1. The spermatid of the house-cricket, Acheta domesticus, has been studied by the use of vital dyes, histochemical tests, and electron microscopy.

  2. The acroblast consists of an outer and an inner part. The former, which contains lipids and a neutral mucopolysaccharide, is seen in the electron micrographs to consist of parallel lamellae. In section these appear to be arranged in the form of a horseshoe. The internal part of the acroblast gives feeble reactions for a neutral mucopolysaccharide and for a lipid. In electron micrographs it is seen to contain minute vesicles.

  3. The acrosome arises within the acroblast. It gives a strong reaction for neutral mucopolysaccharide. It appears to be devoid of internal structure.

  4. The Nebenkern formed by the fusion of all the mitochondria in the young spermatid, exhibits the ‘cristae’ characteristic of ordinary mitochondria. The reactions of this structure with vital dyes and acid fuchsine also parallel those of the mitochondria. Histochemical tests indicate the presence of a lipid.

  5. The periphery of the nucleus of the late spermatid shows a radial structure.

The spermatogenesis of the house-cricket, Acheta domesticus (L.), has been worked out by Nath and Bhimber (1953), who studied living cells by phase-contrast microscopy and also used testes fixed in Lewitsky’s and Champy’s fluids; paraffin sections were stained in iron haematoxylin.

The cricket is a very convenient animal for studies of the cytoplasmic inclusions in spermatogenesis, because the animal lives and breeds readily in the laboratory, the testes are large and very easy to dissect out, and the cytoplasmic inclusions of the male germ-cells easy to study by light microscopy. We have therefore sought to extend the work of Nath and Bhimber by the use of vital dyes, histochemical tests, and electron microscopy. Our attention has been concentrated mainly on the young spermatid, though we have also made some observations on the later stages, including the spermatozoon.

It is necessary at the outset to review briefly the transformation of the spermatid into the spermatozoon. We have confirmed the main features of Nath and Bhimber’ findings.

The young spermatid, just before it starts to elongate, is represented diagrammatically in fig. 1, A. It contains a spherical nucleus, a subspherical Nebenkern formed by the fusion of all the mitochondria in the cell, and an object supposed to be formed by the fusion of most of the lipid droplets or ‘Golgi bodies’. This object is called the acroblast, because the acrosome first appears within it.

FIG. 1.

Diagrams of four stages in the development of the spermatid of the house-cricket.

FIG. 1.

Diagrams of four stages in the development of the spermatid of the house-cricket.

At a later stage (fig. 1, B), an axial filament extends from the centriole, which later moves to the end of the cell that will become the anterior end of the spermatozoon; the tail grows out; the acrosome appears within the internum of the acroblast; the Nebenkern divides, and the two halves extend along the tail.

The acrosome now becomes attached to the nucleus, and the acroblast moves away from it (fig. 1, C); the halves of the Nebenkern spin out into threads lying beside the axial filament; these are swollen here and there into blebs.

Later the acrosome appears at the anterior end (fig. 1, D); probably this change in position is due to rotation of the nucleus through 180°. The residual cytoplasm is thrown off. It contains those lipid granules that did not take part in the formation of the acroblast, and also of the acrosome itself.

The parts of the cell are now arranged as they will be in the spermatozoon, and it only remains for the acrosome, nucleus, and tail to lengthen and become attenuated.

The crickets were cultured by the method of Jordan and Baker (1956). For the study of the living cells, the testes were teased in this solution of insect saline:

The following vital dyes were used: neutral red, Janus green and black, brilliant cresyl blue, thionine, methylene blue, and dahlia violet. All, except methylene blue (0-04%) and brilliant cresyl blue (o-oi%), were dissolved in the saline solution at 0-02%. The cells were dyed for about to 1 hour in covered watch-glasses and then placed on slides, covered, and at once examined.

The mitochondrial technique of Metzner (Metzner and Krause, 1928) and the ‘Golgi techniques’ of Aoyama (1929), Kolatchev (1916), and Weigl (1910) were used.

Details of the above tests and the histochemical tests are given in the appendix (p. 22).

For electron microscopy the testes of the cricket were rapidly dissected out. Small blocks from the anterior region were fixed in 1% osmium tetroxide dissolved in the insect saline mentioned above. The tissue was dehydrated by standard methods, sectioned at about 25 m/z with a Porter-Blum-Servall microtome. Micrographs, taken in a Siemens Elmiskop I at instrumental magnifications 2,600 and 8,000, were enlarged photographically.

