Paper - The early development of the ferret - the zona granulosa, zona pellucida and associated structures (1932)
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Mainland D. The early development of the ferret: the zona granulosa, zona pellucida and associated structures. (1932) J Anat. 66: 586-601. PMID 17104395
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- 1 The Early Development of the Ferret - The Zona Granulosa, Zona Pellucida and Associated Structures
- 1.1 Introduction
- 1.2 Materials and Methods
- 1.3 Zona Granulosa
- 1.4 Structure of Zona Pellucida
- 1.5 Zona Pellucida Bodies
- 1.5.1 Occurrence and structure
- 1.5.2 Association with fixative and stain
- 1.5.3 Association with bodies in zona pellucida of ovarian ova
- 1.5.4 Association with bodies in cytoplasm
- 1.5.5 Association with zona granulosa
- 1.5.6 Association with bodies outside the ovum
- 1.5.7 Relationship of zona pellucida bodies to perivitelline space
- 1.6 Perivitelline Space
- 1.7 Perivitelline Membrane
- 1.8 Summary
- 1.9 Acknowledgments
- 1.10 References
- 1.11 Explanation of Plate I
The Early Development of the Ferret - The Zona Granulosa, Zona Pellucida and Associated Structures
By Donald Mainland
Department of Anatomy, Dalhousie University, Halifax, Canada
- Part of a thesis recently accepted by Edinburgh University for the degree of Doctor of Science.
Doruine the last few years interest in the zona granulosa and zona pellucida of mammalian ova has waned. This is not unnatural, for it appears as if nothing very new could be learned, at least from paraffin sections, regarding the structure and functions of these membranes. The zona granulosa is generally regarded as a means whereby the ovum, at least in the ovary, is nourished (Marshall, 1922). The zona pellucida is a protective coat, and in particular its late disappearance seems (Robinson, 1904) to be associated with the late implantation of the ovum.
To show that there is still something to be gained from the study of ordinary sections is partly the object of the present record. Moreover, most of the detail regarding these membranes has been obtained from a study of nonCarnivores. The present study is of a Carnivore — the ferret.
Materials and Methods
The material used in these investigations was the same as that employed in the observations on the ferret pronuclei (Mainland, 1930) and on the cytoplasm of ferret ova (Mainland, 1931), viz., 101 ferret ova at stages from the second polar spindle to the first segmentation spindle inclusive. The ova were in the form of mounted paraffin sections cut at 10 microns. The fixatives that had been used were Flemming, Mann with and without formol, Perenyi, Zenker, and the staining methods for different ova were Heidenhain’s ironhaematoxylin and Mayer’s haemalum, with either eosin or orange G as counterstain.
The investigations have involved the use of a method that the author feels more and more to be essential wherever there are variations in phenomena, and, perhaps more than usual, where the number of specimens available is limited. This method is the application of biometrical tests, particularly the x* test. In a study of the pronuclei (Mainland, 1930) this method was discussed. Fisher (1930) gives full details, with tables showing the significance of different values of x*. It should be pointed out here that an association between phenomena is said to be “significant” when the odds are 19 or more to 1 against the association being due to chance.
The records of Robinson (1918) have shown the resemblance between the zona granulosa of the ferret and that of other Mammals. It may now be subjected to a more minute examination. Out of a total of 95 ova at the early tubal stage (i.e. at the stages of pronuclei, second polar spindle or first segmentation spindle), 86 showed more or less of the zona granulosa of the follicle from which they had come. The remaining 9 showed no cells of this kind near them.
Amount of zona granulosa
As is well known, the cells of the zona granulosa disappear as the ovum travels down the uterine tube. It appears desirable to demonstrate the rapidity with which this disappearance occurs, and this is of special importance if, as is strongly indicated (see below), the zona granulosa is associated with the nutrition of the tubal ovum. Table I shows the relative amounts of zona granulosa found near the different ova, arranged according to the stage of development of the ovum. These data were grouped to form a fourfold table, and
Table I. Association between amount of zona granulosa and condition of nuclear material in ovum. (The numbers of ova are recorded.)
Amount of zona granulosa
Nuclear material ‘Nil Very small Small Medium Abundant Total Second polar spindle — — — — 15 15 Pronuclei peripheral
or subcentral 4 — 3 7 1 15 Pronuclei central 5 5 13 ll 5 39 First segmentation
spindle _— _— 1 —_— _ 1
Total 9 5 17 18 21 70
the results of the application of the x? test were: x? = 18-6, n = 1. The results were therefore very highly significant, showing that the disappearance of the zona granulosa can be definitely detected as the ovum advances from the | stage of second polar spindle to the stage of first segmentation spindle. Thus there is given a criterion of development apart from the condition of the ovular nuclear material.
