The ultrastructure of germ cell in foetas l and neonatal male rats · The ultrastructure of germ cell in foetas l and neonatal male rats by L. L. FRANCHI and ANITA M. MANDL From the

/ . Embryol. exp. Morph., Vol. 12, Part 2, pp. 289-308, June 1964Printed in Great Britain

The ultrastructure of germ cells in foetal andneonatal male rats

by L. L. FRANCHI and ANITA M. MANDL

From the Department of Anatomy, University of Birmingham

WITH EIGHT PLATES

INTRODUCTION

THE GONADS of the rat undergo sex differentiation on the 14th day post coitum(p.c), the testis becoming clearly distinguishable by the presence of an incipienttunica albuginea. The male germ cells become incorporated into medullarycords (the precursors of seminiferous tubules). In contrast, the germ cells inovaries are scattered in cortical nests.

Recent quantitative studies have shown that at 14- 5 days p.c, the number ofgerm cells is somewhat greater in the male than the female (Beaumont & Mandl,1963; cf. Beaumont & Mandl, 1962). In both sexes mitotic activity ceases atabout 18-5 days p.c Thereafter, the male germ cells remain at prolonged inter-phase; a proportion of them show histological changes frequently associatedwith degeneration. Quantitative estimates, on the other hand, indicate thatnone are eliminated from the testis. In the coeval female, the germ cells enterthe prophase of meiosis, whereafter no further mitotic divisions are possible.Due to several 'waves' of degeneration, the total number of germ cells fallsrapidly (Beaumont & Mandl, 1962, 1963; Franchi & Mandl, 1962).

At 4 to 5 days after birth, the germ cells of the male divide to give rise to thefirst generation of spermatogonia type-A. A cytological study suggests that theprimordial germ cells change their appearance shortly before renewal of mitoticactivity ('transitional' cells; see Beaumont & Mandl, 1963). Shortly before thereappearance of mitoses, a number of germ cells undergo lysis: the cytoplasmseemingly 'streams out' following the breakdown of the cell membrane, and thenucleus becomes highly distorted. The degenerating gonocytes are frequentlysituated in the centre of the seminiferous cords, while the 'transitional' cellstend to be more peripheral in distribution. The latter observation (see also

1 Authors' address: The Department of Anatomy, The Medical School, Birmingham 15,U.K.

290 L. L. FRANCHI and A. M. MANDL

Sapsford, 1962a; Huckins, 1963) suggests that proximity to the basementmembrane is somehow associated with the survival of the germ cell.

Primordial germ cells in the male rat have been shown to be remarkablysensitive to ionizing radiations. As judged by the percentage of normal,'regenerating' and sterile tubules 25 days after birth, sensitivity rises between13-5 and 17-5 days p.c. and remains at a high level throughout the period ofinterphase; with the renewal of mitotic activity, it declines (Beaumont, 1960,1962; Starkie, 1961; Hughes, 1962). These observations suggest that therelationship between mitotic activity and susceptibility to ionizing radiation isthe reverse of that observed in most other cells (see Mandl, 1964).

The present study was undertaken in order to answer the following questions:(1) Do male germ cells undergo ultrastructural changes during the prolonged

radiosensitive phase of mitotic inactivity? If so, can these be correlated withthe apparent' degenerative' changes observed in histological preparations ?

(2) Does the ultrastructure of mitotically active germ cells, shortly after sexdifferentiation, differ between the two sexes ?

(3) Is the renewal of mitotic activity, shortly after birth, associated withmarked changes in the ultrastructure of germ cells ?

(4) By what process do male germ cells undergo degeneration, and how arethey removed from the testis (cf. female: Franchi & Mandl, 1962)?

MATERIALS AND METHODS

Animals

Adult female rats of the Birmingham colony were housed with males, andthe time of mating determined by means of daily vaginal smears. The does werekilled at intervals ranging from 14 • 5 to 22 • 5 days p.c. Some of the testes werederived from foetuses whose litter-mates were used for previous studies offoetal gonads (Beaumont & Mandl, 1962, 1963; Franchi & Mandl, 1962).Neonatal male rats were killed at intervals between 1 and 7 days post partum(p.p.). Some of these were again litter-mates of specimens used for cytologicaland quantitative studies (Beaumont & Mandl, 1963). A total of seventy testeswas examined with the electron microscope.

Autopsy

The pregnant females were killed by means of chloroform vapour. Thefoetuses were rapidly dissected out and decapitated. Neonatal rats were killedeither by decapitation or by a sharp blow on the head.

