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Volume 87, 1959
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Notes on the Electron Microscopy of the Spermatogenesis of Lumbricus herculeus

By J. Bronte Gatenby,
Zoology Department, Victoria University of Wellington

*

Introduction

In an earlier paper (Gatenby and Dalton, 1958) some notes were given on the electron microscopy of the spermiogenesis of Lumbricus. In the present paper some other aspects of the spermatogenesis are treated, mainly covering the maturation phase, the centrioles and the morphology of the Golgi apparatus. Some of the present micrographs (e. g., Pl. 6, fig. 1; Pl. 7, fig. 5) were at a magnification of 70,000 X, thus bringing such a cytological study to sub-molecular level. Previously the best that could be achieved was an investigation at 1,500 X of fixed material by means of the light microscope For such research the latter has been superseded by the electron microscope.

Regarding general literature on the light microscopy of this subject, the review by Vishwa Nath on spermatogenesis gives most of the useful references up to 1956, and some information based on the phase contrast microscope, which owing to its relatively poor magnification is not adequate D. W. Fawcett's papers on the electron microscopy of various vertebrate germ cells are useful, a short summary being available in the Ciba Foundation Volume on “Ageing”, 1956 A general and most useful treatment of the advances in general electron microscopical cytology is given by Dalton & Felix (1956).

Owing to the fact that the spermatazoa of the earthworm are probably the first that all students of biology and medicine are shown in their early years of training, these cells are of more than intrinsic interest. Because of the unusually small size of the cells, this spermatogenesis has not been described successfully by light microscopists, though, as Chatton and Tuzet (1941) remark, it has often attracted the “travaux pratiques des debutants”.

Acknowledgments

Dr. A. J. Dalton, now of the Sloan-Kettering Institute for Cancer Research, New York, gave indispensable help with the electron microscopy, and kindly handed over those micrographs which did not cover our previous joint work on Lumbricus. The present micrographs were made at the Cancer Department of the U.S.A. Institute of Health, Education and Welfare at Bethesda, Maryland. The earlier studies of this material were partly made at the Argonne National Laboratory, Lemont, U.S.A., under the auspices of the U.S. Atomic Energy Commission. Dr. Bernard Nebel, Dr. Roth, and Mrs. Rosemarie Devine of this Laboratory have given some assistance—Dr. Nebel kindly advised me on the maturation phase micrographs, Mrs. Devine has allowed me to refer to her unpublished work on Melanoplus differentialis, and Dr. Roth went over his micrographs of lepidopterous germ-cells with me. The osmium tetroxide used in this research was purchased with a grant from the Irish Medical Research Council. This paper was written in New Zealand during leave granted by the Board of Trinity College, Dublin, as Research Professor of Zoology

[Footnote] * Professor of Zoology and Comparative Anatomy, Trinity College, Dublin, Ireland, Honorary Member of the Royal Society of New Zealand.

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and Comparative Anatomy. Professor Richardson, of Victoria University, kindly gave me all necessary facilities in his laboratory.

Material

The material was fixed in Dublin, Ireland, in Dalton's chrome-osmic buffered fluid for electron microscopy, and carried back to Bethesda by air. During the several days delay in completing the imbedding routine, the material was stored in 75% ethyl alcohol. The micrographs were made with a recent model of the R.C.A., E.M.U. electron microscope Although sections of several hundred cells were photographed, the material dealt with in this and the previous papers leaves many gaps, owing to the ultra-thin sections used.

Previous Work

Reference to previous light microscopical studies on the spermatogenesis of Lumbricus and Allolobophora which, owing to the very small size of these cells, could only be inadequate has been made in the previous paper by Gatenby and Dalton and will not be repeated here. This refers mainly to the work of Chatton and Tuzet (1941, 1942). Gatenby and Dalton (1958), using electron microscopy, have given a general outline of oligochaete spermiogenesis. The early spermatid contains about six or seven mitochondria, the lamellated Gogli apparatus (acroblast), a chromatoid body, the anlage of the acrosome carrier, a hollow centriole, the nucleus, and recognizable ergastoplasmic strands In a later stage, centriole adjunct material appears (Pl. 7, fig. 5 (p, n)). In the present plates, some of these categories are shown as follows: mitochondrion (Pl. 6, fig. 1 (m)); Golgi body (acroblast) (Pl. 7, fig. 5 (g)); acrosome (Pl. 7, fig. 5 (a)); centriole (Pl. 7, fig. 9 (c)); acrosome carrier (Pl. 6, fig. 4 (a), (r)) and Pl. 6, fig. 2 (a); the chromatoid body and the early acrosome carrier anlage do not appear in the present plates Ergastoplasm is seen in Pl. 6, fig. 3 at (ep) The mitochondria (m) become fixed to the elongating nucleus (Pl. 7, fig. 5), the hollow structure formed by them containing the centriole adjunct material (p) subsequently the mitochondria (mitochondrial nebenkern) elongate to form the sperm tail sheath (Pl. 7, fig. 6 (m), and Pl. 6, fig. 2, to left of (er)), a pin (t) in Pl. 7, fig. 7, has formed running forward into the nucleus, arising apparently from the centriole adjunct, and not from the centriole. The latter is at first single in the early spermatid, divides, and one moiety touches the nucleus, guiding the centriole adjunct pin in situ, then retreats behind the mitochondrial sheath or middle piece. The definitive positions of these parts are shown in Pl. 7, fig. 6, (n) nucleus, (m) mitochondrial middle piece, and (c) centriole.

