- Email: [email protected]
Electron microscope studies of spermateleosis in Fasciola hepatica L
1i
Experirneninl Cell Reseurch 22, l-8 (19611
ELECTRON
MICROSCOPE
STUDIES
IN FASCIOLA R. -3. R. GRESSON Department
OF SPERMATELEOSIS
HEP-ATICA
and MARGARET
L. 31. PERRk
of Zoology, The Queen’s University of Belfast, -Northern Ireland, Animal Gmetics: The University of Edinburgh, Scotland Received
and the Institute
of
January 11, 1960
to their slender nature and lack of markecl structural features visible under a light microscope, there is considerable disagreement regarding the morphology of the spermatozoa of trematodes. ,A number of workers beliere that the sperms of these animals are n+olly nuclear in origin, while others conclude that the cytoplasm of the sperrnatids contribute to their formation, Yosufzai [30] considers that the sperm of Fnsciolrr hepticct consists of a head, a middle piece, and a tail region. ?tlore recently Gresson [I41 claims that it consists of an elongate nucleus and a flagellum, and that fnrther details of its structure are beyond the limits of resolution of the light and phase contrast microsc,opes. The present invest&ation was undertaken in the hope that work with an electron microscope might reveal in greater detail the stages of spermformation and the structure of the ripe spermatozoon, hm~
MATERIAL
AND
METHODS
Living flukes (F. hepcctica) were removed from the livers of sheep and dissected in 1. per cent osmium tetroxide buffered with veronal to a pE-I of 7.4. Small pieces of the testes were quickly transferred to tubes containing the fixing agent. The tissue was embedded in a mixture of 85 per cent n-butyl and 15 per cent methyl methacrylate with 1 per cent benzoyl peroxide as a catalyst. Polymerisation was carried out at WC. In order to obtain further information on the structure of the ripe spermatozouu, small pieces of the seminal vesicle were prepared in the same manner as the testes, Sections were cut with a thermal-advance type microtome, mounted on formuarcoated girds and examined with a Siemens Elmiskop I. OBSERVATIONS Secondmy
I-
6017325s
While our work is concerned primarily n,ith of the spermatid and the spermatozoon, certain features of
spermatocytes.-
the fine structure
Ercperimental Crli Research 22
R. A. R. Gresson and Maryaref M. Perry
2
the secondary spermatocyte are worthy of brief mention. The secondary spermatocytes are irregular in shape and possess round or oval nuclei (Fig. 1). The latter contain dense granules distributed throughout the nucleoplasm and a number of small areas devoid of dark material. At least one nucleolus, composed of granules which lie in a material of lesser density, is present. In addition smaller dark bodies, similar in appearance to a nucleolus, occur throughout the nucleus. At high magnifications the nuclear membrane is seen to be composed of two dense components separated by a narrow clear region; it frequently appears to be interrupted by narrow openings or pores. The plasma membrane consists of two dense components separated by a region of considerably less density. It possesses a number of inraginations extending into the cell. blitochondria, in the form of short rods that are sometimes wrved, are scattered through the cytoplasm. Their external and internal membranes are poorly defined and the latter are irregularly spaced and seldom extend completely across the interior of a mitochondrion. Elements of the Golgi complex are not visible in our electron micrographs. Spermat&-It is convenient to divide the history of the spermatid into four stages. Stage 1: This is a young cell possessing a nucleus, nuclear membrane and plasma membrane closely similar in a1)pearanc.e to those of a secondary spermatoc.yte (Fig. a). The majority of the mitochondria are situated in close proximity to the nucleus but, occasionally, a fern may occur at some distance from the latter. Structures identified as elements of the Golgi complex are sometimes visible but are not sufficiently clearly shown to allow a study of their morphology. These elements are, however, clearly shown in electron micrographs of older spermatids and are described below.
Fig. l.-Two
secondary
spermatocgtes.
Fig. 2.-Spermatid,
stage 1. x 7,500.
Fig. 3.-Spermatid,
stage 2. x 22,000.
s 7,800.
Fig. 4.-Stage 2. The nucleus is undergoing elongation and is extending towards the proximal end of the cell. The thickened plasma membrane at the distal end of the cell is visible. On the right a part of the nucleus of a stage 4 spermatid is shown. x 7,800. Fig. 5.-Stage
2. Distal
Fig. 6.-Stage
2. To show Golgi saccules and the two axial filaments
end of slightly
older cell.
x 13,000.