Preparation of tissues

The gonads (together with their adnexa and fragments of mesonephroi inthe youngest specimens) were rapidly dissected out and placed at once in ice-

The ultrastructure of germ cells 291

cold fixative (1 per cent, osmic acid in Michaelis' veronal/acetate buffer atpH 7-6, containing a balanced salt solution with Na, Ca and K ions).The fixative used was recommended by Dr J. D. Robertson in a personalcommunication. It was prepared from the following solutions. Solution 1:Michaelis's Buffer solution, consisting of 19-428 g. sodium acetate; 29-428 g.sodium veronal dissolved in 500 ml. CO2-free distilled water. Solution 2: N/5hydrochloric acid. Solution 3: 100 ml. of solution containing 96-8 g. sodiumchloride per litre; 2-17 ml. of solution containing 3-42 g. calcium chloride perlitre; 1 • 73 ml. of solution containing 2 • 85 g. potassium chloride per litre. Finalfixative: 5 ml. sodium acetate buffer (Solution 1); 5 ml. N/5 hydrochloric acid(Solution 2); 13 ml. distilled water; 2 ml. salt solution (Solution 3). Then addequal parts (i.e. 25 ml.) 2 per cent. OsO4 in distilled water.

The gonads were fixed for 2-3 hr.; they were then rinsed in buffer anddehydrated in an ethanol series. At the 70 per cent, alcohol stage of dehydra-tion, non-gonadal tissues were removed as far as possible by careful trimmingunder a dissecting microscope. Some mesonephric tissue remained, however,in specimens aged 16-5 days p.c. or less; these were trimmed further afterembedding (see below). The tissues were further dehydrated (up to 100 per cent,alcohol); they were then stained in 0- 5 per cent, phosphotungstic acid in 100 percent, alcohol before being finally dehydrated in absolute acetone. The embed-ding medium employed was Vestopal W (see Ryter & Kellenberger, 1958). Theembedded tissues were sectioned on a Porter-Blum microtome. Silver, palegold and occasionally thicker sections were selected for examination under aSiemens Elmiskop I electron microscope.

Specimens aged 14-5 to 16-5 days p.c. could not be sexed accurately atautopsy. In order to determine the sex of the gonads, and to remove non-gonadal tissues, thick sections (1-2 n) were cut after embedding in Vestopal,mounted on microscope slides, and stained with 0 • 5 per cent, aqueous toluidineblue (see Franchi, 1963). The sections were examined under a light microscope,using both normal and phase contrast illumination. The embedded blockswere trimmed progressively by means of a razor blade, cutting away as muchnon-gonadal tissue as possible. The gonads were sexed by the usual histologicalcriteria (presence of tunica albuginea and formation of medullary cords in themale; random distribution of germinal and somatic elements in the female).

RESULTS

Distribution and general appearance of germ cells

At 14-5 days p.c. the majority of male germ cells are situated within theincipient cords in the inner region of the gonad; a few have been observedimmediately beneath the germinal epithelium. Somatic cells within the cords('supporting' cells) show no specific orientation with respect to the germ cells.


In the female, however, oogonia are scattered in rather ill-defined nests, fre-quently associated with somatic cells; the latter may be closely applied to thegerm cells.

The germ cells of both sexes are characterized by their large size, and byrounded nuclei with sharply defined membranes. In the female, some cells areirregular in shape, and extensions of the cytoplasm may pass between adjacentsomatic cells; in general, however, the germ cells are of simple ovoid or sphericalform.

At 15*5 days p.c, male germ cells have a somewhat more angular outlinethan before, while the oogonia remain rounded. Practically all the gonocytesat this and subsequent stages are restricted to the developing medullary cords.Oogonia, tightly packed in nests, are again frequently associated with crescent-shaped somatic cells.

As the seminiferous cords develop, their inner regions become filled withgerm cells of rounded or angular outline. The majority of 'supporting' cellsappear to be attached to the basement membrane, but extending long irregularprocesses of cytoplasm between the gonocytes (Plate 1, Fig. 1). In contrast,oogonia are frequently seen in the immediate vicinity of the germinal epithelium.Nests of somatic and germinal cells become more clearly distinguishable. Theintermingling of the two cell-types is more marked in the female than in the male(Plate 1, Fig. 2). For an account of the further development of female germ cells,see Franchi & Mandl (1962).

