Judging from their sizes and histochemical reactions, three types of lipid bodies in the form of granules and spheres have been observed in the early stages of oogenesis. The minute granules (L,), belonging to the first type appear to consist of phospholipids, triglycerides, and fatty acids, but the presence of proteins cannot be ruled out. They correspond to the so-called granular mitochondria of other authors of papers on the oogenesis of birds. The second type of lipids (L2) are of medium size and they contain phospholipids and a certain amount of triglycerides. They resemble the so-called ‘Golgi bodies’ of earlier authors in appearance and react like them with osmium tetroxide and silver nitrate. They seem to originate from the granules of the first category (LI). The third type of lipids (Lf) are the AH-negative spheres consisting of triglycerides and cholesterol and its esters. They correspond to the ‘fatty yolk’ of earlier authors. Their origin appears to be from the second type of lipid bodies (L2), as intermediate bodies (ini) of varying chemical composition between the L2 and L3 have been observed. The ‘peripheral lipid bodies’ (pl) found in association with vacuoles are larger than the second type of AH-positive bodies (L2), but they seem to contain different types of phospholipids, triglycerides, and some other substances also. Besides these lipid bodies, a mass of lipoprotein bodies has also been observed. At the time of their origin, the yolk globules of the advanced oocytes do not contain lipids but later on they develop triglycerides and phospholipids. The lipid bodies of the follicular cells correspond very closely to the first two categories of sudanophil AH-positive bodies (L1 and L2) of the oocyte proper.

Since very little work has been carried out on the histochemistry of lipids in oocytes of animals by modern histochemical techniques, it is intended to work out the histochemistry of these lipids by techniques whose validity is now well established. This paper on the lipids of the pigeon oocytes is the first of the series.

FIG 1.

A-D, sections of oocytes prepared by the Sudan black B technique of Baker (1949). E, a portion of section of an oocyte prepared as A to D. F, section of an oocyte fixed in Lewitsky and stained with iron haematoxylin. G, a portion of an unstained oocyte prepared by the Kolatchev technique. H, a section of an oocyte showing the mass of lipoprotein bodies left after treatment of the fresh material with cold alcohol for 24 h. LI, L^, L3, lipid globules of the Ist, 2nd, and 3rd categories; int, intermediate bodies.

FIG 1.

A-D, sections of oocytes prepared by the Sudan black B technique of Baker (1949). E, a portion of section of an oocyte prepared as A to D. F, section of an oocyte fixed in Lewitsky and stained with iron haematoxylin. G, a portion of an unstained oocyte prepared by the Kolatchev technique. H, a section of an oocyte showing the mass of lipoprotein bodies left after treatment of the fresh material with cold alcohol for 24 h. LI, L^, L3, lipid globules of the Ist, 2nd, and 3rd categories; int, intermediate bodies.

To the best of my knowledge there are only three previous publications on the subject. Marza and Marza (1936) and Singh (1938) have demonstrated histochemically fats, lipids, and cholesterols in the hen’s egg, and the presence of cholesterol bodies in the pigeon oocytes respectively. But most of the techniques employed by the former authors are not considered valid by modern histochemists such as Cain (1950) and Pearse (1954). Nama (1956) finds that, in the oocytes of Columba livia, the ‘Golgi bodies’ contain phospholipids, lipoproteins, and proteins, associated with triglycerides and cholesterols. It appears that this author has confined himself to the cytochemistry of Golgi elements of a particular stage only in oogenesis. Apart from this, Nama does not seem to be clear with regard to the identity of his Golgi elements and phospholipids scattered in the cytoplasm.

In the present paper three distinctly lipid bodies of different chemical nature have been described in the early oocytes of the pigeon. For the sake of convenience they have been distinguished as LI, L2, and L?. The changes undergone by these bodies in the course of oogenesis have also been followed very closely. The lipid contents of the yolk of advanced oocytes have also been studied. The histochemistry and behaviour of a fourth type of lipid bodies (pl) found at the periphery of the oocyte in close association with some nonlipid vacuoles have also been studied. The lipid contents of the follicular epithelium have been analysed.

FIG 2.

A, a portion of an oocyte prepared by the Sudan black B technique of Baker (1949). B and c, portions of sections of oocytes prepared by the acid-haematein technique of Baker (1946) and coloured with Sudan black B. D, a peripheral portion of an oocyte fixed in Champy and stained with iron haematoxylin. It shows the lipids of the Ist category only. E, a peripheral part of the section of a ripe ovum prepared by the acid-haematein technique of Baker (1946) and coloured with Sudan black B. It shows the infiltration of lipids of 2nd category through the zona radiata into the oocyte from the follicular epithelium (which is mostly a single layer of cells), and also the yolk globules of the periphery. LltL2, L3, lipid globules of the Ist, 2nd, and 3rd categories; pl, peripheral lipid

FIG 2.

