Studies of the fine structure of ovarian interstitial tissue

Z. Zellforsch. l l3 , 111-132 (1971) �9 by Springer-Verlag 1971

Studies of the Fine Structure of Ovarian Interstitial Tissue 4. Effec ts of S t e ro ids on t h e Theca l G l a n d of t he D o m e s t i c F o w l

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

Received September 24, 1970

Summary. The fine structure of ovarian steroid-producing cells of the domestic fowl as seen after administration of steroids is described. Diaethylstilboestrol, estradiol and hydr- oxyprogesterone were given as intramuscular injections for a 28-days period. The main cytoplasmic changes of the steroid-producing cells were an increase in the number and the size of lipid droplets due to a possible expansion of the endoplasmic reticulum and a confluence of the smooth endoplasmic reticulum and the lipid droplets. The mitochondria became paler, markedly reduced in size and number, and contained less cristae. The Golgi apparatus became smaller and tended to disappear. The most prominent nuclear changes were irregular outline of the nuclear membrane, shrinkage of the nucleus, formation of pseudo-inclusions, decreased number of nuclear bodies with a tendency to vacuolization and small nucleoli. These alterations have never been demonstrated in ovarian steroid-producing cells before, and they are consistent with atrophic changes observed in steroid-producing cells in other organs. The present study substantiates the view that the thecal gland must be concerned with endocrine function. The possible functions of the different cell types of the theeal gland are discussed.

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

I n spite of a vas t a m o u n t of l i t e ra ture deal ing with the in te rs t i t i a l cells of the ovary , there is as y e t l i t t le real in format ion as to thei r funct ion (Appelgren, 1967; Bjersing, 1967; Fa lk , 1959; Rennels , 1951). Almos t every t issue componen t of the ova ry has been cla imed to be the source of estrogen and progesterone (Young, 1961).

I n a t t e m p t s to gain fur ther in format ion abou t the funct ional role of the ova ry and i ts in te r s t i t i a l cells, s teroids have been admin i s t r a t ed to exper imenta l animals . Rennels (1951), using a h is tochemieal technique, found t h a t the admin i s t r a t ion of es t radiol or s t i lboestrol resul ted in a m a r k e d inhibi t ion of ovar ian growth in juveni le rats , and p reven ted the normal deve lopmen t of p r i m a r y in te r s t i t i a l t issue, while proges terone was wi thou t effect in the doses employed. According to E v e r e t t (1961), chronic admin i s t r a t ion of estrogen to i n t ac t animals even tua l ly p roduced ovar ian a t r o p h y by suppression of gonado t rop in secretion.

Brcneman (1955, 1956) inves t iga ted the effect of in ject ion of es t radiol into 30 days old pullets , and found no signif icant difference from ovar ian weights of controls. His tochemica l ly , however, there was evidence t h a t 1 ~g es t radiol caused increased cholesterol deposi t ion in the follicle.

Phi l l ips (1943, 1960) in jec ted 12.5 mg d ie thyls t i lbes t ro l per week into 6 weeks old pul le ts and regis tered a 32 per cent increase in ovar ian weight.

Large doses of proges terone admin i s t r a t ed to lay ing hens in pas te or pel le t form in t e r rup t egg p roduc t ion (Adams, 1955, 1956). Ro thch i ld and F r a p s (1949a,

112 E. Dahl:

b) demonst ra ted tha t progesterone injections about 36-38 hr before expected ovulat ion resulted in atresia of the ovar ian follicle. Atresia can be the result of an inhibi t ion of all gonadotropin secretions, or of the release of too small an a m o u n t of the lutein hormone to cause ovulat ion (van Tienhoven, 1961). Morphological studies on the effects of different steroid adminis t ra t ions on the ovary have been rather l imited (Bullough, 1943; Williams, 1940). Since corresponding changes in the fine s t ructure of ovar ian interst i t ial cells after inject ion of steroids, to the authors knowledge, have never been examined, the present s tudy was performed.

The fine s tructure of the normal steroid-producing cells in the thecal gland has been described previously (Dahl, 1970a, b, c). Since the interst i t ial tissue of the hen ovary has a more homogeneous morphology than in the mammal (Dahl, 1970a), the domestic fowl seems to be well suited for experimental studies of ovarian ul t ras t ructure . The invest igat ion was aimed at gaining informat ion about the u l t ras t ructure of the thecal gland caused by adminis t ra t ion of different synthet ic steroids with pharmacological effects similar to those of endogenous steroids. As it is evident tha t estradiol circulated in the egg-laying domestic fowl (O'Grady, 1966) and progesterone is also produced by the hen ovary (Fraps et al., 1948, 1949) the following, synthet ic hormones: 1) estradiol, 2) hydroxy- progesterone, 3) stilbestrol were administered. Originally, this work was p lanned as three separate studies, and was also performed in this way. However, since the observations proved to be of the same type with all the three steroids, the result are described together in this paper.

Materials and Methods

Twenty White Leghorns hens, 18-24 months old, with an average body weight of 1824 g, and six 3 month-old chickens of an average weight of 900 g were used. The animals were housed in individual cages in a well-ventilated, constant-climate room (17 ~ C, controlled illumi- nation, light on 7 a.m., off 7 p.m., relative humidity 60 %). The diet consisted of commercial chicken fodder, cabbage, sand grits and water ad lib. The main constituents of the fodder were proteins (17-19%), fat (24%), calcium (1.1 1.2%), phosphorus (0.7%) and sodium chloride (0.5 % ). The hens were kept for at least l0 days to get adapted to their environment before the experiment started.

The hormone preparations 1 used were: Estradioli valerianas (Primogyn-Depot, "Schering") This will be referred to as estradiol. Diaethylstilbestroli (Stilbestrol, "Nyco"). This will be referred to as stilbestrol. Hydroxyprogesteroni caproas (Primolut-Depot, "Schering"). This will be referred to as oxyprogesterone. Of the 20 hens and 6 chickens employed in this experiment, 5 hens and 3 chickens served as controls.

The steroids were administrated as follows: Three chickens received 3 mg estradiol as an intramuscular injection 7 days before sacrifice. Five hens received 1 mg estradiol as daily intramuscular injections for 28 days. Five hens received 1 mg stilbestrol as daily intramuscular injections for 28 days. Five hens received 25 mg oxyprogesterone as daily intramuscular injections for 28 days. The hens were sacrificed 24 hr after the last injection. The controls were kept under identical conditions except for the intramuscular injections.