From the age of 16- 5 days^.c. onwards, the general distribution of gonocytesremains more or less constant until some 4 days p.p. The most prominentchange within the germ cells consists in the accumulation of mitochondria atone pole of the cell (mitochondrial 'polarization'; see below). The cells remainclosely packed, and cytoplasmic bridges between adjacent gonocytes arecommon; the conjoined cell membranes at these points may or may not haveosmiophilic and/or phosphotungstic acidophilic material associated with them(Plate 2, Fig. 3). Similar observations have been made on the mammalianovary (Franchi & Mandl, 1962) and the avian testis (Nagano, 1961).

By 19-5 days p.c, mitochondria again become distributed more evenlythroughout the cytoplasm of the gonocyte. Extensions of the 'supporting' cellshave infiltrated between the germ cells which thus tend to be 'pushed apart'.Possibly as a result of the growth of somatic cells, the shape of the gonocytesmay become somewhat more irregular.

The first clear-cut signs of degenerative changes were observed at 2 days p.p.;some components of individual gonocytes become swollen and the cell mem-brane discontinuous (see below).

At 4 days p.p., many germ cells are characterized by cytoplasmic processes,relatively free from organelles, which extend towards the basement membraneof the seminiferous cords (Plate 2, Fig. 4). The' supporting' cells, whose nucleusis almost invariably in the basal portion of the cell, still possess long cytoplasmic

J. Embryol. cxp. Aforph. Vol. 12, Part 2

PLATE 1

FIG. 1. Male, 16-5 days/?.c. Gonocytes (G), closely packed in seminiferous cord, showingangular outlines and cytoplasmic processes of 'supporting' cells (S). The mitochondriatend to aggregate at one pole of the cell.

FIG. 2. Female, 16-5 days p.c. Oogonia (O), intermingled with somatic cells (S).

{Facing page 292)L. L. FRANCHl and A. M. MANDL

/ . Embryol. exp. Morph. Vol. 12, Part 2

PLATE 2

FIG. 3. Male, 18-5 days p.c. Cytoplasmic bridge between three adjacent gonocytes. Osmio-philic material is condensed on the cell membranes of the gonocytes at this junction. Noterounded vesicles of the endoplasmic reticulum and groups of ribosomes scattered throughthe cytoplasm. S = 'supporting' cell.

FIG. 4. Male, 4 days p.p. Cytoplasmic extension of germ cell presumed to be 'transitional',reaching towards the basement membrane (BM). S = 'supporting' cell.

L. L. FRANCHI and A. M. MANDL

J. Embryol. exp. Aforph. Vol. 12, Part 2

?f«>Ti»\ ,f"w:

PLATE 3

FIG. 5. Male, 5 days/?./?. 'Transitional' cell (arrow) showing broad contact with the basementmembrane (BM). The nuclear envelope is irregular in shape.

FIG. 6. Male, 5 days p.p. Spermatogonium type-A, whose long axis is parallel to the basementmembrane (BM). Note the regular arrangement of the mitochondrial cristae. Comparenucleolus with that on Plate 4, fig. 10.

FIG. 7. Male, 19-5 days p.c. Portion of nucleus of gonocyte, showing aggregates of granularmaterial in the nuclear sap (arrow).

L. L. FRANCH1 and A. M. MANDL

J. Embryol. cxp. Alorph. Vol. 12, Part 2

PLATE 4

FIG. 8. Male 21-5 days p.c. Portion of gonocyte showing typical nucleolus surrounded by arelatively clear zone of the nuclear sap ('halo'). The cytoplasm contains B-bodies (Biand B3).

FIG. 9. Male, 14-5 days p.c. Nucleolus of young gonocyte.FIG. 10. Male, 6 days p.p. Rod-shaped nucleolus of 'transitional' cell with small areas of

high electron density (cf. Plate 3, Fig. 6).

L. L. FRANCHl and A. M. MANDL (Facing page 293)


processes which, together with the germ cells, fill the inner region of the semini-ferous cords. The latter are therefore still solid.

Between 5 and 7 days p.p., the proportion of cells which have establishedcontact with the basement membrane increases. A few gonocytes remainseemingly unattached to the basement membrane; an unknown proportion ofthese may, however, extend cytoplasmic processes towards it in a differentplane. Both 'attached' and 'unattached' gonocytes fall into two categories:(1) large cells, essentially similar to gonocytes seen at earlier stages, but for theirregularity of the shape of their nucleus (Plate 3, Fig. 5); and (2) smaller cells,characterized by a reduced cytoplasmic/nuclear ratio (Plate 3, Fig. 6). Acomparison with histological preparations suggests that the former correspondto the 'transitional' cells, and the latter to spermatogonia type-A (see Beaumont& Mandl, 1963). In addition, a number of 'unattached' gonocytes retain theirearlier configuration; with increasing age, these become rarer and some showsigns of degeneration (see p. 299).

