Paper - Morphological and physiological studies on the musculature of the mature graafian follicle of the sow (1921)
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Guttmacher MS. and Guttmacher AF. Morphological and physiological studies on the musculature of the mature graafian follicle of the sow. (1921) Johns Hopkins Hospital Bulletin 32: 391-399.
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Morphological And Physiological Studies On The Musculature Of The Mature Graafian Follicle Of The Sow
By M. S. and A. F. Guttmacher
From the Anatomical Laboratory The Johns Hopkins University
Introduction
In the recent literature on the morphology of the ovary, passing mention is made of the occurrence of smooth muscle fibers in the wall of the Graafian follicle. The work reported in this paper was undertaken to verify or disprove these observations, and, if this muscle were proven to exist, to determine the types of autonomic nerve fibers to its cells, and further to investigate the importance of this element in the physiological activity of the follicle.
The authors wish to express to Dr. Corner, of the Department of Anatomy, their appreciation for his generous interest and direction, without which these researches could not have been performed. We also wish to thank Professor Abel and Dr. Macht of the Department of Pharmacology for their advice and help.
History
In the initial vohnne of his famous Journal (1849), Die Zeitschrift fiir WissenschafUiche Zoologie, von Kolliker was . the first to mention smooth muscle as a structural constituent of the ovary. In 1861 C. Aeby by means of the then much relied upon acetic and nitric acid technique made extensive investigations on the character and location of the muscle fibers iu the ovaries of various birds and mammals. Two years later Grohe described the complete course of the muscle fibers in the ovary of the pig, from its hilus to the very edge of the mature follicles, which he found it to encircle.' Grolie also mentioned the unusually generous distribution of smooth muscle fibers about the blood vessels of the ovary. In this he supported the hypothesis formulated by Rouget seven years earlier, that, in contracting, the muscle fibers of the ovary compressed the blood vessels, in consequence of which the congestion from impaired venous return led to the rupture of the mature follicles. For a half century little progi'ess was made in either the histology or physiology of the smooth muscle of the ovary; more refined methods of staining were developed Imt nothing of note was learned. In 1909 H. von Winiwarter and Sainmont announced the occurrence of what they believed to be true smooth muscle fibers in the theca externa of the Graafian follicles of the cat. In the latter part of 1919 Tliompson, in his paper on the himian Graafian follicle, ilwelt at length on the occurrence and purposes of muscle fibers in the theca externa, which he was not able to demonstrate very clearly by a microphotograph. He obtained his best differentiation with safranin and light green. This author stated that the muscular element could not be demonstrated in all of his preparations. Corner, earlier in the same year, briefly mentions the application of the van Gieson technique in distinguishing these fibers. Other than this, the authors have been unable to find mention of the occurrence of muscle in Graafian follicles.
The nerve supply of the Graafian follicle has been a problem long and bitterly disputed. It was early recognized by Fraukenhauser ('ii'i') and Waldeycr ('70) that sympathetic nerves entered the hilus of the ovary with the ovarian artery. This observation was substantiated by all subsequent workers. Riese ('91) showed that the nerve fibers on entering the ovary were separated into two groups — one group accompanying the blood vessels, and the other going directly to the follicles. The workers in this field were soon divided into three camps : Riese ('91), von Herff ('92), von Gawronsky ('94), Winterhalter ('96), Brill ('15) and others believed that nerve fibers independent of the perivascular network penetrate all the layers of the follicle wall, the theca externa, the theca interna and the granulosa. The second group represented by Abel and Mcllroy ('12-'13) claimed that only the two outermost coats of the follicle wall have a nerve supply. The third group wliich numbered among its protagonists Retzius ('93), rle Vos ('94), and Mandl ('95) held that the follicle is not in any way penetrated by nerve fibers, other than those of the perivascular network.
Material Our selection of the sow was based on several considerations. The sow has been found to ovulate spontaneously and to have an oestrous cycle of 21 days. The large size of the ripe fpllicles (.5 to 10 mm. in diameter) offered great advantages, particularly in the physiological work. The existence of two pork packing houses in close proximity to this laboratory assured us twice daily a supply of fresh tissue. Then too, the experience of Dr. Corner made his advice invaluable as to selection of the ovaries of animals in heat, and the rejection of pathological material.