Fig. 7.-Stage 3. To show parts of the nucleus of a spermatid section. x 20,000. Ezperimentnl
Cell Research 22
of an older cell.
cut in transverse
x 36,000.
and longitudinal
Fig. S.-Transverse sections of flagella to show the two axial filaments and the sheath. x 11,700. Fig. S.---To show terminal parts of axial filaments not surrounded by a sheath. x 13,000. Fig. IO.-Stage 4. To show a small part of the nucleus of a sperm. x 20,000. Fig. Il.-Stage 4. Part of the head of a sperm to show the sheaths. The two clear areas towards the top of the mierograph may he artifacts, but these serve to show the two sheaths that surround the head of the sperm. x 24,000. G, Golgi saceufes; I, in~a~na~~on of plasma membrane; X, mitoc~ondria; IV, nucleus; P, plasma membrane; TP, thickened plasma membrane at distal end of cell. Ezperimenial
Cell Research 22
4
R. A. R. Gresson and hdargaret hl. Perry
Stage 2: The cell and its nucleus soon undergo a process of elongation so that the distal (posterior) ends of both become narrow. The nucleus is at first situated distally, but later extends towards the proximal end of the cell. During the early phases of the elongation of a spermatid, the nuclear granules become more conspicuous and gradually are arranged in alignment to form thread-like or filamentous structures. ,4 nuclear membrane consisting of two dense components, visible in electron micrographs of younger spermatids, is no longer present. It now appears to be made up of a single row of dark granules resembling in density the nuclear granules described above. The membrane is not continuous but is interrupted at intervals by the presence of openings or pores (Figs. 3 and 4). Soon, some of the nuclear filaments come to lie more or less parallel to the long axis of the cell (Fig. 5). The nucleolus and some of the smaller granular bodies present in the early spermatid persist for some time. hlitochondria are numerous and are situated chiefly in the proximal (anterior) and middle regions of the cell. Many have increased in size and their internal membranes are often more clearly visible than during the preceding stage (Figs. 3 and 4). Elements of the Golgi complex are frequently shown and consist of bundles of saccules lying in a material of slightly higher density than the surrounding cytoplasm (Fig. 6). Usually one bundle only is visible in a section of a cell, but in rare instances at least two are present. Our electron micrographs show the saccules in both longitudinal and transverse section, the latter indicating that they are roughly c.ircular in outline. The internal diameter of the ends of the sac.cules is slightly greater than that of their other regions. As large vacuoles and minute vesic.les are absent, we conc.lude that the Golgi complex is represented by bundles of saccules alone and is, therefore, unlike that of the cells of vertebrates and of many invertebrate animals. The plasma membrane is composed of an external and an internal dense component separated by a clear area that is wider than that of the membrane of a younger spermatid. Invaginations of the inner dense component extend for some distance into the cell and sometimes reach the neighbourhood of the nucleus. The two dense components of the part of the plasma membrane surrounding the narrow distal region of the spermatid is thicker and much darker than elsewhere (Figs. 4 and 5), and the cytoplasm of the distal region is slightly less dense than that of the middle and proximal regions. At this stage axial filaments, of which there are two in eac.h spermatid, are frequently visible lying between the posterior end of the nucleus and the distal extremity of the cell (Fig. 6). Erperimental
Cell Research 22
Spermateleosis in Fasciola hepatica
5
Stage 3: The nucleus is now a long thread-like, curved and twisted structure that extends almost throughout the length of the cell. In a longitudinal section of a nucleus the dense material resembles in appearance a number of filaments that often run obliquely across the nucleus (Fig. 7). In oblique and transverse sections the filaments are seen as a network enclosing areas of various sizes and of low density (Fig. 5). We conclude that, at this stage, the dense material of the nucleus is in the form of thin sheets or lamellae and that these are folded into scroll-like structures that frequently c.ome into contact with one another, so that in transverse and oblique sections they give the appearance of a network. At frequent intervals the sheets are in contact or continuous with the nuclear membrane which they resemble in general appearance. Occasionally openings or pores are visible in the nuclear mcmbrane. The mitochondria are scattered through the cell except at its distal end, and, as in the preceding stage, are often xTery large. Bundles of Golgi saccules are present in the ytoplasm. The plasma membrane possesses the same structural features as in Stage 2. Invaginations of its inner dense component arcmore conspicuous than at an earlier stage of spermatogenesis, and frequently extend for a considerable distance into the cytoplasm (Fig. i). ,-5 spermatid possesses two axial filaments each of which is composed of two central fibrils and nine peripheral fibrils. In many cases a small fibrillike structure is visible lying between the two axial filaments (Fig. ii). 