Mitotic figures, whose incidence rises at this time, have been observed morefrequently in 'attached' than in 'unattached' cells. The plane of division inrelation to the basement membrane varies; in some cases, one daughter cell isinitially separated from the basement membrane, while the other lies with itslong axis parallel to the basement membrane. Mitoses in centrally-placed germcells are frequently abnormal, the cell contents spilling out of gaps in themembrane (see below).

Individual components of germ cellsNucleus

Shortly after sex differentiation, the nuclei of both gonocytes and oogoniaare typically large and rounded; occasionally they may be pear- or kidney-shaped.The nuclear envelope is sharply defined, particularly the outer membrane. Anarrow layer of electron dense material is closely applied to the inner membrane.Obliquely cut sections show well defined annular nuclear pores.

The nuclear sap of gonocytes contains a heterogeneous assortment of granuleswhich are frequently absent from oogonia. From 17-5 days/?.c. onwards, thenuclear sap in the gonocyte contains small groups of extremely dense particles;these appear to belong to an irregular reticular network formed by other, lessdense particles. In addition, the sap contains one or more areas of particles ofmoderate size (200-250 A) forming groups of 0-7-1 -0 fx in diameter (Plate 3,Fig. 7). Even before the onset of meiosis in the female, the nuclear sap is con-siderably more homogeneous.

With increasing age, the reticular network in the nuclear sap in some—but notall—male germ cells is less pronounced in the immediate vicinity of thenucleolus; a 'halo' or clear zone thus appears (Plate 4, Fig. 8). This change maybe associated with nucleolar 'vacuolation' (see below).

The nuclei of cells believed to be 'transitional' are highly irregular in shape


(Plate 3, Fig. 5), often showing invagination at one point. The nuclear sap bothof these cells, and of spermatogonia type-A, is finely granular (Plate 3, Figs. 5,6). Cells showing peripheral condensation of nuclear material, observed onlyin the oldest age-group studied, may correspond to spermatogonia of theintermediate type (see Clermont & Perey, 1957).

NucleolusAt 14- 5 days/j.c, the nucleoli (probably two or three per cell) are variable in

form. They appear to consist, in both sexes, of loosely arranged knots ofgranular cords; one or more nucleoli in each cell may be attached to the innernuclear membrane. Portions may show two different electron densities (Plate 4,Fig. 9). Within the following 2 days, the nucleoli begin to acquire a moreregular outline; those of some oogonia clearly show zones of two differentdensities (see Franchi & Mandl, 1962). In male specimens aged 17-5 days/?.c.to 4 days p.p., the nucleoli consist of a network of dense granular material withlight areas within the meshes (Plate 4, Fig. 8). They grow without undergoingany marked ultrastructural changes. In some gonocytes with a perinucleolar'halo', the nucleoli appear foamy and vacuolated.

The nucleoli of 'transitional' cells and spermatogonia type-A differ fromthose of typical gonocytes in that they are angular or rod-shaped, rather thanspherical; moreover, they may be larger and contain material of two distinctelectron densities (Plate 4, Fig. 10).

Ribosomes

There appears to be no marked difference between the sexes in the size anddistribution of ribosomes. At early developmental stages, these are abundant inboth gonocytes and oogonia; their incidence is of the same order as that insomatic cells. Subsequently, ribosomes tend to be more regularly arranged ingroups or rosettes, their incidence becoming less pronounced in germinal thanin somatic cells.

Endoplasmic reticulum

The endoplasmic reticulum of mammalian germ cells is known to be verypoorly developed, only isolated vesicular profiles being seen in thin sections.The reticulum develops more rapidly in the female than in the male. Between14-5 and 16-5 days p.c, that of gonocytes is sparse, consisting of a few roundedvesicles and occasional flattened long tubules or cisternae (Plate 1, Fig. 1);the latter may run parallel to the nuclear envelope. Oogonia already containmore abundant endoplasmic reticulum at 14- 5 days; it consists of isolated smallvesicles, and occasional chains of vesicles or long cisternae.