- It is interesting to note that at this time, prior to the great contributions of His ('65) and Waldeyer (70), the follicular layers had been imperfectly differentiated, the two external layers of the wall of the follicle being generally referred to as the theca folliculi.
Our tissue was obtained immediately after slaughter. The ovaries of animals that were in oestrus or Just about to enter oestrus formed the chief source of our material. The oestrous period could be determined by the large size of the follicles, the retrogressing corpora lutea, the great vascularity of the peritubal tissue, and the relative pallor of the uteri. Corner ('19), in a study of the follicles of animals known to have been in heat when killed, stated that these follicles possessed clear, translucent, almost spherical walls, protruding a great part of their bulk from the ovary, as is characteristic of the species; they all measured about 7 mm. in diameter, the measurement ranging from 6 to 8 mm. in some which were distorted by crowding. The surface presented no "stigma" or other sign of impending rupture.
The Musculature of the Graafian Follicle The ovaries used in studying the muscular morphology were promptly immersed in Bouin's fluid. After the Bouin fixation, and the usual paraffin embedding technique, the tissue was sectioned from 3.3 to 5 micra, either by the ordinary microtome technique, or by Professor Huber's wet knife method. The sections were then stained in haemotoxylin with iron alum as a mordant. After decolorization, they were subjected to the van Gieson technique, for the differentiation of smooth muscle and connective tissue fibers. This stain was found to be entirely satisfactory — the bright red connective tissue and the yellow muscle fibers, with their typical niiclei, forming a brilliant contrast.
In the thecaj externte of all our specially stained preparations, we were able to distinguish typical smooth muscle cells, with their characteristic cytoplasm and nuclei, which iu their distribution gave the suggestion of a lamina over the two internal layers of the follicle, in many places incomplete and interrupted by areas of connective tissue and blood vessels. In many of our sections miiscle fibers were found (Figs. 1 and 2), grouped into bundles of rather impressive proportions in juxtaposition to the cells of the theca interna. In many areas these bundles formed pure bands of muscle more than a half-dozen cells in thickness, completely devoid of intercellular connective tissue. In some places, however, only very small scattered groups of muscle cells were present in the follicular wall. Here, as has been noted in the ovarian stroma, these muscle bands seemed to be particularly numerous in the region of the blood vessels. In all of the material sectioned there was no suggestion of the existence of muscle cells in the theca interna. In sections made of a freshly ruptured follicle (the eggs were recovered from the tubes, and two normal, still unruptured, follicles were found in the ovary), the arrangement of the muscle fibers suggests that they played an active role in the rupture of the follicle. That the theca externa did not remain as a mere passive membrane on the periphery of the newly ruptured follicle, but in conjunction with the other layers projected finger-like processes into the recently contracted follicular cavity, is made evident by Fig. 2. Corner ('19) has shown by means of Weigert's elastic tissue stain, that with the exception of the blood vessel walls the follicle is completely devoid of elastic tissue. Since in this species only about 2-5 per cent of the recently raptured follicles contain any blood in their cavities, perhaps, as he suggests, the muscle here by compressing tlie wall plays the mechanical role of a hemostat.
Having followed the advice of Gaskell to found our issue on a firm anatomical basis before resorting to confirmatoij physiological measures, we proceeded to prove the existence of muscle by functional tests. We followed the usiial " in vitro " technique in this phase of our work. While the ovaries were in Locke's solution needles and thread were passed through opposite poles of the follicle, then by pulling on these sutures we were able to cut the projecting portion of the follicular wall free from tlie stroma of the ovary. Great care was exercised so that none of the stroma was included in the tissue removed. This bit of living tissue was suspended in a bath of oxygenated Locke's solution maintained at body temperature. The suture on the one end of the follicle was tied to a glass rod well submerged in the solution, while that on the other end was attached to a very sensitive writing lever. In this way accurate records of the muscular contractions of the wall of the follicle could be obtained on a smoked drum. At times in order to make these records more decisive we combined strips of tissue from two follicles of the same ovary.