1Ye propose to call this structure the middle fibril. Throughout the greater parts of their lengths the axial filaments and the middle fibril are surrounded bp a sheath composed of dense granules enclosing an area that varies in diameter. -1 flagellum terminates in two axial filaments devoid of an external sheath, but united by a central strand or fibril. It may be concluded that the posterior end of a spermatid elongates to form a structure containing two axial fiiaments and a middle fibril surrounded by a sheath. This part of the c.ell becomes narrow distally and finally the axial filaments extend beyond the sheath where they are united by a structure continuous with the middle fibril (Figs. 8 and 9). Stage 4: The nuclear sheets become less clearly defined and are converted into short segments and granules. The nuclear membrane is now very dense and much thicker and more homogeneous than during Stage 3 (Figs. 4 and IO). It is in contact with an external sheath that forms the outer covering of the sperm-head (Fig. 11). The flagellum is closely similar to that of the slightl! younger cell. This stage includes the final phases of the metamorphosis of a spermatid into a spermatozoon. Expcrimentoi
Ceti Research 22
6
R. A. R. Gresson and Margaret
M. Perry
Spermatozoa.-We attempted to study the structure of the spermatozoon in sections of the seminal vesicle. This material did not give as good results as the testes. However, examination of elec.tron micrographs confirms our conclusions regarding the structure of the mature spermatozoon. The head of the ripe sperm is surrounded by two membranous structures or sheaths. We consider that the inner sheath is derived from the nwlear membrane and the outer one in part, at least, from the plasma membrane of the late spermatid. The region of the flagellum provided with a sheath is shown in most of our electron micrographs of the seminal vesicle and, as in the later stages of the spermatid, is provided with a middle fibril and two axial filaments. DISCUSSION
The present study confirms Gresson’s conclusions [14], based on work with light and phase contrast microscopes, that the spermatozoon of F. hepatica consists of an elongate head and a flagellum. M’e found no evidence that mitochondria or elements of the Golgi complex enter into the composition of the flagellum, or that the sperm-tail possesses a region comparable to the middle-piece of a mammalian sperm. Dhingra [5] reports that the sperm of the digenetic trematode, Isoparorchis eurytremum, is provided with two short free flagella that arise from a centriosome and extend for a relatively short distance beyond the posterior pole of the nucleus. He also studied spermateleosis in the digenetic trematodes, Cyclocoelium bivesiculatum [6], Cotylphoron elongatum [7], and Gastrothylax crumenifer [8], and c.laims that the spermatozoa of these animals possess a single flagellum and centrosome. It would seem, therefore, that there is wide variation in the structure of the sperms of digenetic trematodes. In general the structure of each axial filament of F. hepatica follows the usual pattern for those of flagella. The presence of two axial filaments, a middle fibril, and a connecting strand or fibril between the axial filaments at the distal extremity of the flagellum appear to be features not previously recorded. Arms and spoles similar to those observed by Afzelius [l] in association with the fibrils of the flagellum of the sperm of Psammechinus miliaris were not identified. Afzelius, however, used a new method of fixation to demonstrate these structures. It is probable that the thickened region of the plasma membrane that surrounds the narrow distal region of a stage 2 spermatid gives rise to the external sheath of the flagellum. Although a dense body was occasionally observed in the vicinity of the distal pole of the nucleus of a spermatid, it could not be identified with certainty as a centrosome. Experimental
Cell Research 22
Spermuteleosis in Fascioln hepatica
7