As the male foetus develops, the incidence of elongated profiles in the germcells increases; in addition, some discontinuous or complete rings of endo-plasmic reticulum of variable size have been observed. At birth, some gonocytes


show long tracts in the form of chains of vesicular or short tubular form,studded with ribosomes. Groups of micro vesicles or whorls may be associatedoccasionally with mitochondria. The endoplasmic reticulum may also formcomplex structures near the nuclear membrane, reminiscent of multilamellarbodies.

Early post-natal development is not associated with any striking changes inthe endoplasmic reticulum except in cells believed to be undergoing degeneration(see below).

MitochondriaEven at the youngest stages studied, the mitochondria of germ cells differ

strikingly from those of somatic cells. They are generally larger, round or ovoidin shape (some may be elongated) and contain clearly marked cristae. Sphericalmitochondria sometimes show relatively sparse, radially arranged cristae.

While the shape and numbers of mitochondria in gonocytes and oogonia areessentially similar, their distribution diverges between 15-5 and 18-5 days/?.c.They remain randomly scattered throughout the cytoplasm in the female. Inthe male, they begin to aggregate at one pole of the cell at 15-5 days; their'polarization' is most clearly seen at 16-5 to 18-5 days (Plate 1, Fig. 1).

From 19-5 days p.c. onwards, a progressively higher proportion of themitochondria, which become evenly spread again, undergo slight ultrastructuralchanges. Some contain dense granules, while others may show vacuolatedintercristal spaces, 'stubby' cristae or some of villous form (Plate 5, Fig. 11).The majority of the cristae, however, are parallel or radially arranged, as inearlier specimens. Signs taken to indicate mitochondrial budding were observedin specimens approaching full term; some mitochondria are clearly hour-glassshaped (Plate 5, Fig. 11), while others in the neighbourhood are very small.Enlargement of mitochondria associated with irregularity of cristae and a paleinternal matrix, accompanied by swelling of the endoplasmic reticulum, isprobably a sign of degeneration (see p. 299 and Plate 5, Figs. 12, 13).

Cells establishing contact with the basement membrane, presumed to be'transitional', tend to have a higher proportion of mitochrondria with regularlyspaced parallel cristae (Plate 5, Fig. 14). The small germ cells, corresponding tospermatogonia type-A, contain fewer but relatively larger mitochondria whosecristae are typically parallel (Plate 3, Fig. 6). In contrast, large 'unattached'gonocytes, persisting in 5- and 6-day-old animals, frequently contain irregularlyshaped mitochondria of variable size and internal structure.

Golgi apparatus

Between 14-5 and 16-5 days^.c, the Golgi apparatus is similar in the twosexes. It consists of a mass of small rounded vesicles with fairly dense contents;similar material may be seen in occasional stacks of elongated vesicles (Plate 5,Fig. 15). Associated with this organelle are several larger vesicles, some

20


0-25-0-35 ft in diameter, enclosing a small knot of dense material (Plate 5,Fig. 16). Similar structures occasionally occur in oogonia (see Franchi &Mandl, 1962). Multivesicular bodies are rare, while centrioles are seenfrequently.

The complexity of the Golgi apparatus increases with advancing age. By18-5 days p.c, some gonocytes contain a distinctly annular structure, consistingmore of micro vesicles than of sac-like stacks. The greatest degree of com-plexity, again showing an annular arrangement, is seen in germ cells believed tobe 'transitional' and spermatogonia type-A at 5 to 7 days after birth (Plate 6,Fig. 17).

Other cytoplasmic inclusionsOccasional multivesicular bodies, apparently not associated with the Golgi

apparatus, have been observed in both gonocytes and oogonia at the earlieststages examined. They are also observed occasionally in germ cells throughoutthe age-range studied; but they appear more consistently in 'transitional' cellsand spermatogonia type-A.

Between 15-5 and 17 • 5 daysp.c, some germ cells contain small electron densebodies, similar to those in the Golgi apparatus, but only 0-1-0- 11/xin diameter.Dense, osmiophilic fat droplets first appear, though infrequently, in gonocytesof 17-5-day-old specimens; while some are round, the majority are contractedand star-shaped. Larger fat droplets appear in older specimens (Plate 6, Fig. 18).Their incidence is variable, and they tend to aggregate in small groups. Somaticcells sometimes contain similar fatty inclusions.

Two distinct classes of cytoplasmic inclusions, referred to for convenience asA- and B-bodies, were observed in developing gonocytes.

A-bodies. These first appear at 18 • 5 to 19 • 5 days p.c, in the form of granularaggregates without membranes. As one or two are generally seen in each

PLATE 5

FIG. 11. Male, 22 • 5 days p.c. Mitochondria of gonocyte showing details of internal structure(see text, p. 295).