Graph A. — Contraction of wall of Graafian follicle due to stimulation with barium chloride. Time intei-vals 10 minutes.
BaCl/ Bad:"
Barium chloride 1-10,000. Barium chloride 1-10,000.
We first made use of a solution of barium chloride, the classical means of chemically stimiilating smooth muscle regardless of its innervation, with which we obtained most active contractions. Even the addition of 2 c. c. of a 0.5 per cent solution of barium chloride to 25 c. c. of Locke answered admirably for tliis purpose. A relatively long latent period and a gradual protracted "rise" marked the contractions obtained with this drug. With solutions of greater concentrations more marked contractions were obtained than the one recorded in Graph A. At this point we became interested in deteiniining whether the stimulation by barium was due to its toxic effect alone. We found that very definite contractions of the tissue from the wall of the follicle could be obtained by using solutions of barium, made isotonic with Locke's solution, sufficiently dilute to permit the life of amcebje, paramecia, and rotifers for more than 6 hours. The records which were obtained through barium stimulation were typical of smooth muscle and as barium has not been found to cause the contraction of any tissue other than muscle, we may conclude that the wall of the Graafian follicle contains non-striated musclefibers.
The nerves of the Graafian Follicle It is very difficult to stain satisfactorily the nerves of the Graafian follicle. The delicacy of the fibrils makes microtome sections of this material quite unsatisfactory, for at best the sectioned nerve fibers look like a series of fine dots of doubtful structure. It was apparent to us therefore early in our study, that the information we sought could best be obtained from gross material suitably stained. The Golgi and the Bielschowski impregnation methods gave unsuccessful results in our hands, probably because of the tliickness of the follicle wall and the impermeability of the outer layers of the ovary. Good results were obtained by Ehrlich's methylene blue staining, following the modifications of J. G. Wilson. Injections of the ovary, through the ovarian or uterine arteries, with a 1/20 per cent methylene blue solution were made under great pressure, which was necessary to insure a complete injection of the mature follicles. After the tissue had been exposed to the oxidation of the air for from one-half to one hour (the time required to stain the neiwes distinctly) some of the specimens were fixed in ammonium molybdate, and others in ammonium picrate. The picrate fixation made the specimens easier to tea.se apart and had the additional advantage of staining the muscle fibers yellow. On the other hand, the tissue after ammonium molybdate fixation cleared much better, and the specimens were made quite firm by dehydration. In specimens fixed in this way it was possible to peel ofl' the various layers of the follicle and study tliem separately.
Ill the theca externa, the muscle-containing layer of tlie follicle, there is a rich plexus of nerve fibers (Fig. -1). The nerves )im (10 to 15 together) in good sized non-medullated bundles, independent of the perivascular network which is also demonstrable in our preparations. These bundles of non-medullated nerve fibers give off numerous fibrils to the surrounding tissue. The fibrils have the ordinary morphology of the non-medullated nerve. As Fig. 3B shows, the nerve fibrils course among the muscle cells, running parallel with some cells and crossing others. They give a definite impression of bearing a functional relation to the muscle cells of the theca. These nerve fibrils tenninate on muscle cells in .sympathetic motor endings (Fig. 3A), the smaller endings covering one mu-scle coll, while the larger terminations overlie two or three cells. As Fig. 3A shows, these endings are of two distinct types — one ending is large, pale, clear and oval ; while the other is small, dark, full of granules and triangular in form. We have therefore a complete path : nerve fibrils running intimately among muscle fibers, sympathetic motor-endings on muscle cells, and muscle cells capable of functioning. It seems unquestionable that there must be some physiological application for this apparatus. We also thought it desirable to obtain exact information as to the type of autonomic innervation of the ovary and its follicles, since in the abundant literature on the nerves of this organ we have found no mention of such a study. Thompson ('19) records some pharmacological studies made by Gunn at the former's suggestion, to demonstrate by experimental means the smooth muscle of the ovary. In one experiment conducted on the whole ovary of a virgin rabbit, Gunn elicited a response from stimulation with epinephrine (1-200,000) — an observation which led him to maintain that the ovary has true sympathetic innervation. (It is interesting to note, however, that Gunn finds the true sympathetic nerves to be excitatory nerves of the ovarj^ in the rabbit, while we have found them to be inhibitory nerves in the sow's ovaiy.) In 1909 under the direction of Von Winiwarter the ovary of the cat was stimulated chemically and electrically but with negative results. Langley and Anderson ('96), in the cat and rabbit, and Kuntz ('19), in the dog, have shown through dissections and physiological degeneration experi
HCl' Ma.OH' HCrOH "
Graph B. — Contraction of the wall of the Gnuitian follicle due to changea in the hj'drogen-ion concentration, caused by the addition of hydrochloric acid and sodium hydroxide.