The acrosome (a) forms in connection with the Golgi apparatus (g), as in Pl. 7, fig. 5. Later it passes into the acrosome carrier ((a) in Pl. 6, fig. 4), the earlier stages not being shown in the present plates In the early spermatid, the carrier is situated in the position shown in Pl. 6, fig. 4 (a), (r), and later appears at the head of the spermatid as in Pl. 6, fig. 2 (a), and Pl. 9, figs. 13 and 14 (a) and (ac) In other words, the acrosome carrier apparently transports the acrosome in a bead of protoplasm forwards up to the anterior end of the spermatid In Pl. 9, fig. 13, the carrier (ac) has parted with its acrosome, which thereupon fixes onto the head of the nucleus As far as known, in the formation of a special acrosome carrier, the oligochaete spermatogenesis is unique In all other animals described, the acrosome of flagellate spermatozoa is deposited on the head of the spermatid nucleus by the Golgi apparatus or acroblast, which in the case of the Lumbricus spermatid, remains always at the base of the cell, as in Pl. 7, figs. 6, 7, 8 and 9 An outline of this process, according to Gatenby and Dalton, is given in Text-figs. 8–11.

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Observations

The Spermatocyte

In Pl. 6, fig. 3 is part of a spermatocyte showing about eleven mitochondria, mostly chondriomites. In a juxta-nuclear position (n) is a mass of Golgi bodies (g) and small vesicles belonging to the Golgi apparatus. In the lamellated dictyosomes at (g), there are three to five complete lamellae (the so-called collapsed saccules), but these dictyosomes consist largely of aggregated small vesicles of Dalton and Felix (1953). In places, masses of electron opaque vesicles occur, but no lamellae can be seen, as at (g) right. This ill-defined condition persists until spermio-genesis begins, the typical spermatid dictyosome being shown in Pl. 7, fig. 5 (g). In the chondriomites, the cristae run in the length of the mitochondrion, but have sidelines giving a herring-bone effect. This is well shown in Pl. 6, fig. 1 (m), where the matrix is seen to be denser than is usual in the mitochondria of higher animals. Ergastoplasmic strands can be seen in places (in Pl. 6, fig. 3 (ep)), but associated punctate granules are not usually clear in the present material.

In the vicinity of the Golgi apparatus are spherical or ovoid bodies, (x) in Pl. 6, fig. 3, containing fine vesicles, indicating that some form of secretion has already appeared in the spermatocyte. It is considered that these vesicles are proacrosomic granules, and the full grown acrosome (Pl. 7, fig. 5, a) is formed by fusion of such elements.

Second Maturation Division

In Pl. 8, fig. 10, and Pl. 9, fig. 12, are two metaphase stages Other stages are not yet known. In Pl. 9, fig. 12, the metaphase stage is oriented so that the centriole astral region would be at six o'clock, but the centriole has not been cut across. at (g) is part of the Golgi apparatus, the partly vesiculated condition being evident. The object in the lower part of this cell is not a centriole but is a part of the Golgi apparatus. It has been known to light microscopists for many years that, during dictyokinesis, the Golgi bodies in some cases become associated with spindle or astral fibres, and are thus shepherded into the daughter cells in a sub-equal manner, so that each spermatid gets its moiety. In Pl. 8, fig. 10 (h), upper, the centromere attached to the chromosome is clear: it consists of a pyramidal body down the centre of which is a canal (see also Text-figs. 15 and 16, H). The centromere continues on into a hollow spindle fibre, one of which is clearly shown in Pl. 8, fig. 10 (p) lower left. At (h) upper, the spindle canal is shown attached to the centromere, but cut almost transversely. At (p) right, the astral region has been cut across, but the centriole is not in this section. From the study of a number of micrographs, the amphiastral figure has been reconstructed in Text-fig. 15, and the centromere-spindle attachment and chromosome is drawn semi-diagrammatically in Text-fig. 16. In the micrographs, the canal in the centromere does not appear to connect directly with the hollow spindle fibre, for there is a membrane or stopper at (S) In addition, the spindle fibre tube seems to have side branches (ST) in some cases near the astral region which is here reconstructed from such evidence as that in Pl. 8, fig. 10 (p) left, but the extent of the astral fibres (tubes) is not properly known In all cases where cut suitably, the centromere sits in a crater in the chromosome As fixed in Dalton's chrome-osmic mixture, the chromatin is seen to consist of electron opaque granules and micro-vesicles In Lumbricus the ergastoplasm is scanty as in Pl. 6, fig. 3 (ep), and Pl. 9, fig. 12 (ep), and there is no evidence that this cell element is associated with the spindle fibre tubes, but the possibility cannot be completely excluded on the basis of the present micrographs.