A, a portion of an oocyte prepared by the Sudan black B technique of Baker (1949). B and c, portions of sections of oocytes prepared by the acid-haematein technique of Baker (1946) and coloured with Sudan black B. D, a peripheral portion of an oocyte fixed in Champy and stained with iron haematoxylin. It shows the lipids of the Ist category only. E, a peripheral part of the section of a ripe ovum prepared by the acid-haematein technique of Baker (1946) and coloured with Sudan black B. It shows the infiltration of lipids of 2nd category through the zona radiata into the oocyte from the follicular epithelium (which is mostly a single layer of cells), and also the yolk globules of the periphery. LltL2, L3, lipid globules of the Ist, 2nd, and 3rd categories; pl, peripheral lipid

An attempt has been made to correlate the cytoplasmic components after chrome-osmium and silver nitrate techniques with the usual histochemical techniques. Most of the chrome-osmium and silver techniques have been found to preserve only a very small portion of the total lipid contents of the cell, which, in this particular case, are dominated by neutral fats (triglycerides).

FIG 3.

A, B, sections of early oocytes prepared by the acid-haematein technique of Baker (1946). c-F, portions of sections of oocytes prepared as A, B. L1, L2, lipid globules of the Ist and 2nd categories; int, intermediate bodies; pl, peripheral lipid

FIG 3.

A, B, sections of early oocytes prepared by the acid-haematein technique of Baker (1946). c-F, portions of sections of oocytes prepared as A, B. L1, L2, lipid globules of the Ist and 2nd categories; int, intermediate bodies; pl, peripheral lipid

The specimens of the pigeon, C. livia intermedia, were collected from Hoshiarpur proper. Only adult specimens were used. The animals were dissected immediately after the head had been chopped off. The ovary was removed in physiological saline (Baker, 1944). After the blood had been washed off, the ovary was cut into small pieces and placed in various fixatives.

FIG 4.

A—D show the cholesterol bodies in sections of oocytes fixed in formaldehyde-saline, post-chromed as recommended by Baker (1949), and submitted to Schultz’s method of cholesterol as reported by Gomori (1952). D shows the presenceof cholesterol or its esters only in the cortical region

FIG 4.

A—D show the cholesterol bodies in sections of oocytes fixed in formaldehyde-saline, post-chromed as recommended by Baker (1949), and submitted to Schultz’s method of cholesterol as reported by Gomori (1952). D shows the presenceof cholesterol or its esters only in the cortical region

The techniques for morphology

The following techniques were employed for studying the morphology of cytoplasmic components. The material was fixed according to Lewitsky (Flemming without acetic acid), Champy, Bouin, Aoyama, Da Fano, Mann-Kopsch, and Kolatchev techniques and embedded in wax. The sections were either stained with iron haematoxylin or mounted unstained as in silver nitrate and osmium tetroxide techniques. The silver nitrate preparations were also toned with gold chloride to remove the excess of silver.

Histochemical colour tests

For all· the histochemical techniques, gelatine-embedded material fixed according to the methods given below was employed.

Sudan black B in 70% ethanol was used for the demonstration of lipids in the material fixed in formaldehyde-saline (Baker, 1949) and formaldehydecalcium (Baker, 1946) with or without post-chroming. Neutral lipids (triglycerides) were demonstrated by the Nile blue technique of Cain (1947 a, b, 1948). Phospholipids were demonstrated by the acid haematein technique with pyridine extraction control (Baker, 1946, 1947; Cain, 1947 a, b, 1950; Casselman, 1952; Pearse, 1954). Only those lipids that were positive in acid-haematein (AH) and negative in pyridine extraction (PE) will be described as AH-positive.

The Sudan III and Sudan IV method of Kay and Whitehead (1941), the gelatine-Sudan method (Govan, 1944), and Fettrot (of Ciba) in 70% ethanol as described by Pearse (1954), were employed for neutral fats (triglycerides). Only those bodies which gave a pink colour with Nile blue and were stained with these dyes, have been interpreted as containing neutral fats. I used Sudan III and Sudan IV in the gelatine-Sudan method of Govan. The gelatine-Sudan method and Fettrot gave a very clear picture of the lipids as there was no precipitation of the dyes; nor was there any loss of small lipid droplets in the former technique.

Fresh coverslip preparations of the contents of the ripe ovum treated for a short period with 2% osmium tetroxide solution as used by Nath since 1929 (see Nath, 19570, for full references) were also used for the study of lipids. Fischler’s method for fatty acids as reported by Pearse (1954) with usual control was also used. Schultz’s method for cholesterol and its esters as reported by Gomori (1952) was used on gelatine sections oxidized by iron alum.