Fixation 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, at pH 7.3. The perfusion lasted for a minimum 10 min. Details of the perfusion technique

1 Primogyn-Depot and Primolut-Depot were a gift from Schering A.-G., Berlin, Germany, obtained through the Norwegian representative Barfods Farmaceutiske A/, Oslo. Stilbocstrol was a gift from NYCO, Nyegaard & Co. A/S Oslo, Norway. I should like to express my gratitude for both donations.

Effects of Steroids on the Thecal Gland (Fowl) 113

have previously been reported by Kjaerheim (1969). The ovary was then excised and, while kept in a drop of fixative, cut into thin slices under the dissecting microscope. Samples from follicles of different sizes were then fixed separately in glutaraldehyde for an additional period of 2 hr at 4 ~ C. Subsequently the tissue blocks were rinsed for 10 rain in 0.15 M phosphate buffer at pH 7.3 and fixed in 1% osmium tetroxide at 4 ~ C for 2 hr. The blocks were rapidly dehydrated in a graded series of acetone and embedded in Vestopal W. (Ryter and Kellenberger, 1958). Ultrathin sections were cut on an LKB Ultrotome and treated with uranyl acetate for 30 min followed by lead citrate (Reynolds, 1963) for 5 rain to increase contrast. The sections were examined in Siemens Elmiskop Ia electron microscope, equipped with 50 microns platinum objective apertures. Accelerating voltage was 80 kv. One micron thick sections were also cut for light microscopy and stained for 30 sec on a heating stage with 0.1% toluidine blue adjusted to pH 8.5 with M/15 Na2HPO 4.

Results

General

In the domest ic fowl only the left o v a r y reaches a funct ional s ta te . The cor tex of the ova ry possesses l i t t le s t romal tissue, bu t is subjec ted to wide s t ruc tu ra l var ia t ions according to the reproduc t ive s ta te . I n egg-laying hens there is a hier- a rchy of developing follicles of different sizes. I n the theca in t e rna of each follicle, the thcca l g lands are seen as his tological ly well-defined s t ructures . Several g lands are seen th roughou t the whole circumference of the follicle, a lmost a t a regular d is tance from each other. They are composed of two cell types . The vas t m a j o r i t y of the cells of the thecal g land have the fine s t ruc ture analogous with s te ro id-producing cells in o ther organs, wi th smooth endoplasmic re t iculum, l ipid droplets , dense bodies and mi tochondr ia wi th t ubu l a r cristae. These cells will be referred to as s te ro id-producing cells. The o ther type , the enclosing cells, are few in number and are i nva r i ab ly loca ted a t the pe r iphery of the gland. They are charac te r ized by thei r tenuous cy top lasmic extensions, which par t ly , or completely, sur round the s te ro id-producing cells and also enclose the nerve processes essent ia l ly in the same manner as t h a t of the Schwann cell (Dahl, 1970a, b, c). The cy top lasm of the s te ro id-producing cells of the theca l g land was charac ter ized by a large number of l ipid drople t s and mi toc hond r i a wi th t ubu la r cristae. The Golgi a p p a r a t u s was a lways present wi th in the j ux t anuc l ea r region. The smooth- surfaced endoplasmic re t icu lum occurred in a re la t ive ly larger amoun t t han in the adrena l cor tex (Kjaerheim, 1968a), while the rough-surfaced endoplasmic re t icu lum was a lmos t comple te ly absent . The cy toplasmic organellcs revealed a p ronounced po la r i t y in the i r d is t r ibut ion . The m a j o r i t y of the l ipid droplets , the mi tochondr ia and the smooth cndoplasmic rc t icu lum were found in the basal cy toplasm, whereas the Golgi complex, dense bodies, a t t a c h m e n t devices, and rough endoplasmic re t icu lum were observed apical ly . The nucleus conta ined one or two nucleoli and, occasional ly, nuclear bodies. The enclosing cells were morphological ly qui te different from the s te ro id-producing cells. Norma l ly t hey d id no t conta in any l ipid droplets , the mi tochondr ia were smal ler and wi thou t t u b u l a r cristac, the endoplasmic rc t icu lum was of the g ranu la r type , and the nucleus was elongated, rich in chromat in , bu t usua l ly wi thou t any visible nucleolus. The enclosing cell had a close re la t ionship to the basal lamina. A large number of nerve fibres were regular ly found with axon te rmina ls in con tac t wi th the steroid- producing cells.

8 Z. Zellforsch., Bd. 113

Figs. 1 and 2

E. Dahl: Effects of Steroids on the Thecal Gland (Fowl) 115

Experimental Animals

Dai ly in ject ions of the 3 di f ferent s teroids impa i red the general condi t ion of the animals. Af ter some days the egg-laying ceased, the comb became decreased in size and qui te pale, and a t the end of the exper imenta l per iod t hey s t a r t ed to moul t , especial ly the proges terone t r ea ted animals. Their appe t i t e decreased and the i r v i t a l i t y seemed to be genera l ly reduced con t r a ry to the s i tua t ion observed in the control mater ia l . The ovaries were smaller t han normal , wi th numerous, small follicles, all of the same size, giving the ova ry an appearance s imilar to cod roe. L igh t microscopic examina t ion of the s t e ro id - t rea ted mate r ia l revea led a s t r ik ing increase in the a m o u n t of l ipid, wi th an increase in number as well as size of the l ipid droplets . The size of the cell d id not seem to be reduced, bu t the nuclei were i r regular and shrunken, the nucleus looked smaller t han in the controls, and the apical p a r t of the cell appea red reduced in size. Con t ra ry to wha t was found in the normal mater ia l , the s te ro id-producing cells, in the t r e a t e d animals d id no t show the same polar d i s t r ibu t ion of the i r organelles. Very often the nucleus was found d isp laced to the basal par t , and the apical por t ion was often a lmost t o t a l l y occupied by l ipid drople ts .

Changes within the Organelles

No signif icant changes were observed in the chickens af ter a single inject ion, ne i ther af ter examina t ion of the ova ry af ter 7 days . All the a l te ra t ions which will be descr ibed refer to a l te ra t ions seen af ter 28 days , and were ident ica l in all the di f ferent groups.