The exact arrangement of the dense nuclear material of a stage 3 spermatid is difficult to determine. Somewhat similar appearances hare been observed in the nuclei of the spermatids of molluscs [13, 15, 161, grasshoppers [4, 9, Ii, IS, 191, Loczzsfra Ill], and Drosophila [18]. Rebhun [16] interprets the filament-like nuclear structures in the spermatids of Atala lacfea as sheets or plates, and Gibbons and Bradfield [l l] believe that in Loczzsfra migraforin “the chromatin becomes formed into sheets and then into t.ubes running parallel with the long axis” of the nucleus. In sections of the spermatid nucleus of F. hepafica appearances sometimes su,, “Oest that the dense material is in the form of tubes, but after careful study we conclude that the nuclear sheeks do not form tubes but are folded into scroll-like structures. It is worthy of note that the Golgi elements or dictyosomes of the classical techniques are represented by bundles of sac&es alone. According to Dalton and Felix [3] large Golgi vacuoles are not associated with the saccules of the spermatids of Lzzmbricus ferresfris and Helix asperscz. They show that small vesicles are present in the neighbourhood of the saccules and state that there is evidence of the budding of the former from the saccules. hIore recently Gatenby and Dalton [lo] claim that the dictyosome of the spermatid of Lurnbriczrs herculezrs consists of eight to twelve flattened sacs. Associated scattered resicles, they believe, are “probably of the same nature as the lamellae, sinc.e they show a constant and intimate relationship to them”. Large vacuoles are absent. Gras&, Carasso and Farard [13j and Gras& and Carasso 1121 consider that small osmiophiiic vesicles associated with Golgi saccules originate from the latter. In our electron micrographs small vesicle-like structures are often seen at the ends and in the immediate vicinity of the bundles of Golgi saccules. As these are fen- in number and are nearl) always in close proximity to the ends of the saccules, we suggest that the: represent sections through the slightly expanded ends of the saccules. A. Golgi remnant was not seen in our micrographs of late spermatids [2]. Invaginations of the plasma membrane have been obsewed in many types of cells. As the outer sheath surrounding the sperm head of F. hepaficcr probably arises, in part at least, from the plasma membrane, it is likel:; that the inraginations of the inner component of the latter are concerned with the formation of the sheath. It is also possible that the invaginations may provide internal surfaces for metabolic processes accompanying spermateleosis. Elements of the endoplasmic reticulum are few in the spermatocytes and spermatids of F. hepafica.
Experimental
Cell Research 22
R. A. R. Gresson and Margaret Al. Perry
8
SUMMARY and the stages of spermateleosis of Fasciola The Golgi complex of the spermatid is represented by bundles of saccules unaccompanied by large vacuoles. hlitochondria are numerous. The nuclear membrane of the spermatid consists at first of two dense components separated by a region of low density. Later, it seems to possess a granular structure. Pores are present As the spermatid elongates, the dense nuclear in the nuclear membrane. granules give rise to sheets or plates that are folded into scroll-like structures. Some of the sheets are in contact with the nuclear membrane. The nucleus of the spermatozoon is composed of dense granules and rod-like bodies lying in a material of low density. It is surrounded by two sheaths. The spermatozoon consists of a head and a flagellum. The latter consists of proximal and distal regions. The proximal part contains two axial filaments each composed of two central and nine peripheral fibrils. A middle fibril lies between the two axial filaments. An external sheath, probably derived from the plasma membrane, is present. The distal region is devoid of an external sheath and consists of two axial filaments joined by a structure continuous with the middle fibril. There is no evidence that mitochondria or Golgi saccules take part in the formation of the spermatozoon. The secondary
spermatocytes
hepatica were studied by means of electron microscopy.
We wish to thank Professor C. H. Waddington time and facilities to undertake this work.
for giving one of us (M. M. P.)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
AFZELIUS, B., J. Biophys. Biochem. Cytol. 5, 269 (1959). CLERMONT, Y., ibid. 2, No. 4, suppl. 119 (1956). DALTON, i\. J. and FELIX, M. D., ibid. 2, 79 (1956). Dass, C. M. S. and RIS, H., ibid. 4, 129 (1958). DHINGRA, 0. P., Res. Bull. Punjab Lrnio. 44, 21 (1954). __ ibid. 61, 159 (1954). __ ibid. 64, 1 (1955). __ ibid. 65, 11 (1955). GALL, J. G. and BJBRK, L. B., J. Biophys. Biochem. Cyfol. 4, 478 (1958). G~TENBY, J. B. and DALTON, R. J., ibid. 6, 45 (1959). GIBBONS, J. R. and BRADFIELD, J. R. G., ibid. 3,153 (1957). GR.~ssI?, P. P. and C.~R.~SSO,N., Nature 179, 31 (1957). GRAS&, P. P., C~RASSO, N. and FAvaRD, P., ilnn. sci. Nat. 18, 338 (1956). GRESSON, R. X. R., Quart. J. microscop. Set. 98, 493 (1957). KAYE, J. S., J. Morphol. 103, 311 (1958). REBHUN, L. I., J. Biophys. Biochem. Cytcl. 3, 509 (1957). Y.~SUZIJMI, G., ibid. 3, 663 (1957). Yasuzunrr, G., FUGIMURI, W. and ISHIDA, H., Exptl. Cell Research 14, 268 (1958). Y~suzur.~r, G. and ISHID~, H., J. Biophys. Biochem. Cytot. 3, 663 (1957). YOSUFZAI, H. K.,La Cell& 55, 7 (1952).
