Z. Zellforsch. 109, 212--226 (1970) �9 by Springer-Verlag 1970

Studies of the Fine Structure of Ovarian Interstitial Tissue 3. The I n n e r v a t i o n of the Theca l G l a n d of the Domest ic Fowl

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

Received May 25, 1970

Summary. The fine structure of the nerves of the ovarian stroma of the domestic fowl is described for the first time. In the fowl, the nerves are concentrated upon blood vessels, smoth muscles and mainly, the thecal gland with the steroid-producing cells. Myelinat- ed as well as unmyelinated nerve fibers were observed. Numerous axon terminals represent- ing adrenergic and also presumptive cholinergic nerve fibers are regularly seen in membranous contact with steroid-producing cells. In these axon terminals microvesicles are oriented towards the steroid-producing cells indicating a specialization of the surface from axon-to-cell contact. Evidence has been presented that there is a membranous neuro-humoral contact between the peripheral autonomic nervous system and the steroid-producing cells in the ovary. The present investigation has demonstrated that there is morphologic evidence for

a nervous control of steroid-producing cells. The physiological importance of this neuro-humoral contact is discussed.

Key-Words: O v a r y - Interstitial c e l l s - Thecal g l a n d - Innervation - - Domestic fowl.

The ovary is innerva ted main ly through the ovar ian plexus (Kuntz , 1945; Watzka , 1957). This plexus arises from the aortic and renal plexus and consti tutes a meshwork of nerve fiber bundles which invest both the ovar ian ar tery and vein, forming the extrinsic nerves of the ovary (Kuntz , 1945). The intr insic nerves, which enter the ovary from the ovar ian plexus, accompany the ovar ian vessels into the s t roma where larger bundles give rise to branches which accompany the branches of the ovar ian vessels (Kuntz , 1945; Watzka , 1957).

Ear ly invest igat ions (Frankcnhaueser , 1867 ; yon Herff, 1892, and Winterha l te r 1896) described the intr insic nerves of the ovary as supplying the ovar ian follicles as well as the blood vessels. Others (Retzins, 1893; de Vos, 1894; Mandl, 1895) observed nerve fibers in the proximi ty of the follicle, bu t were no t able to determine whether they penet ra ted or t e rmina ted close to the follicles. The occurrence of ganglion cells in the ovary was reported by Bocura (1907). Brill (1915) described an a b u n d a n t supply of nerve fibers to the inters t i t ia l secretory tissue and traced structures, which he regarded as nerve fibers, into the corpus lu t eum to their t e rmina t ion on lutein cells. (For review and references, see K u n t z , 1945.)

K u n t z (1945) main ta ined tha t even though a few of the earlier investigators were led to conclude tha t nerve fibers actual ly penet ra ted the ovar ian follicles and also supplied the inters t i t ia l secretory tissue in the ovary, he did no t regard the evidence as convincing. He was of the opinion tha t the entire efferent nerve supply to the ovary probably was dis t r ibuted solely to the blood vessels and other s t ructures in the ovary which contained smooth muscle.

Inves t iga t ions by Bradley (1950), Biswall (1954), and Gilbert (1965) have revealed t ha t the ovary of the bird is innerva ted like tha t of mammals . Gilbert

Innervation of the Thecal Gland 213

(1965), us ing l igh t m ic roscopy , is t h e o n l y one w h o has e x a m i n e d t h e i n n e r v a t i o n of

t h e foll icle. To t h e a u t h o r ' s k n o w l e d g e , n l t r a s t r u c t u r a l s tud ies of t h e i n n e r v a t i o n of t h e t h e e a l g l a n d h a v e n e v e r b e e n ca r r i ed out , n e i t h e r has t h e f ine s t r u c t u r e of t h e

o v a r i a n n e r v e s a n d t he i r m o d e of c o n t a c t been desc r ibed . D u r i n g t h e i n v e s t i g a t i o n of t h e f ine s t r u c t u r e of t h e t h e e a l g l a n d of t h e d o m e s t i c fowl (Dahl , 1970a, b),

i t was o b s e r v e d t h a t th i s g l a n d has an e x t e n s i v e a n d c o m p l e x i n n e r v a t i o n , w h i c h wil l be desc r ibed in de t a i l in t h i s paper .