A further test was applied to show the relation between the number of hours after insemination of the animal and the amount of zona granulosa remaining near the ovum. It has been shown (Mainland, 1980) that the postinseminal period is to some extent a measure of the ovular development, for the more developed ova were in the main found in those animals that had lived longest after insemination. There was, however, no significant association between the length of the post-inseminal period and the amount of zona granulosa present. The post-inseminal period, as is well realised, is in the ferret not a very satisfactory measure of development, owing probably to the great variation in the lapse of time between insemination and ovulation. In this series, therefore, there could not be demonstratéd any change in the amount of zona granulosa in the first 100 hours after insemination.
Staining and shape of cells
Little difference was noted in the eosinophilic staining of the cells as development proceeded. More instructive results were obtained from examination of the shape.
When the ovum is liberated it has around it the typical corona radiata, with an inner layer of pyramidal cells and two or three outer layers of polyhedral cells. Illustrations are to be found in Robinson’s monograph (1918) and also in the accompanying figure (Plate I, fig. 7). As the ovum proceeds down the tube the zona granulosa cells degenerate. This degeneration, like the degeneration of many of the cells of the ovary, deserves attention, for it takes place in a healthy environment. It occurs in a medium composed partly of follicular fluid, partly of fluid from the tube wall and partly, no doubt, of fluid from the developing ovum itself. The degenerating cells are of various shapes (Plate I, fig. 2). Some are polyhedral, others more irregular, others spherical and still others ellipsoidal. Table II shows the relationship between the stage of ovular development and the shape of the zona granulosa cells. The x? test was applied to the data grouped in a 8 x 2 table, and proved that there was a significant relationship between the shape of cell and the ovular development. Inspection of Table II reveals that the significance of the association is largely if not wholly due to the greater frequency of the pyramidal cells at the polar spindle stage. It was interesting to note that when the post-inseminal period was used as a measure of development, there was a highly significant association between the shape of the zona granulosa cells and the development as thus indicated. This was owing to the greater abundance of pyramidal granulosa cells around the ova at the second polar spindle stage, these ova being found in the animals with the shortest post-inseminal period. ‘Thus the post-inseminal period appears on some occasions a satisfactory indication of development.
Table II. Association between shape of zona granulosa cells and condition of nuclear material in ovum. (The numbers of ova are given. The table is condensed.) Dominant shape of zona granulosa cells Polyhedral Spherical and Nuclear material Pyramidal and irregular ellipsoidal Total Second polar spindle 9 — — 9 Pronuclei peripheral or sub- 2 1 8 ll central Pronuclei central or first segmentation spindle 3 12 16 31 Total 14 13 24 51
The next problem is the elucidation of the order of shape changes. There is no indication from the figures that spherical cells occur more abundantly at the later than at the middle stages of development, and therefore the stages might be: pyramidal, polyhedral, spherical; or: pyramidal, spherical, polyhedral. Examinations of the specimens suggested that the former series was correct. A definite answer to the question was obtained by studying the relationship between the amount of granulosa and cell-shape. It was obvious that, as would be expected, when the shape of cell was pyramidal, the amount was greater than when the cells were of other shapes; but even when all the specimens with pyramidal cells were excluded it could be shown by the x? test that the less abundant the granulosa the more often were its cells spherical or ellipsoidal. It appeared as if more and more of the zona granulosa cytoplasm disappeared and the remainder became a spherical shell around the nucleus.
There was no association demonstrable between differences of fixative and shape of cells.
The zona granulosa of each ovum was classified according to whether its nuclei showed only globules (or granules) of chromatin or whether some or all of its nuclei were homogeneously basiphilic (Plate I, figs. 2-4). It seemed as if the basiphilic nuclei were a sign of degeneration, but to test this supposition use was made of the fact just recorded that the shape of the granulosa cells changed from pyramidal to polyhedral and then to spherical. It was then shown that there was almost, but not quite, a significant association between nuclear structure and cell-shape. The nuclei, therefore, are of little value as an index of degeneration. There was no significant association between the nuclear structure and the stage of development of the ovum as indicated by its polar spindle or pronuclei.
