Z. Zellforsch. 119, 188-194 (1971) �9 by Springer-Verlag 1971

The Effects of Clomiphene on the Granulosa Cells of the Domestic Fowl

EI~IK DAHL

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

Received April 5, 1971

Summary. The present paper describes for the first time the fine structure of ovarian granulosa cells as seen after administration of clomiphene to the domestic fowl for a 28 days period.

The main cytoplasmic changes of the granulosa cells seemed to be an increase in the number of mitochondria, dense bodies and complex bodies. The Golgi apparatus became enlarged, and there was an increase in the endoplasmic retieulum, annular desmosomes and cytoplasmic processes.

All the observations made are similar to those made after administration of steroids and gonadotropins. In conclusion, therefore, the present study has demonstrated that administra- tion of clomiphene exerts a stimulating effect on the granulosa cells. The mechanism of this effect is discussed.

Key-Words: Granulosa cells - - Fowl - - Influence of clomiphene - - Ultrastructure.

The abi l i ty of clomiphene to s t imulate ovar ian funct ion and to cause ovula t ion in women suffering from infer t i l i ty is well established (e.g. Sherman, 1966). So far, invest igat ions of the effect of clomiphene have main ly been confined to bioassay or clinical studies. I n previous reports it has been demons t ra ted tha t the ovary of the domestic fowl possesses an endocrine gland, the thecal gland, which was subjected to u l t ras t ruc tura l a l terat ions after admin is t ra t ion of clomi- phene (Dahl, 1970c, 1971a). The present invest igat ion is concerned with the u l t ras t ruc tu ra l aspects of the influence of clomiphcne on the gTanulosa cells. A series of studies on the ovar ian fine s t ructure serves as a base line for these

exper iments (DAM, 1970a--c , 1971a--f) . By comparing the series of experiments of these two cell types which were

t aken together from the same follicles under exactly the same exper imental condit ions, it was the aim to achieve new informat ion about the morphology and the func t ion of the different ovar ian cell types.

Materials and Methods

Ten White Leghorn hens, 18-24 months old, with an average body weight of 1820g, were used. The animals were housed in individual cages in a well-ventilated, constant climate room (I7 ~ C, controlled illumination, light on 7 a.m. and of 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 %), fats (24%), calcium (1.1-1.2 %) phosphorus (0.7%) and sodium chloride (0.5%). The hens were kept for at least 10days to get adapted to the environment before the experiment started.

Effects of Clomiphene on Granulosa Cells 189

Fig. 1. A granulosa cell after treatment with elomiphene for 28 days. The nucleus (Z r) has small indentations, and is small in proportion to the cytoplasm. The cell is in mutual contact with the neighbouring cells, resting at a thick basement membrane (Bm). There is an increase in the number of dense bodies (Db) and complex bodies (Cb). Note the microbody-like

mitochondria (M). x 24000

The clomiphene preparation used was: 40 mg clomiphene per ml in 8% gelatine. Of the 10 animals employed in the experiment, 5 served as controls. Since experiments concerned with steroids (Dahl, 1971b) and gonadotropins (Dahl, 1971e) had been over a period of 28 days with daily injections, five hens received 10 mg of clomiphen as intramuscular injec- tions daily for 28 days. All hens were sacrificed 24 hr after the last injection.

190 E. Dahl: Effects of Clomiphene on Granulosa Cells

Fig. 2. There is an increased number of mitochondria reflected by many small ones (M). Note the large microbody-like mitochondrion (M1) and the lipid droplets (L). • 37500

Fig. 3. Lipid droplets (L) were seen more frequently together with smooth endoplasmic reticulum (SER) and an enlarged Golgi area (G). • 24000

Fig. 4. The Golgi apparatus is markedly increased (G). Within the same area there seems to be an increased number of complex bodies (Cb) dense bodies (Db) and occasionally lipid

droplets (L). • 37500

Fig. 5. The number of annular desmosomes is markedly increased (Ad), especially towards the oocyte. • 24000

:Fig. 6. In some areas there are large collections of granular endoplasmic reticulum (RER) together with mitochondria (M) and complex bodies (Cb). Towards the oocyte there is also

an increase in cytoplasmic processes (arrows). • 24000

192 E. Dahl:

Fixation was performed as an intracardial perfusion of dextran under nembutal anasthesia 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 of ten rain. The ovary was then excised, and while kept in a drop of fixative, it was cut into thin slices under the dissecting microscope.