Experimental
Cell Reseurch 22
Experirneninl Cell Reseurch 22, l-8 (19611
ELECTRON
MICROSCOPE
STUDIES
IN FASCIOLA R. -3. R. GRESSON Department
OF SPERMATELEOSIS
HEP-ATICA
and MARGARET
L. 31. PERRk
of Zoology, The Queen’s University of Belfast, -Northern Ireland, Animal Gmetics: The University of Edinburgh, Scotland Received
and the Institute
of
January 11, 1960
to their slender nature and lack of markecl structural features visible under a light microscope, there is considerable disagreement regarding the morphology of the spermatozoa of trematodes. ,A number of workers beliere that the sperms of these animals are n+olly nuclear in origin, while others conclude that the cytoplasm of the sperrnatids contribute to their formation, Yosufzai [30] considers that the sperm of Fnsciolrr hepticct consists of a head, a middle piece, and a tail region. ?tlore recently Gresson [I41 claims that it consists of an elongate nucleus and a flagellum, and that fnrther details of its structure are beyond the limits of resolution of the light and phase contrast microsc,opes. The present invest&ation was undertaken in the hope that work with an electron microscope might reveal in greater detail the stages of spermformation and the structure of the ripe spermatozoon, hm~
MATERIAL
AND
METHODS
Living flukes (F. hepcctica) were removed from the livers of sheep and dissected in 1. per cent osmium tetroxide buffered with veronal to a pE-I of 7.4. Small pieces of the testes were quickly transferred to tubes containing the fixing agent. The tissue was embedded in a mixture of 85 per cent n-butyl and 15 per cent methyl methacrylate with 1 per cent benzoyl peroxide as a catalyst. Polymerisation was carried out at WC. In order to obtain further information on the structure of the ripe spermatozouu, small pieces of the seminal vesicle were prepared in the same manner as the testes, Sections were cut with a thermal-advance type microtome, mounted on formuarcoated girds and examined with a Siemens Elmiskop I. OBSERVATIONS Secondmy
I-
6017325s
While our work is concerned primarily n,ith of the spermatid and the spermatozoon, certain features of
spermatocytes.-
the fine structure
Ercperimental Crli Research 22
R. A. R. Gresson and Maryaref M. Perry
2
the secondary spermatocyte are worthy of brief mention. The secondary spermatocytes are irregular in shape and possess round or oval nuclei (Fig. 1). The latter contain dense granules distributed throughout the nucleoplasm and a number of small areas devoid of dark material. At least one nucleolus, composed of granules which lie in a material of lesser density, is present. In addition smaller dark bodies, similar in appearance to a nucleolus, occur throughout the nucleus. At high magnifications the nuclear membrane is seen to be composed of two dense components separated by a narrow clear region; it frequently appears to be interrupted by narrow openings or pores. The plasma membrane consists of two dense components separated by a region of considerably less density. It possesses a number of inraginations extending into the cell. blitochondria, in the form of short rods that are sometimes wrved, are scattered through the cytoplasm. Their external and internal membranes are poorly defined and the latter are irregularly spaced and seldom extend completely across the interior of a mitochondrion. Elements of the Golgi complex are not visible in our electron micrographs. Spermat&-It is convenient to divide the history of the spermatid into four stages. Stage 1: This is a young cell possessing a nucleus, nuclear membrane and plasma membrane closely similar in a1)pearanc.e to those of a secondary spermatoc.yte (Fig. a). The majority of the mitochondria are situated in close proximity to the nucleus but, occasionally, a fern may occur at some distance from the latter. Structures identified as elements of the Golgi complex are sometimes visible but are not sufficiently clearly shown to allow a study of their morphology. These elements are, however, clearly shown in electron micrographs of older spermatids and are described below.
Fig. l.-Two
secondary
spermatocgtes.
Fig. 2.-Spermatid,
stage 1. x 7,500.
Fig. 3.-Spermatid,
stage 2. x 22,000.
s 7,800.
Fig. 4.-Stage 2. The nucleus is undergoing elongation and is extending towards the proximal end of the cell. The thickened plasma membrane at the distal end of the cell is visible. On the right a part of the nucleus of a stage 4 spermatid is shown. x 7,800. Fig. 5.-Stage
2. Distal
Fig. 6.-Stage
2. To show Golgi saccules and the two axial filaments
end of slightly
older cell.
x 13,000.