Mate r ia l s and Methods

Eleven White Leghorn hens, 18---24 months old, and three White Leghorn chickens, 3 months old, were used in this study. Two of the animals received intramuscular injections of 2 mg/kg metaradrini bitartras aeqv. (Aramine bitartras 1% "MSD", March Sharp and Dohme), which is a va~opressor substance with protracted effect, 18 and 4 hr before sacrifice. 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~ and a relative humidity of 60%. The hens were fed pelleted commercial chicken fodder, cabbage, sand grits, and water ad lib. The principal constituents of the fodder were proteins (17--19%), fats (2--4%), calcium (1.1--1.2 % ), phosphorus (0.7 % ), and sodium chloride (0.5 % ). Before sacrifice, the hens were kept for a minimum of 12 days for adaption to their new environment.

The hens 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 was performed us an intraaortic perfnsion with 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 either 0.1 phosphate buffer (two hens and all the chickens), 1% Tyrode (four hens), and 0.1 M cacodylate buffer (three hens), all at pH 7.3. The perfusion technique was used according to specifications given in detail by Kjaerheim (1969).

After perfusion for 10 min the ovary was excised and cut into thin slices under the dissecting 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 ~z/up to 5 mm. The tissues were then fixed for an additional period of 2 hr by immersion in the pcffusion fixative. Twenty blocks were processed from each ovarium. Subsequently, the specimens were rinsed for l0 min in 0.15 M phosphate buffer at pH 7.3 and post-fixed in 1% osmium tetroxide at 4~ for 2 hr (Millonig, 1961). After fixation the blocks were rapidly dehydrated in graded series of acetone solutions and embedded in VestopaI W (Ryter and Ketlenberger, 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 min, followed by lead citrate (Reynolds, 1963) for 5 rain. The sections were examined in a Siemens Elmiskop Ia electron microscope operaatcd at 80 kv, and equipped with 50 microns platinum objective apertures. From the same plastic blocks, sections one micron 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 pH 8.9 with 0.067 M Na2HP04.

In addition, 35 hens and 3 chickens, primarily used in stimulation and inhibition experi- ments in other studies (Dahl, 1970a), were also examined with regard to the innervation of the thecal gland and the ovarian stroma.

Observations

E x a m i n a t i o n of t h e o v a r i a n s t r o m a a n d t h e fol l icles of t h e d o m e s t i c fowl r e v e a l e d t h e m to be e x t r e m e l y wel l i n n e r v a t e d (Figs. 1 - - 5 , 17, 19). Seve ra l l a rge nerves , c o m p o s e d of a 100 or m o r e i n d i v i d u a l n e r v e f ibers , were r e g u l a r l y

15"

214 E. Dahl: Innervat ion of the Thecal Gland

Fig. 1. Survey electron micrograph of the thecal gland of the domestic fowl. The steroid- producing cells (SC) are characterized by their content of lipid droplets (L). The enclosing cells (EC) surround portions of the steroid-producing cells. The whole gland is surrounded by a basement membrane (Bin). Numerous nerve fibres (arrows) are seen at the periphery of the gland. Note portion of a peripheral nerve (Ne) adjacent to the gland, x 12,000

Fig. 2. Portion of a peripheral nerve (Ne) adjacent to a steroid-producing cell (SC) and some axon terminals (A). Groups of granular (arrow) and small agranular vesicles (double arrows) are discernible in some axons. Note the contact between the axon terminals and the steroid-

producing cell (upper left), x 18,000

Fig. 3. Portion of a large nerve (Ne) in the theea interna adjacent to the granulosa cell layer (GC). One large nucleated Sehwann cell (Sw) is seem in the centre of the nerve. Note the basement membrane (Bm) of the granulosa cell layer and the absence of nerve fibers

in this area. x 12,000

Fig. 4. Portion of a nerve in the wall of a capillary (Ca). Several axon terminals with agranular and granular vesicles are seen (arrows). Basement membrane (Bin) surrounding the pericytes

of the capillary, x 24,000

Fig. 5. Portion of a small nerve in an area of smooth muscle cells (MC). Some terminal varicosities with agranular and granular vesicles are seen (arrows). x 16,000