The Structure of the Dictyosome

As will be seen from Pl. 6, fig. 3, and Pl. 9, fig. 12, the dictyosome of the pre-spermatid stages is irregular and shows no more than three or four organised lamellae. The main part of the dictyosome is formed of microvesicles of the same

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nature and diameter as the lamella in transverse section. As will be seen, this condition persists during the maturation divisions where, as in Pl. 9, fig. 12 at (e), imperfect lamellae and masses of microvesicles together make up the dictyosome body.

In Pl. 7, fig. 5, in the original photograph at 71,000 X, the very regular lamellated condition of the dictyosome is seen. Assuming that the position of the acrosome (a) marks the ventral surface of the dictyosome, this micrograph would be cut transversely From a study of many examples, it appears that the spermatid dictyosome is formed of a pile of somewhat oval or rectangular plates tending to be of slightly different sizes Text-fig. 17 gives a somewhat inadequate impression of the construction of the dictyosome, Pl. 7, fig. 5 being cut through A-B and Pl. 6, fig. 1 through C-D.

The dictyosome in Pl. 7, fig. 5 appears to be oriented in the sense that the upper region (o) is different from the lower (a), in which lies the acrosome (a) and the proacrosomic material. The presence of the microvesicles (o) cannot always be ascertained, and it is quite certain that the Lumbricus dictyosome is never provided with the elaborate double cover seen in gryllid dictyosomes (Beams, Tahmisian, Devine, and Anderson, 1954), and shown at C1 and C2 in Text-fig. 12.

Nevertheless, in the present material the acrosome always appears on that side of the dictyosome turned partly or wholly away from the nucleus. It will appear to the observer that a model of the Lumbricus late spermatid dictyosome could be made by a pile of eight to twelve irregularly oval or rectangular pieces of three-ply wood. The pieces may be flat, but in such a figure as Pl. 7, fig. 9 (upper right) the plates may be conical or warped That the plates are independent is shown in the acroblast remnant, where (Pl. 7, fig. 7, g) individual parts often drift apart as happens when a pile of dinner plates begins to fall over This phenomenon has been shown to be the case in numbers of other micrographs not published in this or the previous paper with Dalton: this is a matter of considerable importance in trying to assess the function of the dictyosome Previous electron microscopists, and the author and his colleagues (Gatenby, Tasmisian, Devine, and Beams, 1958) have tentatively accepted the idea that the lamellae are really collapsed sacs, such as would be formed when a pile of empty grain sacs is thrown on the ground In Pl. 7, fig. 5, the parts (e) appear to be broken, but as if originating, from the same lamella At (d) in this dictyosome the edges of the lamellae are becoming blebbed, and at both (b) and (d) the blebs appear to originate from single electron opaque lamellae We know from such examples as that in Pl. 6, fig. 2 (g), Pl. 7, fig. 9 (right-upper), and Pl. 7, fig. 7, that the lamellae are independent, and the condition existing in Pl. 7, fig. 5, may be due to bad fixation In Lumbricus, as in gryllids studied, the lamella is undoubtedly formed like three-ply wood, the central layer being electron translucent, the upper and lower layers electron opaque Thus in the micrographs, two dark lines represent the upper and lower layers of a single lamella or saccule.

The Centriole

It is now known that the animal cell centriole is a hollow bead or collar as viewed by the electron microscope. In the present micrographs centrioles appear in Pl. 7, figs. 6, 7, 8 and 9 (c). In all cases they are hollow, and show different stages in the 9 + 1 subdivisions now known to occur universally in cilia and flagella. The centriole of the spermatocyte in Pl. 7, fig. 9 (c) is believed to be elongating, preparatory to binary fission. The subsequent stages have been given by Gatenby and Dalton in the previous paper In the lengthening spermatid, there is evidence that the centriole (c, in Pl. 7, fig. 6) passes forward into a tube which either is, or arises from, the centriole adjunct Inspection of Figs. 6 and 7 will show that at this stage, the centriole is a mere shell, from the back of which emerges the 9 +1 (or 9 + 2) flagellar filaments In Pl. 7, fig. 8 the centriole has become

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subdivided into its 9 parts, the central filament still being undeveloped. In Pl. 6, fig. 1 the middle filament has appeared. The method of origin of this filament is unknown. The middle filament is clear in the flagella cut across in Pl. 8, fig. 11, left and upper In Pl. 7, figs. 6 and 7, the middle filament appears to originate from the bottom of the centriole and not from its hollow centre. The two stages intermediate between centriole and flagellum are shown at (c) and (f) in Pl. 7, fig. 8. Where the centriole flagellum complex emerges from the cell, as in (c) in Pl. 7, fig. 5, a thickening of the surrounding cell wall takes place. The peculiar position of the head centriole with reference to the middle-piece is mentioned in the Discussion.

The Acrosome Carrier

In Pl. 9, fig. 14 (a), the acrosome in situ has been cut in transverse section. all the other sections in this figure have cut across the spermatozoa somewhat lower down, that is through the nucleus. In Fig. 13, at (a) upper, the acrosome is passing out of its carrier into the upper part of the cell, preparatory to fusing with the nucleus, at (a) lower, the acrosome is still in the carrier, and just above this and at the right upper (ac) are two spent acrosome carriers At Pl. 8, fig. 11 (a) a normal unspent acrosome carrier is shown.