Fig. 7.-Stage 3. To show parts of the nucleus of a spermatid section. x 20,000. Ezperimentnl
Cell Research 22
of an older cell.
cut in transverse
x 36,000.
and longitudinal
Fig. S.-Transverse sections of flagella to show the two axial filaments and the sheath. x 11,700. Fig. S.---To show terminal parts of axial filaments not surrounded by a sheath. x 13,000. Fig. IO.-Stage 4. To show a small part of the nucleus of a sperm. x 20,000. Fig. Il.-Stage 4. Part of the head of a sperm to show the sheaths. The two clear areas towards the top of the mierograph may he artifacts, but these serve to show the two sheaths that surround the head of the sperm. x 24,000. G, Golgi saceufes; I, in~a~na~~on of plasma membrane; X, mitoc~ondria; IV, nucleus; P, plasma membrane; TP, thickened plasma membrane at distal end of cell. Ezperimenial
Cell Research 22
4
R. A. R. Gresson and hdargaret hl. Perry
Stage 2: The cell and its nucleus soon undergo a process of elongation so that the distal (posterior) ends of both become narrow. The nucleus is at first situated distally, but later extends towards the proximal end of the cell. During the early phases of the elongation of a spermatid, the nuclear granules become more conspicuous and gradually are arranged in alignment to form thread-like or filamentous structures. ,4 nuclear membrane consisting of two dense components, visible in electron micrographs of younger spermatids, is no longer present. It now appears to be made up of a single row of dark granules resembling in density the nuclear granules described above. The membrane is not continuous but is interrupted at intervals by the presence of openings or pores (Figs. 3 and 4). Soon, some of the nuclear filaments come to lie more or less parallel to the long axis of the cell (Fig. 5). The nucleolus and some of the smaller granular bodies present in the early spermatid persist for some time. hlitochondria are numerous and are situated chiefly in the proximal (anterior) and middle regions of the cell. Many have increased in size and their internal membranes are often more clearly visible than during the preceding stage (Figs. 3 and 4). Elements of the Golgi complex are frequently shown and consist of bundles of saccules lying in a material of slightly higher density than the surrounding cytoplasm (Fig. 6). Usually one bundle only is visible in a section of a cell, but in rare instances at least two are present. Our electron micrographs show the saccules in both longitudinal and transverse section, the latter indicating that they are roughly c.ircular in outline. The internal diameter of the ends of the sac.cules is slightly greater than that of their other regions. As large vacuoles and minute vesic.les are absent, we conc.lude that the Golgi complex is represented by bundles of saccules alone and is, therefore, unlike that of the cells of vertebrates and of many invertebrate animals. The plasma membrane is composed of an external and an internal dense component separated by a clear area that is wider than that of the membrane of a younger spermatid. Invaginations of the inner dense component extend for some distance into the cell and sometimes reach the neighbourhood of the nucleus. The two dense components of the part of the plasma membrane surrounding the narrow distal region of the spermatid is thicker and much darker than elsewhere (Figs. 4 and 5), and the cytoplasm of the distal region is slightly less dense than that of the middle and proximal regions. At this stage axial filaments, of which there are two in eac.h spermatid, are frequently visible lying between the posterior end of the nucleus and the distal extremity of the cell (Fig. 6). Erperimental
Cell Research 22
Spermateleosis in Fasciola hepatica
5
Stage 3: The nucleus is now a long thread-like, curved and twisted structure that extends almost throughout the length of the cell. In a longitudinal section of a nucleus the dense material resembles in appearance a number of filaments that often run obliquely across the nucleus (Fig. 7). In oblique and transverse sections the filaments are seen as a network enclosing areas of various sizes and of low density (Fig. 5). We conclude that, at this stage, the dense material of the nucleus is in the form of thin sheets or lamellae and that these are folded into scroll-like structures that frequently c.ome into contact with one another, so that in transverse and oblique sections they give the appearance of a network. At frequent intervals the sheets are in contact or continuous with the nuclear membrane which they resemble in general appearance. Occasionally openings or pores are visible in the nuclear mcmbrane. The mitochondria are scattered through the cell except at its distal end, and, as in the preceding stage, are often xTery large. Bundles of Golgi saccules are present in the ytoplasm. The plasma membrane possesses the same structural features as in Stage 2. Invaginations of its inner dense component arcmore conspicuous than at an earlier stage of spermatogenesis, and frequently extend for a considerable distance into the cytoplasm (Fig. i). ,-5 spermatid possesses two axial filaments each of which is composed of two central fibrils and nine peripheral fibrils. In many cases a small fibrillike structure is visible lying between the two axial filaments (Fig. ii). 