Ground cytoplasm

In the electron micrographs this appears to have the usual characters (figs. 2, A, B; 3; 4, A-C; 5, A, B). The background is spotted with small granules. Laminae with double membranes appear to be dispersed at random throughout the cytoplasm, without orientation. It is clear that the laminae must have considerable extension, because great lengths of them appear in all sections. The laminae show a general resemblance to the endoplasmic reticulum of Palade (1956), but no granules have been noticed on the surfaces of the laminae. The laminae are about 30 thick. They never seem to swell into vesicles.

FIG. 2.

(plate), A, spermatid, early stage. Mitochondria (m); nuclei (n) with nuclear membrane (nm), and electron-dense object (ni) inside the nucleus. B, spermatid, more advanced stage. Laminae (mb) in the ground cytoplasm; acroblast (aa) showing laminated structure of the externum and associated vesicles, also vesicles in the internum; acrosome (a); Nebenkern (nk) with double membranes (mm); axial filament (f). surrounded by a membrane; nuclei (n).

FIG. 2.

(plate), A, spermatid, early stage. Mitochondria (m); nuclei (n) with nuclear membrane (nm), and electron-dense object (ni) inside the nucleus. B, spermatid, more advanced stage. Laminae (mb) in the ground cytoplasm; acroblast (aa) showing laminated structure of the externum and associated vesicles, also vesicles in the internum; acrosome (a); Nebenkern (nk) with double membranes (mm); axial filament (f). surrounded by a membrane; nuclei (n).

FIG. 3.

(plate). Acroblast before formation of acrosome, showing laminated structure of externum.

FIG. 3.

(plate). Acroblast before formation of acrosome, showing laminated structure of externum.

FIG. 4.

(plate). A, spermatid, more advanced stage. Double laminae (mb) in the ground cytoplasm; Nebenkern (nk) with double membranes (mm); nucleus (n). B, spermatid, more advanced stage. Nebenkern (nk) dividing; double membranes (mm); nucleus (n) with electron-dense object (ni); laminae (mb). C, spermatid, more advanced stage. Nebenkern (nk) dividing, double membranes still present; nuclei (n) with electron-dense object (ni).

FIG. 4.

(plate). A, spermatid, more advanced stage. Double laminae (mb) in the ground cytoplasm; Nebenkern (nk) with double membranes (mm); nucleus (n). B, spermatid, more advanced stage. Nebenkern (nk) dividing; double membranes (mm); nucleus (n) with electron-dense object (ni); laminae (mb). C, spermatid, more advanced stage. Nebenkern (nk) dividing, double membranes still present; nuclei (n) with electron-dense object (ni).

FIG. 5.

(plate). A, spermatid, later stage. Wall of cyst (w); acrosome (a) with electron-dense posterior end; nuclei (n) with electron-dense object (ni). B, spermatid, later stage. Wall of cyst (w); nuclei (n) with electron-dense object (ni). C, axial filament (/); transverse section (left side) and longitudinal section (right side). Two filaments (nk) into which the Nebenkern spins out; cytoplasmic covering (cc); wall of cyst (w). D, spermatid, late stage. Longitudinal section of nucleus showing radial structure and attachment of tail (t); dorsal (d); ventral (v).

FIG. 5.

(plate). A, spermatid, later stage. Wall of cyst (w); acrosome (a) with electron-dense posterior end; nuclei (n) with electron-dense object (ni). B, spermatid, later stage. Wall of cyst (w); nuclei (n) with electron-dense object (ni). C, axial filament (/); transverse section (left side) and longitudinal section (right side). Two filaments (nk) into which the Nebenkern spins out; cytoplasmic covering (cc); wall of cyst (w). D, spermatid, late stage. Longitudinal section of nucleus showing radial structure and attachment of tail (t); dorsal (d); ventral (v).

Cell membranes have not been distinctly seen. The possibility exists that at certain stages the spermatids form a syncytium, limited only by the cyst wall. Later, however, the spermatids are obviously separate from one another and from the wall (figs. 5, C, D; 6, A, B).

Acroblast

The acroblast consists of two regions: an outer laminated part, the externum, which appears horseshoe-shaped in section, and an inner area, the internum, which is apparently structureless.

The externum of the acroblast was coloured by the vital dyes dahlia and Janus black. The externum was coloured red by Metzner’s method for mitochondria, and was blackened by osmium in the Baker (Hermann-Kopsch) and Weigl (Mann-Kopsch) techniques, and by silver in Aoyama’s method. All these techniques left the internum negative or less positive than the externum.