FIG. 12. Male, 2 days/?./?. Gonocyte showing early signs of degeneration characterized bygrossly enlarged B-body (B3) and mitochondria (M).

FIG. 13. Male, 2 days p.p. Cytoplasm of gonocyte at more advanced stage of degeneration.Note the highly distorted enlarged mitochondria (M), swollen endoplasmic reticulum(e.r.) and numerous B-bodies (B3); the limiting membrane of some of the latter is in-complete (arrow).

FIG. 14. Male, 7 days/?./?. 'Transitional'cell which has established contact with the basementmembrane. The mitochondrial cristae are regular and parallel. The cytoplasm containsan A-body (A); cf. Plate 6, Fig. 19.

FIG. 15. Male, 15-5 days/?.c. Portion of Golgi apparatus of young gonocyte, showing astack of lamellae.

FIG. 16. Male, 14-5 days p.c. Portion of Golgi apparatus of young gonocyte, showingmicrovesicles and larger vesicles containing electron dense material.

J. Embryol. cxp. Morph. Vol. 12, Part 2

PLATE 5

L. L. FRANCHI and A. M. MANDL (Facing page 296)

/. Embryo!, exp. Morph. Vol. 12, Part 2

PLATE 6

F I G . 17 . Male, 7 days/?./?. Golgi appara tus of spermatogonium type-A showing centriole (C). F I G . 18 . Male, 2 days p.p. Cytoplasm of gonocyte containing group of fat droplets ( F ) and

B-bodies (Bj , B 2 and B 3 ) . F I G . 1 9 . Male, 2 0 - 5 days p.c. Granules composing A-body (A) in close association with

endoplasmic reticulum (e.r.). F I G . 2 0 . Male, 1 8 - 5 days p.c. Port ion of gonocyte containing B-bodies (Bj). F I G . 2 1 . Male, 2 1 - 5 days p.c. Port ion of gonocyte showing groups of B-bodies (B 3 ) . F I G . 2 2 . Male, 2 2 - 5 days p.c. A n enlarged B 3 -body.

L. L . F R A N C H I and A . M . M A N D L {Facing page 2 9 7 )


sectioned gonocyte, the number per cell is liable to be high. The A-bodies arecommonly seen in the vicinity of the nuclear membrane, and are sometimesintimately associated with vesicles of the endoplasmic reticulum. The granuleswhich compose them are finer than ribosomes and less well defined (Plate 6,Fig. 19). In comparison, the collection of granules in the nuclear sap, to whichthe A-bodies are superficially similar, are coarser and less compact.

The incidence of A-bodies appears to rise with age up to 1 • 5 or 2 days p.p.Thereafter it declines, as does the frequency with which gonocytes themselvesoccur. Only rarely were they found in germ cells believed to be 'transitional'(Plate 5, Fig. 14) or in spermatogonia type-A. In some post-natal specimens,occasional A-bodies were found to contain dense patches or to have a palecentre with a somewhat foamy rim.

B-bodies. Three varieties are included under this heading (see Plate 4, Fig. 8;Plate 6, Figs. 18, 20, 21). Although the individual organelles differ somewhat inappearance, they occur intermingled in clusters within the cytoplasm. All aremembrane-bound. Transitional forms are common. B-bodies show no spatialrelationships with the Golgi apparatus or other cytoplasmic organelles. Thefirst to be encountered were in specimens aged 18-5 days/?.c.

TYPE BJ. Small oval or comma-shaped bodies consisting of a single limitingmembrane and a condensed electron opaque core of material sometimes showingsigns of internal organization (Plate 6, Figs. 18, 20). Their size is of the sameorder as that of vesicles of the endoplasmic reticulum. They usually occur ingroups in germ cells. Organelles of similar appearance have occasionally beenseen in 'supporting' cells.

TYPE B2. Intermediate in size between Bx and B3, usually bounded by a singlemembrane. These bodies have a fairly electron dense core of tightly packedgranular or vesicular material which, as in type B1} is separated from the limitingmembrane by a narrow comparatively clear zone (Plate 4, Fig. 8; Plate 6,Fig. 18). This relatively scarce organelle appears in groups, in close associationwith other B-bodies; occasionally flocculent material develops in its core.