Solubility tests

Acetone was used to dissolve neutral fats and cholesterols, but the phospholipids were removed with alcohol and ether. Boiling alcohol was used to extract the lipoproteins.

For solubility tests, sections of material fixed in formaldehyde-calcium was used, with or without post-chroming.

Pieces of fresh ovary were also extracted for 24 h with acetone to dissolve triglycerides and cholesterols, or with alcohol and ether to dissolve phospholipids. Some of the extracted material in each case was fixed in formaldehydecalcium, post-chromed, and embedded in gelatine for the cutting of sections, while the rest of the material after extraction was taken directly to water by passing it quickly through various grades of alcohol. From water it was embedded in gelatine for sectioning. After extraction of the fresh material, fixation in formaldehyde-calcium with post-chroming did not make any difference in the preservation of lipids in the material under investigation. The lipids that resisted cold acetone also resisted hot acetone.

For the extraction of lipoproteins (which resisted cold acetone, alcohol, and ether) boiling alcohol was used for 16 h and gelatine sections were prepared.

The cold and hot ether did not give good results in the case of fresh material. This may be attributed to its inability to penetrate the tissue. For the solubility of various lipids I have not noticed any difference between material fixed in formaldehyde-calcium and fresh tissue, but the great contraction of the oocytes and the displacement of various lipid bodies are disadvantages in using fresh tissue. In the case of material fixed in formaldehyde-calcium and post-chromed, the phosphlipids resist the action of fat solvents named above.

The sudanophIl lipids in the early oocytes of the pigeon are present in the form of granules and spheres which can be divided roughly into three categories according to their size and histochemical reactions.

I. The minute granules (If). These are sudanophIl and AH-positive, give a light pinkish tinge with Nile blue, and appear orange-red with Sudan III and Sudan IV, pinkish-red with Fettrot, and deep blue after Fischler’s method for fatty acids. After treatment with acetone these granules appear to lose some lipids as they colour comparatively less intensely as compared with untreated sections coloured with Sudan black B. They are still AH-positive but do not give a pinkish tinge with Nile blue. With Sudan HI, Sudan IV, and Fettrot the colour that they take up is so pale that the reaction may be considered as negative. Some of these granules seem to lose their lipids completely in acetone. In alcohol and ether all these granules lose their lipid contents as they do not stain after Sudan black B (see Z,I in figs, I, A—G; 2, A-D ; and 3, A-F).

2. The second type of granules (Lf. These are bigger than the first ones, intensely sudanophIl, AH-positive, are coloured pink with Nile blue, and appear deep orange-red after Sudan III and Sudan IV, and pinkish-red with Fettrot (see L2 in figs, i, B-G; 2, A-C; and 3, A-E). They dissolve completely in cold and hot acetone, alcohol, and ether, as they do not appear at all with Sudan black B after such treatments.

However, a mass of sudanophIl material has been observed after treatment with these fat solvents at a particular stage of oogenesis (lip, fig. 1, H). This mass is only colourable with Sudan black B but not with other Sudans and Fettrot. It is also not stained with acid-haematein. When the material is boiled with alcohol, its sudanophilia is lost completely as it cannot be seen even after colouring with Sudan black B. This mass of sudanophIl material will be referred to again in the discussion.

3. The third type of lipids (If). These generally occur as spheres of comparatively large size, are deeply sudanophIl and AH-negative, colour red with Nile blue, Sudan III, and Sudan IV, and pinkish-red with Fettrot. These bodies are soluble in acetone, alcohol, and ether, whether the extractions are tried on fresh or fixed and post-chromed materials (see L3, figs. 1, B-E ; and 2, A-C).

Many varieties of the lipid bodies intermediate between these three forms have also been observed, which will be described in the developmental history of the oocyte.

In the earliest oocyte examined, the sudanophilia occurs in the form of a few separate granules of the first category (If. These granules are scattered in the cytoplasm around the nucleus, which occupies the centre of the oocyte (fig. i, A). Development of the oocyte from now onwards reveals a considerable increase in the size of the nucleus as well as the cytoplasm, although during the later period of growth the cytoplasm outstrips the nucleus in its growth. With the growth of the oocyte the nucleus becomes eccentric and the amount of sudanophilia increases gradually. Fig. i, B represents an oocyte in which there is present a considerable amount of sudanophil bodies. The largest of these, which more or less dominate the picture, belong to the lipids of the third category (Lf). The granules that are somewhat smaller than these can be assigned to the second category (Z2), whereas the minutest granules are of the first category (L^).

