Z. Zellforseh. 119, 58-67 (1971) �9 by Springer-Verlag 1971

The Fine Structure of the Granulosa Cells in the Domestic Fowl and the Rat

ERIK DAHL

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

Received April 5, 1971

Summary. The granulosa cells of the ovarian follicle of the rat and the domestic fowl have been studied with the light and electron microscope. The nuclei of the granulosa cells were irregular with indentations and large in proportion to the cytoplasm of the cell. The mito- chondria had a dense, dark matrix with only few cristac. The Golgi apparatus was moderately developed, located towards the oocyte in a juxtanuclear position. The endoplasmic reticulum was rather sparse. Lipid droplets were only occasionally encountered. Microtubules were regularly observed. The functions of the granulosa cells are discussed. Compared with the steroid-producing cells of the theca interna of the same follicles, the granulosa cells primarily are the nursing cells for the growing oocyte and mainly have the characteristics of protein forming cells.

Key-Words: Ovary - - Rat Fowl - - Granulosa cells - - Ultrastructure.

The actual site of product ion of the different hormones in the ovary has no t been determined with cer tainty, and remains controversial (Young, 1961; Eck- stein, 1962; Bjcrsing, 1967). I t is generally accepted tha t the ovar ian follicle is impor t an t in the elaborat ion of steroid hormones. I n a series of experiments it was demons t ra ted t ha t the theca in te rna contains cells of similar morphology as steroid-producing cells in other organs (Dahl, 1970 a, b, 1971a). Fur thermore , these cells were also subjected to u l t ras t ruc tura l al terat ions after admin i s t r a t ion of hormones and drug (Dahl, 1970 c, 1971 b, c). However, the role of the granulosa

cells is debatable (Bjersing, 1967). The present s tudy was under t aken in order to ascertain whether granulosa

cells generally have the characteristic u l t ras t ruc tura l features of steroid-producing cells as seen after vascular perfusion with glutaraldehyde, and also in order to ob ta in a base line for subsequent experiments with hormones and drugs (Dahl,

1971 d-f). By comparing the series of experiments of these two cell types which were

t aken together from the same follicles under exact ly the same exper imental con- ditions, it was the aim to achieve new informat ion about the morphology and the funct ion of the different ovar ian cell types.

Materials and Methods

Ten White Leghorn hens, 18-24 months old, three female White Leghorn chickens, 3 months old, and four female albino rats, 75 days old, were used for this study. The average body weight of the hens was 1627 g, of the chickens 850 g, and of the rats 210 g. The animals were kept on a 12-hr light-dark cycle in individual cages in a well-ventilated, air-conditioned room with a constant temperature of 17 ~ C and a relative humidity of 60%. The hens were

Granulosa Cells in the Fowl and the Rat 59

fed pelleted commercial chicken fodder, cabbage, sand grits, and water ad lib. The rats were fed pelleted commercial rat fodder and water ad lib.

Before sacrifice, the animals were kept for a minimum of 12 days for adaption to their new environment. They were sacrificed between 10.00 a.m. and 1.00 p.m. to minimize possible structural changes due to diurnal variations in ovarial activity.

Fixation: In three chickens and two hens fixation was performed as an intra-aortic perfusion of dextran (intradex "Nyco", Nyegaard & Co., Oslo, Norway) under nembutal anesthesia (Nembutal sodium, 5%, Abbott laboratories S.A., Brussels, Belgium) followed by 1.7 % glutaraldehyde (Fluka AG, Buchs SG, Switzerland) in 0.1 M phosphate buffer at pH 7.3. The perfusion technique was used according to specifications given in detail by Kjaerheim (1969).

In six additional hens fixation was performed in the same way except that in the first three hens 0.1 M cacodylate was used and in the remaining three 1% Tyrode buffer instead of the phosphate buffer. In two rats fixation was performed as on intra-aortic pedusion, with 1.7% glutaraldehyde in 0.1 M phosphate buffer at pH 7.3.

After perfusion for l0 min the ovary was excised and cut into thin slices under the dissect- ing microscope. The left ovary was chosen in all animals. In all the hens samples were taken from the cortex, and efforts were usually made to get more than one follicle in a single sample. The size of the follicles could vary from 20 ~ up to 5 mm. The tissues were then fixed for an additional period of 2 hr by immersion in the perfusion fixative.

Ovaries from two rats and two hens were fixed by immersion in glutaraldehyde 1.7 % 0.1 M phosphate buffer for 20 hr.