The caution which is necessary in applying a x? test is indicated by the results obtained when the association between the post-inseminal period and the nuclear condition of the granulosa was investigated. This test showed a highly significant association between the two, that is, more granular nuclei were found associated with the ova early after insemination than later on. This association appeared difficult to explain, since, as has just been stated, there was no apparent association between ovular development and the condition of the granulosa nuclei. The explanation was revealed, however, when the effect of staining was observed. For each section a record was made of the depth of the basic staining of the ovum and the surrounding tissues, e.g. tube and ovary, the classification being “dark,” “‘medium,” “pale.”’ It was then shown that there was a highly significant association between the condition of the granulosa nuclei and the depth of staining. The homogeneous nuclear appearance was much more common among deeply stained sections than among the less deeply stained. Moreover, it was found that, for some reason or another, deeply stained sections were much commoner among the ova taken at the late post-inseminal periods. This would account for the result mentioned above. The more deeply stained sections had more homogeneous nuclei. These sections were more abundant at the later post-inseminal periods, and therefore a spurious association was produced between granulosa nuclear structure and the post-inseminal period.
Since the relation between depth of staining and homogeneity of the nuclei is so strong, one cannot help feeling that this may mask a relationship between the nuclear structure and the degeneration of the cells. This suggestion was, however, not confirmed by any tests. A test, for example, was made by using the amount of granulosa present as an index of its progress towards degeneration. There was no significant association between this amount and the nuclear structure. The conclusion is that, so far as can be shown, the nuclear structure is not an index of the degeneration.
The x? test showed no significant association between the fixative (Mann, Zenker or Perenyi) and the nuclear structure of the granulosa cells.
Relationship of zona granulosa and ovum
In 10 ova of the collection some of the cells of the granulosa demonstrably penetrated the zona pellucida (text-fig. 1). In these specimens the granulosa was in the form of a corona radiata. In 9 other ova, in which the zona granulosa also formed a corona, no penetration of the zona pellucida was detected. Owing to the smallness of the numbers no conclusion could be reached regarding the influence of fixative and staining on the demonstration of this penetration. In some specimens the penetration when visible was not through the whole thickness of the zona pellucida.
The interest of the proximity of the zona granulosa to the ovum lies partly in a consideration of the factors that influence the disappearance of the granulosa—dissolution by degeneration, removal by the cilia of the tubal wall and removal by the muscular contractions of the tubal wall, if, as Sobotta (1922) considers to be the case, the muscular activity of the tube is the cause of the passage of the ovum. There was a definite association between the amount of granulosa near the ovum and its proximity to the ovum, i.e. whether it was in close contact, at a little distance or at a greater distance. This was to be expected if the removal of the granulosa is largely mechanical. If degeneration alone were responsible for its disappearance, it would be expected that the cells would be near to the ovum throughout the whole course of the degeneration. They do not degenerate to the stage of forming an amorphous mass before being removed.
Structure of Zona Pellucida
Since these investigations on the ferret were confined to ova after ovulation, no attempt is made to demonstrate the origin of the zona pellucida. Its structure and staining reactions, however, deserve attention, because of the different theories of its formation and also because of the “‘zona pellucida bodies” The Early Development of the Ferret 591
discussed below. In the attempt to elucidate this question it was necessary to consult the various records of zona pellucida development. Among other records are those of Cattaneo (1922), Dubreuil and Regaud (1908), Fischer (1905), O. van der Stricht (1923) and Mjassojedoff (1923).
Text-fig. 1. Ovum no. 24. At points. astrand of a zona granulosa cell penetrates the entire thickness of the zona pellucida, z.; z.g., zona granulosa; pun. sp., perivitelline space containing granular material; ov. ovum. (Mann with formol; Heidenhain’s iron-haematoxylin and orange G; camera lucida drawing; magnification approx. 1000.)
Text-fig. 2. Ovum no. 2. Tangential section through zona pellucida, z. » Showing the pitted or “cellular” appearance of its outer part; z.g., zona granulosa; sp., spermatozoon in zona pellucida. (Zenker; Heidenhain’s iron-haematoxylin and eosin; camera lucida drawing; magnification approx. 1000.)
Texture of zona pellucida
Tangential and oblique sections through the zona pellucida demonstrated the pitted or “cellular” structure of its outer part (text-fig. 2). This was demonstrated by haematoxylin, but was observed also in specimens merely tinted with eosin. It was independent of the difference in fixatives. On transverse section the zona pellucida was smooth at its inner surface, rather more rough at its outer surface, in agreement with the cellularity observed on tangential section.
Heidenhain’s iron-haematoxylin with a light eosin counterstain gave a grey, bluish grey, blue-black or deep purplish colour to the zona pellucida. If the basic stain was less deep as in the ova stained by Mayer’s haemalum, the eosin gave the membrane a dull pink stain or an orange colour. When the specimens (fixed in Perenyi’s fluid) were stained with Heidenhain’s iron-haematoxylin and counterstained with orange G, the outermost part (say 1/5 or 1/6) of the zona pellucida was pale yellow, the intermediate part (the largest portion) was of a darker yellow colour, while the innermost part was a dark grey line. This suggests a difference in texture of the different parts, especially a lack of density in the parts adjacent to the zona granulosa.