The S te ro id-Producing Cells

Mitochondria. Several a l t e ra t ions were regular ly encountered in the mi tochond- r ia (Figs. 5-8). There seemed to be a reduc t ion in number of the whole mi tochondr ia l popu la t ion wi th a p ronounced decrease in size (Fig. 5). There was l i t t le va r i a t ion in size, and most of the mi tochondr ia were spherical . E longa ted forms were se ldom encountered (Figs. 6, 7) and division of mi tochondr ia only occasion- a l ly observed. P robab ly , the mos t p ronounced a l t e ra t ion was the s ignif icant ly

Fig. 1. Survey electron micrograph of the normal thecal gland of the domestic fowl. The gland is surrrounded by a well-defined basement membrane (BM). The major part of the gland is made up of the steroid-producing cells (SC) which are characterized by lipid droplets (L), dense bodies (Db) and a spherical nucleus (N) with nucleolus (Nu). The lipid droplets are located mainly in the basal parts of the cells, i.e. the end adjacent to the connective tissue (CT) or the capillaries (Ca). The dense bodies and the Golgi apparatus (G) are located in the opposite portion of the cells. The enclosing cells (EC) are seen at the periphery of the gland, surrounding part of the steroid-producing cells. Capillaries and nerve fibres (Ne) are always seen adjacent to the gland. Granulosa cells (GC) with their basement membrane (BM) are seen

in the upper left corner. Mitochondria (M). • 6525

Fig. 2. Survey electron micrograph of the thecal gland of a hen injected with progesterone for 28 days. The most typical changes are the increase in number and size of the lipid droplets (L), reduction in size and number of the mitochondria (M), dense bodies and the Golgi area. Enclosing cells (EC) and nerve fibres (arrows) are seen at the periphery of the gland. Nucleus (N). Capillary (Ca). Connective tissue (CT). Steroid-producing cell (SC).

• 5 040

8*

Figs. 3-5


decreased density of the matr ix which appeared pale with a marked decrease in the number of cristae which had also lost their pronounced tubular form (Fig. 8). There was a definite increase in dense material within the matr ix (Fig 7), and in some mitochondria this material occupied substantial par t of the organelle. Blebs or vacuoles of the outer membrane were never observed. On the contrary, there often seemed to be a condensation of the outer membrane (Fig. 6). The mitochondria were generally markedly smaller than the lipid droplets (Figs. 3, 5).

The Golgi Apparatus. The Golgi appara tus was smaller than normal with its dichtyosomcs dispersed among the lipid droplets (Figs. 3, 4). The dichtyosomes consisted of just a few small cisternae and vesicles, and were usually barely visible at low magnification. Membrane-bounded dense bodies, coated vesicles and centrioles were only seldom encountered as compared to the controls (Figs. l, 2). The dense bodies appeared less osmiophilic, and the coated vesicles less pronounced than in the normals. The Golgi area, located in the juxtanuclear region, occupied only a small port ion of the apical par t of the cell (Fig. 3), and relatively often only a few remnants of the Golgi complex were found (Fig. 4).

Smooth-Sur/aced Endoplasmic Reticulum. The smooth-surfaced endoplasmic re- t iculum (smooth endoplasmic reticulum) was present in a small amount of the basal port ion of the cell adjacent to lipid droplets or mitochondria (Fig. 4). Para- somes were never encountered. In some cells, part icularly in the stflbestrol- t reated animals, the smooth endoplasmic reticulum, which was still present, was expanded as compared with the normals (Fig. 9). These expanded tubules of the smooth endoplasmic reticulum had contact with the lipid droplets, and sometimes they appeared as if they were in open communicat ion with the lipid droplets (Fig. 13). Furthermore, in some areas it was difficult to recognize differences between the morphology of large expanded tubules and lipid droplets. There also seemed to be confluence of expanded tubules to the same size as lipid droplets, and morphologically they appeared to be s tructural ly identical with lipid droplets (Fig. 13). Together with the decrease of smooth endoplasmic ret iculum there was also a marked decrease in the volume of the ground cytoplasm and a reduction of free ribosomes (Figs. 6, 13). The rough-surfaced endoplasmic reticulum was still present, bu t not so often encountered as in the control material. Coated vesicles and pinocytic vesicles were significantly reduced in numbers.

Fig. 3. Following injections of diaethylstilbestrol, the juxtanuclear portion of the steroid- producing cells is invariably occupied by lipid droplets (L), with a reduction of the Golgi apparatus (G). There is also a reduction in the size and number of mitochondria (M), dense bodies (Db) and depletion of smooth endoplasmic reticulum. Ribosomes (R). Microtubules

(Mr). Nucleus (N). • 21600

Fig. 4. High magnification from the Golgi area (G) with only a few cisternae in the steroid- producing cell in an estrogen-treated hen. There is almost no smooth-surfaced endoplasmic

reticulum (SER), while free ribosomes (R) are present. Lipid (L). • 54000

Fig. 5. Note the general increase of lipid droplets (L), the depletion of smooth cndoplasmic reticulum and the reduction in size and number of mitochondria (M). Note the difference in

size of the lipid droplets and mitochondria. Nucleus (N). • 21600

Figs. 6-8


Lipid Droplets and Dense Bodies. Perhaps the most striking alterations in the steroid-treated animals was the considerable increase in the amount of lipid in the steroid-producing cells (Fig. 2). The increase comprised both the number and size of lipid droplets (Figs. 2, 14). Generally, the droplets were markedly larger than the mitochondria, and in some cells they might fuse, forming droplets larger than the nucleus (Fig. 14). Areas which normally were more or less devoid of lipid now appeared to be filled with lipid droplets, such as the apical portion of the cell, which normally contains the Golgi complex (Figs. 2, 4). The smooth endoplasmic reticulum was only sporadically found surrounding the lipid droplets, independent of the size of the droplets (Fig. 4). Dense bodies were significantly decreased in number and found only infrequently. As in the control material, they were located in the apical portion of the cell (Fig. 3). Contrary to the normals, membrane-bounded dense bodies were only sporadically encountered in the Golgi areas. From the apical to the basal portion of the cell a gradual change in the dense bodies was observed. In the apical region small lipid droplets were found in the centre of the dense bodies. In the para-nuclear areas lipid droplets surrounded by parallel myelin-like membranes (Fig. l l ) were seen, and in the basal portion there were translucent lipid droplets surrounded by only a small osmiophilic brim (Fig. 12). There seemed to be a spatial dissociation of dense bodies and lipid droplets according to their morphological categories. Their distri- bution seemed to occur in a gradual fashion in a apical-basal direction (Fig. 12). As in the controls, the dense bodies sometimes appeared in contact with the lipid droplets (Fig. 10).