Samples from follicles of different sizes were then fixed separately in 1.7 % glutaraldehyde for an additional period of 2 hr at 4 ~ C. Subsequently, the tissue blocks were rinsed for ten rain in 0.15 M phosphate buffer at pPI 7.3 and fixed in 1% osmium tetroxide at 4 ~ C for 2 hr (Millonig, 1961). 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 min to increase the contrast. The sections were examined in a Siemens Elmiskop Ia electron microscope, equipped with 50 micron platinum objective apertures. The accelerating voltage was 80 kV. From the same blocks, sections one micron thick were cut for light microscopy and stained 30 sec on a heated stage with 0.1% toluidine blue adjusted to pH 8.9 with 0.067 M.

Observations

In jec t ions of c lomiphene left the hens a lmos t unaffec ted for the f irst few days . L a t e r the i r p lumage increased, and the comb became s l ight ly larger and a t t a i n e d a deep, fresh, r ed colour. Compared with the controls , the change in the colour of the comb was pronounced. The an imals also ga ined weight s teadi ly , and thei r v i t a l i t y increased dur ing the exper imen ta l period. A t the t ime of sacrifice, the hens were in excel lent phys ica l condi t ion.

Electron microscopic examina t i on of the c lomiphene- t rea ted ma te r i a l r evea led the granulosa cells of the p r i m a r y follicles to the bo th cuboidal and columnar . The nuclei were i r regulare wi th inden ta t ions (Fig. 1) and in the co lumnar cells

loca ted ad j acen t to the oocyte. The Golgi a p p a r a t u s was well deve loped (Figs. 1, 3, 4) and seemed to be

enlarged compared with the controls. I n the columnar cells i t was loca ted t ow a rds

the basemen t membrane . Dense bodies and complex bodies (Figs. 1, 3, 4) were found in an increased

number , mos t of ten ad j acen t to the Golgi appa ra tu s . Endop la smic re t icu lum of the smooth t y p e was found more of ten t h a n in the

controls (Figs. 3, 4) and the rough endoplasmic re t i cu lum also seemed to be increased in a m o u n t (Fig. 6). A d j a c e n t to the endoplasmic re t i cu lum l ip id drople t s were

encounte red (Figs. 2, 3). The mi tochondr ia (Fig. 2) were dense, and microbody- l ike mi tochondr ia were

of ten found. The number of desmosomes (Fig. 5) was increased, of ten toge ther wi th an

increased n u m b e r of cy top lasmic processes (Fig. 5).

Discussion Many s tudies have been conduc ted in order to e lucidate the physiological

ac t ion of clomiphene. The presence of an ant ies t rogenic effect of the drug on h u m a n subjects is suppor t ed b y a series of d a t a (e.g. Dahl , 1970c) and an increase in u r i na ry excre t ion of gonado t rop ins and estrogens has been r epor t ed (Roy et al., 1963; Greenb la t t et al., 1962). However , the mode of ac t ion and the si te of ac t ion is stil l unknown. The effects of c lomiphene on the fine s t ruc ture of the granulosa cells have never been demons t ra t ed . Dahl (1970c) de mons t r a t e d t h a t

Effects of Clomiphene on Granulosa Cells 193

administration of clomiphene induces a stimulating effect on the steroid-producing cells of the ovary. In the present study, the granulosa cells of exactly the same follicles have been examined. Comparing the present observations with those from previous studies (Dahl, 1970a-c, 1971 a-f), it was evident that the grannlosa cells do show alterations after administration of clomiphene. The enlarged Golgi apparatus, the increased amount of dense bodies, number of mitochondria, annular desmosomes, enlarged cells and the proximal position of the nucleus, are all alterations similar to those observed after administrations of steroids and gonadotropins (Dahl, 1971e, f). In conclusion, therefore, the present study seem to have demonstrated that administration of clomiphene exerts a stimulating effect on the grannlosa cells of the small follicles, and in this way probably initiates the stimulating effect on the ovulation which is observed clinically.

