hypertrophy of the enclosing cells (EC) with smooth endoplasmic reticulum (SER). Nuclear body (Nb). Mitochondrion (M). Insert: High magnification of a nuclear body with vesicles of

similar morphology as the coated vesicles. • 10800

Figs. 5-7


The Golgi Apparatus. The Golgi complex increased markedly in size following administrat ion of both PMSG and HCG (Fig. 6). Although the basic organization of parallel arrays of membranes and vesicles were retained, both components were more plentiful and more extensively distr ibuted in the cell (Fig. 6). I t was located on the apical side of nucleus and occupied a substantial port ion of the cytoplasm. Cilia were occasionally encountered (Fig. 6). There was a significant increase in the number of dense bodies, sometimes with small f ragments within the same membrane (Fig. 5). These small f ragments seemed to be sequestered from the larger ones. Giant membrane-bound bodies were never found; they usually seemed to increase to a certain size, and then small f ragments were sequestered.

Dense Bodies. As in the controls, dense bodies were usually found in the apical region of the cells, part icularly in the Golgi region or adjacent to it (Figs. 2, 3, 7). However, in long-term treated animals dense bodies were also seen towards the basal par t of the cells where the lipid droplets were few in number (Fig. 4). Transitional forms between dense bodies and lipid droplets were regularly found (Fig. 3). I n cells which contained small amounts of lipid, the dense bodies were generally increased in number (Fig. 4), and they were also smaller than normal with a dense homogeneous granular structures. These changes were seen especially in long-term treated animals with PMSG. Generally there was a significant increase in the number of dense bodies, especially after 28 days.

Ribosomes and Rough - Sur/aced Endoplasmic Reticulum. There seemed to be an increase of free ribosomes after l0 days and part icularly after 28 days in the HCG-trea ted animals. I n the PMSG-t rea ted animals no significant changes could be demonst ra ted after 28 days. On the contrary, in some areas, especially in the juxtanucleolar region and in the basal peripheral par t of the cell, there seemed to be a decrease in the ribosomes (Fig. 18). The rough-surfaced endoplasmatic ret iculum was as scanty as in the normal material and seemed to be unaffected by the gonadotropins.

There seemed to be a general increase of fibrillar material, especially in the PMSG-t rea ted animals (Fig. 16).

The Nucleus. Even though the gonadotropins did not induce t remendous alterations in the nucleus, changes were observed (Fig. 2). The nucleus became more spherical, slightly enlarged and, contrary to what was observed in the control material, indentat ions of the cell membrane were extremely seldom encountered. The amoun t of chromat in was scarce. Nuclear bodies occurred more frequently than in the control animals (Figs. 4, 7), and, in addition, their internal s tructure seemed to be more complex (Fig. 4). Nuclear bodies of the lipid form were never seen.

Fig. 5. An area of a steroid-producing cell illustrating the close relationship between the lipid droplets (L), mitochondria (M) and dense bodies (Db). A complex body (Cb) is seen in the upper right corner. Note the small fragment of a dense body (arrow) which is about to be

sequestered, lV[icrotubules (Mr). Smooth endoplasmic reticulum (SER). x 54000 Fig. 6. Administration of gonadotrophin is followed by a marked increase in the Golgi area (G). Cilium (Ci) is occasionally observed. Note all the coated vesicles (CV) in the Golgi

area. Lipid droplets (L). Dense bodies (Db). • 21600 Fig. 7. The nucleolus becomes enlarged especially following administration of PMSG, and consists of a granular (Gr) and a filamentous (Fi) part. Nuclear bodies (Nb) are seen more

frequently. • 54000

Figs. 8-12


Despi te extens ive examina t ion mitosis was never encountered. The Nucleolus. The nucleolus was cons iderably enlarged, especial ly following

P M S G admin is t ra t ion . As in the normal cells, the nucleolus consisted of two pa r t s (Fig. 7). The larger pa r t conta ined granules abou t 150 A in d iamete r and the smal ler pa r t was made up to fine f i laments abou t 40 A in d iameter . The two pa r t s were a lways segregated as seen in Fig. 7. Spherical vacuoles, as prev ious ly demon- s t r a t ed in the adrena l cor tex (Kjaerheim, 1968c), were only occasional ly encountered.

The Cell Surlace. A t the cell surface an increase of cy top lasmic projec t ions was one of the s t r ik ing features. P inocy t ic vesicles were seen in some areas a lmos t a t a regular d is tance f rom each o ther (Fig. 17) and vesicles, often p rov ided with an ex t raneous coating, were found in ra the r large numbers in the cytoplasm, ad jacen t to the cell membrane .