Pre-Secretion in the Spermatocyte Golgi Field

In Pl. 6, fig. 3 spherical or ovoid bodies marked (x) are found associated with the Golgi apparatus field Identical bodies have been found by Gatenby and Roth in micrographs of the spermatocytes of the snail (Helix aspersa). In the Mammalia, light microscopists have previously described in spermatocytes what have been called intra-archoplasmic spherules, which have been traced into the spermatid as the pro-acrosomic material. In Pl. 9, fig. 12, x, one of these spherical bodies can be seen at (x) imbedded in a cloud of small vacuoles, which shows that such bodies are carried through the maturation divisions presumably a number of these fuse, or in some other way contribute to the formation of the acrosome (Pl. 7, fig. 5 (a)). This is a matter of some importance, for the presence of such presumed pro-acrosomic bodies in the spermatocyte implies that aggregation, but not secretion, of the acrosome has taken place in the spermatid stage, as for example in Pl. 7, fig. 5; it follows that the lamellae of the Golgi apparatus must now have some function other than secretion at the spermatid stage.

The Nuclear Fin

It was shown in the previous paper, that as the ripening spermatid nucleus begins to pass from the spherical to the ovoid condition an aggregation of granules on one side appears and eventually stretches from the anterior to the posterior end of the elongated spermatid. The position of this fin is put in Text-figs. 9–11 (F). In transverse section the fin is shown at (n) in Pl. 9, fig. 14. Its function is not known, but similar structures have been described in spermatozoa of other animals by light microscopists.

Discussion

The Lamellae or Saccules of the Golgi Apparatus

Dalton and Felix and Sjöstrand (for reference see Dalton and Felix, 1956) first showed that the Golgi apparatus is made up of plates or lamellae; previously light microscopists believed that the vesicles, crescents and rods forming this cell organella were single walled. For a time it was believed that the multiple lines seen in electron micrographs of the Golgi apparatus represented bundles of strings or rods cut lengthwise. This view has more recently given way to the conclusion that the Golgi apparatus is formed of a series of plates forming a pile in juxtaposition. The multiple rod-like appearance is due to the sectioning of such a pile of plates in a transverse direction. As has already been explained, in Pl. 7, fig. 5 the plates are

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cut transversely, while in Pl. 6, fig. 1 the section is mainly horizontal. It is pointed out by Gatenby and Roth (in press) that in the gryllid Nemobius, the number of plates can be accurately counted. For example, there are almost always seventeen in the acroblast in the spermatid of this animal, and ten in the primary spermatocyte dictyosomes.

It is well known from light microscopy that vacuoles or canals, presumably fluid-filled, are usually associated with the Golgi apparatus. That this is so was confirmed by Dalton and Felix (1953) in their well-known study of the cells of the epididymis. The position of the vacuoles with reference to the lamellae is still a matter of opinion. It is now universally admitted that each lamella consists of three layers, upper and lower electron opaque layers enclosing a third inner, electron translucent layer. The inner layer, according to some electron microscopists, is to be regarded as a hollow space, which can be expanded at intervals by vacuoles (VE) as shown in Text-fig. 14. On the other hand there seems to be considerable evidence from electron microscopy, that free vacuoles (VF) can lie both between and beside the piles of lamellae.

This brings one to the question of the significance of the pile of lamellae as a functional unit in the animal cell. It probably will be readily agreed that the pile of lamellae is some kind of filter (Text-fig. 13). That this is so appears very likely from a study of the pile of oriented lamellae with which the formation of the acrosome is associated. In the case of the Gryllidae, the acroblast has a cover on the obverse side, consisting of two presumed sealing layers (C1, C2 in Text-fig. 12), and the acrosome makes its appearance in a nidus formed on the reverse side by the bent over lamellae (Text-fig. 12, A). On the other hand, as will have been noted, the acroblast of Lumbricus (Pl. 7, fig. 5) has no large vacuoles associated with it, and except for the appearance of the acrosome on one side, there is no elaborate cap or cover as occurs in gryllids. In the case of the Lumbricus acroblast, it thus seems difficult to understand how this pile of lamellae could function as a filter. In all cases, however, it seems that the upper and lower layers of the single lamellar plate are formed by oriented molecules, but the relationship of piles of such plates to the ground cytoplasm is today quite obscure. That each plate is independent of the others in the pile can be ascertained by inspection of such micrographs as those in Pl. 6, fig. 2, and Pl. 7, fig. 7, where, in the elongating spermatids, the pile of plates spills over in such a way as to show this independence. One of the clearest demonstrations of the independence of each lamella or saccule of the Golgi apparatus of protozoan cells has been given by Noirot Timothée (1957) in the case of ophryoscolecid infusorians. She has found chains of such lamellae, sometimes more than a dozen in number, all more widely separated than in lumbricids, but still parallel to one another.