216 E. Dahl:

Fig. 6. Nerve fibres (Ne) embedded in the cytoplasm of enclosing cells (EC). Some axons contain granular and agranular vesicles, while others contain only granular vesicles. Note the basement membrane (Bm), which surrounds the enclosing cell and the contact between the

enclosing cell and the steroid-producing cell (SC). • 24,000

Fig. 7. A small portion of the thecal gland surrounded by basement membrane (Bin). An axon terminal (A) is seen in contact with the steroid-producing cell (SC) and the enclosing cell (EC). Note the different types of vesicles in the axon terminal. Connective tissue cells (CT)

to the left. • 24,000

Fig. 8. Par t of an axon terminal (A) in membranous contact with a steroid-producing cell (SC). Note the membranous contact between the axon and the cell, the content of vesicles of different types, and the mitochondria (M) in the axon. Basement membrane (BM). • 24,000

Innervation of the Thecal Gland 217

Figs. 9--11. Serial section of a naked axon terminal (A) invaginated into the cytoplasm of a steroid-producing cell (SC). Note the membranous contact and the vesicular content of the

axon. Nucleus (N). Basement membrane (BM). • 24,000

Fig. 12. High magnification of the same axon terminal as seen in Figs. 9--11. Axon (A). Basement membrane (BM). Steroid-producing cell (SC). • 60,000

218 E. Dahl:

Figs. 13--16

Fig. 13. Axon terminals (A) par t ly ensheathed by enclosing cell cytoplasm (EC) in contact with steroid-producing cells (SC). Some of the axons are ensheathed only by the basement membrane (BM) and are in contact with the steroid-producing cells (SC). Note t ha t some of the axons contain only agranular vesicles while others have bo th agranular and granular

vesicles, x 24,000

Imaervation of the Thecal Gland 219

encountered (Fig. 3). These large bundles seemed to branch towards the thecal glands (Fig. 1) and areas of smooth muscle cells (Figs. 5, 19). Nerve fibers were also regularly seen in the wall of the vessels, even the capillaries (Fig. 4). I n the theca interna numerous small nerves were seen approaching and more or less surrounding the thecal gland (Fig. 1).

The following structures of neurogenic origin were found: unmyel ina ted nerve fibers, myel inated nerve fibers and axon terminals (nerve endings).

Unmyelinated Fibers. These fibers were regularly seen as bundles th roughout the ovarian s t roma together with myel inated fibers (Fig. 17). I n the theca interna they formed more delicate nerves, and cross sections revealed closely packed axons, which approached and surrounded the thecal gland in the absence of vessels and smooth muscle cells (Fig. 1). After passing th rough the basement membrane and indentat ions of the enclosing cells (Figs. 2, 6, 7), the nerves ended up as naked fibers in contact with the steroid-producing cells (Figs. 2, 8). Many fibers were seen surrounding every single gland (Fig. 1), and as far as could be ascertained, all the steroid-producing cells seemed to be in contact with a nerve fiber or axon terminal. Some naked fibers were also seen penet ra t ing the gland and were the only non-glandular tissues intervening between steroid- producing cells deep in the gland. Usually, the nerve fibers and the axon terminals both had contact at the per iphery of the steroid-producing cells, bu t occasionally int racytoplasmic (invaginated) axon terminals were found (Figs. 9--12) . Two kinds of unmyel ina ted fibers were encountered (Figs. 6, 13, 15). One type was characterized by vesicles with a central, dense osmiophilic granule, whereas the other one lacked such granuales.