Increase of the in terce l lu lar spaces or in tercel lu lar subs tance was never encountered.

The Enclosing Cells

Even though the a l t e ra t ions seen in these cells were no t as p ronounced as in the s te ro id-producing cells, there was no doub t t h a t admin i s t r a t ion of gonado- t ropins had some influence on the i r morpho logy (Figs. 14, 18-20).

The most conspicuous changes were the a l t e ra t ions f rom rough- to smooth-surfaced endoplasmic re t i cu lum (Fig. 20), increase of the dens i ty of the m a t r i x in the mi tochondr ia (Fig. 20), and accumula t ion of l ip id drople t s (Fig. 19).

The nucleus a t t a i n e d a more spherical form, and the a m o u n t of ch romat in seemed to decrease.

Genera l ly the organelles of the cell became more l ike the organelles of the s te ro id-produc ing cells, bu t the i r morphological differences were still as pronounced as in the normal cells. The mi tochondr ia never became tubular . The differences be tween mi tochondr ia as well as in the ground cy top lasm are c lear ly d e m o n s t r a t e d in Fig. 20.

I n t e r s t i t i a l Tissue and Cells

As men t ioned prev ious ly (Dahl, 1970b, c, d), the theca in t e rna in add i t ion to the theca l gland, possessed some cells which seemed to be ra the r undi f ferent ia ted . However , admin i s t r a t i on of gonadot ropins , and especial ly PMSG, induced m a r k e d a l te ra t ions in these cells. Normal ly , t hey possessed only rough-surfaced endoplasmic

Figs. 8-13. Demonstrate alterations within the mitoehondria of the steroid-producing cells of the thecal gland seen after treatment of gonadotrophins for varying periods

Fig. 8. Generally there is an increase in number as well as in size and density of the mitoehondria of the steroid-producing cells. Smooth endoplasmie reticulum (SER). Mitoehon-

drion (M). X 14400

Fig. 9. Giant mitochondria (M) with closely packed cristae were regularly encountered. Arrow indicates a small, rodshaped mitochondrion. Lipid droplets (L). x 14400

Fig. 10. Deposition of dense material within the matrix surrounded by concentric membranes is seen more frequently than in the control material, x 36000

Fig. 11. Elongated forms of mitoehondria (M) are seen enlarged, x 14400

Fig. 12. Sometimes the mitochondria (M) had indentations which almost divided them. x 21600

Fig. 13. High magnification of the mitochondrion in Fig. 12 illustrating vacuoles or blebs (short arrow) at the outer membrane. Note the continuity of the outer membrane and the

cytoplasmic vacuole (long arrow), x 54000 Fig. 14. The illustration demonstrates the differences in the steroid-producing cell (SC) and the enclosing cell (EC) with their membranous contact without any intervening basement membrane (arrows). In these portions of the cells there is a pronounced difference in the mitochondria (M1-M~) and the ground cytoplasm. Note the amount of rough endoplasmic reticulum (RER) in EC and the amount of smooth endoplasmic reticulum (SER) in SC.

• 54 000

Fig. 15. Following gonadotrophin injections there is a marked hyper t rophy of the smooth- surfaced endoplasmic reticulum (SER) in the basal cytoplasm. In the enclosing cells (EC) there

are lipid droplets (L). Mitochondrion (M). • 21600 Fig. 16. In some of the steroid-producing cells there is an increased amount of fibrillar material (F) in the PMSG- t reated animals after 28 days. Lipid droplets (L). Smooth endo-

plasmic reticulum (SER). • 21600 Fig. 17. Pinocytic vesicles (PV) are considerably increased in number in the steroid-producing cells. The vacuoles are coated with a fuzzy material. Lipid droplets (L). x45000

10a Z. Zellforsch., Bd. 113

Figs. 18-20


ret iculum in a rather large amount , elongated, small mitochondria wi thout tubu- les and only occasionally lipid droplets. After administrat ion of gonadotropins, however, their rough-surfaced endoplasmic ret iculum changed to smooth (Figs. 21, 22), and a direct communicat ion between rough- and smooth surfaced endoplasmic ret iculum was regularly encountered (Fig. 22). There was an increase of lipid droplets (Figs. 21, 22) and enlargement of the mitochondria (Fig. 24), which also seemed to be tubular (Fig. 22). Generally, they seemed to be much like the steroid-producing ceils of the thecal gland with almost the same organelles, though not as pronounced as in those cells. Cilium was also observed (Fig. 23).