It appears that the large AH-negative spheres (L3) originate by the growth and transformation of the smaller ones (Z2). Such a conclusion is strengthened by the fact that many granules which are intermediate in size between the AH-positive (L2) and AH-negative (L3) sudanophil bodies, give only a slight AH-positive reaction. Usually with acid-haematein these intermediate granules (ini) appear in the form of duplex structures the rim of which gives a slight AH-positive reaction while the medulla is completely AH-negative (see int, fig. 3, A, B). With Sudan black B these bodies appear uniformly blue-black. These duplex vesicles have also been observed in Kolatchev and Aoyama preparations (int, fig. i, G), although in the latter case they are greatly contracted. When gelatine sections of material fixed in calciumformaldehyde and post-chromed are treated with cold acetone, alcohol, and ether, and coloured with Sudan black B, these bodies continue to appear in the form of duplex vesicles. These are not preserved when living material is extracted with these solvents. This observation leads to the conclusion that the intermediate bodies (int) are composed of a cortex rich in phospholipids and a medulla rich in triglycerides.

The increase in the number of all the three different categories of sudanophil bodies keeps pace with the growth of the oocyte.

The next stage (fig. i, c) initiates the peripheral disposition of the large AH-negative spheres (Lf). To begin with these large spheres become arranged in two groups occupying each side of the nucleus, which has come to lie on one side of the oocyte. In between these groups of large AH-negative spheres lies a concentrated mass of AH-positive sudanophil bodies belonging to both the first (L1) and the second (L2) categories. The intermediate forms between the second (L2) and third (L2) categories of lipids occur mostly around the concentrated mass of AH-positive sudanophil bodies. The large spheres (L3) do not react positively to the AH-test (fig. 3, A, B).

The large AH-negative sudanophil spheres (L3) now move still further towards the periphery of the oocyte and become arranged in the form of a ring immediately below the follicular epithelium (fig. I, D, E). The number of these bodies is increased greatly at this stage. It has also been observed that during the later stages of growth some of these large spheres do not colour homogeneously with Sudan black B, thus appearing in the form of rings with varying thickness of their cortices. This appearance may be attributed either to incomplete fixation of the more centrally situated lipids, or to the presence of lipids in the centre solid at ordinary temperatures, which remain uncoloured with Sudan black B (figs, I, c-E; 2, A). This ring-like appearance of large spheres (L2) is of very rare occurrence and has only been observed in material fixed in formaldehyde-saline and coloured with Sudan black B (after Baker, 1949). A similar appearance of fat droplets has also been recorded by Bradbury (1956).

Simultaneously with the peripheral disposition of the lipids of the third category the AH-positive granules (fI and f2) become dispersed in the cytoplasm. The granules now appear in more or less irregular patches, the intermediate spaces between these patches containing widely distributed granules (fig. i, E).

The large AH-negative sudanophIl spheres (f3) are not preserved in any of the chrome-osmium or silver nitrate techniques. But when unstained Lewitsky preparations are examined immediately after mounting in Canada balsam, most of these AH-negative spheres are seen as black globules. When such slides have been dried in the oven, these black globules are completely washed out and appear in the form of vacuoles.

The smaller AH-positive sudanophIl granules and f2), on the other hand, are preserved in chrome-osmium and silver nitrate techniques (fig. 1, F, G), and in unstained Lewitsky and Champy preparations they are seen as dark granules.

In the chrome-osmium and silver nitrate techniques the nucleus and the cytoplasm of the oocytes undergo great contraction and distortion.

The oocyte now enters a stage when all the three categories of the sudano-phil bodies (fI, f2, and f3), which were hitherto easily distinguishable from one another by their size and distribution, now become intermingled and come to occupy the whole of the cytoplasm uniformly. At the same time the size of the large AH-negative spheres (f3) becomes so much reduced that it is not possible to distinguish them from the larger AH-positive bodies (f2) in Sudan black B preparations. However, the granules of the first category (fI) can still be distinguished from those of the other two types (fig. 2, A, B, C). If comparison is made between oocytes in the corresponding growth periods in Sudan black B and acid-haematein preparations, the very much reduced number of the larger sudanophIl bodies (f3) in the latter betrays the presence of AH-negative spheres (figs. 2, A-C; 3, C-E). Intermediate, slightly AH-positive bodies, can be made out at these stages also.