Myelinated Nerve Fibers. The major i ty of the fibers in the bundles of inter- glandular connective tissue were unmyel inated, but some myel inated fibers were always present (Fig. 17). I n the peripheral layer of the theca interna several myelinated fibers were observed within a single nerve. Even after the nerve bundles had branched as they approached the proximi ty of the theeal gland, myel inated fibers were found, bu t then usually as a single fiber surrounded by several unmyel ina ted ones (Fig. 17). No myel inated nerve fibers, however, were ever seen penetra t ing the basement membrane of the thecal gland. I n the peri- glandular region both myel inated and unmyel ina ted fibers were surrounded by a Schwann cell sheath. As the fibers penetra ted the gland, they not only lost

Fig. 14. Axon terminal (A) in longitudinal section in membranous contact with a steroid- producing cell (SC). The vesicles within the axon are located towards the steroid-producing cell. The orientation of the vesicles in the axon, the membranous contact and the density of the ground cytoplasm in SC (arrow) may indicate a specialization of the surface for axon-to-cell

contact. Mitochondria (M). • 45,000

Fig. 15. Portion of a nerve adjacent to a steroid-producing cell (SC) with vesiculated varicosi- ties (arrows). Note that some of the axons possess only agranular vesicles while others have

agranular as well as granular vesicles. • 40,000

Fig. 16. A basket type axon terminal (A) between steroid-producing (SC) and enclosing cells (EC). Nucleus (N). • 15,000

220 E. Dahl :

Fig. 17. Portion of a peripheral nerve with numerous nerve fibres (Ne) and a myelinated nerve (arrow) in the theea interna, x 12,000

Fig. I8. Axon terminals (A) seen after t reatment with metaraminol. Note the depletion of granules (cfr. Figs. 4, 5, 19). Enclosing cell (EC). Basement membrane (BM). Steroid-producing

cell (SC). x 24,000

Fig. 19. Axon terminals (A) from a control animal. Note the amount of granular and agranular vesicles within the nerve terminals (A) compared with the metaraminol-treated animals

(Fig. 18). Smooth muscle cells (MC). X 18,000

Innervation of the Thecal Gland 221

their myelin, but also their sheaths. Instead, intra-glandular axons were supported by enclosing cells in much the same manner as do the Schwann cells elsewhere.

A xon Termina l s . I t was not always possible to distinguish clearly between nerve fibers, axon terminals, and true nerve endings. As long as the axons were invested by the enclosing cells, they were considered to be fibers, but as naked proscesses it might sometimes be difficult to make any distinction. The content of granules or vesicles of the axons were often to some help in the effort to distinguish the different parts of the axon, but it was not possible to distinguish a fiber from an axon terminal by the use of these organelles alone (Figs. 4, 13, 15). However, round or oval naked processes, found in a recess of a steroid- producing cell, were regarded as axon terminals (Fig. 8). Nerve processes in contact with a steroid-producing cell with a bulbous and broad, more irregular outline and with accumulation of mitochondria and vesicles of the size commonly identified as synaptie vesicles, were presumed to be expansions, axon beadings, of the terminal nerves (Figs. 7, 8, 15). Furthermore, longitudinal sections of axon terminals sometimes revealed them in membranous contact with the steroid- producing cells, with the intra-axonal vesicles located towards the steroid-produc- ing cell (Fig. 14). The orientation of the vesicles, the membranous contact and the density of the ground cytoplasm of the steroid-producing cell (Fig. 14) may indicate a specialization of the surface for axon-to-cell contact and, probably, represents a form of true nerve ending.

Three kinds of these axon terminals were seen, one type characterized by containing vesicles with central dense osmiophilic granules of an average size of 500/~, and the second devoid of granules (Fig. 13). Each steroid-producing cell seemed to be in contact with several of these terminals. Injections of metaraminol resulted in partial or complete depletion of these granules (Fig. 18).

In contrast to the two types of endings mentioned above, there was a third type which made a much more extensive contact along the surface of the steroid-producing cells (Fig. 16). These endings were characterized by having a relatively empty cytoplasm with few microtubules, fibrils, and synaptic vesicles. They were found underneath the enclosing cell and in contact with it. These features, and because they could also be found as fibers in the nerves, they were considered to be nerve endings. In some cases they almost completely surrounded the steroid-producing cell (Fig. 16) like a basket, within a given section. However, since this was a rather infrequent finding with the other endings encountered, it is conceivable that these endings do not completely surround the steroid cell in three dimensions, but only in one plane. Compared with the other axon terminals, they were very seldom encountered.