Discussion

General

Opinions on the role of the various ovarian cells in steroid production have differed from time to t ime (Falck, 1959, Bjersing, 1967). Although the basic dependence of the ovarian cells on the secretion of p i tu i tary gonadotropins has long been established, no studies have documented the ul t ras t ructural changes in the thecal gland by exogenous gonadotropic stimulation. I n previous reports (Dahl, 1970b, c, d), it has been revealed tha t in the theca interna of the domestic fowl, the thecal gland is the main site of steroid-producing cells. I t therefore seemed reasonable to examine the influence of gonadotropins on these cells.

The first question which arises in a s tudy of the fine structure of the ovarian tissues is whether mammal ian pi tu i tary hormones exert any st imulating action on the ovarian cells of the domestic fowl. Tha t administrat ion of mammal ian hormones m a y induce ul t ras t ructural changes in the fowl has recently been documented by Kjaerheim (1968 c) who demonst ra ted alterations in the adrenocortical cells after administrat ion of ACTH. Wi th regard to the gonadotropflls, Opel and Nalbandov (1961) demonst ra ted the macroscopic effect of mammal ian F S t t as well as P M S G on the ovary of the fowl.

I n the present investigation there was general evidence of effect th rough increase to the comb and of body weight. Over a longer period there atso seemed to be symptoms of overst imulation or toxic effect. There was a marked progressive

Fig. 18. In some steroid-producing cells there is an increase of fibrillar material (F) at the periphery, with a decrease of smooth-surfaced endoplasmic reticulum. Note the paucity of free ribosomes (R). Note the hypertrophic enclosing cell (EC) with nucleolus (Nu) and lipid drop-

lets (L). Desmosome (D) characteristic for this cell type. • 16200

Fig. 19. Survey illustration showing portions of several cells from the thecal gland with steroid-producing cells (SC) to the left and enclosing cells (EC) to the right. The micrograph illustrates the transformation of the enclosing cell to a cell type with a more spherical nucleus and nucleolus containing lipid droplets (L). There is still a difference m the endoplasmic reticulum between the two cell types. Smooth endoptasmic reticulum (SER). Rough

endoplasmic reticulum (RER). • 10800 Fig. 20. Portions of a steroid-producing cell (SC) and enclosing cell (EC) as seen after gonadotrophin treatment. Note the differences in the ground cytoplasm and the organeIles. The mitochondria are dense (M1-M~) and differ in size and appearance of cristae. Arrow indicates transformation of rough endoplasmic reticulum (RER) to smooth endoplasmic reticulum (SER).

Lipid droplets (L). Nucleus (N). • 21600

lob Z, Ze]Norsch., Bd. 113

Figs. 21-24


increase in the total number of follicles entering the phase of rapid growth and a significant accumulation of small follicles. HCG was less able to induce growth of large follicles than PMSG. However, long treatment with PMSG only caused the follicles to undergo degeneration, and, obviously, this hormone did not support the growth of the follicle to mature size. On the other hand, the administration of HCG over a long period generally seemed to stimulate all the normal functions of the ovary compared with the control animals.

The interpretation of changes in the fine structure after administration of bio- logically active substances has previously been hampered by fixation difficulties. With the introduction of improved fixation techniques and by using an experimental animal with a well-developed steroid-producing gland in the theca interna of the follicle, some of these difficulties seem to have been circumvented. The findings presented in this study have revealed that administration of gonadotropins can induce alterations in the fine structure of the theca interna, and especially in the steroid-producing cells of the thecal gland.

Changes within the Organelles

The Steroid-Producing Cells

The Endoplasmic Reticulum. The endoplasmic reticulum is now established as the site of a large number of steps in steroid biosynthesis (Christensen, 1965; Kjaerheim, 1968b, c). The increase in the quantitiy of this organelle, as demonstrated in this study, therefore supports that the thecal gland is involved in the steroid production. Furthermore, combined and compared with previous biochemical and physiological investigations on hypophysectomized animals (Opel and Nalbandov, 1961), the present study seems to disclose that steroid production is regulated directly by the influence of the gonadotropins. An interesting phenomenon was that the increase of smooth endoplasmic reticulum was combined with depletion of lipid. Furthermore, lipid droplets could be demonstrated in the interstitial cells and enclosing cells previous to the formation and the increase of smooth endoplasmic reticulum. These observations seem to indicate that a certain amount of lipid, as precursor, is necessary for the formation and the increase of smooth endoplasmic reticulum. Finally, the increase of smooth endoplasmic reticulum which

Figs. 21-24. Illustrate alterations of the connective tissue cells in the thcca interna following long-term treatment with gonadotrophins

Fig. 21. Survey illustration of a portion of the theca interna showing the hypertrophy of the connective tissue cells after gonadotrophin-treatment. The nucleus (N) becomes spherical, the population of mitochondria (M) increases and lipid droplets (L) are seen in the cells.