It appears to the present author that in the years gone by, studies of the remarkable behaviour of Golgi bodies and mitochondria have tended to divert attention from the apparent fact that the ground cytoplasm is the more important part of the cytosome, and not those bodies floating in it. Presumably the materials which ultimately become segregated within the acrosome (lipolytic and/or proleolytic enzymes) originate in the ground protoplasm and the lamellae of the Golgi apparatus in some way not understood filter and concentrate it.

The resemblance between the lamellar pile of the Golgi apparatus, and the voltaic pile will be apparent to all, except that separate alternate layers have not been found in the former. There is no evidence that the lamellar pile of the Golgi apparatus is either a hydrostatic or natant organ, because the acrosome of Lumbricus is brought up to the head of the elongate nucleus not by the Golgi apparatus but by a special protoplasmic bead (or carrier). So far as is known, the lumbricid dictyosome is the only one described which is free of internal or peripheral large vacuoles; this case makes it difficult to sustain the osmotic hypothesis of Golgi

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Text-figs. 1–11. Figs. 1–3—Acrosome formation as found in Homo, Cavia, Mus, Gryllus (Acheta), Helix, etc Figs. 4–7—Acrosome formation in Melanoplus differentialis (for Figs. 6 and 7, see text) Figs. 8–11—Acrosome formation in Lumbricus herculeus
Lettering: A, acrosome; AL, acrosome carrier; C, centriole; CJ, centriole adjunct region; F. nuclear fin, G, Golgi apparatus (acroblast); K, attachment of sperm cell to central nutrient mass, N, nucleus; P, cell wall cover of acrosome.

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apparatus function, and the filtration hypothesis is equally difficult to accept in such a case.

The Gross Morphology of the Golgi Apparatus

The earlier workers such as Perroncito (references in Wilson's “Cell”) considered the Golgi apparatus of Invertebrata such as the pulmonate Mollusca, to be short rods, often crescentic in appearance, which he named “dittosomi” (dictyosomes) from the Greek, meaning a stick-like body. More recently, light microscopists came to believe that, in germ cells, the dictyosome is a sphere, one side of which is thickened to give a crescentic appearance. These opinions were based on observation of these very small objects with the inadequate resolution of the glass lens of the optical microscope. The electron microscope micrographs of Golgi bodies have added a new interpretation. In micrographs studied at Argonne National Laboratory by Gatenby and Roth it has been shown that the pulmonate molluscan dictyosome is formed of slightly bent rectangular plates, and is not a spherical body as more recently assumed by light microscopists Thus the older drawing of the dictyosome as a short rod was more nearly exact than that of a sphere. The lamellar and proacrosome parts shown in Text-fig. 12 would with the poorer magnification and resolution of the optical microscope appear spherical In the case of the oligochaete Golgi apparatus shown in Pl. 7, figs. 6–9, the appearance seen with the light microscope is spherical or more usually helmet-shaped. In the Lepidoptera the dictyosomes of the male germ cells have for many years (Gatenby, 1921) been known to be ovoid or spherical. Recently Roth (unpublished) has produced micrographs of such spherules, which show that the wall is multilamellar. These dictyosomes are true spheres just as they appear under phase contrast.

Spindle Fibres

The older light microscopists universally described spindle fibres during mitosis and meiosis. The best preparations of these fibres were obtained by fixatives which were strong protein coagulents. As often happens in such cases, the presence of spindle fibres intra vitam was denied by subsequent workers until the centromere attachment of the chromosome was discovered, when a reversal of view took place. The first electron micrographs of dividing cells have been disappointing in this respect In fact, in mammalian mitosis spindle fibres in dividing cells have only recently been shown by the electron microscopist. Dr. Bernard Nebel, working on mouse cells Dr. Nebel's cells were, it is understood, purposely fixed in a moribund state. The present micrographs of lumbricid spermatocyte divisions clearly show both spindle fibres and centromeres.

Acrosome Formation

In Mammalia, Insecta, and Mollusca, the acrosome, after its appearance, is placed upon the nucleus in the manner shown in Text-figs. 1–3. The applies to Homo, Cavia, Mus, and Oryctolagus, and probably to all other vertebrates. After the second maturation division the spermatid becomes oriented, possibly by the position of the centriole, or possibly by the position of the whole cell with reference to the spermatic tube lumen, the latter possibility is the more likely. The position of the Golgi apparatus or acroblast with its pro-acrosome or acrosome is variable. If it lies away from the now anterior end of the cell, it migrates up to the anterior end, and deposits the centriole usually in the correct place. Slight deviations from this do take place, but, either by movement of the nucleus or of the acrosome, the latter comes to he in the long axis of the cell, as in Text-fig. 3. In Insecta. which have been much studied, there are sometimes several Golgi bodies—e g, in Lepidoptera, and these may secrete and deposit a number of acrosomes which tend to lie in the correct position and later fuse and become exactly oriented in the long axis of the nucleus, probably in some cases by migration over the nuclear membrane.