Specimens fixed in Mann’s or Zenker’s fluid and stained with Mayer’s haemalum and eosin showed in some cases a purple outermost zone while the rest of the zona pellucida, larger in extent, was orange. Similarly, among 4 Flemming-fixed specimens, 2, stained moderately deeply with Heidenhain, showed the outer part of the zona pellucida blue-black, the inner part yellowish grey.
There was no difference in staining between the Perenyi and Zenker specimens when stained with Heidenhain’s iron-haematoxylin, nor between Zenker and Mann specimens stained with Mayer’s haemalum and eosin, nor between Perenyi specimens stained with Heidenhain and orange G and those fixed in Mann’s fluid and formol and stained in the same way.
Under the subject of zona granulosa reference is made to the presence of strands of the corona radiata penetrating the zona pellucida. Except for these, no striae were seen passing through the entire thickness of the zona pellucida. On the other hand the majority of the specimens, whatever the fixative or stain, showed numerous fine radial striae in the peripheral part of the zona pellucida (Plate I, fig. 5). Even Flemming specimens tinted with eosin showed these. They were present irrespective of the presence or absence of corona radiata. They presumably corresponded to the attachments of the pointed ends of the corona radiata cells.
Size Exact determination of the thickness of the zona pellucida was not made.
It is indicated in the figures of ova accompanying the text, and may be stated as roughly 4-6 p.
Zona Pellucida Bodies
Occurrence and structure
In the zona pellucida of 16 out of 99 tubal ova there were discovered spheroidal or ellipsoidal bodies (Plate I, figs. 1 and 2). These bodies stained usually more darkly than the zona pellucida, but rather similarly to this structure, i.e. grey or bluish grey or dark blue in the specimens stained by Heidenhain’s iron-haematoxylin and a little eosin, and a dull red in the ova stained by Mayer’s haemalum and strongly counterstained with eosin. Their structure was homogeneous except that a few had each a central homogeneous basiphil globule. It appeared unnecessary to determine very exactly the size of these bodies. Their diameters were frequently of about the thickness of the zona pellucida. In some specimens they were less than even the nuclei of the neighbouring granulosa cells.
The 16 ova that showed these bodies were from 4 different animals (1 tube from each animal). Other ova from the same animal or tube did not necessarily show such bodies.
Association with fixative and stain
These bodies were found only in specimens fixed in Zenker’s fluid. Thus out of 26 specimens fixed in Zenker and stained by Heidenhain’s iron-haematoxylin and eosin, as many as 12 definitely showed such bodies and 2 showed them doubtfully. Out of 15 specimens fixed in Zenker and stained by Mayer’s haemalum and eosin, 4 showed such bodies. Similar bodies were not found in the 23 specimens fixed with Perenyi’s fluid and stained with Heidenhain, nor in the 19 Mann-fixed specimens, nor in the 15 Flemming-fixed specimens. The association with Zenker’s fixative is therefore of extremely high significance.
With regard to staining it may be pointed out that the central basiphil globule was present only in the zona pellucida bodies in specimens stained with Heidenhain’s iron-haematoxylin, and not in all of these. In this connection it should be recalled that the zona pellucida itself can, as mentioned above, take on a deep basic stain, and there is no need to suggest that the basiphil centre of the zona pellucida bodies is of the nature of a nucleus.
There was no significant difference between the power of Heidenhain’s iron-haematoxylin and Mayer’s haemalum to demonstrate the zona pellucida bodies in Zenker-fixed ova.
The demonstration of these bodies by the use of Zenker’s fluid and the paraffin section technique shows that they do not consist wholly of lipoid material. The failure of Flemming’s fluid to demonstrate them shows that if any lipoid is present in them, it must have less than 50 per cent. non-saturated fatty acid (Parat, 1927). They are presumably of protein origin. The difference between the effects of Zenker’s fluid, which demonstrates them, and Perenyi’s fluid, which does not, is more difficult to explain. Zenker’s fluid resembles Perenyi’s in possessing a chromic acid compound, but Zenker contains also mercuric chloride, which may be the cause of the demonstration of the bodies. The fact that one protoplasmic fixative and not others demonstrates them suggests that they are products of the cytoplasm rather than cytoplasm themselves. They may be either degeneration products or secretory products.
Association with bodies in zona pellucida of ovarian ova
In the preparation of most of the specimens the tube and ovary had been cut and mounted together and consequently the slides containing the tubal ova contained in many cases also sections of medium-sized or even large ovarian ova. Out of the 16 instances in which there were bodies in the zona pellucida of tubal ova, there were 11 in which medium or large ovarian ova showed similar bodies. There was shown to be a very definite association between the two occurrences.