Rough-surfaced endoplasmic reticulum (RER). • 10800

Fig. 22. High magnification of one of the cells in Fig. 21 where a large amount of smooth- surfaced cndoplasmic reticulum (SER) is seen. Arrow indicates continuity of smooth endoplasmic reticulum (SER) and rough-surfaced endoplasmic reticulum (RER). Nucleus (N). Lipid

droplets (L). Ribosomes (R). Mitochondrion (M). • 21600

Fig. 23. Contrary to the normal material, cilium (CI) is also seen in these cells after gonadotrophin-treatment. •

Fig. 24. There is an increase in the size of the mitochondria (M) as well as in the amount of free ribosomes (R) in these cells after gonadotrophin-treatment. •

150 E. Dahl:

was found in the basal part of the cell, may suggest that the smooth endoplasmic reticulum, in addition to taking an active part in the steroid biosynthesis, also functions as a storing place for precursors of the steroid hormones.

Mitochondria. The most prominent alterations in the mitochondria were an increase in number and in density, mitochondrial gigantism, and a more hetero- geneous mitochondrial population. These features generally seem to indicate a hyperfunetion (Kjaerheim, 1968 c). The different types of alterations seen in this study are also described in the interstitial cells of testes after administration of gonadotropins (de Kretser, 1967). Since preservation of mitochondria was no pro- blem in the present investigation, it seems reasonable to presume that the different changes reflect biological alterations rather than fixation artifacts. The alterations are also consistent with changes seen in adrenocortical cells after stimulation with ACTH (Kjaerheim, 1968e).

Toren et al. (1964) demonstrated that the mitochondria of the interstitial cells of rat testes were involved in the transformation of cholesterol to pregneolone. The increase in number of mitochondria in the steroid-producing cells of the thecal gland, as seen in this study, most likely reflects an increased metabolic activity, together with an increased demand for the mitochondrial enzymes which cleave the cholesterol side-chain in its conversion to pregneolone (Christensen, 1965). In this context, the increase in size and surface area of the mitoehondria presumably allows a larger reactive area for oxidative phosphorylation and cholesterol conversion (De Kretser, 1967). The observation in the stimulated cells in this study of an increased number of mitochondria containing accumulation of intramitoehon- drial electron-dense matrix, supports the postulation of Christensen (1965) that the side-chain cleaving enzyme is located within the mitochondria rather than on its surface, and that an accumulation of substrates within the mitochondria will result in a dense matrix. Furthermore, the increase in number of mitochondria containing lipid may be evidence of a building up of substrate in the mitochondrial matrix. The same intra-mitochondrial lipid was previously observed by Kjaerheim (1968 e) in adrenoeortical cells after stimulation with ACTH, by De Kretser (1967) in interstitial cells of human testes after stimulation with gonadotropins, and by Gordon et al. (1964) in their study of the gonads in testicular feminization syn- drome. Common for all these studies is the fact that they are dealing with cells which are under higher hormonal stimulation than normally, and it therefore seems more reasonable to presume these forms of mitochondria to be exhaus- ted mitochondria in the process of being broken down, as suggested by Kjaerheim, (1968c). This is also supported by the observation that in degenerated thecal glands the same intra-mitoehondrial lipid accumulation is regularly found (Dahl, 1970e).

I t is well known that the mitochondria participate in the steroidogenesis, but little is known about the ultrastructural changes which are combined with the elaboration of steroid hormones or how this hormone leaves the mitochondria. The increase in blebs and vacuoles on the surface of the mitoehondria seems to indicate the possibility that the steroid hormones, or their precursors, may leave the mitoehondria via these membrane structures. The presence of membrane- bound vesicles, of the same morphology as the outer membrane of the mitochondria, adjacent to the mitochondria, may support this suggestion.


The Golgi Apparatus. The Golgi complex increased significantly in size following administration of gonadotropins, especially PMSG. Membrane-bounded bodies were observed in increased numbers in relation to the increase of the Golgi complex and, furthermore, the membrane-bound bodies were found to be increased in number within the Golgi apparatus. These findings seem to support the view of Crabo (1963) and De Kretser (1967) suggesting that the Golgi region is impli- cated in the formation of the membrane-bound bodies found in the interstitial cells of testes. No connection could be traced between the increased Golgi region and the increase of the endoplasmic reticulum. Fishman et al. (1967) suggested tha t enzyme proteins may be ordered in arrangements similar to the electron system in mitochondria, and with their substrates may contribute to the structure of organelles such as the endoplasmic reticulum. They also suggest that fragments of organelles containing these enzymes are sequestered in lysosomes. As revealed in this study, membrane-bounded dense bodies reached a certain size, and then small fragments seemed to be sequestered. This concept leads to the consideration tha t metabolic activity produced by the stimulation of gonadotropins may result in an increased turnover of organelles and may, consequently, also result in a rise in the number of lysosomes (De Krctscr, 1967). On this basis, the increase in lysosomes seen in the stimulated cells in the thecal gland may simply be a reflec- tion of the high metabolic activity associated with the increased steroid biosynthesis due to gonadotropin stimulation, linked to an increased protein synthesis within the cell, or uptake of substances from outside the cell rather than directly involved in steroid secretion (Kjaerheim, 1968).