The Nucleus. The nucleus was more or less displaced from its normal location in the apical part of the cell. Usually it was found more peripherally in the basal portion, sometimes even in contact with the cell membrane. The nuclei were significantly reduced in size (Fig. 2), often with an irregular outline with indentation of the nuclear membrane due to pressure from the lipid droplets (Fig. 14). There was an increased amount of chromatin, especially in the peri- pheral par t of the nucleus. The nuclear bodies were reduced in number, and there seemed to be alterations in their morphology (Fig. 15a--d). Instead of consisting of a fibrillar material, they appeared as homogeneous, spherical, translucent droplets, surrounded by a dense halo of finely granular material, while others were similar to lipid droplets. Genuine lipid inclusions, of the same morphology as the cytoplasmic lipid droplets, were also encountered, and often lipid droplets were

Figs. 6-8. Demonstrate different changes seen in the mitochondria after long-term treatment with steroids

Fig. 6. The mitochondria (M) are generally smaller than the lipid droplets (L). The outer membranes of the mitochondria are without blebs or vacuoles, and in fact denser than

normally (arrow) (progesterone) x 54000 Fig. 7. An increase amount of dense material is found within the mitochondrial matrix.

Mitochondria (M). Lipid droplets (L). (Diaethylstilbestrol). X 54000 Fig. 8. The mitochondrial cristae are simple and markedly decreased in number. The matrix is paler than normal. Lipid droplet (L). Mitochondrion (M). (Diaethylstilbestrol). x54000

Figs. 9-12


found in indentat ions of the nuclear membrane, par t ly embraced by the nucleus (Fig. 14). The nucleolus was smaller than normally and often found located towards the periphery of the nucleus. Mitoses were never encountered.

The Enclosing Cells

Although the changes in the enclosing ceils were not as pronounced as in the steroid-producing cells, alterations in the fine structure were regularly encountered (Fig. 16). The mitochondria were usually smaller than normally with a less dense matrix. There also seemed to be a decrease in the rough-surfaced endoplasmic ret iculum (Fig. 18), the Golgi appara tus (Fig. 17), and the density of the ground cytoplasm. The nucleus appeared smaller than in the controls with an increased amount of chromatin, and the cell appeared to be reduced in size.

The Interst i t ial Tissue of the Theca In te rna

There were only slight alterations in the other cells of the theca interna except for the smooth muscle cells with appeared generally atrophic with a decreased number of fibrils. The connective tissue cells in the theca interna seemed to be decreased in number as well as in the size of their different organelles. There seemed to be an increased number of mast cells.

Discussion

General

The avian ovary secretes three hormones, estrogen, androgen and progesterone. Extensive reviews have been published on the various aspects of steroids on birds (van Tienhoven, 1961). Estrogen, in addit ion to effecting the secondary sex organs in the female, also caused marked changes in the composition of the blood (van Tienhoven, 1961).

Progesterone seems to function largely in regulating the ovula tory cycle, at least in the chicken, and it is also presumed to act synergistically with estrogen to stimulate oviductal development and secretory act ivi ty (van Tienhoven, 1961).

Fig. 9. In the steroid-producing cells there is a close relationship between the mitochondria (M), the smooth endoplasmic reticulum (SER) and the lipid droplets (L) (cfr. Figs. 6-8). The smooth endoplasmic reticulum (SER) is expanded, and arrows indicate the possible confluence of smooth endoplasmic reticulum (SER) and lipid droplets. There is a reduction in the number of free ribosomes (R) and a increase of fibrillar material (F) in the ground

cytoplasm. (Diaethylstilbestrol). x 54000

Figs. 10-12. Illustrate different types of complex bodies (Cb) and lipid droplets (L) in the steroid-producing cells. (Estradiol)

Fig. 10. Complex bodies (Cb) are encircled by parallel, myelin-like membranes with an electron-lucent interior in the paranuclear region. Note the close relationship to the lipid

droplets (L). x 54000 Fig. 11. High magnification of a complex body (Cb) with myeline-like membranes. • Fig. 12. Towards the basal part of the cell the lipid droplets are large, occasionally with a dense periphery (L1), more often with an electron-lucent interior and dense periphery (L~), but generally completely electron-lucent (L3). Mitochondria (M). Dense body (Db). • 18900

Figs. 13 and 14


In the present investigation relatively large doses of three different synthetic hormones were used in an a t tempt to induce alterations in the fine structure of the ovary. There was general evidence of their effect by cessation of egg-laying, reduced vitali ty and moulting. Most likely this is related to the general atresia of the ovary which was found. This will result in a reduced production of all the three different hormones normally produced by the ovary and is reflected by the moult, which is suggested to be precipitated when the ovary becomes atretic (Himeno and Tanabe, 1957). The decrease in size of the comb, which is presumably due to the reduced secretion of androgen (Witschi and Fugo, 1940), is con- sidered as further evidence of reduced hormone production.

Changes within the Organelles

The Steroid-Producing Cells

Lipid Droplets and Dense Bodies. One of the most remarkable observations in this investigation was the general increase in the amount of lipid within the steroid-producing cells. A similar effect was demonstrated by Kjaerheim (1968b) in his study of adrenocortical a t rophy after steroid treatment, and it is also consistent with adrenocortical a t rophy in rodents as seen after hypophysectomy. According to Ichfi et al. (1967) the major part of the lipid droplets in steroid- producing cells in the adrenals consists of cholesterol which is synthesized in the mitochondria and part ly also derived from the blood plasma. Kjaerheim (1968c) suggested that the increased amount of lipid in adrenals following hypophysectomy or corticosteroid-treatment probably implies tha t cholesterol is synthesized at a normal or increased rate, while the conversion of cholesterol to corticosterone is decreased. He did not observe any changes within the mitochondria which should presumably be able to function normally. The lipid increase, as found in this study, seems to be due to the same mechanism as suggested by Ichfi et al. (1967) and Kjaerheim (1968c). The marked degenerative changes, as seen in the mitochondria in the present investigation, indicate tha t they were almost non-functioning as to the steroid synthesis. That the smooth endoplasmic reticulum was also found to be expanded and, in some places, in membranous contact and even open communication with the lipid droplets, supports the previous investigations concerning the interaction between lipid, smooth cndoplasmic reticulum and the mitochondria (Dahl, 1970d; Kjaerheim, 1968c). I t may also indicate tha t cholesterol is normally transported to the mitochondria by way of the tubules of the smooth endoplasmic reticulum, but when the mitochondria stop functioning, the smooth endoplasmic reticulum will be filled with lipid and