1Ye propose to call this structure the middle fibril. Throughout the greater parts of their lengths the axial filaments and the middle fibril are surrounded bp a sheath composed of dense granules enclosing an area that varies in diameter. -1 flagellum terminates in two axial filaments devoid of an external sheath, but united by a central strand or fibril. It may be concluded that the posterior end of a spermatid elongates to form a structure containing two axial fiiaments and a middle fibril surrounded by a sheath. This part of the c.ell becomes narrow distally and finally the axial filaments extend beyond the sheath where they are united by a structure continuous with the middle fibril (Figs. 8 and 9). Stage 4: The nuclear sheets become less clearly defined and are converted into short segments and granules. The nuclear membrane is now very dense and much thicker and more homogeneous than during Stage 3 (Figs. 4 and IO). It is in contact with an external sheath that forms the outer covering of the sperm-head (Fig. 11). The flagellum is closely similar to that of the slightl! younger cell. This stage includes the final phases of the metamorphosis of a spermatid into a spermatozoon. Expcrimentoi
Ceti Research 22
6
R. A. R. Gresson and Margaret
M. Perry
Spermatozoa.-We attempted to study the structure of the spermatozoon in sections of the seminal vesicle. This material did not give as good results as the testes. However, examination of elec.tron micrographs confirms our conclusions regarding the structure of the mature spermatozoon. The head of the ripe sperm is surrounded by two membranous structures or sheaths. We consider that the inner sheath is derived from the nwlear membrane and the outer one in part, at least, from the plasma membrane of the late spermatid. The region of the flagellum provided with a sheath is shown in most of our electron micrographs of the seminal vesicle and, as in the later stages of the spermatid, is provided with a middle fibril and two axial filaments. DISCUSSION
The present study confirms Gresson’s conclusions [14], based on work with light and phase contrast microscopes, that the spermatozoon of F. hepatica consists of an elongate head and a flagellum. M’e found no evidence that mitochondria or elements of the Golgi complex enter into the composition of the flagellum, or that the sperm-tail possesses a region comparable to the middle-piece of a mammalian sperm. Dhingra [5] reports that the sperm of the digenetic trematode, Isoparorchis eurytremum, is provided with two short free flagella that arise from a centriosome and extend for a relatively short distance beyond the posterior pole of the nucleus. He also studied spermateleosis in the digenetic trematodes, Cyclocoelium bivesiculatum [6], Cotylphoron elongatum [7], and Gastrothylax crumenifer [8], and c.laims that the spermatozoa of these animals possess a single flagellum and centrosome. It would seem, therefore, that there is wide variation in the structure of the sperms of digenetic trematodes. In general the structure of each axial filament of F. hepatica follows the usual pattern for those of flagella. The presence of two axial filaments, a middle fibril, and a connecting strand or fibril between the axial filaments at the distal extremity of the flagellum appear to be features not previously recorded. Arms and spoles similar to those observed by Afzelius [l] in association with the fibrils of the flagellum of the sperm of Psammechinus miliaris were not identified. Afzelius, however, used a new method of fixation to demonstrate these structures. It is probable that the thickened region of the plasma membrane that surrounds the narrow distal region of a stage 2 spermatid gives rise to the external sheath of the flagellum. Although a dense body was occasionally observed in the vicinity of the distal pole of the nucleus of a spermatid, it could not be identified with certainty as a centrosome. Experimental
Cell Research 22
Spermuteleosis in Fascioln hepatica
7