Lipid and Dense Bodies. Normally there seems to be a balance between the lipid droplets, the dense bodies and the smooth endoplasmic rcticulum, probably regulated by the influence of the gonadotropins. Since the lipid is a precursor in the steroid synthesis, an increased influence of the gonadotropins wilt result in an increased mobilization of the lipid. At the same time, however, there seems to be an increase in number of dense bodies. Furthermore, in areas where the lipid droplets completely disappear, the dense bodies do not seem to be confined to the apical part, but are also found more towards the basal area. The normal morphological transition from the apical to the basal part seems to be changed, which points to the fact that there may be a biochemical interaction between the dense bodies, the lipid droplets and the endoplasmic reticulum.

Ribosomes, Coated Vesicles, Pinocytosis. Administration of gonadotropins leads to a rapid synthesis of proteins and RNA in the ovary {Reel and Gorski, 1968a, b). Correspondingly, the increased number of ribosomes, as found in this study, indicates an increased protein synthesis necessary for the growth of the cell. Mitosis were not observed. The last phenomenon may indicate that there is no increase in DNA, but that the cell only increases in volume (Kjaerheim, 1968 c).

Coated pinocytic invaginations and vesicles are thought to be involved in the cellular uptake of protein (Kjacrheim, 1968b, c). In this s tudy there was a significant increase in pinocytic vesicles. This phenomenon most likely reflects the increased activi ty of the stimulated cell and an increased demand for proteins (Kjaerheim, 1968 c).

The Nucleus. The demonstration in this s tudy that the size of the nucleus of the steroid-producing cells in the thecal gland increased following administration of gonadotropins is similar to what has previously been observed in adrenocortical

152 E. DaM:

cells as seen after ACTH administration (Kjaerheim, 1968c). In the electron micrographs of this s tudy the nuclear membrane was evenly and regularly curved in the hypertrophic cells, contrary to the invaginations of the small nuclei seen after t reatment with different steroids (Dahl, 1970e). The growth of the nucleus most likely reflects the increased metabolic activity of the whole cell in the increase protein production required by the cell and the increased demand for enzymes involved in the steroidogenesis. One of the striking features of the nucleus was the amount of nuclear bodies randomly distributed in the nucleus outside the nucleolus. Such bodies have previously been described in the domestic fowl by Kjaerheim (1968b), and later observed in a variety of cell types in this species (Dahl, 1970a). There seemed to be no doubt that nuclear bodies are normal organelles of the cells without any etiological or pathological significance (Dahl, 1970a). However, the most interesting finding in this s tudy was the fact the internal structure of the nuclear bodies seemed to be more complex, which is consistent with previous suggestions that their morphology is dependent on, and reflects the metabolic activity of the cell (Dahl, 1970a, f).

The Nucleolus. In the fowl the granular and fibrillar parts of the nueleolus in adrenocortieal cells seem to be more segregated than in most mammalian cells. This has also been found in the adrenals (Kjaerheim, 1968b, e). A previous investigation related to the nuclear bodies (Dahl, 1970a) revealed the same phenomenon and it was observed in several organs. After stimulation with gonadotropins this segregation became more pronounced in the steroid producing cells of the thecal gland. Segregation of nucleolar elements has been reported following administration to toxic substances (Svoboda etal., 1967). However, since the nucleolar elements of the domestic fowl are normally segregated, and because the same observation was made in the adrenoeortical cells following ACTH stimulation, this change is considered to be an indication of hyperact ivi ty rather than toxicity (Kjaerheim, 1968c).

The Enclosing Cell

Administration of gonadotropins obviously had some influence on the morphology of the enclosing cells. The most interesting phenomen was that these cells, from having the appearance of a kind of supporting cell, a sustentacular cell, changed more towards a steroid-producing cell with smooth-surfaced endoplasmic reticulum, lipid droplets and decrease in fibrillar material. Furthermore, the matrix of the mitochondria became more dense, even ff tubular formations could not be demonstrated. Compared with the steroid-producing cells and the undifferentiated cells of the theca interna, these cells ended up as an intermediary type. All these observations point to increased activity in both of these cell types, but definite interaction between the steroid-producing cells and the enclosing cells could not be demonstrated from the morphological point of view, except for their close spatial relationship. Whether their qualitative changes were only dependent on the stimulation or there exists a functional interaction between these two cell types could not be elucidated.