Ganglion cells were never seen in the theca interna. Finally, it should be added tha t nerves were found scattered regularly between smooth muscle cells (Fig. 5) and adjacent . to the pericytes and endothelial cells of the capillaries underneath the basement membrane of the vessels (Fig. 4).

Unmyelinated nerves were also found adjacent to the granulosa cell layer (Fig. 3), but never in membranous contact with the granulosa cells or the oocyte. Furthermore, nerves were never seen penetrating the basement membrane of the granulosa cell layer.

Intramuscular injections of gonadotropins (Dahl, 1970d) and clomiphene (Dahl, 1970e) did not affect the structure of the neurogenenic tissue.

222 E. Dahl:

Discussion Difficulties in identifying the different gland tissues of the ovary have made

it problematic to map out the innervation of the various structures by using ordinary histological methods. Differences in the species examined have not ~ z e d the divergences, and therefore the results obtained have also been conflicting. Pines and Sehapiro (1930) believed tha t the nerves entered the ovary as a fine plexus between the epithelial cells of the ovarinm surface and ended in the theca interna. StShr {1954) suggested tha t the nerve endings were intracellu- larly and tha t the ovarian stroma was dependent on some vegetative function. Furthermore, he reported having observed nerve endings at the surface of the ovum in the monkey. Koppen (1952) maintained tha t nerve endings were in contact with the pr imary follicle of the rabbit. However, Goecke and Beaufays (1935), Goecke (1938) and Kladetzky (1951) were not able to find the nerve endings as described by StShr (1954).

According to Bradley (1950), the ovary of the bird is innervated from the abdominal plexus and from the posterior continuation of the sympathetic trunk. Gilbert (1965), in a study of the nerves of the ovary of the domestic fowl, found the follicle to be innervated. In addition to nerves and nerve endings, he also stated tha t neurons in varying numbers, proportional to the size of the follicle, could be seen in the theca interna. He never found myelinated nerves and indicated tha t the adrenergic component of the innervation of the follicle, if present, was small.

The present s tudy has revealed the theca interna of the ovarian follicle of the domestic fowl to be extremely well innervated. Furthermore, this innervation seems to include small, as well as larger, follicles.

Many authors have described beadings of autonomic nerves in their terminal distribution in a wide variety of methylene blue-stained preparations (e. g. Wollard, 1926 ; Millen, 1948). Electron microscopy has confirmed that the autonomic termi- nal nerves show successive narrowings and expansions where accumulations of mitochondria and microvesicles may be found (Thaemert, 1966). Adrenergic (Lever et al., 1968) and cholenergic (Esterhuizen et al., 1968) beadings have been described and it is accepted, that in adrenergic nerves, t ransmitter release may occur at these expansions (Hertting and Axelrod, 1961 ; Lever et al., 1965, 1968 ; Spriggs et al., 1966). Terminal axon varicosities have been demonstrated in which both large (600---1,000 A) and small (300--500 A) vesicles are located, and some of the smaller vesicles possess a dense central granule surrounded by a ring of less dense material (Ruskell, 1967). Chemical analysis of tissue fractions (Michael- son et al., 1964), and electron microscopic autoradiography (Wolfe et al., 1962) have revealed tha t nor-epinephrine is stored in granular vesicles measuring about 500 A in diameter, demonstrable electron microscopically in adrenergic nerves (Richardson, 1964; Bondareff, 1965). Such granular vesicles in adrenergic nerve fibers can be depleted by administration of metaraminol (Bondareff and Gordon, 1966). Ruskell (1967), in his s tudy of axon terminals of the lacrimal gland in monkeys, found that axon terminals populated with small granular vesicles (300--500 A) are sympathetic, while large granular vesicles (650--1000 A) are present in both sympathetic and parasympathet ic terminals.