HCr Hydrochloric acid, 0.2 c. c. of 0.3 per cent to 25 c. c. of Locke's solution.
ments that the gonads are innervated by rami from the third, fourth, fifth, and very rarely from the sixth lumbar prevertebral sympathetic ganglia. We have verified this for the sow in a dissection of a large embryo prepared in formalin. However, the important work of Gaskell, Langley and Dickinson, has indisputably shown that the only reliable method of studying autonomic innervation is by means of drug's. By sucli studies one is enabled to differentiate sympathetic (thoracolumbar) from para-sympathetic (bulbar-sacral) and inhibitory from excitatory fibers. We, therefore, undertook such studies on the nerve supply of the mature Graafian follicle. In this work we followed the ordinary " in ~vitro " technique already described in this paper. Before the work had progressed far, we noticed that the smooth muscle of the follicle wall is very sensitive to any variation of the hydrogen-ion concentration either to the alkaline or acid side. The addition of such a minute amount as 0.2 c. c. of 0.3 per cent HCI to 25 c. c. of Locke's solution gave the first rise reproduced
in Graph B. It was therefore imperative to use neutral salts of the alkaloids or, if no neutral salts were available, to make the solutions neutral with NaHCOj.
On the addition of pure epinephrine, furnished by Dr. Abel, a definite relaxation of the follicle wa.s recorded (Graph C).
Gr-Aph C. — Relaxation of wall of the Graafian follicle due to stimulation with epinephrine and contraction due to the addition of acid. Ep Epinephrine 1-5000.
HCI Hydrochloric acid.
The extent of tlie relaxation was jiractically the same with a 1-10,000 solution as with a 1-2,000,000, the amount of relaxation diminishing slightly with the increased dilution. Thus the nervous mechanism of the Graafian follicle is shown to respond to the action of a drug which stimulates fibers of the true sympathetic system. In this case the action of the drug causes relaxation of the smooth muscle fibers.
Through the addition of physostigmine sulphate (eserine) and atropine sulphate we showed that the follicle also has para-sympathetic innervation. A 1-10,000 solution of physostigmine caused a very marked contraction of the tissue (Graph D). The proof of a para-sympathetic innervation was further substantiated by the action of atropine, which paralyzed the myoneural junction of the para-sympathetic nerve fibers, so that on the addition of physostigmine no contraction was elicited ; this drug also relaxed the tissue previously contracted by phj-sostigmine (Graph D). It is clear then that the follicle has a para-sympathetic innervation, thase fibers acting as contractors of the follicle wall. Similar results in all respects were obtained by us with experiments on the ovarian stroma — these drugs which stimulate the sympathetic system acting as inhibitors and the para-sympathetic stimulants contracting the ovarian musculature. All
"Phys' Vhys" W "Atf"
• si I • J I I Graph D. — Contraction of wall of Graafian follicle due to stimulation with phj'sostigmine sulphate and subsequent relaxation on the addition of atropine sulphate.
Phys' Physostigmine sulphate 1-10.000.
Phys" Physostigmine sidphate 1-10,000.
Atro' Atropine sulphate 1-1500.
Atro" Atropine sulphate 1-1500.
of our results demonstrate that the ovary and the follicles of the sow have an innervation analogous to that of the intestines.