The Interstitial Cells

In addition to the thecal gland, the theca interna also possesses cells of connective tissue origin. Obviously these cells changed after administration of


gonadotropins. As demonstrated, these cells originally had the appearance of fibrocytes, but after hormonal stimulation they contained the morphological features characteristic of steroid synthesis: Smooth-surfaced endoplasmic reticulum, lipid inclusions and dense mitochondria. These findings are all consistent with the previous report of Merker and Diaz-Encinas (1969) who, after administration of PMSG to juvenile rats and rabbits, observed the same alterations in fibrocytes in the theca intcrna. One of the striking features in the present study was the regularly encountered phenomenon of the rough-surfaced endoplasmic reticulum being continous with the smooth-surfaced endoplasmic reticulum, indicating that the latter organelle in this situation is transformed directly from the first. Furthermore, there appeared to be a necessity for lipid droplet formation before this transformation, which seems to support the view that lipid droplet together with smooth endoplasmic reticulum are needed in steroid-producing cells.

Correlation o[ Structure and Function

Correlation of structure and function of steroid-producing cells can be based on general knowledge regarding the function of the organelles, morphological comparison between the normal and the hypo- or hyperactive states, and biochemical data (Kjaerheim, 1968a, b, c).

Concerning the ovary, particular advantage is offered by the domestic fowl, as compared with the mammal, because the population of the steroid-secreting cells seems to be more homogeneous (Dahl, 1970b). The functional significance of the different organelles has been discussed above. By comparing these findings with general knowledge of function and morphology (Young, 1961 ; Zuckerman, 1962), there seems to be doubt that gonadotropins induce a hyperactive state in the steroid-secreting cells when administrated in large doses. This is reflected in the electron micrographs as lipid depletion and hypertrophy of smooth endoplasmic reticulum.

Since Claesson et al. (1947a, b, 1948, 1953, 1954) applied a number of histochemical tests designed to visualize lipid and steroid materials in the ovary (Claesson et al. 1947a, b, 1948, 1953, 1954; Rennels, 1951), several workers have reported enhanced steroid-production following administration of gonadotropins (Rcnncls, 1951; Zuckerman, 1962). However, there have been divergences of opinion about the site of production of the estrogens in the ovaries. While the theca interna cells were postulated to be active in hormone production in the early histochemical literature (Appelgren, 1967), the granulosa cells were presumed to play little, if any, role (Appelgren, 1967). Claesson et al. (1947a, b, 1948, 1953, 1954) in their work found that the variation in the cholesterol content of the theca interna cells could be correlated to the secretion of estrogens. Falck (1959), however, suggested that the layers of both the granulosa cells and the theca cells were necessary for the estr )ten biosynthesis. Bjcrsing (1967) suggested that C-19 precursor steroids are elaborated by the theca interna cells and transferred to the granulosa cells for production of testerone. Aromatization to estrogen, particularly oestradiol-17 •, may then be carried out by both granulosa cells and theca cells. Finally, Appelgren (1967), using labelled cholesterol, supports the findings of Claesson et al. (1947a, b, 1948, 1953, 1954) concerning the distribution of chole-

154 E. Dahl:

sterol in the ovary, but radioactivity, although in a low concentration, was also found in the granulosa cells and even in the ovum.

In the present investigation it has been demonstrated that gonadotropins, and especially PMSG caused hypertrophy of the steroid-producing gland in the theca intcrna, with depletion of lipid and increase in all the other organelles. The findings seem to support those of Claesson et al. (1947a, b, 1948, 1953, 1954). Concerning the two-cell theory of Falck (1959) and Bjersing (1967) mentioned above, the close relationship revealed between the enclosing cells and steroid- secreting cells leads to the speculation as to whether they may have some functional interaction or be dependent on each other, since both types react to the gonadotropins. There seems to be little evidence that some precursor from the steroid- secreting cell should be transferred to the enclosing cells and from these to the granulosa cells. In this way, an interaction between the steroid-secreting cells and the granulosa cells would require the precursor to pass through the enclosing cells, the basal membrane of the thecal gland, theca interna, and the membrane of granulosa cells, such an interaction seems less likely in the domestic fowl.