In the present study numerous bulbous axons, identical with the beadings and axon varicosities mentioned above, were regularly found, and they seemed to be

Innervatioa of the Thecal Gland 223

in extensive contact with the steroid-producing cells. The naked axon beadings without Schwann cell, the membranous contact between the axon and the steroid- producing cell, the presence of microvesieles oriented towards the steroid-produc- ing cell, the mitochondria, and the density of the ground cytoplasm in the steroid- producing cell suggest a specialization of the surface at the site of axon-to-cell contact. In conclusion, evidence is presented that in the ovary of the domestic fowl there is a direct neuro-humoral contact between the autonomic nervous system and the steriod-producing cells in the theea interna. As far as can be ascertained, this kind of membranous contact between the peripheral autonomic nervous system and steroid-producing cells has never been demonstrated. In the axon terminals, granules measuring about 500 A were regularly encountered. Furthermore, injection of metaraminol resulted in a depletion of these granules. One may therefore conclude that the ovarian stroma with the theeal gland are furnished with an autonomic innervation rich in adrenergic nerve fibers, and that their varieosities represent adrenergie axon terminals. I t also seems reasonable to suggest that the axon terminal with vesicles and mitochondria in the absence of osmiophilic granules represent cholinergic nerves and efferent axon terminals.

The basket-type endings described did not show more than a few vesicles and are more similar to the sensory endings of the hair cell {Spoendlin, 1966). They also resemble the numerous endings between the cells of the taste bud, which are presumably sensory {Murray and Murray, 1960, 1967). I t is therefore reasonable to suggest that the steroid-producing cells of the theeal gland have an efferent as well as an afferent innervation.

The function of this complex nervous system is unknown. Regarding the physiology of the different nerves and axon terminals, no direct experimental evidence, except for the fact that some of them are adrenergie nerves, is available at present. Therefore, any discussion about their function must be speculative. Steroid-producing cells are generally accepted as being under the influence of hormonal control exclusively. However, the present investigation has revealed that the function of the thecal gland from an anatomical point of view can be subjected to some nervous control. Since it seems reasonable to assume that there are two kinds of efferent nerve endings, it is tempting to presume that their function will be antagonistic, viz. stimulation and inhibition. That the basket cells may be sensory endings seems reasonable, though nothing is known regarding their physiology or the effective stimulus which may activate them. Examination of the brain of the domestic fowl {unpublished results) revealed the presence of central adrenergie nerves, which may support the conception that the autonomic nervous system may have central connections relating to, and dependent upon, the function of peripheral organs. This seems to be supported by observations reported by Rothchild and Fraps (1944), and Wood- Gush and Gilbert (1964), who found that removal of the most recent postovula- tory follicle affects nesting and oviposition. Since nerves are observed in the vessels and smooth muscles, it is also possible that the rupture of the follicle may be under neurogenic control by affecting the smooth muscles directly or by affecting the blood flow, as suggested by Gilbert (1965). This may explain the presence of islets of smooth muscle cells in the ovary.

The steroid-producing cells of the thecal gland were the only hormone- secreting cells of the ovary which were found to be innervated. This may possibly

224 E. Dahl:

be a key to fur ther invest igat ions related to the controversies as to the origin of the steroid hormones. The possibility exists t ha t the steroid cells of the thecal gland, as a ma in source of steroid hormones, are under central control both hormonal ly and neurogenically, bu t eventua l ly hormonal product ion of other cells of the ovary, viz. granulosa cells, arc secondary as a source and only under hor- monal influence.

The possibili ty also exists t ha t the autonomic innerva t ion of the steroid- producing cells in the theca in te rna may be involved in the regulat ion of the seasonal ac t iv i ty of the reproductive behaviour which take place in the birds. Some external factors, especially the increase and the decrease in hours of sunshine, which is characteristic in the spring and the au tumn , arc proposed to be of impor tance (Marshall and Coombs, 1957). However, the influence of dayl ight is unknown. I t might be tha t the fight in some way exerts an influence on the central nervous system, which secondarily through the direct neuro- hormonal contact s t imulates or inhibi ts the steroid-producing cells.

Exogenous adminis t ra t ion of steroids, gonadotrophins and clomiphene, to be reported in later papers, did no t seem to affect the fine s tructure of the nerves or nerve endings (Dahl c, d, e).