In older oocytes these AH-negative sudanophIl bodies (f?) once more become concentrated in the form of a cortical ring (fig. 2, c). A few of these bodies are of considerable size. Since this ring is composed mostly of AH-negative bodies (f?), it does not appear in acid-haematein preparations (fig· 3, E

With further growth of the oocyte there comes a period of great physiological change during which all the large AH-negative (f?) as well as larger AH-positive bodies (f2) gradually disappear from view. The phenomenon of disappearance starts from the nuclear region and extends ultimately to the whole of the cytoplasm. The AH-negative bodies (f?) in the cortical ring are the last to disappear. All that remains now in the cytoplasm of the oldest oocyte examined by me are the minute sudanophil, AH-positive granules of the first category (ZI, fig. 2, D). These granules do not give pinkish tinge with Nile blue, but are stained uniformly blue with it. This means that at this late stage of growth of the oocyte the lipid granules of the first category (L^) get completely shorn of their neutral fat contents. These granules can be observed in all the preparations, viz. Sudan black, acid-haematein, Nile blue, Kolatchev, Mann-Kopsch, and silver nitrate.

Once the larger sudanophil, AH-negative bodies (Z3) have disappeared more or less completely from view, the yolk globules start appearing in the centre of the oocyte. These are at first small spherical bodies, not coloured at all with Sudan black B, acid-haematein, Nile blue, &c.; they are completely osmiophobe and argentophobe but are stained blue in Bouin / iron haematoxylin preparations. As the development of the oocyte proceeds further, these yolk globules grow and increase in number (fig. 3, F), and correspondingly the number of minute AH-positive granules (Lj) decreases and ultimately they disappear.

With maturity the yolk globules acquire a very complex chemical nature and start colouring with Sudan black B, and also with acid-haematein; they appear pink with Nile blue, orange-red with Sudan III and Sudan IV, and pinkish red with Fettrot. They are also blackened by 2% osmium tetroxide solution in fresh coverslip preparations. At this time some sudanophil and AH-positive bodies belonging to the second category (Z2) can also be observed lying in the cytoplasm between the yolk globules at the periphery of the oocytes. These appear to have been infiltrated from outside the oocyte, as lipids of similar chemical composition have been observed lying in the zona radiata and follicle cells (fig. 2, E).

In the early oocytes, when the three types of sudanophil bodies are still in the form of a juxta-nuclear mass, some vacuoles start making their appearance in the peripheral cytoplasm (fig. I, B). With the growth of the oocyte these vacuoles grow in number and size. At the same time some intensely AH-positive sudanophil bodies (pl) of comparatively large size appear inside as well as outside these vacuoles (fig. 3, B, c). The vacuoles as well as the AH-positive bodies (pl) increase considerably in size and in number up to a certain stage, but remain restricted to the immediate periphery of the oocyte (fig. 3, D, E). As far as histochemical reactions of the peripheral lipid bodies (pl) are concerned, they resemble the AH-positive sudanophil bodies of the second category (L1), except that they are larger and are not retained in material fixed in formaldehyde-saline and coloured with Sudan black B (figs. 1, B-E; 2, A). They are clearly visible and are coloured with Sudan black B in the material fixed in formaldehyde-calcium and post-chromed, as recommended by Baker in 1946 (pl, fig. 2, B, c). I have not observed these bodies in material prepared by the chrome-osmium or silver nitrate techniques. These bodies as well as the vacuoles disappear from view gradually (fig. 3, F) and do not appear again. It has not been possible to discover the significance of these bodies.

The follicular epithelium of the oocytes is a single layer of cells in early stages of development (figs. I, D, E; 3, A, C). The first two categories of AH-positive sudanophilic bodies (ZI and Lf) described in the oocytes can also be found in the cytoplasm of these cells. The size-difference between these two types of AH-positive granules exists here also.

With the growth of the oocyte, the follicular epithelium becomes multilayered (fig. 3, E, F), and there is an increase in the amount of sudanophilia corresponding with the increase in the oocyte. But when the zona radiata is fully developed the follicular epithelium again becomes single-layered (fig. 2, E).

When Schultz’s cholesterol reaction (as given by Gomori (195 2)) is applied to gelatine sections of material fixed in formaldehyde-calcium or formaldehyde-saline, blue-green bodies of various sizes and shapes are seen in the oocytes. The colour of these bodies fades after some time. They are soluble in acetone, alcohol, and ether, and do not appear when sections are treated with these solvents. In the earliest oocytes, the cholesterol elements are present near the nucleus (fig. 4, A). With the growth of the oocyte the number of these cholesterol bodies increases, but there is a decrease in the size of the individual bodies (fig. 4, B, c).

When the AH-negative sudanophil material (L3) forms a cortical ring in the late stages, the cholesterol bodies of various sizes are seen in this region (fig. 4, D). There is no cholesterol material in any other region of the oocyte at this stage. Ultimately, however, the cholesterol material also disappears from view along with the AH-negative spheres (Z3). Thus from their size, distribution, and reactions it appears that cholesterol and its esters are present in the AH-negative spheres (L3) themselves.