Biochemical studies of immature rats have revealed that estrogen is liberated 6 to 8 h after injection of PMSG (Reel, 1968a, b). In the present investigation no ultrastructural alterations could be demonstrated even after 18 h, which is in accordance with the results obtained by Christensen (1959), who failed to demonstrate any ultrastructural changes in the interstitial cells of testes of rats treated with a single intravenous dose of HC G. Release of lipid droplets into the blood-stream as suggested by Guraya et al. (I964, 1967) has not been observed. I t therefore seems probable that the ovarian steroids or their precursors, must be stored in such large amounts that a " m o m e n t a r y " release will not have any influence on the cytological structure. Regarding the pituitary-ovarian interrelationship, the role of the hypothalamus and neuronal mechanism must be included as a necessity in the considerations, the fact that the steroid-secreting cells are furnished with a complex innervation may be of some functional significance as to the releasing or the synthesizing factors. Considering to what extent the gonadotropins on the one hand, and the steroids on the other comprise a closed circuit with reciprocal interaction, the present investigation has only confirmed that the gonadotropins, directly or indirectly have the ability to induce morphological alterations which are consistent with stimulation of the production of steroids. Presuming tha t the effect of the gonadotropins on the steroid-secreting cells is a direct one, it has not been possible to demonstrate the entry of the gonadotropic molecule into the steroid- secreting cell. Reel and Gorski (1968a, b) found a significant increase in cystidine and amino acid incorporation into RNA and protein respectively, 4 h after PMSG treatment of mature rats. They propose tha t gonadotropins act primarily at early stages by increasing precursor delivery to the immature ovary. Presumably this increase in precursor delivery is mediated via an expansion of the microcirculation, which, in turn, result in an enhanced perfusion of blood through the ovary. In the domestic fowl there seem to be anatomical structures which can support this view. The fact that the thecal gland, as the site of steroid-producing cells, is in close contact with the capillaries, indicates that especially this structure must either be dependent on, or related to the microcirculation in a more important way than the other structures of the follicle. Furthermore, the hypertrophy of the Golgiapparatus


the increased size of the nucleus and nucleolus, and the increased n u m b e r of nuclear bodies are consistent with RNA and protein accumulat ion, steroid production, and enhanced ac t iv i ty of the cell. Regarding the al terat ions demonst ra ted in the connective tissue cells in the theca in terna , these are most probably due to the same factors which act on the thecal gland. The cells mus t be accepted as mult i- potent , and the relat ively high doses adminis t ra ted have s t imula ted the cells to be t ransformed to the steroid-producing type, a f inding which is consistent with observations made by Merker and Diaz-Encinas (1969). However, since adequate evidence exists to show tha t the avian ovary secretes three different hormones, estrogen, androgen and progesterone, the possibili ty may exist tha t the different cell types described may produce specific hormones (Dahl, 1970e).

I n conclusion, the present s tudy seems to present a morphologic basis for the physiological impor tance of the theca interna. There seems to be no doubt tha t in the theca in te rna of the domestic fowl, the thecal gland with the steroid-producing cells is the s t ructure which is the target for the effect of gonadotropins. This demons t ra t ion therefore support the other reports in this series of exper iments (DAM, 1970b-f) t ha t the thecal gland is the ma in site of steroid product ion in the theca interna.

R e f e r e n c e s

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Belterman, R., Stegner, H. E. : Elektronenmikroskopische Untersuchungen an den Ovarien neugeborener MKuse nach Behandlung mit humanem hypophys~rem Gonadotropin (HH G) Acta endoer. (Kbh.) 57, 279-288 (1968).

Bjersing, L. : On the morphology and endocrine function of granulosa cells in ovarian follicles and corpora lutea. Acta endoer. (Kbh.), Suppl. 125, 1-23 (1967).

Christensen, A. K. : The fine structure of the interstitial tissue of the rat testis at various ages and after experimental treatment. Anat. Rec. 183, 367 (abst.) (1959).

- - The fine structure of testicular interstitial cells in guinea pigs. J. Cell Biol. 26, 911-935 (1965).

Claesson, L. : Quantitative relationship between gonadotrophic stimulation and lipid c~anges in the interstitial gland of the rabbit ovary. Acta physiol, seand. 81, Suppl. 113, 23-51 (1954).

- - D i c z f a l u s y , E., Hillarp, N./~., HSgberg, B.: The formation mechanism of oestrogenic hormones. III. Lipids of the pregnant rabbit ovary and their changes at gonadotrophic stimulation. Acta physiol, scand. 16, 183-200 (1948).

- - Hillarp, N. A. : The formation mechanism of oestrogenic hormones. I. The presence of an oestrogen-precursor in the rabbit ovary. Acta physiol, scand. 18, 115-129 (1947a).