Referenees

Biswall, G. : Additional histological findings in the chicken reproductive tract. Poult. Sci. 88, 843--851 (1954).

Boeura, K. : Nachweis von chromaffinem Gewebe und wirklichen Ganglienzellen in Ovar. Wien. klin. Wschr. 20, 695--699 (1907).

Bondareff, W. : Submicroscopic morphology of granular vesicles in sympathetic nerves of rat pineal body. Z. Zellforsch. 67, 211--218 (1965).

- - Gordon, B. : Submicroscopic localization of norepinephrine in sympathetic nerves of rat pineal. J. Pharmacol. exp. Ther. 153, 4 2 4 7 (1966).

Bradley, O. C. : The structure of the fowl, 3rd ed., p. 60. Edinburgh-London: Oliver and Boyd 1950.

Brill, W. : Untersuchungen fiber die Nerven des Ovariums. Arch. mikr. Anat. 86, 338--344 (1915).

Dahl, E. : 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. Z. Zellforsch., to be published (1970a).

- - Studies of the fine structure of ovarian interstitial tissue. 2. The ultrastrueture of the thecal gland of the domestic fowl. Z. Zellforsch., to be published (1970b).

- - Studies of the fine structure of ovarian interstitial tissue. 4. Effects of steroids on the thecal gland of the domestic fowl. Z. Zellforsch., to be published (1970c).

- - Studies of the fine structure of ovarian interstitial tissue. 5. Effects of gonadotropins on the thecal gland of the domestic fowl. Z. Zellforsch., to be published (1970d).

- - Studies of the fine structure of ovarian interstitial tissue. 6. Effects of clomiphene on the theeal gland of the domestic fowl. Z. Zellforsch., to be published (1970e).

Esterhuizen, A. C., Spriggs, T. L. B., Lever, J. D. : Axon beadings in autonomic cholinergic nerves. J. Cell Biol. 38, 454--457 (1968).

Frankenhaueser, F. : Die Nerven der Geb~rmutter und ihre Endigung in den glatten Musket- fasern. 82 pp. Jena: F. von Mauke 1867.

Gilbert, A. B. : Innervation of the ovarian follicle of the domestic fowl. Quart. J. exp. Physiol. 50, 437 445 (1965).

Goecke, H. : Die Endausbreitung des vegetativen Nervengewebes im menschlichen Ovarium und ihre Bedeutung fiir die Funktion des Ovariums. Arch. Gyn~k. 166, 187--189 (1938).

- -Beaufays, J.: Neurohistologische Untersuchungen am Ovarium. Arch. Gyn~k. 16{}, 571--579 (1935).

Innervation of the Thecal Gland 225

Herff, O. von: l]ber den feineren Verlauf der Nerven im Eierstocke des Menschen. Z. Geburtsh. Gyn~k. 24, 289--308 (1892).

Hertting, G., Axelrod, J . : Fate of triated noradrenaline at the sympathetic nerve-endings. Nature (Lond.) 192, 172--173 (1961).

Kjaerheim, A. : Studies of adrenocortical ultrastructure. 1. Aldehyde perfusion fixation of the domestic fowl. Acta anat. (Basel) 74, 424 453 (1969).

Kladetzky, J . : ~be r die Innervation des Ovarinms. Nach Untersuehungen an einigen S~ugern. Arch. Gyn~k. 179, 363--383 (1951).

Koppen, K. : Die vegetative Irmervation der weiblichen Genitalorgane beim Menschen und ihre psychophysische Problematik. Acta neuroveg. (Wien) 8, 833--345 (1952).

Kuntz, A. : The autonomic nervous system, 3rd ed. 687 pp. Philadelphia: Lea & Febiger 1945. Lever, J . D., Graham, J . D. P., Irvine, G., Chick, W. J. : The vesiculated axons in relation

to arteriolar smooth muscle in the pancreas. A fine structural and quantitative study. J . Anat. (Lond.) 99, 299--313 (1965).

- - Spriggs, T. L. B., Graham, J. D. P. : A formol-fluorescence, fine-structural and autoradio- graphic study of the adrenergie innervation of the vascular tree in the intact and sympa- thectemized pancreas of the cat. J . Anat. (Lond.) 108, 15--34 (1968).

