Z. Zellforsch. 135, 553--561 (1972) © by Springer-Verlag 1972

Studies of the Fine Structure of Ovarian Interstitial Tissue 7. The P o s t n a t a l E v o l u t i o n of the Theca l G l a nd in the Domest ic Fowl

Erik Dahl

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

Received August 28, 1972

Summary. The post-natal evolution of thecal gland in the domestic fowl has been explored, and a hypothesis for this development is proposed. I t is suggested that the surface epithelium of the ovary forms crypts and submicroscopic clefts (ingrowing cords) which contribute with epithelial elements required for the regeneration of some of the ovarian elements, including the thecal glands. Complexes of these ingrowing epithelial cells are forming small islets which, after hormonal stimulation, are transformed into well defined structures, the thecat glands, with a specific function, viz. steroid bio-synthesis.

Key words : Ovary - - Domestic fowl - - Thecal gland - - Postnatal evolution - - Electron microscopy.

Introduction

Ovarian inters t i t ia l cells were first described by Pfliiger (1863), and the term " in ters t i t ia l g land" was later applied collectively to them. Evidence has been presented tha t they are involved in steroid biosynthesis (e.g. Brandau, 1970). Numerous views have been expressed on the origin of these cells, and almost every cell type of the ovary has been implicated (e.g. Watzska, 1957; Harrison, 1961; Merker and Diaz-Eneinas, 1969).

Previous studies of the fine s t ructure of ovar ian inters t i t ia l tissue have es- tabl ished tha t in the theca in te rna of the domestic fowl, there is a well defined Fautologie structure, the thecal gland, with endocrine funct ion (DAM, 1970, 1971, 1972). The present observations, made in the course of these investigations, have been recorded because they may indicate a pos tnata l evolut ion of this inters t i t ia l s tructure.

Materials and Methods

Sixty-two White Leghorn hens, 18-24 month old and six White Leghorn chickens, 3 month old, some of which were treated with hormones and drug (for details see Dahl, 1972) were used for this study. Fixation was achieved under nembutal anaesthesia by intra-aortic perfusion of dextran followed by 1.7% glutaraldehyde in 0.l M phosphate buffer at pi t 7.3, or 0.1 M cacodylate or Tyrode buffer. The tissues were then fixed for an additional period of 2 h by immersion in the perfusion fixative.

All the specimens from the different animals were rinsed for 10 min 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 h (Millonig, 1961). After fixation the blocks were rapidly dehydrated in graded series of acetone solutions and embedded in Vestopal W (Ryter and Kellenberger, 1958). Ultrathin sections were cut on an LKB Ultratome III. The sections were treated with uranyt acetate for 30 min, followed by lead citrate (Reynolds, 1963) for 5 min. The sections were examined in a Siemens Elmiskop Ia electron microscope. From the same plastic blocks, sections 1 y,m thick were cut for light microscopy. These sections were stained on a heating stage with an aqueous solution of 0.1% toluidine blue adjusted to pt-I 8.9 with 0.067 hI-Na~HPOa.

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554 Erik Dahh

Observations

Gross Anatomy. The size of the ovary in the domestic fowl is subjected to wide structural variation according to the reproductive state of the individual, in chicken and in the quiescent state of the adult it is a flat organ with small follicles. In the active state it is a large organ composed of five to seven large follicles filled with yellow yolk, and a large number of smaller follicles of a paler colour. The larger follicles are graded in size, whereas the smaller follicles are more uniform. Parenteral administration of hormones interferes with this natural development (Dahl, 1972).

Light Microscopy. In light microscopy the peripheral part of the ovary, the cortex, is seen with rather deep Ioldings which may form regular crypts partly encircling the growing follicles (Fig. 1). The surface of this folding cortical layer is covered by a continuous sheet of epithelium (the germinal epithelium) which con- sists of simple, cuboidal or columnar cells. In some areas it has a stratified appear- ance, most likely due to the cutting angle of the surface (Fig. 1). Growing follicles of different sizes are seen in close proximity to the cortical surface. In the theca interna of larger follicles, islets of epithelial cells, the thecal glands, are seen (Fig. 1). Compared with the tissue components mentioned above, there is little stromM tissue in the cortex. The medulla is built up by an extensive network of connective tissue, forming lacunae of different sizes (Fig. 1). Thecal glands are also seen scattered in the medulla, both in chicken and in adults.