Association with bodies in cytoplasm
As mentioned above, 15 ova had been fixed in Zenker’s fluid, stained by Mayer’s haemalum and strongly counterstained with eosin. Out of these, 13 showed bodies in the cytoplasm resembling in size and staining qualities the zona, pellucida bodies. The relationship between the occurrence of these cytoplasmic bodies and the zona pellucida bodies is shown as follows (the numbers representing ova):
Bodies in zona pellucida
Total Bodies present in cytoplasm 3 10 13 Bodies absent from cytoplasm 1 1 2 Total 4 ll 15
None of the other ova, fixed either in Zenker’s or other fluid, showed bodies that were at all comparable to these cytoplasmic bodies. There was therefore a significant association between the occurrence of the zona pellucida bodies and that of the cytoplasmic bodies, inasmuch as they only occurred after Zenker fixation. There was however a definite difference between them in relation to staining. Mayer’s haemalum and deep eosin staining demonstrated the cytoplasmic as well as the zona pellucida bodies, whereas Heidenhain’s ironhaematoxylin demonstrated only the zona pellucida bodies. Among the Zenker-fixed ova stained by Mayer and eosin there was a significantly greater number of ova with cytoplasmic bodies than with zona pellucida bodies.
The relationship of the cytoplasmic bodies to the zona pellucida bodies will be discussed later. At present it is desirable to give more detail regarding the structure of the cytoplasmic bodies (Plate I, fig. 6). Thus, in ovum No. 55 there lay in the cytoplasm near its edge an irregular ellipsoidal body stained orange with eosin. The surface facing the zona pellucida was more irregular in contour than the surface facing the centre of the ovum. In another part of the same ovum were three similar bodies. One of these, nearer the zona pellucida, had a The Early Development of the Ferret 595
smooth surface facing the zona pellucida; the two deeper in the ovum were more irregular all round and less homogeneous in texture. The irregularities of these bodies corresponded fairly closely to the alveolar spaces of the cytoplasm itself, spaces which in the living ovum were filled with lipoid material. If the bodies can so accommodate themselves to the masses of deutoplasm, it appears that the cytoplasmic bodies must be fairly fiuid before fixation.
Differences in size between the bodies in the peripheral part of an ovum and those in the central part were not marked, and there was no evidence of absorption in the sense of a diminution in the size of the bodies as a result of a dissolution progressing from the edges towards the centre.
Table III. Association between amount of granulosa and presence of zona pellucida bodies. (Zenker specimens only. Numbers of ova.)
Zona pellucida bodies Amount of granulosa None Doubtful Present Many Total
Nil 3 — _ —_ 3 Very small 1 _— 3 _— 4 Small 8 — — — 8 Medium 6 _ 6 3 15 Large . 3 1 4 _ 8
Total 21 1 13 3 38
Table IV. Association between amount of granulosa and presence of bodies in cytoplasm. (Numbers of ova.)
Cytoplasmic bodies None or Present or Amount of granulosa doubtful numerous Total Small 3 7. 10 Medium or abundant 9. 6 15 Total 12 13 25
Association with zona granulosa
The Zenker-fixed ova were arranged according to the presence or absence of the zona pellucida bodies and according to the amount of zona granulosa present (Table III). There was shown by the x? test to be a significant association between the two classifications. Ova with a medium or large amount of granulosa near them more often presented zona pellucida bodies than did ova where the zona granulosa was absent or small in amount. Two possible interpretations might be placed upon this. It might be claimed as a proof that the zona pellucida bodies were products of the zona granulosa; or the amount of granulosa might be taken simply as an indication of the development of the ovum (see above), and then the relationship of granulosa and zona pellucida bodies would indicate that these bodies disappeared as the ovular development progressed.
It should be noted at this point that there was no significant association between the occurrence of the bodies in the cytoplasm and the amount of zona granulosa near the ova. If anything there was a tendency for these bodies to be more often found where the zona granulosa was small (Table IV). There was therefore a distinct difference between the zona pellucida bodies and the cytoplasmic bodies in this respect. It was thought possible that this was a spurious difference due to the difference between Mayer’s staining and Heidenhain’s in demonstrating the bodies in the cytoplasm, but x? tests showed no indication that this was so. The conclusion is therefore that, while the zona pellucida bodies get less frequent as the ovum progresses and the zona granulosa disappears, the bodies in the cytoplasm remain, or perhaps even increase in frequency.