Mandl, L. : l]ber Anordnung und Endigungsweise der Nerven im Ovarium. Arch. Gyn~k. 48, 376--392 (1895).

Marshall, A. J. , Coombs, C. J . F. : The interaction of environmental, internal and behavioural factors in the rook, Corvus F. frugilegus Linnaeus. Proc. zool. Soc. (Lond.) 128, 545--589 (1957).

Michaelson, I. A., Richardson, R. C., Snyder, S. N., Titus, E. 0. : The separation of catechol- amine storage vesicles from rat heart. Life Sci. 8, 971--978 (1964).

Millen, J . W . : Observations on the innervation of blood vessels. J. Anat. (Lond.) 82, 68--80 (1948).

Millonig, G. : The advantages of a phosphate buffer for OsOa solutions in fixation. J . appl. Physiol. 82, 1637 (1961).

Murray, R. G., Murray, A. : The fine structure of the taste buds of rhesus and cynomalgus monkeys. Anat. Rec. 188, 211--233 (1960).

- - - - Fine structure of taste buds of rabbit foliate papillae. J . Ultrastruct. Res. 19, 327--353 (1967).

Pines, L., Seh~piro, B. : Uber die Innervation des Eierstockes. Z. mikr.-anat. Forsch. 20, 327--372 (1930).

Retzins, G.: ]~ber die Nerven der Ovarien und ttoden. Biol. Unters., N . F . 5--31 (1893). 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). Richardson, K. C. : The fine structure of the albino rabbit iris with special reference to the

identification of adrenergic and cholonergic nerves and nerve endings in its intrinsic muscles. Amer. J . Anat. 114, 173--205 (1964).

Rotchild, I., Fraps, R. M. : On the function of the ruptured ovarian follicle in the domestic fowl. Proc. Soc. exp. Biol. (N. u 56, 79--82 (1944).

Ruskell, G. L. : Vasomotor axons of the lacrimal glands of monkeys and the ultrastructural identification of sympathetic terminals. Z. Zellforsch. 88, 321--333 (1967).

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

Spoendlin, H. : The organization of the cochlear receptor. Fortsehr. Hals-Nas.-Ohrenheilk. 18, 185 (1966).

Spriggs, T .L . , Lever, J . D . , Rees, P.M., Graham, J . D . P . : Controlled formaldehyde- catecholamine condensation in cryostat sections to show adrenergic. Stain Technol. 41, 323--327 (1966).

St6hr, Ph., J r . : Zusammenfasssende Ergebnisse fiber die Endigungsweise des Vegetativen Nervensystems. Acta neuroveg. (Wien) 1O, 21--109 (1954).

Thaemert, J. C. : Ultrastrueture of cardiac muscle and nerve contigiutes. J. Cell Biol. 29, 156--162 (1966).

Vos, J. de: ]~tude de l ' innervation de l'ovaire. Bull. Acad. roy. M6d. Belg. Ser. IV, 8, 552--558 (1894).

226 E. Dahl: Innervation of the Thecal Gland

Watzka, M.- Weibliche Genitalorgane. Das Ovarinm. In: Handbueh der mikroskopisehen Anatomie des Menschen (W. Bargmann ed.), Bd. 7, T. III . 178 pp. Berlin-GSttingen-Heidel- berg: Springer 1957.

Winterhalter, E. H. : Ein sympathisches Ganglion im mensehlichen Ovarium. Arch. Gyn~k. 51, 49--55 (1896).

Wolfe, D. E., Potter, L. T., Richardson, K. C., Axelrod, J. : Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science 138, 440~442 {1962).

Wood-Gush, D. G. M., Gilbert, A. B. : The control of the nesting behaviour of the domestic hen. II. The role of the ovary. Anim. Behav. 12, 4 5 1 ~ 5 3 (1964).

Woollard, H. H. �9 The innervation of blood vessels. In: Heart (T. Lewis ed.), vol. 13, p. 319--336. London: Shaw & Sons Ltd. 1926.

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