The histochemical use of Sudan black B has demonstrated that lipids are abundant and widely distributed in the oocytes of the pigeon. The oocyte shows a progressive increase in the amount of sudanophil lipids up to a certain stage in the course of its growth. This stage is followed by disappearance of most of the lipids. They reappear in the yolk globules during the late stages of yolk formation. During the early stages of oogenesis the lipids occur in the form of granules and spheres of various sizes, which can be divided roughly into the following three types according to their size and histochemical reactions.

I. Lipids of the first category (ÄI). Very minute sudanophil granules (Lf) belonging to the first category give a positive reaction with acid-haematein controlled by a negative reaction in pyridIne-extracted material; this shows that they contain phospholipid. With Nile blue they give a pinkish tinge; this indicates the presence of neutral fats in them. Their neutral fat content is further revealed by the fact that they colour orange-red with Sudan III and Sudan IV and pinkish red with Fettrot. Their dark blue colour with Fischler’s method shows that they contain fatty acids. Besides phospholipids, neutral fats (triglycerides), and fatty acids, the presence of other material (especially proteins) cannot be ruled out, as they appear in the form of corroded granules in Bouin / iron haematoxylin preparations and in pyridine extracted material. In the latter they appear slightly yellow.

After treatment with acetone, the neutral fats (triglycerides) are lost and, therefore, the intensity of colouring with Sudan black B decreases. The loss of triglycerides from these granules is confirmed by the lack of any red tinge with Nile blue and the failure of Sudan III, Sudan IV, and Fettrot to colour these granules after such treatment. But their phospholipid contents are still intact as they are coloured with acid-haematein. Their phospholipids are removed with cold alcohol and ether, as they do not colour with Sudan Black B after treatment with these fat solvents.

They resist wax-embedding after chrome-osmium fixation and are stained blue with iron haematoxylin. They might thus be said to correspond with the granular mitochondria of other workers on the oogenesis of birds. Their dark appearance in unstained Kolatchev, Mann-Kopsch, and Lewitsky preparations, studied immediately after mounting, reveals that they contain unsaturated lipid.

2. Lipids of the second category (L2). The second type of sudanophIl lipid bodies (f2) are of medium size. They contain phospholipids and neutral fats, as they are stained blue-black with acid-haematein and pink with Nile blue. Their deep orange-red colour with Sudan III and Sudan IV and pinkish red with Fettrot further confirms the presence of neutral fats (triglycerides) in them. The intensity of colouring with the red Sudan colouring agents and with Fettrot depends upon the amount of triglycerides present in these lipid bodies. Thus the colour varies between the orange-red and red. These bodies differ from the minute granules of the first category (fI), in being larger and containing comparatively more triglycerides. They also lack fatty acids and proteins, which appear to be present in the minute granules (Lj).

The lipid bodies of the second category (f2) dissolve out completely in acetone because of the presence of triglycerides along with phospholipids. Cain (1950) and Krishna (1950) have stated that a small amount of triglycerides will cause lipids to dissolve completely in acetone. Such a possibility has also been described by Lovern (1955). Thus their solubility in acetone also suggests that they are a mixture of phospholipids and triglycerides. Here the question arises why the minute granules of the first category (fI), which also contain phospholipids and triglycerides, do not lose their lipids completely in acetone. First, it may be possible that a thin film of triglycerides, which dominate the total sudanophilia of the oocyte, surrounds the granules of the first category (fI). If that is so, this film is easily washed away in acetone, while the phospholipid contents remain intact. Secondly, the amount of triglycerides in these granules of the first category (fI) may be so small that it is dissolved in acetone without affecting the presence of phospholipids. The first view is supported by the fact that with the disappearance of triglyceride spheres (f?) from the advanced oocytes, these minute granules (fI) also lose their triglycerides, as they no longer give a pinkish tinge with Nile blue (fI in figs. 2, D; 3, F).

The lipid bodies of the second category (f2) also resist wax-embedding after chrome-osmium fixation, and they are stained blue by iron haematoxylin, but they are completely removed in Bouin / iron haematoxylin preparations. They turn jet black with silver nitrate, and in Kolatchev, Mann-Kopsch, and unstained Lewitsky preparations. Their black appearance in Kolatchev, Mann-Kopsch, and in unstained Lewitsky preparations studied immediately after mounting, shows that the lipids present in them are unsaturated. They always appear in the form of granules in these preparations. Whenever there is a collection of a few granules, the silver is deposited in the spaces between these granules and a network-like mass is produced. Thus their granular form is entirely concealed. These lipid bodies (Z2) probably correspond to the so-called ‘Golgi bodies’ of earlier authors on bird oogenesis.