Fig. 13. Expanded tubules of the smooth endoplasmic reticulum (SER) are seen in contact with lipid droplets (L). Arrows indicate the confluence of smooth endoplasmic reticulum (SER) and lipid droplets (cfr. Fig. 9). Note the size of the mitochondrion (M) compared with the

lipid droplets. (Diaethylstilbestrol). • 54000

Fig. 14. Following administration of steroids the nucleus (N) is shrunken with an irregular outline of the nuclear membrane. Sometimes the nucleus contains lipid-like inclusions (L1). The lipid droplets (L~) may be as large as, or even larger than the nuclei. The enclosing

cell (EC) is almost devoid of organelles. (Estradiol). • 10800

Figs. 15-18


then expand. In this way the smooth endoplasmic reticulum might be transformed to lipid droplets.

The presence of morphological intermediary stages between lipid droplets and dense bodies, as revealed in this study, has also been observed in normal ovarian interstitial cells of rabbits (Davies and Broadus, 1968) and in the adrenal cortex of the domestic fowl after steroid treatment (Kjaerheim, 1968b). Davies and Broadus (1968) suggested that the lipid droplets are transformed into electron- dense bodies and that the empty areas in these intermediary stages represent poor penetration of the fixative. Kjaerheim (1968a), however, suggested that this morphological transition may point to a biochemical interaction between these two organelles, that intermediary forms represent fusion of these structures, and, finally, that lipid droplets can be degraded by lysosomes which are located in the dense bodies. That dense bodies must be important for the function of the steroid-producing cell is revealed by the fact that they are present normally and that their number is increased significantly after stimulation with gonado- trophins (Dahl, 1970d). There seems to be no doubt that there must be some relationship between dense bodies and the lipid droplets. However, in the present study it was not possible to obtain any new significant information concerning their interaction. The decrease in number of dense bodies may be due to at least some of the following facts: dense bodies of the steroid-producing cells of the ovary may, like dense bodies of the adrenocortical cells, contain the hydrolytic marker enzyme acid phosphatase and therefore be included in the lysosome series (Idelman, 1966; Penney and Barrnett, 1938; Szabo et al., 1967). Lipid which is not necessary for steroid production, is degraded by lysosomal enzymes. The increase in the amount of lipid droplets on account of decreased steroid- synthesis will then result in an increased demand for lysosomal enzymes, i.e. dense bodies. However, a decreased function of the cells and the other organelles of the cells which form the dense bodies, results in a decreased production of dense bodies, reflected by a reduction in their numbers.

Mitochondria. Fixation difficulties have previously greatly complicated the ana- lysis of the true form of the mitochondria in ovarian steroid-producing cells (Davies and Broadus, 1968) as well as mitochondrial changes in other steroid- producing cells (Kjaerheim, 1969). Davies and Broadus (1968) using immersion

Fig. 15a-d. Various aspects of intranuclear structures assumed to be nuclear bodies with a lipid-like appearance, a Diaethylstilbestrol, b Progesterone, c Estradiol, d Progesterone.

• 21600

Fig. 16. Enclosing cell (EC) showing a reduction in the number and size of the mitochondria (M), and in the number of free ribosomes (R) and depletion of rough endoplasmic reticulum. Steroid-producing cell (SC) to the right. Basement membrane of the thecal gland (Bm).

Lipid droplets (L). (Estradiol). • 21600

Fig. 17. A reduction of the Golgi area (G) was regularly found in the enclosing cells following steroid treatment. Nucleus (N). Steroid-producing cell (SC). (Estradiol). • Fig. 18. Note the low amount of rough endoplasmic reticulum (RER) in the enclosing cell (EC). Note the membranous contact (arrow) between the enclosing cell and the steroid-producing cell (SC) with differences in mitochondria (M1-M~). Dense body (Db). Nucleus (N).

(Estradiol). • 21600

126 E. Dahl :

fixation, found regression of degenerated interstitial cells in normal animals associated with shrinkage of the mitochondria and increased density of their matrix. Compared with the normals (Dahl, 1970b) and the gonadotropin-treated animals (Dahl, 1970d), the mitochondria in the present study showed a simplification of the eristae, which lost their typical tubular appearance. Furthermore, the density of the matrix decreased and the mitochondria became markedly reduced in size as well as in number. Kjaerheim (1968b) using the same fixation method as in this study, did not observe any changes within the mitochondria of the adrenocortical cells after steroid treatment, while there were pronounced alterations in the mitochondria of the steroid-producing cells in the present study. Possibly the reasons for this may be the time and the doses of hormones used in the different experiments.

The alteration of the mitochondria, as observed in the present study, indicates a significant decrease in function, not only of the mitochondria, but also of the entire cell. One may speculate as to whether the decrease in size represents a shrinkage of mitochondria already present or an establishment of a new genera- tion. From their structural appearance, the latter assumption seems to be the most likely one.

The reduced function of the mitochondria is also most likely the reason for the expansion of the smooth endoplasmic reticulum and the confluence of lipid droplets, because cholesterol is no longer utilized to the same degree as under normal conditions.

The Golgi Apparatus. Decrease of the Golgi complex has previously been reported in a t rophy of the adrenal cortex (Kjaerheim, 1968b), and the marked reduction observed in this investigation confirms previous observations. The Golgi apparatus probably reflects the synthesizing and secreting activity of the steroid- producing cells. This is not only supported by this study, in which a decrease in size was found, but also by the fact that stimulation with gonadotropins induces an increase of the Golgi area, which is also found after administration of clomiphene (Dahl, 1970e). The decrease in dense bodies, as found in this study, and the increase of the same organelle, which was seen in this area in the gonadotropin- and clomiphene-treated hens, supports the suggestion tha t dense bodies are produced in the Golgi apparatus.