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- - - - Sterol content of the interstitial gland and corpora lutea of the rat, guinea pig and rabbit ovary during pregnancy, parturition and lactation. Acta anat. (Basel) 5, 301-305 (1948).

- - - - HSgberg, B. : Lipid changes in the interstitial gland of the rabbit ovary at oestrogen formation. Acta physiol, scand. 29, 329-339 (1953).

Crabo, B. : Fine structure of the interstitial cells of the rabbit testes. Z. Zellforsch. 61, 587-604 (1963).

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structure of the ovarian interstitial tissue in the rat and the domestic fowl. J. Anat. (Lond.) (accepted for publication) (1970).

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156 E. Dahl: Effects of Gonadotropins on the Thecal Gland (Fowl)

Dahl E. : Studies of the fine structure of ovarian interstit ial tissue. 3. The innervation of the thecal gland of the domestic fowl. Z. Zellforsch. 109, 212-226 (1970).

- - Studies of the fine structure of ovarian interstit ial tissue. 4. Effects of steroids on the thecal gland of the domestic fowl. Z. Zellforsch. (accepted for publication) (1970).

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Falck, B. : Site of production of oestrogen in ra t ovary as studied in micro-transplants. Acta physiol, scand. 47, Suppl. 163, 1-101 (1959).

Fishman, W. H., Goldman, S. S., De LeIlis, R.: Dual localization of B-Glucuronidase in endoplasmic reticulum and lysosomes. Nature (Lond.) 218, 4 5 7 4 6 0 (1967).

Gordon, G. B., Miller, L. R., Bensch, K. G. : Electron microscopic observations of the gonad in the testicular feminization syndrom. Lab. Invest . 13, 152-160 (1964).

Guraya, S. : Cytochemical observations concerning the formation, release, and t ranspor t of lipid secretory products in the interstitial (thecal) cells of the rabbi t ovary. Z. Zellforsch. 83, 187-195 (1967).

- - Greenwald, G. S. : Histochemical studies on the interstit ial gland in the rabbi t ovary. Amer. J. Anat. (Lond.) 114, 495-519 (1964).

Kjaerheim, A. : Studies of adrenocortical ul trastructure. 2. The interrenal cell of the domestic fowl, as seen after glutaraldehyde perfusion fixation. Z. Zellforsch. 91, 4 2 9 4 5 5 (1968).

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Kretser, de D. M. : Changes in the fine s tructure of the human testicular intersti t ial cells after t rea tment with human gonadotrophins. Z. Zellforsch. 88, 344-358 (1967).

Merker, H. J., Diaz-Encinas, J. : Das elektronenmikroskopische Bild des Ovars juveniler l~at- ten und Kaninchen nach Stimulierung mit PMS and HCG. Z. Zellforsch. 94, 605-623 (1969).

Millonig, G. : The advantages of a phosphate buffer for OsO 4 solutions in fixation. J. appl. Phys. 32, 1637 (1961).

Opel, H. L., Nalbandov, A. V. : Follicular growth and ovulation in hypophysectomised hens. Endocrinology 69, 1016-1028 (1961).

Reel, J . R., Gorski, J . : Gonadotrophic regulation of precursor incorporation into ovarian RNA, protein and acid-soluble fractions. I. Effects of pregnant mare serum gonadotrophin (PMSG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Endocrinology 88, 1083-1091 (1968).

- - - - Gonadotrophic regulation of precursor incorporation into ovarian RNA, protein and acid-soluble fractions. II . Changes in nucleotide labeling, nuclear RNA synthesis, and effects of RNA and protein synthesis inhibitors. Endocrinology 88, 1092-1100 (1968).

Rennels, E. G. : Influence of hormones on the histochemistry of ovarian interstit ial tissue in the immature rat. Amer. J . Anat. 88, 63-100 (1951).

Reynolds, E. S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963).

Ryter, A., Kellenberger, E. : L'inclusion au polyester pour l 'ultramicrotomie. J. Ultrastruct . , Res. 2, 200-214 (1958).

Svoboda, D., Racela, A., Higginson, J. : Variations in ul t rastructural nuclear changes in hepa- tocarcinogenesis. Biochem. Pharmacol. 16, 651-657 (1967).

Toren, D., Menon, K. M., Forcheilli, E., Dorfman, R. I. : In vitro enzymatic cleavage of the cholesterol side-chain in ra t testis preparations. Steroids 8, 381-386 (1964).

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Dr. Erik Dahl Depar tment of Anatomy Dental Facul ty Universi ty of Oslo Blindern Oslo 3, Norway

studies of the fine structure of ovarian interstitial tissue

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