Of all the histochemical tests tried, only those for lipids and polysaccharides gave positive results. The externum was blackened both by Sudan black and by acid haematein, but it was found not possible to analyse further the lipid constituent responsible for these reactions. Both the internum (feebly) and the externum (strongly) gave positive reactions to the periodic acid / Schiff (PAS) test, especially after fixation in Bouin’s fluid. The reaction was even more positive after the sections had been treated by salivary amylase. No part of the acroblast was spontaneously metachromatic, but treatment on the slide with sulphuric acid (Lison, 1953) resulted in a positive reaction by both internum and externum. These facts indicate the presence of a lipid and may indicate the presence of a neutral polysaccharide, especially in the externum.

The externum and the internum are clearly differentiated in the electron micrographs. The externum appears in the form of parallel laminae with double membranes, mostly in close apposition to one another, all bent in the form of a horseshoe (figs. 2, B; 3). Vesicles are occasionally seen in the externum. These reach a diameter of about 150 mju,. In fig. 2, B they are confined to one side of the organelle. The double membranes of the externum are electron-dense. This may perhaps be due to reduction of osmium tetroxide by unsaturated lipid.

The internum contains numerous, randomly dispersed vesicles, about 30 , in diameter. The intervening material is as transparent to electrons as the background of the cytoplasm.

Acrosome

Neutral red was the only vital dye that coloured the acrosome. It was also coloured red by acid fuchsine in Metzner’s method. It was negative to all tests for lipids except to the acid haematein technique; it responded positively to the PAS test even after treatment with saliva. It became metachromatic after treatment on the slide with sulphuric acid. Thus the histochemical tests gave some Results similar to, and others at variance to, those shown by the acroblast.

Under the electron microscope the acrosome appears at first to be almost structureless internally, though bounded by an electron-dense envelope (fig. 2, B). When it has reached the front end of the nucleus, its posterior end is very dense to electrons (fig. 5,A).

Nebenkern

The Nebenkern of the living cell, like the separate mitochondria of earlier stages, coloured strongly with dahlia and Janus green and black. Like the mitochondria it was coloured red by acid fuchsine in Metzner’ technique.

It was darkened by osmium in the ‘Golgi techniques’, and with silver in the Aoyama technique. It reacted positively to Sudan black and feebly positive with acid haematein and PAS even after salivary extraction. The Nebenkern was feebly basiphil with basic fuchsine even after treatment with chromic acid.

The young Nebenkern, which, as we have seen, is formed by the aggregation of all the mitochondria in the cell, still retains something of the internal structure of the mitochondria (figs. 2, B; 4, A-C). Double membranes (‘cristae’) are seen within it, and some of them are clearly seen to extend to the wall of the organelle (fig. 4, A). Some of the electron micrographs show the Nebenkern in the act of dividing (figs. 4, B, C). The cristae are still present at this stage.

Fig. 5, C shows, in transverse section, the thin filaments into which the Nebenkern spins out at a later stage.

Axial filament

The electron micrographs show that the structure is the usual one for flagella and cilia: that is to say, there are two fibres near the centre and nine near the periphery (figs. 2, B; 5, c; 6, A). In one micrograph of the early stage (fig. 2, B) the eleven threads are clearly surrounded by a membrane. There is some indication that the fibres may be hollow.

Nucleus

The nucleus of the young spermatid (figs. 2, A, B; 3; 4, A-C) shows the granular structure usual in electron micrographs. The granules appear to be uniformly distributed. Some of the sections of the nuclei show a rounded electron-dense object, which may be the X-chromosome or possibly the nucleolus. The nucleus is surrounded by a double membrane (figs. 2, A; 4, B).

When the nucleus begins to elongate, the outer part of it becomes electrondense (figs. 5, A, B). When the nucleus is greatly elongated in the late spermatid, an unusual radial structure appears in its outer part. This radial structure is well seen in a transverse section through the nucleus (fig. 6, A). At a still later stage the radial structure still appears in longitudinal sections of the nucleus (fig. 5, B). It is difficult to reconcile the appearance given in transverse and longitudinal sections. Longitudinal sections suggest the presence of transverse partitions extending into the interior from the nuclear membrane. The radial structure is not always seen in the nucleus of the late spermatid (fig. 6, B).

FIG. 6.

(plate). A, spermatid, late stage. Transverse section of nucleus showing curious structure. Axial filament (f); filaments (nk) into which Nebenkern spins out. B, spermatid, late stage. Cytoplasmic covering (c).

FIG. 6.

(plate). A, spermatid, late stage. Transverse section of nucleus showing curious structure. Axial filament (f); filaments (nk) into which Nebenkern spins out. B, spermatid, late stage. Cytoplasmic covering (c).