From the observations of Nama (1956) I have not been able to understand which bodies he has called ‘Golgi bodies’, because he has stated that the small bodies which looked and reacted like ‘Golgi elements’ are not really the ‘Golgi elements’ but are phospholipids scattered in the cytoplasm. He further says that after treatment with alcohol and ether, the ‘Golgi elements’ colour with Sudan black B and he has attributed this colouring to the presence of lipoproteins in these bodies. As far as my observations are concerned the total lipid contents of the oocytes dissolve in alcohol and ether, except a mass of sudanophil bodies observed in the juxta-nuclear area at a particular stage of the oogenesis only (fig. i, H, lip). This mass of sudanophil material loses its sudanophil nature when the material is treated with boiling alcohol only. This mass can be interpreted, by the use of selective staining and solubility tests (Krishna, 1950, 1953; Nama, 1956), as lipoprotein bodies, which, owing to the vigorous action of fat solvents, have formed one compact mass. It is, therefore, probable that Nama has described this juxta-nuclear mass of lipoproteins as the ‘Golgi bodies’. These lipoprotein bodies appear to be separate from other lipid bodies (ZI, L2, and Z?).

3. Lipids of the third category (Z?). The third type of lipid bodies (Z?) are large, intensely sudanophil spheres which remain completely negative after the AH-test. Their reactions suggest the absence of phospholipids, which are present in the first two categories of lipid bodies (ZI and Z2). Their deep pink colour with Nile blue shows that they are droplets of neutral fats. Their red coloration with Sudan III and Sudan IV and deep pinkish red colour with Fettrot further reveal that they are made up of triglycerides. Their triglyceride nature is also confirmed by their solubility in acetone. They are not preserved in Bouin / iron haematoxylin preparations. Their black colour in unstained Lewitsky preparations, studied immediately after mounting, indicates the unsaturated nature of the triglycerides of these bodies (Z?).

It has already been pointed out in observations that these lipid bodies (Z?) give a positive reaction with Schultz’s method after oxidation in iron alum, but not without it. Thus, besides triglycerides, cholesterol or its esters also form a part of their fatty contents. Marza and Marza (1936) have also found that the fatty balls of early oocytes of the hen and the ‘cortical’ granular layer of fat in the advanced oocytes are rich in cholesterol.

Singh (1938) and Nama (1956) have not been able to state clearly whether cholesterol exists in separate globules or is present in the neutral fat globules. They have simply mentioned the presence of cholesterol in the oocytes of the pigeon. The various forms and shapes of the cholesterol bodies described by Singh (1938) can be attributed first to the drying effect on the neutral fat bodies and secondly to the quick dehydration by the acids used in the Schultz’s reaction.

Although it is rather difficult to correlate the various lipid bodies of the pigeon oocytes with each other, one is tempted to conclude that the larger neutral fat bodies (L2) arise by the growth and chemical change of the lipid bodies of medium size (i2). This conclusion is strengthened by the fact that intermediate forms (figs. 3, A, B; 1, a) of various sizes, with a chemical composition varying between these two, occur quite commonly.

The differential staining of these intermediate bodies (int’) with acid-haematein suggests that the medullary region contains neutral fats (triglycerides), as it is negative to AH-test, while the cortical region contains triglycerides masked with phospholipids, as it gives a feeble positive reaction with acid-haematein. As these bodies grow, the rim is attenuated with the result that the whole of the sphere becomes AH-negative. Thus it appears that with growth in size the phospholipid contents of the large AH-positive bodies (L2) are either transformed into neutral fats (triglycerides) or disappear from these bodies. The duplex structure of the intermediate bodies (int’) in the material prepared by the Kolatchev and silver nitrate methods, and in the post-chromed material fixed in formaldehyde-calcium and treated with acetone, alcohol, and ether, can be explained by the fact that the triglycerides of the central part are dissolved by the fat solvents, while the fixed phospholipids of the rim resist the action of these fat solvents.

This growth and chemical transformation of the large AH-positive bodies (the so-called ‘Golgi bodies’) into AH-negative spheres (L^ (the so-called ‘fatty yolk’ of earlier authors) appears to be exactly like the origin of ‘fatty yolk’ from the ‘Golgi vesicles’ as described by Nath since 1929. References may be made to Nath (1957) for full bibliographies on this subject.

It also appears that the larger AH-positive bodies (L2, the ‘Golgi bodies’ of other authors) arise from the smallest AH-positive granules (Z,I, the ‘mitochondria’ of other authors), as suggested by Hirsch (1939). Reference may also be made to Nath (1956, 1957) for full references on the subject.