Ribosomes and Ground Cytoplasm. The decreased amount of ribosomes and ground cytoplasm is consistent with the observations of Morrow et al. (1967) tha t protein synthesis in adrenocortical cells is inhibited by steroids. I t is also in agreement with the opposite observation that they increase in number after stimulation with gonadotropins (Dahl, 1970d). The morphological alterations observed in the present study, induced by the administration of steroids, indicate a pronounced inhibition of the function of the cell. Any secretory activity is most likely at a minimum, and there will therefore be little need for the protein synthesis normally required for the production of enzymes and organelles. The decreased amount of pinocytic vesicles also supports the impression of decreased activity of the steroid-producing cell.

Smooth-Sur/aced Endoplasmic Reticulum. Several investigations have demonstrated tha t the amount of smooth endoplasmic reticulum tubules reflects the


level of secreting activity of steroid-producing cells (Enders and Lyons, 1964; Kjaerheim, 1968c; Rennels, 1966). This observation is explained by the fact that smooth endoplasmic reticulum is the main site of steroid biosynthesis (Siekevitz, 1963). Further support for this concept is lent by the observation of hyper- t rophy of the smooth endoplasmie reticulum in the interrenal cells of the hen following ACTH administration (Kjaerheim, 1968c) and in the steroid-producing cells in the thecal gland following gonadotropin administration (Dahl, 1970d). Similar observations of hypertrophic smooth endoplasmic reticulum were made in the pig about 7 days after ovulation when the production of progesterone was high (Bjersing, 1967), and also in the granulosa lutein cells following treatment with a mammotrophie factor (Enders and Lyons, 1964) and chorion gonadotropin (Reynolds, 1963). On the other hand, the amount of smooth endoplasmic reticulum was found to be markedly decreased in the granulosa lutein cells after hypophysectomy (Enders and Lyons, 1964; Rennels, 1966).

The pronounced decrease in the number of smooth endoplasmic reticulum tubules following steroid treatment, as seen in this study, supports previous studies of this organelle as the main site of steroid biosynthesis. The study also seems to indicate that the smooth endoplasmic reticulum is more dependent on the activity of the cell than the other organelles. Even if the secretory activity is markedly decreased or has ceased completely, the other organelles, such as the mitochondria, dense bodies and Golgi complex, persist, at least to a certain extent. The smooth endoplasmic reticulum, however, seems to be the organelle which, during low secretory activity, will decrease or disappear. The main reason for this is, probably, that the synthesizing processes in the mitochondria are reduced after administration of steroids. Since the cholesterol is no longer utilized to the same degree as in the normals, the transport of cholesterol through the smooth endoplasmic reticulum will more or less stagnate, the lipid will expand the tubules, and they will either be transformed into lipid droplets or fuse with the lipid droplets, which in this way will increase in size and number while the smooth endoplasmic reticulum decreases.

Nucleus and Nucleolus. Like the other organelles of the cells there were re- gressive changes in the nucleus following treatment with steroids. The most striking features were the reduced size and the increase of chromatin, especially at the periphery of the organelle. The nuclear membrane was often wavy and irregular. This seems to be mainly due to pressure from the surrounding lipid droplets and not to any primary shrinkage of the nucleus itself. Similar observations were made by Kjaerheim (1968 b) in the study of adrenocortical cells after treatment with steroids. The decreased number of nuclear bodies, as seen in this study, is consistent with the increase of this organelle as observed after administration of gonadotropin (Dahl, 1970d) and in the adrenocortical cells (Kjaerheim, 1968c). In the present study, however, morphological alterations were also observed in the nuclear bodies. Changes in the internal structure of this organelle were also observed after the administration of gonadotropin (Dahl, 1970d) and clomiphene (Dahl, 1970e). These findings indicate that nuclear bodies are normal organelles of the cells and that their morphology is dependent on the functional activity of the cell (Dahl, 1970).

128 E. Dahl:

The true fat inclusions seen within the nucleus were clearly demonstrated in micrographs to be of cytoplasmic origin. They were engulfed by the nucleus of the steroid-producing cell by way of a process similar to pinocytosis, and they were observed in cells containing enormous amounts of lipid. The demonstration of a reduced size of the nucleolus clearly indicates that this organelle is closely associated with the secretory activity of the cell. This observation is also consistent with previous findings in adrenocortical cells after t reatment with steroids (Kjaerheim, 1968b). Furthermore, the increase of the same organelle, as seen in gonadotropin- and clomiphene-treated animals (DAM, 1970d, e), supports this suggestion.

The Enclosing Cell

Administration of steroids obviously had some influence on the morphology of the enclosing cells. The alterations observed point to a decreased activity of the cell. This can be due to the general atresia of the ovary which was observed, but may also be caused by a specific inhibitory effect of the steroids used. Since administration of gonadotropins (Dahl, 1970d) and clomiphene (Dahl, 1970e) also induces alterations in the enclosing cells as well as in the steroid-producing cells, the enclosing cell may, at least under special conditions, have some specific function in relation to the synthesis of the ovarian steroids.

The Interstitial Cells

Even though only minor alterations in the fine structure of the interstitial cells were observed, the findings indicate a generally decreased activity also in these cells. In view of the considerable alterations seen in the gonadotropin- and clomiphene-treated animals, there is a possibility that these cells may change their morphology, as well as function, under hormonal influence.

Correlation of Structure and Function

In the present study it has been demonstrated that in the theca interna of the domestic fowl there are cells which alter their ultrastructure specifically after t reatment with synthetic hormones. The cells comprise mainly the steroid-producing cells of the thecal gland.

The functional significance of the different organelles of these cells has been discussed above, and also in connection with administration of gonadotropins (Dahl, 1970d). Decreased secretory activity is reflected in the electron micrographs as increased amount of lipid, mitochondrial alterations, and a decreased amount of smooth endoplasmic reticulum, which confirms the close association of these structures. Previous studies have shown similar alterations in steroid- producing cells in other organs after administration of steroid (Kjaerheim, 1968 b). I t is therefore reasonable to conclude that the alterations observed in the present study represent atrophic changes of the steroid-producing cells of the thecal gland. I t is generally accepted tha t the atrophy caused by steroid t reatment is due to a feed-back mechanism via the anterior pituitary gland (van Tienhoven, 1961).