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Fig. 1.—Part of spermatid of Lumbricus, showing cristae (cr) in the mitochondrion (m), the centriole with the central filament (c) developed. The Golgi apparatus has been cut horizontally (refer to Text-fig. 17, D-E). Fig. 2.—Somewhat oblique longitudinal section of the nearly ripe spermatozoon. At the head (a) is the acrosome carrier, with the acrosome passing out to the head of the spermatozoon. At (nx) the nuclear fin is cut, and at (un) is the nucleus containing a space possibly arising from the nucleolus At (g) the Golgi apparatus at the posterior end of another sperm is cut. The separate parts or lamellae of the Golgi apparatus can be seen at (e). Fig. 3.—Part of a spermatocyte Golgi field (g) showing the supposed pro-acrosomic vesicles (x). The lamellae are less clear, the Golgi bodies being mainly formed of small vesicles as at (g) lower right Ergastoplasm (ep) is scanty. It conforms to the usual type Fig. 4.—A spermatid showing the acrosome carrier belonging to this cell, but not cut quite across its centre Compare with its position in Fig. 2. The acrosome carrier shows the acrosome (a), some granules (r) and the vacuole above Compare with the spent carrier in Pl. —, fig. 13 (ac). The mitochondrial middle-piece (m) and the lamellated Golgi apparatus (g) are well shown

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Fig. 5.—High power micrograph of Golgi apparatus (g) with about eight lamellae, at (o) are vesicles which may represent the dictyosome cap of the grvllid the acrosome (a). The mitochondrial middle-piece (m) is formed around the centriole adjunct material or post-nuclear region (pn). The centrioles are not properly cut across but lie at (C) where the cell wall is thickened. The nature of the vesicle (/) is not known but may be part of the forming acrosome carrier. Figs. 6–8—Parts of the post-nuclear region of the lengthening spermatid showing the hollow head centriole (C) tail flagellum with central fibre and in Fig. 7 spilled over lamellae of the Golgi apparatus reject (g). Fig. 9.—On the left a spermatocyte showing ovoid hollow centriole (c) probably preparatory to division On the right the Golgi apparatus (upper) is cut transverselv below horizontally

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Fig. 10.—A group of four cells in metaphase of second maturation division. At (h) upper, a centiomere. to the left of which is the oblique section of the spindle fibre tube. At (p) lower left the spindle fibre tube (p) is cut longitudinally. At (p) right a part of the astral fibres are cut across. The mitochondria (m) become scattered during meiotic divisions (Compare with Text-figs. 15 and 16.) Fig. 11—Neck of protoplasm (k) by which all sister spermatic cells are attached to the non-nucleated central mass, partly cut on left. This mass contains fat etc. and mitochondria (m). An unspent acrosome carrier on right containing the acrosome (a). Two flagella (f) cut across showing internal fibres nine peripheral and one central.

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Fig. 12.—Lower part of meiotic metaphase probably the first. (Compare with Text-figs. 15 and 16). This micrograph shows well the Golgi apparatus (g), spindle fibre tubes (p). At (ep) ergastoplasm. The crater-like attachment of the centromere (h) appears well in some of the chromosomes. At (px) is a hollow spindle fibre tube. At six o'clock is a part of the Golgi dictyosome. Figs. 13–14—Sections across the upper end of nearly ripe spermatozoa across the region (a) in Pl. — fig. 2 Full and spent acrosome carriers are cut across in Fig. 13 Acrosome (a). In Fig. 14, the nuclear fin at (n), and at (a) higher up across the acrosome.

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Text-figs. 12–18.—Fig. 12—Golgi apparatus (dictyosome) of Nemobius sp. (Gryllidae). Fig. 13—Suggested function of lamella. Fig. 14—Plan of association of fluid vacuoles with lamellae. Fig. 15—Semi-diagrammatic reconstruction of second maturation division in Lumbricus. Fig. 17—Semi-diagrammatic drawing of pile of lamellae in Lumbricus Golgi body (acroblast). Fig. 18—Golgi body of lepidopteran (Roth)

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In other insects, such as the Gryllidae, the acroblast or Golgi body is single, as in Mammalia, and the acrosome is usually deposited very little outside its definitive position. If it is out of position it appears to migrate over the nuclear membrane.

In all these cases the anterior centriole has already become fixed to the nucleus, and, so far as is known, does not move, and always constitutes the one fixed point in the long axis of the nucleus and cell.

In Text-figs. 1–11, from stages 1–3, this almost universal simple type of acrosome formation is given. At stage 2 slight rotation of the nucleus, or short migration of the acrosome to its definitive position, has been assumed to occur, but it is certain that in the majority of cases migration upwards of the Golgi apparatus (acroblast) with its acrosome in stage 1 occurs. It is at this period also that the hitherto scattered mitochondria (M) pass down to the back or post-nuclear region. How these differential movements occur is unknown.