TYPE B3. Irregular, but generally rounded bodies, initially about half thesize of mitochondria. They are bounded by single or double membranes, butoccasionally a double structure over part of their circumference coexists with asingle one over the remainder (Plate 4, Fig. 8; Plate 6, Figs. 18, 21). Rarely,and only in specimens of more advanced age, do they become multilamellate.B3-bodies show variable contents: some enclose distorted internal membranesand small vesicular structures, frequently associated with deposits of denseflocculent material applied to the inner limiting membrane; others contain smallgranules of variable density. B3-bodies are preceded in appearance by type Bj,but they seem to arise in close association with them in clusters.

The incidence of B-bodies rises as development proceeds. Between birth and2 days p.p., many gonocytes contain one or more clusters consisting of all threetypes. Thereafter their incidence, particularly of types Bj and B2, declines fairly


rapidly. By 3 to 4 days p.p., Bj bodies are no longer present, while B2 are rare.As noted above, the number of 'unattached' gonocytes declines also. In themeantime, both the appearance and the incidence of B3-bodies change con-siderably. Between 21-5 days p.c. and term, the average diameter of B3-bodiesincreases to that of typical mitochondria (0 • 6 pi). While the majority are smallerthan this, some are 0-8-1-5/A in diameter and a few may even reach 2-3 /x.These enlarged organelles contain membranous material, partially obscured byamorphous deposits and occasionally also by a granular component (Plate 5,Fig. 12; Plate 6, Fig. 22). Some B3-bodies of average size show some resem-blance to mitochondria; others, however, are somewhat reminiscent of struc-tures in the uterine epithelium, described by Nilsson (1962) and purported to belysosomes. The larger varieties of B3-bodies resemble the 'lysosome-like'organelles found in the epithelium of the rat prostate by Brandes, Groth &Gyorkey (1962). Although all enlarged B3-bodies are basically similar inappearance, the nature and complexity of their internal constituents varyconsiderably.

By 3 to 4 days/?.;?., such B3-bodies as persist are largely, though not entirely,restricted to 'unattached' gonocytes. No characteristic B-bodies of any typewere observed in normal germ cells in 6- and 7-day-old animals. In contrast,germ cells believed to be undergoing degeneration appear to contain abundantB-bodies, particularly large and irregular forms of B3 whose limiting membranesare frequently incomplete. Cells of this type were observed between 1 • 5 and3 days;?./?, and were also characterized by swelling of the endoplasmic reticulumand/or abnormalities of the mitochondria (see below).

'Transitional' cells and spermatogonia type-A in the oldest specimens studiedoccasionally contained the following cytoplasmic inclusions: (a) large bodiescontaining membranes and some finely granular or flocculent material; theseare more like mitochondria than B3-bodies; (b) vesicles, resembling those of theendoplasmic reticulum, but containing granules; (c) inclusions similar to butnot identical with B2-bodies; and (d) multivesicular bodies.

Organelles showing some resemblance to Brbodies have occasionally beenobserved in the basal region of 'supporting' cells of 6- and 7-day-old animals.

Degenerative changes

In view of the widely accepted belief that many primordial germ cells degene-rate during late foetal and early neonatal life (see Beaumont & Mandl, 1963),particular attention was paid to any ultrastructural changes which mightindicate the onset of cell-death.

Occasional pyknotic nuclei and other degraded cellular components, sur-rounded by abnormal cytoplasm, were observed in early foetal testes; it wasimpossible, however, to decide whether these were of germinal or somaticorigin. The debris appeared to be intimately surrounded by adjacent somatic

Vol. 12, Part 2

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PLATE 7FIG. 23. Male, 2 days p.p. Gonocyte showing swelling of mitochondria and of nuclear

membrane. Note occasional B-bodies (arrows).FIG. 24. Male, 2 days p.p. Cytoplasm of gonocyte similar to above, showing swollen mito-

chondria and endoplasmic reticulum as well as B-body (B3).

L. L. FRANCH1 and A. M. MANDL (Facing page 298)

J. Embryol. exp. Morph, Co/. /2, /V/r/ 2

V .

PLATE 8FIG. 25. Male, 5 days p.p. Central region of seminiferous cord showing intermingling of

somatic and germinal organelles. GM = mitochondria of lysing gonocyte; SM =mitochondria of somatic cells.

FIG. 26. Male, 6 days p.p. Region similar to above, showing swelling of endoplasmicreticulum and breakdown of the cristae of germinal mitochondria (GM). SM = mito-chondria of somatic cells.

FIG. 27. Male, 5 days p.p. Somatic cells apparently enclosing cellular debris, of uncertainorigin, bounded by membranes (see text, p. 299).