All the physiological work reported in this paper was done with the same apparatus, and yet there were marked variations in the results obtained. With some ovaries the contractions were relatively great; while with others only feeble contractions were noted. From our observations we think perhaps that the amplitude of the contraction depends on the exact moment in the oestrous cycle at which the animals were slaughtered. Those ovaries containing large distended follicles giving the impression of imminent rupture, produced the greatest contractions, while those specimens less advanced in the cycle generally seemed to give contractions of much less magnitude.
Observations Relative to the Method of EurTUKE of THE Follicle
Since the early part of the last century various speculations have been made as to the method of rupture of the Graafian follicle. Suffice it to say that the theories have been almost as numerous as the theorizers. Thompson in 1919 was the first to suggest that the smooth muscle in the wall of the follicle plays a major role in this process. It is of interest to note that it had been maintained, as early as 18.59, by Pfliiger that the peristaltic movements of the frog's ovaries caused rupture of the follicles.
When we found with what readiness the smooth muscle of the Graafian follicle of the sow could be stimulated to contract in vitro, we had high hopes of producing these contractions, with the ovaries still attached to the excised uteri, and thus perhaps induce rupture. We adopted several methods of procedure with the isolated organs — injections of barium chloride, physostigmine, etc., were made into the uterine and ovarian arteries, in some of which high pressure (350 mm. of Hg.) was produced by a hydrostatic apparatus, while in others a low pressure (21 mm. of Hg.) was maintained for a long period of time. Separate follicles were excised from the ovai7 and direct injections were made into the follicular cavity by means of a hypodermic syringe. Experiments were also performed in which a constant low head of pressure was maintained in these excised follicles, which were immersed in warm oxygenated solutions.
The results of all of these experiments were either negative or so inconstant, because of the experimental difficulties in carrying out these preccdures in excised organs some time after death, that we were unable to produce any conclusive evidence as to the role of muscle in the rupture of the follicle. We feel confident that experiments conducted on this problem in the living animal would give fruitful results. We believe that our experiments on this phase of the problem have definitely shown that the pure mechanical hypothesis of Hensen which has been so widely accepted, that rapture is induced solely through increased arterial tension, is false. This theory is supported by a single case of rupture induced by Clark ('00) in a human ovary during an intravascular injection of carmine gelatin. In connection with our work 72 injections of the ovarian and uterine arteries of ovaries with mature follicles were made imdor great pressure. The follicular vessels could be seen to wash out clearly but rupture did not occur in a single instance, even though one braced himself against a wall and pushed the piston of the injection syringe with all of the physical strength available. A constant head of pressure of over 300 mm. of Hg. was exerted through these arteries for hours, but again without inducing rupture. It is indeed difficult to conceive of such enormous pressures existing in the living animal. Dr. Comer tells us that in his previous work in the injections of the follicular vessels of the sow he has never obtained rupture under increased arterial tension.
Conclusions
- The theca externa of the Graafian follicle of the sow contains an abundance of typical smooth muscle cells.
- Autonomic nerves with typical motor endings are fotaid in juxtaposition to these muscle cells.
- The musculature of the ovary and its follicles has a double innervation ; the true sympathetics acting as inhibitory and the para-sympathetics as excitatory nen'es. This innervation is similar to that of the musculature of the intestine.
- The rupture of the follicle is not produced solely by an increase in the arterial tension of the follicular vessels.
Bibliography
1. Abel, W., and Mcllroy, A. L.: The arrangement and distribution of nerves in certain mammalian ovaries. Proc. Royal Soc, 1912-13, VI, Obst. and Gyn. Sec, 240.
2. Aeby, C: Die glatten Muskelfasem in den Eierstocken der Wirbelthiere. Arch. f. Anat. u. Phys., 1861, 635.
3. Brill, W. : Untersuchungen iiber die Nerven des Ovariums. Arch. f. raikr. Anat., 1915, LXXXVI, Sec. 1, 338.
4. Clark, J. G.: The blood vessels of the ovaiy. Johns Hopkins Hospital Reports, 1900, IX, 593.
5. Corner, G. W.: On the origin of the corpus luteum of the sow from both the granulosa and the theca interna. Amer. Jour. Anat.,
1919, XXVI, 117. Idem: Cyclic changes in the ovaries and uterus of the sow. Publications of the Carnegie Institution, No. 276 (Contributions to Embryology, No. 64).