Fifteen blocks were processed from each animal, and all the specimens from the different animals were rinsed for 10 rain in 0.15 M phosphate (or cacodylate/Tyrode) buffer at pH 7.3 and post-fixed in 1% osmium tetroxide at 4 ~ C for 2 hr (Millonig, 1961). After fixation the blocks were rapidly dehydrated in graded series of acetone solutions and embedded in Vest- opal W (Ryter, 1958). Ultrathin sections were cut on an LKB Ultrotome III, equipped with glass knives, collected on formvar-coated copper grids; the formvar being backed with carbon. The sections were treated with uranyl acetate for 30 rain, followed by lead citrate (Reynolds, 1963) for 5 rain. The sections were examined in a Siemens Elmiskop Ia electron microscope operated at 80 kV. and equipped with 50 microns platinum objective apertures. From the same plastic blocks, sections were cut at one micron for light microscopy. These sections were stained on a heating stage with an aqueous solution of 0.1% toluidine blue adjusted to pH 8.9 with 0.067 M Na~HPO 4.

It should be added that the author in a study of the domestic fowl (Dahl, 1970c) has used vascular perfusion and phosphate buffer. Five animals from previous studies (Dahl, 1970c) in which vascular perfusion and phosphate buffer had been used, were employed as controls for evaluation of the results in the present study.

Observations

Both in the ra t and in the domestic fowl the oocyte of the p r imary follicles were surrounded by f la t tened granulosa cells (Figs. 1-4). In a larger p r imary

follicles the granulosa cells were found to be cuboidal (Figs. 5, 6). They were in

mutua l contac t to each other and to the oocyte and connected to each other by

means of desmosomes which occupied only small segments of the surface (Figs. 6,12).

Annular desmosomes were also occasionally encountered. A dis t inct basement

membrane (Figs. 5, 6) separated the granulosa cell layer f rom the ad jacent theca interna.

The nuclei of the granulosa cells were irregular with indentat ions , and large in propor t ion to the cytoplasm of the cell (Figs. 5, 6, 9). The contac t between the oocyte and the granulosa cells is pa r t ly a mutua l contact , and par t ly an a t tach-

men t of processes f rom the granulosa cells to the surface of the oocyte by means of desmosomes (Figs. 6, 12).

Fig. 1. Survey photomicrograph of the ra t ovary. A growing follicle with an t rum is seen in the centre surrounded by the interstitial gland (IG). The arrow indicates the hypertrophic

theca interna with lipid laden-cells. Granulosa cells (GC). x 60

Fig. 2. Section of the wall of a follicle under higher magnification. Granulosa ceils (GC), theca interna (TI) and the interstit ial gland (IG). The arrows indicate lipid-laden cells in the theca in terna (cf. Fig. 1). Note the difference between the lipid-laden cells in the theca interna and

the cells of the interstitial gland, x 330

Fig. 3. Survey photomicrograph of the hen ovary with several follicles. The arrows indieate lipid-laden cells in the theca interna, x 68

Fig. 4. Par ts of two follicles from the hen ovary under higher magnification. The arrows indicate collections of the lipid-laden cells which form the thecal glands. Note the absence of

the interstit ial gland (cf. Fig. 2). Granulosa cells (GC), theca interna (TI). x 366

E. Dahl: Granulosa Cells in the Fowl and the l~at 61

Fig. 5. Granulosa cells from the rat follicle. The nuclei (N) are large in proportion to the cytoplasm. The cells are in membraneous contact, resting at a basement membrane (Bin). The mitochondria (M) are rod-shaped with a dense, dark matrix. Note that lipid droplets arenot

present. G Golgi area. • 13025

Fig. 6. Granulosa cells from the follicle of the domestic fowl. The nucleus (N) has indentations. The Golgi area (G) is seen adjacent to the cell. Note the microbody-like mitochondria (M).

Bm Basement membrane. • 10500

The m i t o c h o n d r i a were e v e n l y d i s t r i b u t e d in t h e c y t o p l a s m . T h e y h a d a dense , d a r k m a t r i x w i t h o n l y few cr is tae (Figs. 7, 8) a n d a q u i r e d s o m e t i m e s t h e appea r -

ance of m ic robod ie s (Fig. 8). T y p i c a l t u b u l a r e r i s tae were n o t obse rved .

62 E. Dahl :

Fig. 7. Mitochondria from the granulosa cells of the rat. Note the dense matrix and the few cristae. Tubular cristae were never observed. R E R Rough endoplasmic reticulum. X 52 500

Fig. 8. Mitochondria from the granulosa cells of the domestic fowl. Note the few cristae. (cf. Fig. 7). X 52500

The Golgi appara tus was moderately developed, usual ly located towards the oocyte in a jux tanuc lear posit ion (Figs. 6, 10). Dense bodies were regularly found in this region (Fig. 6).