The avian ovary secretes three groups of hormones, i.e. estrogen, progesterone and androgen (van Tienhoven, 1961). Estrogen secretion from the ovary was demonstrated by Marlow and Richert (1940), and Marshall and Coombs (1957)


believed that estrogen was produced in the thecal gland. As pointed out by Dahl (1970a, b, c), this structure is the only one which possesses all the cyto- logical features necessary for steroid production. The present study, combined with observations made in the normals and after administration of gonadotropins, seems to leave little doubt tha t the theca interna is the main source of steroid production. Since estrogen is the ovarian hormone which is produced in the larg- est amount (van Tienhoven, 1961), it is most likely that it is produced by the thecal gland.

That the avian ovary secretes progesterone is proved by detection of bio- logically active progestin in the blood of egg-laying- and non-laying hens (Fraps et al., 1948; Fraps, 1955) and by chemical identification of progesterone in extracts from ovaries of egg-laying hens (Layne et al., 1957). The source of progesterone in the ovary has not been established.

In all probability it is not the ruptured follicle because the amount of progesterone in it is small (van Tienhoven, 1961). Fraps (1955) proposed that the maturing follicle is a possible, but not the exclusive, source of progesterone, whereas Marshall and Coombs (1957) favoured certain amorphous non-cellular aggregations of cells with cholesterol positive lipid in atretic follicles. Neither of these tissues has been proven to be the source of progesterone. Young (1961) suggests that it may be futile and unrealistic to a t tempt to identify specific cell types as the source of hormones in the ovary. He points to the view that the various tissue involved in steroid hormone biosynthesis may be subject to metabolic aberrations which change their hormone production either in rate or in kind. In the present study it has been revealed that estrogens, as well as a progesterone derivate, have induced the same specific alterations in the steroid- producing cells. This evidence may support the theory that the same cell may be the source of different hormones. However, it must also be recognized that, in the thecal gland, Dahl (1970b) has demonstrated cells, the enclosing cells, which changed their structure following the administration of gonadotropins (Dahl, 1970d) and clomiphene (Dahl, 1970e). In the present investigation these cells also altered their morphology after steroid treatment. Their close spatial relationship to the typical steroid-producing cells may indicate some interaction between these two cell types. Since the domestic fowl does not possess any corpus luteum, the possibility may exist that the enclosing cell may be of some importance to the production of progesterone, either by elaborating some steroid precursor, or releasing factors which have a special influence on the hormone production of the steroid-producing cells.

Evidence of secretion of androgen by the avian ovary can be found in the red vascular comb of the chicken before and during egg production (Taber, 1951; Witschi and Fugo, 1940). Marshall and Coombs (1957) have suggested that the interstitial cells of the ovary, or more specifically, the interstitial cells arising from the connective tissue cells of the ovarian stroma, represent the source of androgen in female birds. In the present study a reduction in the size and colour of the comb was observed, which may indicate a reduced production of andro- gens. There were no marked changes in the fine structures of the connective tissue cells, though alterations were observed indicating a reduced function of

9 Z. Zellforsch., Bd. 113

130 E. Dahl :

these cells. However, st imulation with gonadotropin (Dahl, 1970d) and adminis- t rat ion of clomiphene (Dahl, 1970e) resulted in significant alterations, especially in these cells, and there was a marked increase in the size of the comb, its colour changing t o deep red. Since these cells seem to be able to change their morphology under certain circumstances and take on an appearance almost consistent with steroid-producing cells, the possibility exist tha t these cells may be involved in the product ion of the androgen which takes place in the female bird.

Generally, one m a y conclude tha t the present study, with administrat ion of three different hormones, has confirmed tha t the theca interna mus t play an impor tan t role in the product ion of the different ovarian hormones. I t seems most conceivable to assume tha t estrogen is produced by the thecal gland. Since the other cells in the theca interna, viz. the enclosing cells of the thecal gland and the connective tissue cells, also reveal alterations of the fine structure following administrations of hormones and drugs, it is possible t ha t these cells may be involved in the product ion of progesteron and androgen, either by interaction with other cells or independently. As mentioned previously, however, the possibility also exists tha t there are metabolic aberrations in the steroid-producing cells in the thecal gland which change their hormone production. I n fact, the alterations observed in the enclosing cells and connective tissue cells m a y only be due to a local influence by the hormones which are produced.

Enzyme biochemistry and histochemistry combined with ul t rastructural morphology seems to be the necessary approach in order to solve this problem.

The present s tudy is the first demonstrat ion of the ul t ras t ructural changes of ovarian cells in the fowl following administrat ion of hormones. The similarity of the fine structural changes, following the t rea tment of the three different hormones, support the concept tha t the pathogenetic mechanism may be identical in all three cases, viz. an inhibition of the gonadotropins f rom the hypophysis. Since steroids are used therapeutically in various gynaecologic disorders and also for contraception, one must be aware of the fact tha t long-term use of steroids in high doses may induce morphologic alterations of the ovary.

References

Adams, J. L. : Progesterone-induced unseasonable molt in single comb White Leghorn pullets. Poult. Sci. 34, 702-707 (1955).

- - A comparison of different methods of progesterorfe administration to the fowl in affecting egg production and molt. Poult. Sci. 35, 323-326 (1956).

Appelgren, L.-E. : Sites of steroid hormone formation. Autoradiographic studies using labelled precursors. Acta physiol, scand., Suppl. 301, 1-108 (1967).

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

- - On the ultrastructure of granulosa lutein cells in porcine corpus luteum. With special reference to endoplasmic reticulum and steroid hormone synthesis. Z. Zellforsch. 82, 187- 211 (1967).

Breneman, W. R. : Reproduction in birds: the female. In: comparative physiology of reproduction. Mcm. Soc. Endocr. 4, 94-110 (1955).

- - Steroid hormones and the development of the reproductive system in the pullet. Endo- crinology 58, 262-271 (1956).

Bullough, W. S. : The effects of oestrone on the ovary of the mouse. J. Endocr. 8, 235-243 (1943).


Dahl, E. : Studies of the fine structure of ovarian interstit ial tissue. 1. A comparative s tudy of the fine s tructure of the ovarian interstit ial tissue in the ra t and the domestic fowl. J. Anat. (Lond.) (1970) (accepted for publication).

- - Studies of the fine s tructure of ovarian intersti t ial tissue. 2. The ul t ras t ructure of the thecal gland of the domestic fowl. Z. Zellforsch. 109, 195-211 {1970).

- - Studies of the fine s tructure 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 s tructure of ovarian interstit ial tissue. 5. Effects of gonadotropins on the thecal gland of the domestic fowl. Z. Zellforsch. (1970) (accepted for publication).