Previous studies have revealed a series of general effects of clomiphene (Dahl, 1970c), with an increase in the size of the ovary and an increase in the number as well as the size of the follicles. Degenerated, immature follicles as observed after administration of pregnant mare serum gonadotropin, were never encountered. When these different series of experiments on the ovary are com- pared and combined (Dahl, 1970a-c, 1971a-f), it is obvious that clomiphenc has a stimulating effect, both on the steroid-producing cells in the theca interna and also on the granulosa cells. The alterations demonstrated in the steroid- producing cell are of the same category as those seen after administration of gonadotropin, while the alterations observed in the granulosa cells are similar to those found after administration of steroids or gonadotropins. The present study seems to support the view that the sequence of events probably is as follows: Primarily clomiphene induces a stimulating effect on the gonadotropins which secondarily is followed by an increase in the estrogen due to a stimulation of the steroid-producing cells of the theca intcrna. The raise in estrogen then probably results in a stimulating effect on the granulosa cells of the corresponding follicle. This is also supported by the bioassay studies of Roy et al. (1963) who found a peak in FSH secretion attained prior to changes in estrogens.

However, the present studies have not been able to yield exact information if this stimulating effect of clomiphcne is due to a direct stimulation of the hypo- thalamic-pituitary axis, or by mitigating the inhibitory influence of estrogens on this axis because of the competitive anticstrogenic action of the compound (Roy et al., 1963; Dahl, 1970c).

References

Dahl, E. : Studies of the fine structure of ovarian interstitial tissue. 2. The ultrastructure of the thecal gland of the domestic fowl. Z. Zellforsch. 109, 195-211 (1970a).

- - Studies of the fine structure of ovarian interstitial tissue. 3. The innervation of the thecal gland of the domestic fowl. Z. Zellforsch. 109, 212-226 (1970b).

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

- - Studies of the fine structure of ovarian interstitial tissue. 1. A comparative study of the fine structure of the ovarian interstitial tissue in the rat and the domestic fowl. J. Anat. (Lond.) 108, 275-290 (1971a).

- - Studies of the fine structure of ovarian interstitial tissue. 4. Effects of steroids on the thecal gland of the domestic fowl. Z. Zellforsch. ll3, 111-132 (1971 b).

194 E. Dahh Effects of Clomiphene on Granulosa Cells

Dahl, E. : Studies of the fine structure of ovarian interstit ial tissue. 5. Effects of gonadotropins on the thecal gland of the domestic fowl. Z. Zellforsch. 118, 133--156 (1971e).

- - The fine structure of the granulosa cells in the domestic fowl and the rat. Z. Zellforsch. 119, 188-194 (1971d).

- - The effects of steroids on the granulosa cells in the domestic fowl. Z. Zellforsch. 119, 179- 187 (1971 e).

- - The effects of gonadotropins on the granulosa cells of the domestic fowl (Submit ted for publication) (1971 f).

Greenblat t , t~.B., Roy, S., Mahesh, V. B., Baifield, W . E . , Jungck, E .C. : Induct ion of ovulation. Amer. J. Obstet. Gynec. 97, 120-123 (1962).

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

Roy, S., Greenblatt , R., Mahesh, V., Jungck, E. C. : Clomiphene citrate: fur ther observations on its use in induction in the human and on its mode of action. Fertil. and Steril. 14, 575-595 (1963).

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

Sherman, 1~. P. : Induct ion of ovulation. Aust. Ann. Med. 15, 266-280 (1966).

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