Association with bodies outside the ovum
In 15 specimens spheroidal bodies resembling those of the zona pellucida in shape, size and staining reactions were discovered outside the ovum, some in contact with the zona granulosa near the tubal ovum, some in the tube at a distance from the ovum. These bodies appeared fairly homogeneous in structure. Similar bodies were found in the liquor folliculi of some moderate sized follicles in the ovary corresponding to ovum No. 65 which itself showed zona pellucida bodies. In one specimen corresponding to ovum No. 67 somewhat similar bodies were found among the granulosa cells of a recently ruptured follicle, i.e. one about to become a corpus luteum.
Relationship of zona pellucida bodies to perivitelline space
In one ovum a zona pellucida body projected into the perivitelline space causing a depression of the adjacent surface of the ovum. In ovum No. 11 (Plate I, fig. 8) several bodies were seen partly inside the zona pellucida, partly outside, while some lay in the perivitelline space. The part against the ovum did not depress the latter, but was spread out on its surface. The staining of the bodies in the space was sometimes a little lighter, sometimes a little darker than that of the zona pellucida itself. There was no evidence that the zona pellucida was deficient near the bodies that lay in the space. The zona pellucida did not seem to have lost any of its substance although the bodies so closely resembled it in staining reactions.
The above data have been recorded without the introduction of any suggested explanation. The question of origin and destiny may now be discussed.
Origin and destiny of zona pellucida bodies Before proceeding with an attempt to elucidate the genesis and fate of these bodies, it is desirable to note the references to closely similar structures in
other animals. The author is acquainted with only two such descriptions. Sobotta (1908) recorded the occasional appearance of small nucleated cells, not polar bodies, in the perivitelline space of mouse ova at the early tubal stages of development, but these bodies could not be classified definitely as follicular epithelial cells or as abnormalities in the structure of the zona pellucida. A few years later Sobotta and Burckhard (1911) described in regard to the tubal ova of the rat some roundish bodies readily stained with acid aniline dyes. These bodies were sometimes in the zona pellucida, sometimes between the zona granulosa cells, and commonly appeared first in the later stages of fertilisation. The authors could not decide whether the bodies were products of tubal secretions or of hyaline degeneration of the zona granulosa cells. At first sight it appears rather remarkable that a structure that is so common in the tubal ova of the ferret should be so seldom mentioned in connection with the ova of other animals, but technique may well be the cause of this. O. van der Stricht and his collaborators, for example, were so much concerned with cytoplasmic structure that Zenker’s fluid was seldom used in their preparations, and it has been shown above that this fixative is the only one that demonstrated the bodies in the present investigations.
A brief recapitulation of the facts so far demonstrated in the ferret may now be made.
The results of fixation strongly suggested that the zona pellucida bodies were not cytoplasmic in structure, but were rather the products of degenerated or secretion products. Similar bodies were found in relation to ovarian ova, and were demonstrated by Mayer’s haemalum and eosin in the cytoplasm of tubal ova. The zona pellucida bodies were less frequently met where the amount of zona granulosa near the ovum was small. There was if anything a tendency in the opposite direction with regard to the cytoplasmic bodies. In a number of instances structures somewhat similar to the zona pellucida bodies were found outside the tubal ova among the granulosa or elsewhere in the tube.
The bodies in the perivitelline space suggest stages of a progression into or out of the zona pellucida and out of or into the ovum. No adequate transition stages were observed between the cytoplasmic bodies and the zona pellucida bodies, but there appeared no reasonable ground for doubt of the identity of these two. It is, of course, obvious that, since they were found near the ovarian ova they could not be products of the uterine tube. The question next arises whether the bodies are products of the ovum or of the zona granulosa, i.e. the question of direction in which the bodies are passing. This is partly answered by the fact that as the zona granulosa cells get smaller in number, i.e. as the ovum advances in development, the zona pellucida bodies are less frequently met, while the cytoplasmic bodies are as abundant, or perhaps even increase in number.
One of the ovarian ova corresponding to the tubal ovum No. 17 was very instructive in regard to the origin of the zona pellucida bodies. The specimen was a medium-sized healthy ovarian ovum, fixed in Zenker and stained with Heidenhain. In its zona pellucida small basiphil granules were detected, and by careful focussing it appeared possible to detect a connection between these and the strands of the zona granulosa around the ovum. No other example of this was observed, but the numbers of available ovarian ova of fair size were small.
When the zona granulosa is in the form of degenerating rounded cells it is sometimes very close to the zona pellucida, and may be somewhat imbedded in the outer surface of the latter. It may therefore be asked whether the zona pellucida bodies and those found in the ovular cytoplasm are not degenerate granulosa cells. This suggestion is further supported by the presence in some of the zona pellucida bodies of a central area stained blue-black like a nucleus by Heidenhain’s stain. On the other hand, this area was absent from most of the bodies, even when stained by this method. Moreover, while some of the bodies were large enough to be granulosa cells, others could at most be fragments of granulosa cells. The presence of bodies in relation to ovarian ova is also strongly against this possibility, and it should be recalled that the fixation phenomena indicated that they were not actually cytoplasm.