- - Studies of the fine structure of ovarian interstit ial tissue. 6. Effects of clomiphene on the thecal gland of the domestic fowl. Z. Zellforsch. 109, 227-244 (1970).

- - The fine s tructure of nuclear inclusions. J . Anat. (Lond.) 106, 255-262 (1970). Davies, J., Broadus, C. D. : Studies on the fine structure of ovarian steroid-secreting cells in

the rabbit . I. The normal interstit ial cells. Amer. J. Anat. 128, 4 4 1 4 7 4 (1968). Enders, A. C., Lyons, W. R. : Observations on the fine structure of lutein cells. II . The effects

of hypophysectomy and mammotrophie hormone in the rat. J . Cell Biol. 22, 127-141 (1964).

Everet t , J . W. : The mammalian female reproductive cycle and its controlling mechanisms. In: Sex and internal secretions, 3rd ed., vol. I, (W. C. Young ed.), p. 497-555. Balt imore: Williams & Wilkins Co. 1961.

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

Fraps, R. M. : The varying effects of sex hormones in birds. In : Comparative physiology of reproduction. Mem. Soc. Endocr. 4, 205-219 (1955).

- - Hooker, C. W., Forbes, T. R. : Progesterone in blood plasma of the ovulating hen. Science 108, 86-87 (1948).

- - - - - - Progesterone in blood plasma of cocks and nonovulat ing hens. Science 109, 493 (1949) Himeno, K., Tanabe, Y. : Mechanism of molting in the hen. Poult. Sci. 86, 835-842 (1957). Iehii, S., Kobayashi, S., Yago, N., Omata, S. : Role of plasma cholesterol in ra t adrenal cortico-

steroidogenesis in vitro. Endocr. jap. 14, 138-142 (1957). Idelman, S.: Contribution a la cytophysiolgic infrastructurale de la corticosurr~nale chez le

Ra t albinos. Ann. Sci. nat . Zool. 8, 205-362 (1966). Kjaerheim, A. : Studies of adrenocortical ul trastrneture. 2. The interrenal cell of the domestic

fowl as seen after glutaraldehyde perfusion fixation. Z. Zellforsch. 91,429-455 (1968a). - - Studies of adrenocortical ul trastructure. 3. Effects of dexamethasone and medroxy-pro-

gesterone on interrenal cells of the domestic fowl. Z. Zellforsch. 91, 456-474 (1968b). - - Studies of adrenocortical ul trastructure. 4. Effects of ACTH on interrenal cells of the

domestic fowl. J. Microscopie 7, 715-738 (1968c). - - Studies of adrenocortical ul trastructure. 1. Aldehyde perfusion fixation of the domestic

fowl. Acta anat . (Basel) 74, 424-453 (1969). Layne, D. S., Common, R. H., Maw, W. A., Fraps, R. M. : Presence of progesterone in extracts

of ovaries of laying hens. Proc. Soc. exp. Biol. (N.Y.) 94, 528-529 (1957). Marlow, H. W., Richert, D. : Estrogen of the fowl. Endocrinology 26, 531-534 (1940). Marshall, A. J., Coombs, C. J. F. : The interaction of environmental , internal and behavioural

factors in the rook, Corvus /rugilegus Linnaeus. Proc. Zool. Soc. Lond. 128, 545-589 (1957).

Morrow, L. B., Burrow, G. N., Mulrow, P. T. : Inhibi t ion of adrenal protein synthesis by steroids in vitro. Endocrinology 80, 883-888 (1967).

O' Grady, J. E. : Oestrogens in plasma from the domestic fowl. In : Physiology of the domestic fowl. (C. Horton- Smith and E. C. Amoroso, eds.), p. 23-29. Edinburgh and London: Oliver & Boyd 1966.

Penney, D. P., Barrnet t , R. J., The fine structural localization and selective inhibit ion of nucleosidephosphatases in the ra t adrenal cortex. Anat. Rec. 70, 543-556 (1938).

Phillips, R. E. : Ovarian response of hens and pullets to injections of ambinon. Poult. Sci. 22, 368-373 (1943).

- - Endocrine mechanisms of the failure of Pintails (Arias acuta) to reproduce in captivity. Diss. Abstr. 20, 3033 (1960).

9*

132 E. Dahl: Effects of Steroids on the Thecal Gland (Fowl)

RenneIs, E. G. : Influence of hormones on the histochemistry of ovarian interstit ial tissue in the immature rat. Amer. J. Anat. 88, 63-108 (1951).

- - Observations on the ul t ras t rncture of luteal cells from PMS and PMS-HCG treated immature rats. Endocrinology 79, 373-386 (1966).

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).

Rothchild, I., Fraps, R. M. : The interval between normal release of ovulating hormone and ovulation in the domestic hen. Endocrinology 44, 134-140 (1949a).

- - - - The induction of ovulating hormone release from the pi tui tary of the domestic hen by means of progesterone. Endocrinology 44, 141-149 (1949b).

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

Siekevitz, P. : Protoplasma: endoplasmic retieulum and microsomes and their properties. Ann. Rev. Physiol. 25, 1 5 4 0 (1963).

Szabo, D., Stark, E., Varga, B. : The localisation of acid phosphatase activity changes in lysosomes in the adrenal zona fasciculata of intact and hypophysectomized rats following ACTH administration. Histochemie 10, 321 328 (1967).

Taber, E. : Androgen secretion in the fowl. Endocrinology 48, 6-16 (1951). Tienhoven, A. van: Endocrinology of reproduction in birds. In : Sex and internal secretions,

3rd ed., vol. II, (W. C. Young ed.), p. 1088-1169. Balt imore: Williams & Wilkins Co. 1961. Williams, P. C. : Effect of stilboestro] on the ovaries of hypophysectomized rats. Nature (Lond.)

145, 388-389 (1940). Witschi, E., Fugo, N. W. : Response of sex characters of the adul t female starling to synthet ic

hormones. Proc. Soe. exp. Biol., (N.Y.) 45, 10-14 (1940). Young, W. C. : The mammalian ovary. In : Sex and internal secretions (W. C. Young, ed.),

3rd ed., vol. I, p. 449496. Baltimore: Williams & Wilkins Co. 1961.

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

Documents

Studies of the fine structure of ovarian interstitial tissue