Electron Microscopy. The cortical surface consists of epithelial cells, characteri- zed by a large, elongated nucleus and a distinct nucleolus (Figs. 2, 3). Large in- dentations in the nucleus are usually seen, and nuclear bodies are often encounte- red (Fig. 2). A striking feature of this surface epithelium is the partial lack of membranous contact with neighbouring cells (Fig. 2). Furthermore, in the depth of the crypts, a surface-to-surface continuation of the epithelium is seen as deep, submicroscopic clefts in the cortex (Fig. 3). A distinct basement membrane is separating the surface epithelium from the underlying tissue (Fig. 3). Underneath of this basement membrane, epithelial cells may be traced as an apparent con- tinuation of epithelial elements from the surface towards the theca interna of the adjacent growing follicles (Fig. 3). Towards the more basal part of the cortex, groups of 5-7 rather undifferentiated epithelial cells are forming small islets, surrounded by a distinct basement membrane (Fig. 4). The cells are in membra- nous contact, and have a morphology similar to the surface epithelium, except for a more spherical nucleus (Fig. 4). Islets of different sizes are found. With increasing size, the epithelial cells become more differentiated, with small lipid droplets, mitochondria with a dense matrix, and increased amount of endoplasmic reticulum

Fig. 1. Survey photomicrograph of the peripheral part of the hen ovary, with the cortex and the underlying medulla. The surface is covered by epithelial cells (E) in close proximity to growing follicles (F). Cortex is folded with crypts (C) of different sizes, partly encircling the growing follicles. The medullary part is built up by an extensive network of connective tissue forming large lacunae (L). Islets of epithelial, lipid containing cells, the thecal gland (TG) are seen in the theca interna of a relatively large follicle (LF). The circle indicates a similar area of the cortex

as shown in Figs. 2 and 3. x 372 (Normal hen)

Postnata l Evolut ion of the Thecal gland 555

Fig. 2. Electron micrograph of the surface epithelium from a small crypt (C). The cells are characterized by a large, elongated nucleus (N) with a distinct nucleolus (Nu), and relatively small mitoehondria (M). Nuclear bodies (Nb) are regularly seen. Note t ha t the epithelial cells in some areas are separated by distinct spaces (arrows) which form deep, narrow clefts in the

epithelial layer. × i1200 (Normal chicken, 3mth old)

Fig. 3. Survey electron micrograph of the surface epithelium (E) from a crypt (U) in the proximity of two growing follicles (F-upper right and lower left corner). In the bottom of the crypt, the epithelial layer is continued as a narrow cleft deep into the cortex with surface-to- surface epithelium partly in membranous contact, partly clearly separated (arrows). Typical epithelial cells at the surface, resting on a distinct basement membrane (BM). Underneath of this basement membrane, epithelial cells (MC, "migrating cells") may be traced as a continu- ation of epithelial elements from the surface towards the theca interna. GC granulosa cells.

× 5 600

Fig. 4. Complex of epithelial cells (E) deep in the cortex. The nucleus is spherical. The cells are in membranous contact surrounded by a common distinct basement membrane (arrows)

5

which gives the complex of cells the appearance of a small islet. Note the small nerve fiber (Ne) and the capillary (Ca), structures which are usually seen adjacent to the fully developed

thecal gland. × 5 600 (Normal chicken)

Fig. 5. Islet of epithelial cells (E) in the theca in terna of a growing follicle. The cells, which still are ra ther undifferentiated, have a spherical nucleus (N). The amount of cytoplasm seems to be increased compared with the cells in Fig. 4. Small lipid droplets (L) are scat tered throughout the cells. The mitochondria (M) have a dense matrix, bu t still wi thout any typical t ranslucent cristae. Note the distinct basement membrane (BM) whieh surrounds the islet completely, the small areas of t igh t junctions (arrows) and the endoplasmic reticulum (ER). × 10000 (Normal

chicken 3mth old)