The attachment of the tail to the nucleus is shown in fig. 5, D. The nucleus is indented at the attachment and overhangs the base of the tail much further on one side than on the other.

The nucleus is enclosed by a thin cytoplasmic covering (fig. 6, B).

In an electron microscope study of the male germ-cells of the cricket, Nemobius, Beams and others (1956) found that the acroblast consisted of double-membraned lamellae and constantly associated vesicles (externum) and an internal vacuolated ground substance (internum).

Although our study is concerned with the acroblast of a different species (Acheta), a strong correspondence exists between the structure of this cellular inclusion and that of the species mentioned above. Both exhibit a somewhat comparable shape in the early spermatid stage; both consist of parallel double-membraned lamellae and an internal finely vesicular region.

However, the number of lamellae in Nemobius is higher than in Acheta and their arrangement is more regular. In the former species, the external vacuoles are larger than those shown by Acheta, in which species they may be lacking.

Beams and others suggest that the lamellar region in Nemobius is probably comparable to the osmiophil region and the inner vacuolar area to the osmiophobe region of classical description. We have shown in Acheta that this suggestion has largely been verified by a combination of electron microscopy, histochemistry, and the so-called classical * Golgi techniques* all carried out on the same tissue.

It is a pleasure to record our thanks to Professor M. M. Swann for his helpful discussions, and to Dr. J. R. Baker for supervising this work and his kind assistance in writing this paper. We also wish to thank Professor Sir A. C. Hardy, F.R.S., in whose department the histochemical work was done.

Dr. B.-P. Clayton and Dr. K. Deutsch would also like to express their appreciation of the financial help for this investigation from a grant from the Melville Trust for Cancer Research. The electron micrographs were printed by Mr. R. A. Fox, of the Department of Zoology, Edinburgh University.

Aoyama
,
F.
,
1929
.
Z. wiss. Mikr
.,
46
,
489
.
Baker
,
J. R.
,
1944
.
Quart. J. micr. Sci
.,
85
,
1
.
Baker
,
J. R.
,
1946
.
Ibid
.,
87
,
441
.
Baker
,
J. R.
,
1947
.
Ibid
.,
88
,
115
.
Baker
,
J. R.
,
1949
.
Ibid
.,
90
,
293
.
Baker
,
J. R.
,
1956
.
Ibid
.,
97
,
621
.
Baker
,
J. R.
,
1957
.
Ibid
, (in press).
Bauer
,
H.
,
1933
.
Jahrb. Morph, mikr. Anat., 2 Abt
.,
33
,
143
,
Beams
,
H. W.
,
Tahmisian
,
T. N.
,
Devine
,
R. L.
, and
Anderson
,
E.
,
1956
.
J. biophys. bio-chem. Cytol
.,
2
, no.
4
, suppl.,
123
.
Cain
,
A. J.
,
1947
.
Quart. J. micr. Sei
.,
88
,
383
.
Feulgen
,
R.t
and
Rossenbeck
,
H.
,
1924
.
Z. phys. Chem
.,
135
,
203
.
Herxheimer
,
G. W.
,
1901
.
Deut. med. Woch
.,
36
,
607
.
Jordan
,
B. M.
, and
Baker
,
J. R.
,
1955
.
Quart. J. micr. Sci
.,
96
,
177
.
Jordan
,
B. M.
, and
Baker
,
J. R.
,
1956
.
Entomol
.,
89
,
126
.
Kolatchev
,
A.
,
1916
.
Arch, russes d*anat. d’hist. et d’emb
.
1
,
383
.
Lison
,
L.
,
1953
.
Histochimie et cytochimie animates
.
Paris
(
Gauthier-Vi 11 ars
).
Metzner
,
R.
, and
Krause
,
R.
,
1928
.
In Abderhalden’s Handbuch der biologischen Arbeits-methoden, Abt. v, 2
:
1
,
325
.
Nath
,
V.
, and
Singh Bhimber
,
B.
,
1953
.
Res. Bull. East Punjab Univ
.,
37
,
145
.
Palade
,
G. E.
,
1956
.
J. biophys. biochem. Cytol
.,
2
, no.
4
, suppl.,
85
.
Pearse
,
A. G. E.
,
1954
.
Histochemistry, theoretical and applied
.
London
(
Churchill
).
Weigl
,
R.
,
1910
.
Bull, internat. Acad. Sci. Cracovie, Sér. B
. (no vol. no.),
691
.

APPENDIX

A summary of the histochemistry of the spermatid of the house-cricket, Acheta domesticus