Granulosa Cells in the Fowl and the Rat 63

Fig. 9. Part of a granulosa cell of the domestic fowl with granular endoplasmic reticulum (RER), Golgi area (G) and typical mitochondria. Note the position of the Golgi in relation to

the nucleus (N) (cf. also Fig. 8). • 13125

Fig. 10. Part of the Golgi area from a granulosa cell of the domestic fowl. In addition to lipid droplet (L), annular desmosomes (Ad), and microtnbules (Mr), there is also some smooth

endoplasmic reticulum (SER) and mitochondria (M). x 39375

The endoplasmic re t iculum was rather sparse in a m o u n t and was main ly of the rough type (Figs. 7, 12) bu t the smooth type was also observed, especially in the domestic fowl (Fig. 10).

64 E. Dahl :

Fig. 11. High magnification of the microbody-like mitoehondria (M) of the granulosa cells of the domestic fowl. Note the granular endoplasmic reticulum (RER), lipid droplets (L) and

annular desmosomes (Ad). • 26250

Fig. 12. Part of an annular desmosomes (Ad) with their cytoplasmic processes. Note the dense membrane and the granules at this membrane. M Mitochondria RER Rough endoplasmic

reticulum. • 52 500

Lipid droplets were only occassionaly encountered (Figs. 10-12) and could be both of the t rans lucent and of the dense type (Fig. 10). Microtubules (Fig. 10) were regu]arly seen, while fibrils never were observed. Centrioles were also regu- larly observed.

Granulosa Cells in the Fowl and the Rat 65

Discussion

Steroid-producing cells have been found to have certain ultrastructural fea- tures in common (Enders, 1962; Blanchette, 1966; Dahl, 1970a, b). They are characterized by their contents of lipid droplets, mitochondria with typical tubular cristae, smooth endoplasmie reticulum, dense bodies, Golgi apparatus and usually a spherical nucleus with a distinct nucleolus. The ovary is a heterogeneous tissues containing three endocrinologieally active structural subunits, including: the corpus luteum (the luteal gland, the pregnancy gland), the interstitial gland, and the thecal gland (Mossman, 1964; Dahl, 1971 a). Most investigators have assumed that the theca interna are the source of the hormones (Westman, 1929, 1934; Falek, 1959 ; Ryan, 1965). Previous comparative investigations of the rat and the domestic fowl have also demonstrated that in the theea interna there are collec- tions of cells, the thecal glands, which fullfill the morphological criteria of steroid- producing cells (Dahl, 1970a, 1971a). Opinions on the role of the granulosa cells in steroid hormone production by the ovarian follicle, however, have differed from time to time (Bjersing, 1967). Falek (1959) found that both theca and granu- losa cells were necessary for the production of estrogens, but the nature of the interaction between the cells was not evident from his studies. Bjersing (1967) as a working hypothesis suggest a modified two-cells type theory where C-19 precursor steroids are elaborated by the theea interna cells through 17a-hydroxy- lation and side-chain cleavage, and transfered across the basement membrane of the folhcle to the granulosa cells for the production of testosterone by these cells. Aromatization to estrogens, particularly estradiol--17b, may then be carried out by both the granulosa cells and the theca cells. His hypothesis is based on bio- chemical, histoehemical and ultrastructural studies on granulosa cells of folhcles and corpora lutea.

In the present study the granulosa cells of the same species as those examined in previous investigations have been studied (Dahl, 1970a, b, 1971a). Generally the granulosa cells from the rat and the domestic fowl were of similar morpho- logical structure. The nuclei were elongated with indentations and had a well developed Golgi apparatus, and mainly granular endoplasmic reticulum. The mitoehondria were of the rod-shaped type without typical tubular cristae, and lipid droplets were only occasionally found. Dense bodies were also seldom encoun- tered. By comparing the granulosa cells of these two species with the lipid- containing cells in the theea interna of the same follicles, there were striking differences, both in the types and in the amount of organelles. I t may therefore be reasonable to suggest that in the follicles examined in this study, the granulosa cells do not seem to be able to produce steroids of any significant amount.

Indication of steroid synthesis in follicular granulosa cells has been obtained in ovarian sections of various species by histochemical demonstrations of hydroxy- steroid dehydrogenase activities, by visualization of androgenic steroids with a modified fluorecent antibody technique, and by autoradiographic steroids using labelled precursor, and biochemical methods (Woods and Domm, 1966; Appelgren, 1967; Bjersing, 1967). However, Wyburn and Baillie (1966) conclude that it might seem to be overrating the functional capacity of granulosa cells to suggest that they are responsible for the secretion of ovarian hormones. What has been demon- strated histochemically is that they are equipped with the enzymes necessary to

5 Z. Zellforsch., Bd. 119

66 E. Dahl :

p r o d u c e t h e h o r m o n e s , a n d n o t t h a t t h e y in fac t a re p r o d u c i n g t h e m a t a n y one

t ime .