The strong resemblance between the bodies and the zona pellucida is striking and important, and the conclusion of these investigations is that the zona pellucida bodies are products of the zona granulosa, in a very similar fashion to part, at least, of the zona pellucida. The bodies are absorbed by the ovum,
There is thus indicated a function for the zona pellucida beyond that of protection. During the process of development of the ovum in the uterine tube the zona pellucida contains material that is probably utilised by the ovum and passes this material on to the ovum.
The space between the zona pellucida and the ovum proper varied in extent from ovum to ovum. It was impossible to say whether this variation was due entirely to differences in the amount of shrinkage during preparation of the ova. This space always contained at least a small quantity of a finely granular material similar to the neighbouring cytoplasm (Plate I, fig. 4). In some specimens, as in Plate I, fig. 5, the space was large and the granular material very definite. There seemed to be no reason to regard this granular material as a product of deutoplasmolysis. It could arise equally well from the precipitation of some albuminous periovular fluid.
In the various photographs shown in Plate I the edge of the ovum proper will be noticed as marked by a line that is denser than the rest. No further evidence of the nature of this “perivitelline membrane” was obtained from a study of the sections. The most profitable way of studying this structure, like the structure bearing the same name in Invertebrate ova, is to investigate the fresh unfixed ovum in which, 50 years ago, van Beneden (1880) working on the rabbit demonstrated that the membrane could be isolated.
Investigations have been made of the zona granulosa, zona pellucida and associated structures of 101 ferret ova in stained paraffin sections, biometrical tests being applied to show the significance of the results. The stages examined were from the second polar spindle stage to that of the first segmentation spindle.
In spite of the great variation between different ova, the disappearance of zona granulosa could be detected as the ova advanced from the first to the last of these stages. Probably owing to the great variation in the lapse of time between insemination and ovulation it was not possible in this series to demonstrate any change in the amount of zona granulosa near an ovum in the first hundred hours after insemination.
The eosinophilic staining of the cells did not change detectably as the ova advanced in development.
There was a significantly greater number of ova with pyramidal granulosa cells at the second polar spindle stage than later in development. The order of shape changes is indicated as: pyramidal, polyhedral, spherical.
There was no indication that the nuclear structure of the granulosa cells altered as the ova developed, or that the differences in nuclear structure indicated degeneration. The more intense the basiphilic staining of the sections, the more abundant were homogeneously stained granulosa nuclei.
The greater the amount of zona granulosa near an ovum, the nearer to the ovum did the cells tend to lie, as would be expected if the cause of their disappearance were mechanical removal rather than mere degeneration and dissolution.
The structure of the zona pellucida was studied (p. 590). The majority of the ova showed radial striae in the zona pellucida, but these were only in its outermost part except in those ova where cells of the corona radiata demonstrably penetrated far into or through the zona pellucida.
The thickness of the zona pellucida was roughly 4-6 p.
Sixteen of the ova showed spherical or ellipsoidal bodies in the zona pellucida. These were demonstrable only in Zenker-fixed ova, not at all in Perenyi-, Flemming- or Mann-fixed ova. They were shown either by staining with Heidenhain’s iron-haematoxylin or by staining with Mayer’s haemalum. There was a significant association between the occurrence of these bodies and the occurrence of similar bodies in medium-sized or large ovarian ova. Similar bodies were found in the cytoplasm of 18 out of 15 Zenker-fixed tubal ova stained with Mayer’s haemalum and eosin, but these cytoplasmic bodies were not demonstrated by Heidenhain’s iron-haematoxylin.
The zona pellucida bodies got less frequent as the ova progressed in development and the zona granulosa disappeared, whereas the similar bodies in the cytoplasm remained or perhaps even increased in frequency.
In a number of instances structures somewhat similar were found outside the tubal ova in the tube and in the liquor folliculi of the ovary.
It was concluded that the zona pellucida bodies were products of the zona granulosa arising in a very similar fashion to part, at least, of the zona pellucida, and that the bodies were absorbed by the ovum. The zona pellucida is therefore more than a mere protection to the ovum.
The perivitelline space varied in extent in different ova.. It always contained some finely granular material, not necessarily a product of ‘“‘deutoplasmolysis.”’
The study of the sections did not give evidence of the nature of the perivitelline membrane.