558 Erik Dahl:

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Fig. 6. Survey electron micrograph of the fully developed thecal gland in the theca interna of the domestic fowl. The cells contain the organelles typical for steroid-producing cells, predo- minated by lipid droplets (L), dense bodies (Db), complex bodies (Cb), dense mitochondria (M) with tubular cristae, spherical nucleus (N) and nuclear body (Nb). The whole gland is surroun- ded by a distinct basement membrane (BM), lying in close proximity to the granulosa cells (GC), inmembraneous contact with nerve fibers (arrows) and in close contact with a capillary (Ca). bm basement membrane of the granulosa cells; Nu Nucleolus. x 7 250 (Normal, adult hen)

(Fig. 5). Character is t ica l ly , these islets are found in membranous contac t wi th nerve fibres and ad jacen t to capil lar ies (Fig. 4), s t ruc tures which regular ly are found incorpora ted wi th the ful ly developed theea l g land (Fig. 6). F ina l ly , islets of t yp i ca l ly s teroid producing cells, the theea l glands, are sca t t e red in the theca

Postnatal Evolution of the Thecal gland 559

interna and the medulla (Fig. 6). The whole gland, which is found in close proximity to the gra~ulosa cells and capillaries, is surrounded by a distinct basement mem- brane, and supplied with an extensive innervation (Fig. 6). Different types of at tachment devices and other characteristics of epithelial cells, together with specific details of this structure, have previously been described (Dahl, 1970).

There are no marked differences of the evolution between chicken and adults as to the fine structure described, neither in the normMs, nor in the hormone and drug-treated animals.

Discussion

The identity and origin of the interstitial cells of the ovary has been the subject of considerable debate for over 60 years. Most investigators feel convinced that this ovarian cellular component originates from mesenchymal cells, while others believe them to differentiate from germinal epithelium, or even follicle epithelium (see Watzka, 1957; Merker and Diaz-Encinas, 1969). O'Donoghue (1916) consider it to be composed of a definitive cell type in its own right, l~ennels (1951) and Dawson and McCabe (1951) find that the interstitial tissue of the rat ovary is of dual origin: a primary type which is present during early juvenile period, closely associated with granulosa outgrowths and ingrowing cords from the germinal epithelium; and a secondary type, which is formed later from the theca interna of atretic follicles. On the other side, Merker and Diaz-Eneinas (1969) are of the opinion that these cells are of connective tissue origin. Primarely they are fibro- cytes which later luteinize after hormonal stimulation. Finally Stegner (1970) finds that in immature mice, the main mass of the stroma consists of undifferen- tiated stromal cells. At least some of these are potential stem cells for both thecal and interstitial elements.

In birds, Marshall and Coombs (1957) believe the secretory components of the ovary to be derived partly from migrating Iibroblasts, partly from connective tissue cells in the ovarian stroma. Studies on the fine structure of ovarian inter- stitial tissue have revealed that in the domestic fowl, the steroid producing cells consist of epithelial cells with a structure similar to steroid producing cells in other organs of the hen (DAM, 1970, 1971). In the present investigation it has been demon- strated that the surface epithelial cells (the germinal epithelium) both in chicken and adults are forming relatively deep crypts in close proximity to the growing follicles and partly encircling them too. Furthermore, a continuation of these epithelial crypts is seen as submicroscopic clefts, corresponding to what pre- viously has been described as ingrowing cords deep in the cortex. The significance of these clefts is difficult to assess. The observations made seem to indicate that the epithelial ingrowth continues throughout the adult life, depending on the active state of the ovary. The present study, therefore, supports the view (Harrison, I961) that this epithehal ingrowth reflects a natural propensity of the surface epithelium to contribute with epithelial elements to the cortical region. According to Narbaitz and Adler (1966) epithelial cells surround germ ceils and become follicular cells. However, in the present study, islets of epithelial cells, suggested to be a transitional intermediate cell type in the differentiation of the thecal gland is observed. The possibility may therefore exist, that the epithelial ingrowth also contributes with epithelial elements to the steroid producing cells, the thecal