I n t h e p r e s e n t s t u d y i t has been d e m o n s t r a t e d t h a t t h e g r a n u l o s a cells m a i n l y

h a v e t h e cha rac te r i s t i c s of p r o t e i n s y n t h e s i z i n g cells, w i t h r e l a t i v e l y a b u n d a n t

Golg i m a t e r i a l , g r a n u l a r e n d o p l a s m i c r e t i c u l u m a n d m i t o c h o n d r i a of d i f f e ren t

sizes a n d forms . These o b s e r v a t i o n s a re also cons i s t en t w i t h p r e v i o u s s tud ies of

t h e r a t o v a r y ( B j S r c k m a n , 1962) and t h e hen o v a r y ( W y b u r n a n d Bai l l ie , 1966).

I t seems r ea sonab le to p ropose t h a t t h e g ranu losa cells p r i m a r i l y are t h e nu r se

cells fo r t h e g r o w i n g oocy te , a n d t h e s t e ro id p r o d u c t i o n , if any , is s econda ry .

A d m i n i s t r a t i o n of e s t rogens a n d g o n a d o t r o p i n s also seem to s u p p o r t th is v i e w

(Dahl , 1971d, e).

R e f e r e n c e s

Appelgren, L. E. : Sites of steroid hormone formation. Autoradiographic studies using labelled precursors. Acta physiol, scand., Suppl. 801, 1-108 (1967).

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

Bj6rckman, N. : A study of the ultrastructure of the granulosa cells of the rat ovar. Acta anat. (Basel) 51, 125-147 (1962)i

Blanchette, E. J. : Ovarian steroid cells. 1. Differentiation of the lutein cell from the granulosa follicle cell during the preovulatory stage and under the influence of exogenous gonado- trophins. J . Cell Biol. 31, 501-516 (1966).

- - Ovarian steroid cells. II . The lutein cell. J. Cell Biol. 31, 517-542 (1966). Daht, 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 (1970) a. - - 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. 113, l l 1-132 (1971 b).

- - Studies of the fine structure of ovarian interstitial tissue. 5. Effects of gonadotropins on the thecal gland of the domestic fowl. Z. Zellforsch. 113, 133-156 (1971 c).

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

- - The effects of gonadotropins on the granulosa cells of the domestic fowl. Acta Endo- erinologia (in press) (1971 e).

- - The effects oi clomiphene on the granulosa cells of the domestic fowl. Z. Zellforsch. 1 1 9 , 188-194 (1971f).

Eekstein, P.: The ovary. In: Zuckerman, S. (ed.), p. 311. New York: Academic Press 1962. Enders, A. C. : Observations on the fine structure of [utein cells. J. Cell Biol. 12, 101-113 (1962). Falck, B. : Site of production of ocstrogen in rat ovary as studied in microtransplants. Acta

physiol, scand. 47, Suppl. 163, 1-101 (1959). Kjaerheim, :~. : Studies of adrenocortical ultrastructure. 1. Aldehyde perfusion fixation of the

domestic fowl. Acta anat. (Basel) 74, 424-453 (1969). Millonig, G. : The advantages of a phosphate buffer for OsOa solutions in fixation. J. appl.

Physiol. 32, 1637 (1961). Mossmann, H. W., Koering, M. J. , Ferry, D., Jr. : Cyclic changes of interstitial gland tissue

of the human ovary. Amer. J. Anat. 115, 235-256 (1964). 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).

Granulosa Cells in the Fowl and the Rat 67

Ryan, K. J. , Smith, O. W.: Biogenesis of steroid hormones in the human ovary. Recent Progr. Hormone Res. 21, 367409 (1965).

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

Westman, A. : Experimentelle Studien fiber die funktionelle Bedeutung der Theca-interna- Zellen. Acta obstet, gynec, scand. 8, 290-306 (1929).

- - Untersuchungen fiber die Abh~ngigkeit der Funktion des Corpus luteum von den Ovarial- follikeln und fiber die Bildungsst~tte der Hormone im Ovarium. Arch. Gyn~k. 158, 476-504 (1934).

Woods, J. E., Domm, L. V. : A histochemical identification of the Androgen-producing cells in the gonads of the domestic fowl and albino rat. Gem comp. Endocr. 7, 559-570 (1966).

Wyburn, G. H., Baillie, A. H.: In: Horton-Smith, C., and Amoroso, E. C. (ed.), Physiology of the domestic fowl, p. 30. Edinburg: Oliver and Boyd 1966.

Young, W. C. : The mammalian ovary. In: Sex and internal secretion, 3rd. ed., (W. C. Young, ed.), p. 449. Baltimore: Williams and Wilkins Co. 1961.

Dr. Erik Dahl Department of Anatomy Dental Faculty University of Oslo Blindern, Oslo 3, Norway

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