To Prof. Arthur Robinson of Edinburgh University I am very much indebted for the loan of the specimens on which these investigations were carried out. To the trustees of the Moray Fund I wish to express my thanks for financial assistance in the preparation of some of the specimens. The observations were carried out in the Anatomy Department of Manitoba University and the work was completed in Dalhousie University. To Miss Nason of the Pathology Department of Manitoba University I am much obliged for the preparation of the photographs.
No attempt has been made to give a complete list of the literature necessarily consulted in such a study.
vAN BENEDEN, E. (1880). Arch. Biol. t. 1, p. 137. CatTANEO, DonaTo (1922). Ric. di morfologia, vol. u, Fasc. 3, pp. 185-95. Dusrevun, G. et Recaup, Cx. (1908). Verh. Anat. Gesellsch. XXII Versamml. Berlin, S. 152-6. Fiscoer, ApourF (1905). Anat. Hefte, Abt. 1, Bd. xxrx, S. 555-89. FisuEr, R. A. (1930). Statistical Methods for Research Workers. London: Oliver and Boyd. Marntanp, Donatp (1930). J. Anat. vol. LxIv, pp. 262-87.
—— (1931). J. Anat. vol. Lxv., pp. 411-26. MarsHat., F. H. A. (1922). The Physiology of Reproduction. London: Longmans, Green and Co. Msassoseporr, S. W. (1923). Arch. Mikr. Anat. Bd. xcvu, S. 72-135. ParatT, MavRIcE (1927). Biol. Reviews, vol. , pp. 285-97. Rostinson, ARTHUR (1904). J. Anat. and Physiol. vol. xxxvmt.
—— (1918). Trans. Roy. Soc. Edin. vol. Lu, pp. 303-62. Soporta, J. (1908). Anat. Hefte, Bd. xxxv, S. 493-552. . —— (1922). Deutsch. Med. Wochschr. Bd. xtvi1, S. 1088 (quoted from Anat. Ber. Bd. 1). Sosorta, J. and BuRcKHARD, G. (1911). Anat. Hefte, Bd. xim, S. 433. VAN DER STRIOHT, O. (1923). Arch. Biol. t. xxx1u, pp. 229-300.
Explanation of Plate I
c.b. Cytoplasmic body. t. Tube (uterine). ov. Ovum. 2.ge Zona granulosa. prn. Pronucleus. zp. Zona pellucida. pun.sp. Perivitelline space. z.p.b. Zona pellucida body.
Fig. 1. Ovum no. 17, showing zona pellucida bodies and zona granulosa. Zona granulosa nuclei are mostly homogeneously basiphil. (Zenker; Heidenhain’s iron-haematoxylin and eosin; magnified by 375.)
Fig. 2. Ovum no. 17, a section nearer the end of the ovum than that of Fig. 1. Numerous zona pellucida bodies of various sizes; zona granulosa with polyhedral cells and many homogeneous nuclei. (Zenker; Heidenhain’s iron-haematoxylin and eosin; magnified by 375.)
Fig. 3. Ovum no. 3. Granular matter in perivitelline space; zona granulosa cells massed together; many homogeneous nuclei. (Zenker; Heidenhain’s iron-haematoxylin and eosin; magnified by 450.)
Fig. 4. Ovum no. 6. Zona granulosa with polyhedral cells; nuclei granular. (Zenker; Heiden hain’s iron-haematoxylin and eosin; magnified by 425.)
Fig. 5. Ovum no. 93. Irregular areas in outer part of zona pellucida, less dense than the rest of it, suggesting radial striae; granular matter in perivitelline space. (Perenyi; Heidenhain’s ironhaematoxylin and eosin; magnified by 1200.)
Fig. 6. Ovum no. 55. Bodies in cytoplasm, one appearing dense in the part facing zona pellucida, more alveolar in the part of it facing centre of ovum. (Zenker; Mayer’s haemalum and eosin; magnified by 650.)
Fig. 7. Ovum no. 10. Zona granulosa of pyramidal cells, apices pointing towards zona, pellucida. (Perenyi; Heidenhain’s iron-haematoxylin and eosin; magnified by 750.)
Fig. 8. Ovum no 11. Zona pellucida bodies, two in zona pellucida, two in perivitelline space. The paler granular matter in perivitelline space is probably a polar body. Remnants of zona granulosa visible. (Zenker; Heidenhain’s iron-haematoxylin and eosin; magnified by 275.)
Cite this page: Hill, M.A. (2020, July 15) Embryology Paper - The early development of the ferret - the zona granulosa, zona pellucida and associated structures (1932). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_early_development_of_the_ferret_-_the_zona_granulosa,_zona_pellucida_and_associated_structures_(1932)
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