In Text-figs. 4–7 is shown the curious case of Melanoplus differentialis discovered by M. Devine at the Argonne National Laboratory. In Text-fig. 4, the acrosome is deposited usually quite near the presumably fixed centriole (C), and therefore well away from its future position at the anterior end of the nuclear axis. Soon the Golgi apparatus parts from the acrosome, which now becomes detached from the nucleus, and is next found stuck to the cell wall opposite (Text-fig. 3). During its time on the cell wall, the acrosome causes the development of an extra-cellular cap (P), which later becomes part of the ripe spermatozoon acrosome. The subsequent stages which bring about the deposition of the acrosome in its definitive position on the nucleus are not fully understood, and there are several possible explanations. The important point that in all the cases known the anterior centriole (C) is fixed has been mentioned. This is assumed to be true, because once the centriole becomes adherent to the nuclear membrane, a reaction takes place in this region which produces the so-called centriole seat of Gatenby (1931). In other cases a pin or peg, is produced in this region, which would appear to anchor the centriole permanently. Thus some explanation of the ultimate arrangement of acrosome and centriole in the long axis of the nucleus must be advanced. In Text-fig. 6, there is, first, one explanation—namely, that the nucleus plus centriole and mitochondrial body all rotate to meet the acrosome. Re-orientation of the whole spermatid with reference to the testicular lumen presumably follows. A second possible explanation is that given in Text-fig. 7, where the acrosome and its outer cap (P) moves up to the anterior end of the cell: a small rectification of the position of the nucleus and acrosome or both would then bring the acrosome into its definitive position.

It is interesting to note that the gryllid Nemobius sp., and Gryllus (Acheta), have the type of acrosome formation shown in Text-figs. 1–3, whilst the locustid Melanoplus differentialis, also an orthopteran, has the quite different system given in Text-figs. 4–7.

Still another method of acrosome formation has now been found in the oligochaete Lumbricus by Gatenby and Dalton (1958), and is given in Text-figs. 8–11. In the spermatid, the usual Golgi apparatus plus acrosome (G, A) is found. In addition a small hollow sphere appears in this region (AL). This is the acrosome carrier anlage, which swells to form a bowl-like structure (Text-fig. 9) into the mouth of which passes the acrosome. The spermatid nucleus has become elongated (Text-fig. 10), marking a point of development at which in other spermatids, such as in Homo, Mus, Cavia, and Invertebrata in general, the acrosome has already become attached to the nucleus as in Text-fig. 3. It has been assumed by Gatenby and Dalton, that the bead of protoplasm containing the acrosome carrier now moves upwards and finally reaches a point where the acrosome can pass over to its correct position as in Text-fig. 11. Examination of stages 9–11 suggests that another explanation of the manner of arrival of the acrosome at the head of the cell might be put forward. If the anterior end of the cell elongated so as to leave the bead

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(A, AL) in its position this elongation might eventually produce the condition found in Text-fig. 11. It is not possible at present to decide this point, but an attempt to do so by light microscopy is being made.

The Position of the Centrioles

Reference to Text-fig. 11 shows the two centrioles lying behind the mitochondrial middle-piece (M). In all other spermatozoa of the flagellate type the head centriole is depicted by light microscopists as lying between the post-nuclear region and the upper end of the mitochondrial middle-piece, as in Text-fig. 3. It has been concluded by the present author and Dalton, that just before the stage shown in Text-fig. 8 the head centriole retreats behind the middle piece Convincing micrographs of this phenomenon have been given in the previous paper. It is not believed that there are three centrioles in the stage of Text-fig. 8 and in subsequent stages. The area between C1 and C2 in Text-fig. 11 is a post-middle-piece region.

Summary

  • (1) The Golgi apparatus of the spermatocyte consists of a juxta-nuclear field consisting of separate dictyosomes mainly formed of microvesicles surrounding more or less ill defined lamellae about four in number. The mitochondria tend to be outside this field. There are secretory vesicles (x) in Pl. 6, fig. 3, associated with and probably arising from the Golgi bodies. The ergastoplasm (ep) consists of a reticulum with micro-granules.

  • (2) The Golgi bodies during the maturation divisions become located mainly on the amphiaster, near the astral region (Pl. 9, fig. 12, g), and still consist of masses of microvesicles surrounding a few lamellae.

  • (3) The spindle fibres are tubular, and are attached to the chromosomes by a hollow pyramidal centromere situated in a crater in the chromosome (Pl. 8, fig. 10, h).

  • (4) After the formation of the spermatids the Golgi apparatus becomes almost entirely lamellar, the microvesicles having run together to form plates (Pl. 7, figs. 5–8, (g)). There are about eight lamellae.

  • (5) The mitochondria are sub-equally divided among the spermatids, ultimately becoming collected behind the nucleus to form the mitochondrial middle-piece (Pl. 7, fig. 5, m).

  • (6) On the basis of the morphology of the Lumbricus dictyosome, which has no large adherent fluid filled vacuoles, it seems impossible to sustain the osmotic hypothesis of the function of the Golgi apparatus.

  • (7) The Golgi apparatus lamellae probably act as some form of filter or concentration device; the manner of their action is not understood.

  • (8) The classical blackened osmium tetroxide and silver Golgi apparatus preparation is due to the deposition of the metals or their salts in the lamellae, and is therefore a valid method.

  • (9) In the living dictyosomes viewed by the phase contrast microscope, the Lumbricus Golgi apparatus is usually helmet-shaped, the lepidopterous Golgi apparatus spherical, and the gryllid Golgi apparatus sub-spherical or bun-shaped according to its orientation. Fixation by Palade and Dalton's fluids does not disturb the gross morphology of these variously shaped bodies.