560 Erik Dahl:

gland. I t seems reasonable to suggest t ha t th roughout the adult life the thecal gland m a y originate as follows: 1) By morphogenetie growth, the cortipal layer is folded and forms crypts and submicroscopic clefts (ingrowing cords) around the growing follicles of the cortex. 2) F rom the depth of these clefts, epithelial cells of the surface migrate towards the theca interna of the adjacent follicles. 3) In turn, these migrating cells become embedded among the cells of the theca interna or the interfollicular tissue. 4) Groups of these cells are forming small islets, which, after hormonal stimulation, are t ransformed into well defined structures, the thecal glands with a specific funct ion viz. biosynthesis (e. g. Dahl, 1972).

A postnatal evolution of the thecal gland is also favoured by the wide structural var ie ty of the hen ovary according to the reproductive state of the individual. Such variations of the size of the organ most likely require regeneration of some of the ovarian elements, including the thecal glands. By growth, the surface epithelium will be able to contribute the epithelial elements needed for this purpose.

I t should be added, however, tha t the cellular components of the ovary, apar t from the germ cells themselves, appear to be capable of undergoing considerable modification under the influence of different hormones (e. g. Dahl, 1972). F rom the information available, it is impossible to say which particular cells, including the non-specific stromal cells, can become transformed under what part icular stimuli. On account of the apparent mult ipotent ial i ty of ovarian stromal cells, and the striking variat ion in different groups, views about the embryological origin, regeneration and t ransformation can not be accurately predicted from separate histological studies, but should still be t reated with reserve.

References

Brandau, H.: Histochemical localization of enzyme activities in normal and gonadotrophin stimulated mouse ovaries. In: Gonadotrophins and Ovarian Development (cd. W. R. Butt, A.C. Crooke, M. Ryle), p. 307-311. London: Livingstone 1970.

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 (1970).

Dahl, E.: Studies of the fine structure of ovarian interstitial t.issue. 1. A comparative study of the fine structure of the ovarian interstitial tissue in the rat and the domestic fowl. J. Anat. (Lond.) 108, 275490 (1971).

Dahl, E.: The effects of gonadotrophins on the granulosa cells of the domestic fowl. Acta endocr. (Kbh.) 69, 298-308 (1972).

Dawson, A.B., McCabe, M.: The interstitial tissue of the ovary in infantile and juvenile rats. J. Morph, 88. 543-571 (1951).

Harrison, R.J.: Cells in reproduction and reproductive organs. In: Recent advances in ana- tomy, (ed. F. Goldby and R.J. Harrison), vol. 2, p. 78-104. London: Churchill 1961.

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Merker, H.J., Diaz-Encinas, J.: Das electronenmikroskopische Bild des Ovars juveniler Ratten und Kaninchen nach Stimulierung mit PMS und HCG. Z. Zellforsch. 94, 605-623 (1969).

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iNarbaitz, R., Adler, 1%.: Submicroscopical observations on the differentiation of chick gonads. J. Embryol. exp. Morph. 16, 4147 (1966).

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Pfliiger, E .F .W. : Uber die EierstScke der Siiugethiere und des Menschen. 124 S. Leipzig: Engelmann 1863.

t~ennels, E. G.: Influence of hormones on the histochemistry of ovarian interstitial tissue in the immature rat. Amer. J . Anat. 88, 63 108 (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 ' inchsion au polyester pour l 'ultramicrotomie. J. Ultrastruct. Res. 2, 200 214 (1958).

Stcgner, H.E. : Electron microscope studies of the interstitial tissue in the immature mouse ovary. In: Gonadotrophins and ovarian development (ed. W.R. Butt, A.C. Crooke and M. Ryle), p. 232-238. London: Livingstone 1970.

Watzka, M.: Die interstitiellen Zellen. In: Handbuch der mikroskopischen Anatomie des Menschen (ed. W. Bargmann), S. 107-116. Berlin-G6ttingen-Heidelberg: Springer 1957.

Dr. Erik Dahl Department of Anatomy Dental Faculty University of Oslo P. O. Box 1052 Blindem Norway