The exact arrangement of the dense nuclear material of a stage 3 spermatid is difficult to determine. Somewhat similar appearances hare been observed in the nuclei of the spermatids of molluscs [13, 15, 161, grasshoppers [4, 9, Ii, IS, 191, Loczzsfra Ill], and Drosophila [18]. Rebhun [16] interprets the filament-like nuclear structures in the spermatids of Atala lacfea as sheets or plates, and Gibbons and Bradfield [l l] believe that in Loczzsfra migraforin “the chromatin becomes formed into sheets and then into t.ubes running parallel with the long axis” of the nucleus. In sections of the spermatid nucleus of F. hepafica appearances sometimes su,, “Oest that the dense material is in the form of tubes, but after careful study we conclude that the nuclear sheeks do not form tubes but are folded into scroll-like structures. It is worthy of note that the Golgi elements or dictyosomes of the classical techniques are represented by bundles of sac&es alone. According to Dalton and Felix [3] large Golgi vacuoles are not associated with the saccules of the spermatids of Lzzmbricus ferresfris and Helix asperscz. They show that small vesicles are present in the neighbourhood of the saccules and state that there is evidence of the budding of the former from the saccules. hIore recently Gatenby and Dalton [lo] claim that the dictyosome of the spermatid of Lurnbriczrs herculezrs consists of eight to twelve flattened sacs. Associated scattered resicles, they believe, are “probably of the same nature as the lamellae, sinc.e they show a constant and intimate relationship to them”. Large vacuoles are absent. Gras&, Carasso and Farard [13j and Gras& and Carasso 1121 consider that small osmiophiiic vesicles associated with Golgi saccules originate from the latter. In our electron micrographs small vesicle-like structures are often seen at the ends and in the immediate vicinity of the bundles of Golgi saccules. As these are fen- in number and are nearl) always in close proximity to the ends of the saccules, we suggest that the: represent sections through the slightly expanded ends of the saccules. A. Golgi remnant was not seen in our micrographs of late spermatids [2]. Invaginations of the plasma membrane have been obsewed in many types of cells. As the outer sheath surrounding the sperm head of F. hepaficcr probably arises, in part at least, from the plasma membrane, it is likel:; that the inraginations of the inner component of the latter are concerned with the formation of the sheath. It is also possible that the invaginations may provide internal surfaces for metabolic processes accompanying spermateleosis. Elements of the endoplasmic reticulum are few in the spermatocytes and spermatids of F. hepafica.
Experimental
Cell Research 22
R. A. R. Gresson and Margaret Al. Perry
8
SUMMARY and the stages of spermateleosis of Fasciola The Golgi complex of the spermatid is represented by bundles of saccules unaccompanied by large vacuoles. hlitochondria are numerous. The nuclear membrane of the spermatid consists at first of two dense components separated by a region of low density. Later, it seems to possess a granular structure. Pores are present As the spermatid elongates, the dense nuclear in the nuclear membrane. granules give rise to sheets or plates that are folded into scroll-like structures. Some of the sheets are in contact with the nuclear membrane. The nucleus of the spermatozoon is composed of dense granules and rod-like bodies lying in a material of low density. It is surrounded by two sheaths. The spermatozoon consists of a head and a flagellum. The latter consists of proximal and distal regions. The proximal part contains two axial filaments each composed of two central and nine peripheral fibrils. A middle fibril lies between the two axial filaments. An external sheath, probably derived from the plasma membrane, is present. The distal region is devoid of an external sheath and consists of two axial filaments joined by a structure continuous with the middle fibril. There is no evidence that mitochondria or Golgi saccules take part in the formation of the spermatozoon. The secondary
spermatocytes
hepatica were studied by means of electron microscopy.
We wish to thank Professor C. H. Waddington time and facilities to undertake this work.
for giving one of us (M. M. P.)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
AFZELIUS, B., J. Biophys. Biochem. Cytol. 5, 269 (1959). CLERMONT, Y., ibid. 2, No. 4, suppl. 119 (1956). DALTON, i\. J. and FELIX, M. D., ibid. 2, 79 (1956). Dass, C. M. S. and RIS, H., ibid. 4, 129 (1958). DHINGRA, 0. P., Res. Bull. Punjab Lrnio. 44, 21 (1954). __ ibid. 61, 159 (1954). __ ibid. 64, 1 (1955). __ ibid. 65, 11 (1955). GALL, J. G. and BJBRK, L. B., J. Biophys. Biochem. Cyfol. 4, 478 (1958). G~TENBY, J. B. and DALTON, R. J., ibid. 6, 45 (1959). GIBBONS, J. R. and BRADFIELD, J. R. G., ibid. 3,153 (1957). GR.~ssI?, P. P. and C.~R.~SSO,N., Nature 179, 31 (1957). GRAS&, P. P., C~RASSO, N. and FAvaRD, P., ilnn. sci. Nat. 18, 338 (1956). GRESSON, R. X. R., Quart. J. microscop. Set. 98, 493 (1957). KAYE, J. S., J. Morphol. 103, 311 (1958). REBHUN, L. I., J. Biophys. Biochem. Cytcl. 3, 509 (1957). Y.~SUZIJMI, G., ibid. 3, 663 (1957). Yasuzunrr, G., FUGIMURI, W. and ISHIDA, H., Exptl. Cell Research 14, 268 (1958). Y~suzur.~r, G. and ISHID~, H., J. Biophys. Biochem. Cytot. 3, 663 (1957). YOSUFZAI, H. K.,La Cell& 55, 7 (1952).
Experimental
Cell Reseurch 22