  • (10) By electron microscopy, the Lumbricus spermatid dictyosome lamella is an irregular rectangular plate (Pl. 6, fig. 1), or a series of double lines when cut transversely (Pl. 7, fig. 5).

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Key to Lettering on Plates

a, acrosome; b, lamella of Golgi apparatus; c, centriole; cr, cristae; e, lamella of Golgi apparatus (see text); ep, ergastoplasm (endoplasmic reticulum); f, flagellum of spermatozoon; g, Golgi apparatus dictyosome or acroblast; h, crater in chromosome; K, protoplasmic bridge to central nutrient mass; m, mitochondrion; n, nucleus; nx, nuclear fin; O, possible cap vesicles; p, pX, spindle fibre tubes; pn, post-nuclear or centriole adjunct material; r, granules in acrosome carrier; t, possible intra-nuclear tube; un, unknown vesicle, probably part of forming acrosome carrier; z, vesicles in Golgi field of spermatocyte probably pro-acrosomic.

Lettering: A, acrosome; C1, C2, acroblast (dictyosome) covers; CR, centriole; CH, chromosome; G, Golgi apparatus (dictyosome); H, centromere; K, attachment of spermatic cell generations to central nutritive mass; L, lamellae; M, mitochondrion; N, nucleus; P, spindle fibre tube; PA, proacrosomic region; S, membrane; ST, side tube; V, vacuole; VF, free vacuole between lamellae; VE, vacuole inside lamella.

References

Beams, H. W., Tahmisian, T. N., Devine, R. L., and Anderson, E., 1956. Phase and Electron Microscope Study of the Spermatogenesis of Nemobius sp. Jour. Roy. Micr. Soc., 76, p. 98.

Beams, H. W., Tahmisian, T. N., Devine, R. L., and Roth, L. E., 1954. Phase-Contrast and Electron Microscope Studies on the Nebenkern of the Grasshopper. Biol. Bull., 107, p. 47.

Chatton, E., and Tuzet, O., 1942. Production par certains individus de Lombriciens de Spermatides normales et de Spermatides nucleolées en parité numerique. C. R. Acad. Sci., 214, p. 894.

—1941. Sur quelques faits nouveaux de la spermiogénèse du Lumbricus terrestris. C. R. Acad. Sci., 213, p. 26.

Dalton, A. J., and Felix, M. D., 1953. Studies on the Golgi Substance of the Epithelial Cells of the Epididymis and Duodenum of the Mouse. Amer. Jour. Anat., 92, p. 277.

—1956. The Electron Microscopy of Normal and Malignant Cells. Ann. N. Y. Acad. Sci., 63, p. 117.

Fawcett, D. W., 1956. The Fine Structure of Chromosomes in the Meiotic Prophase of Vertebrate Spermatocytes. Jour. Bioph. and Biochem. Cytol., 2, p. 403.

Gatenby, J. Bronte, 1921. The Cytoplasmic Inclusions of the Germ Cells: Lepidoptera. Quart. Jour. Micr. Sci., 65, p. 55.

—1922. The Cytoplasmic Inclusions of the Germ Cells: Saccocirrus. Quart. Jour. Micr. Sci., 66, p. 1.

—1931. The Post-Nuclear Granule in Anasa tristis. Amer. Jour. Anat., 48, p. 7.

Gatenby, J. Bronte, and Dalton, A. J., 1958. The Spermiogenesis of Lumbricus An Electron Microscope Study. Jour. Biophys. and Biochem. Cytol. (In press.)

Gatenby, J. Bronte, and Roth, L. E., 1958. Measurements of the Lamellae of Dictyosome and Acroblast of the Cricket Nemobius sp. (Argonne Laboratory Publications.)

Gatenby, J. Bronte, and Tahmisian, T. N., 1958. Centriole Adjunct, Centrioles, Mitochondria, and Ergastoplasm in Orthopteran Spermatogenesis: an Electron Microscope Study. (Argonne Laboratory Publications.)

Gatenby, J. Bronte, Tahmisian, T. N., Devine, R. L, Beams, H. W., 1958. The Orthopteran Acrosome, an Electron Microscope Study. La Cellule, in press. (Argonne Laboratory Publications).

Harven, E., and Bernhard, W., 1956. Etude au microscope electronique de l'ultrastructure du centriole chez les Vertébrés. Zeit. f. Zellforch. Mikr. Anat., 45, p. 378.

Hirschler, J., 1935. Ueber die Beziehung des Fusomproblems zur Vererbungs-Lehre. Zool. Polon., 1, p. 31.

Meves, F., 1897. Ueber Centralkorper in Mannlichen Geschlechtzellen von Schmetterlingen. Anat. Anz., 14, p. 57.

Nath, V., 1956. Cytology of Spermatogenesis. Internat. Rev. Cytology, New York: Acad. Press, 5, p. 395.

Timothee, Noirot, 1957. L'Ultrastructure de l'appareil de Golgi des infusoires ophryo-scolecidae. Compt. rend. Acad. Sci., 244, p. 2847.

Professor J. Bronte Gatenby,


Trinity College,
Dublin,
Ireland.

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