6. de Vos, J.: Etude sur I'innei-vation de I'ovaire. Bull. Acad, do Med. Beige, 1894, IV, t. 8, 552.
7. Frankenhauser: Die Nerven der Gebarmutter und ihre Endigungen in den glatten Muskelfasern., 1867, Jena.
8. Gawronsky (von), N.: Ueber Verbreitung und Endigung der Nei-ven in den weiblichen Genitalien. Arch. f. Gyn., 1894, XL VII, 271.
9. Grohe, F. : Ueber den Bau und das Wachstum des menschlichen Eierstocks. Virchows Arch., 1863, XXVI, 271.
10. Hei-ff (von) : Ueber den feineren Verlauf der Neri'en im Eierstocke des Menschen. Ztschr. f. Geb. u. Gyn., 1872, XXIV, 289.
11. His, W.: Beobachtungen iiber den Bau des Saugethier-Eierstockes. Arch. f. mikr. Anat., 1865, I, 151.
12. Kolliker (von). A.: Beitriige zur Kenntnis der glatten Muskeln. Ztschr. f. Wiss. Zoologie, 1849, I, 30.
13. Kuntz, .A..: Innervation of the gonads of the dog. Anat. Rec,
1920, XVII, 203.
14. Langley, J. N., and Anderson, H. D.: The innervation of the pelvic and adjoining viscera. Jour, of Phys., 1896, XX, 372.
15. Mandl, L.: Ueber Anordnung und Endigungsweise der Nerven im Ovarium. Arch. f. Gyn., 1895, XLVIII, 376.
16. Pfliiger, E.: Ueber die Bewegungen der Ovarien. Arch. f. Anat. u. Phys., 1859, 1, 30.
17. Retzius: Ueber die Nerven der Ovarium und Hoden. Biol. Untersuch., 1893, V, 31
18. Riese, H.: Die feinsten Nervenfasem und ihre Endigungen im Ovarium der Siiugethiere und der Menschen. Anat. Anz., 1891, VI, 401.
19. Robinson, A.: The formation, rupture, and closure of ovarian follicles in ferrets and ferret-pole cat hj-brids and some associated phenomena. Trans. Roy. Soc. Edin., 1918, LII. No. 137, 303.
20. Rouget, C: Recherches sur les organes erectiles de la femme et sur I'appareil musculaire tubo-ovaricn dans leur rapports avec I'ovulation et la menstnjation. Jour, de la phys. de I'homme et des animaux, 1858, 320.
21. Thompson, A.: The ripe human Graafian foUiole together witli some suggestions as to its mode of rupture. Jour, of Anat., 1919, LI\', 1.
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23. Wilson, J. G.: Intra-vitam staining with methylene blue. Anat. Rec, 1910, n^ 267.
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Description of Plate
Fig. 1.— Microphotograph of the three normal layers of a mature Graafian folhcle of the sow. X 90. gr. granulosa,
th.int. theca interna.
th. ext. theca externa (the muscle-bearing layer of the follicle).
Fig. 2. Drawing of Graafian follicle immediately after rupture, showing bundles of smooth muscle cells. Van Gieson stain. X 540. gr. granulosa, th.int. theca interna. th. ext. theca externa, containing numerous smooth muscle cells.
Fig. 3a.— Drawing of nerve fibrils with two types of sympathetic motor nerve endings in the theca externa of the follicle. Methylene Blue. X 975.
Fig. 3b.— Drawing of nerve fibril coursing among the muscle cells of the theca externa. Methylene Blue. X 1125.
Fig. 4.— Drawing of teased preparation of theca externa after ammonium molybdate fixation, showing rich nerve plexus and network. Methylene Blue. X 280.
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