4. Yolk globules. Before the appearance of yolk globules nearly all the lipids disappear from view except some small neutral fat granules in the cortical region (L?), the minute AH-positive granules (Lf), and the peripheral lipids (pl).

The yolk globules, when they first appear, are completely sudanophobe, but as they grow in size they correspondingly attain an appreciable amount of sudanophilia, which is diffused throughout the globule. This sudanophilia gives positive reaction with the AH-test and a negative reaction with PE, which indicates the presence of phospholipids. Their pinkish coloration with Nile blue, orange-red with Sudan HI and Sudan IV, and pinkish red with Fettrot, shows that triglycerides are also present in the yolk globules. The lipid nature of these yolk globules is further revealed by their going black quickly with 2% osmium tetroxide solution in fresh coverslip preparations. This shows that the lipids present in these yolk bodies are unsaturated. All these reactions show their compound lipid nature with phospholipids and triglycerides present. That proteins or carbohydrates or both may be present is suggested by the fact that they resist pyridine extraction and stain blue with iron haematoxylin after Bouin. A similar type of compound yolk globules has also been described by Marza and Marza (1936) in the hen’s egg. They found that the transitional forms between the primordial yolk (my sudanophobe yolk globules) and the yellow yolk of the ripe egg contain various quantities of neutral fats and lipids.

5. Peripheral lipid bodies (pl). From the colour tests and solubility tests it appears that the lipid bodies (pl) found in connexion with the vacuoles of the periphery are of more or less the same chemical composition as the larger AH-positive bodies (L2), i.e. they contain phospholipids and some neutral fat (triglyceride), but they are larger than the latter.

These peripheral lipid bodies (pl) differ from the larger AH-positive bodies (L2) as they are not preserved in material fixed in formaldehyde-saline.. They also do not show well in material fixed in formaldehyde-calcium without postchroming. They appear as intensely sudanophIl bodies in the sections fixed and post-chromed by the acid-haematein technique of Baker (1946). This thus indicates the different chemical nature of the phospholipids of these bodies from those of the second category (L2). It appears that the calcium ions in the preliminary fixation and the post-chroming with calcium-dichromate are necessary for the retention of the phospholipids of these bodies. This observation is in conformity with the view expressed by Cain (1950) and Baker (1956). Similar observations of the use of calcium ions in tire preliminary fixation have also been made by Gupta (unpublished) in this laboratory on the male germ-cells of ticks.

I wish to express here my deep sense of gratitude to Professor Vishwa Nath for suggesting to me this line of research, for his keen interest and constant encouragement throughout the progress of this work, and also for correcting the manuscript for the press; to the Editors of this Journal for sending me valuable suggestions and criticisms; to B. L. Gupta for helpful discussion; and to the University Grants Commission and the Panjab University for the financial help to carry out the work.

Baker
,
J. R.
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1944
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Quart. J. micr. Sci
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85
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1
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Baker
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J. R.
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1946
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Ibid
.,
87
,
441
.
Baker
,
J. R.
,
1947
.
Ibid
.,
88
,
463
.
Baker
,
J. R.
,
1949
.
Ibid
.,
90
,
293
.
Baker
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J. R.
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1956
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Ibid
.,
97
,
621
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Bourne
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G. H.
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1942
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Cytology and cell physiology
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Oxford
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Bradbury
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1956
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Quart. J. micr. Sci
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499
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Cain
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A. J.
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1947a
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Ibid
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88
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383
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Cain
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A. J.
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1947l
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Ibid
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88
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467
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Cain
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A. J.
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1948
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Ibid
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89
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429
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Cain
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A. J.
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1950
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Biol. Rev. Camb. Phil. Soc
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73
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Casselman
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1952
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Quart. J. mIcr. Sci
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Gomori
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Govan
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1944
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J. Path. Bact
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Hirsch
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G. C.
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1939
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Formtend Stojfwechsel der Golgi-Körper. As quoted by Bourne, 1942
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Kay
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W. W.
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Whitehead
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R.
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1941
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J. Path. Bact
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53
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Krishna
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1950
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Proc. Nat. Acad. Sci. India
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60
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Krishna
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1953
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Quart. J. mIcr. Sci
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315
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Lovern
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Marza
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Marza
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Quart. J. mIcr. Sci
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Nama
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1956
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Annals of Zoology (published by the Academy of Zoology, Agra, India)
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1
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171
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Nath
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1929
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Quart. J. mIcr. Sci
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72
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277
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Nath
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Internat. Rev. Cytol
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447
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Nath
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Res. Bull. Panj. Univ. (India)
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127
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Nath
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Ibid
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98
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145
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Pearse
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A. G. E.
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Singh
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Proc. Roy. Irish Acad
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33
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