L. L. FRANCHI and A. M. MANDL {Facing page 299)


cells, but there was no unequivocal evidence that it had been engulfed (cf.Franchi & Mandl, 1962).

Germ cells showing signs of degeneration were sporadically observed in afew specimens aged 21-5 days p.c. to 2 days p.p. In some cases, swelling of theendoplasmic reticulum was evident in both germinal and adjacent somatic cells.Montages of several electron micrographs indicated that these seminiferouscords were in the immediate vicinity of a trimmed edge of the block. Thepossibility that the 'degenerative' changes were due to mechanical damagecannot, therefore, be discounted.

In some other instances, where the proximity of apparently degenerating cellsto the trimmed edge of the block was uncertain, swelling of the endoplasmicreticulum in gonocytes was accompanied by excessively large numbers ofB-bodies (Plate 5, Fig. 13), or unusually large (2-3 ft) mitochondria (Plate 5,Fig. 12) or marked swelling of the nuclear envelope (Plate 7, Fig. 23). Otherabnormal cells were characterized by a reduction in the number of normalmitochondria and an increase in both unusual forms and of ruptured B-bodiescontaining dense flocculent material (Plate 5, Fig. 13; Plate 7, Fig. 24). Indivi-dual germ cells showing these changes were particularly noticed in specimensaged 1 • 5 and 2 days p.p. In the absence of evidence to the contrary, it is highlyprobable that the few cells showing these striking changes were in effect atearly stages of degeneration.

Clear-cut signs of more widespread degeneration first occur in 5-day-oldspecimens. The affected germ cells are situated in the centre of the seminiferouscord and show no contacts with the basement membrane. Their nuclear andcell membranes are incomplete, and their mitochondria, still recognizable asgerminal, disperse over a wide area (Plate 8, Figs. 25, 26). The cell membranesof neighbouring somatic cells also fragment in the vicinity of the gonocyte,though closer to the basement membrane they are bounded in a normal manner.The debris in the centre of the cord consists of a mass of protoplasm in whichsomatic and occasional germinal nuclei, although distorted, can still be distin-guished. The available space is filled by somatic and germinal mitochondria,interspersed with swollen vesicles of the endoplasmic reticulum, occasionalmultivesicular bodies, and small profiles consisting of islands of cytoplasm,with scattered vesicles and perhaps mitochondria enclosed by a single membrane.Clear signs of canalization of the seminiferous cords are as yet absent. It wasnot possible to detect any evidence indicating that the debris was being activelyremoved or absorbed.

The incidence of lysis appears to rise between the 5th and 6th days p.p. Atthe latter age, it was observed that apical regions of 'supporting' cells fre-quently contain rounded masses of membranous and flocculent material (Plate 8,Fig. 27), similar in appearance to engulfed debris in foetal ovaries (Franchi &Mandl, 1962). These masses, however, are invariably bounded by doublemembranes.


DISCUSSION

Gametogenesis is at all times a dynamic process whose interpretation fromfixed material is notoriously hazardous. A systematic study of serial sections,together with estimates of cell populations, partially overcome the inherentdifficulties of interpretation. Such procedures, however, are hardly applicableto electron microscopy. Even though the present work was preceded by aquantitative histological study of the developing testis in the same strain of rat(Beaumont & Mandl, 1963), it was frequently difficult to equate individualdevelopmental stages (e.g. 'transitional' cells) with those seen under the lightmicroscope. Whether or not a cell seen under the electron microscope is cutthrough its largest diameter is always uncertain. Thus cells largely characterizedby differences in size are hard to classify under the electron microscope. More-over, there may be some unavoidable bias in selecting cells for photographicrecord. These pitfalls were constantly borne in mind in the course of the presentstudy; and whenever new features first appeared in a proportion of germ cells,every effort was made, by repeated observations, to confirm their generaloccurrence.

The present findings indicate that shortly after sex differentiation, only minordifferences are detectable between male and female germ cells. The moststriking divergence, namely their topographical distribution in the gonad andtheir relationship to adjacent somatic cells, had already been established fromlight microscopic studies. Electron microscopy has revealed that the develop-ment of the oogonial endoplasmic reticulum is more advanced than that ingonocytes; that the latter possess a more angular outline; and that the incidenceof desmosome-like contacts is higher in the female. At 15-5 days p.c, whengerm cells in both sexes are still mitotically active (Beaumont & Mandl, 1962,1963), the mitochondria in the male begin to aggregate at one pole of the cell.This 'polarization' becomes maximal at 16-5 to 18-5 days p .c , when mitoticactivity begins to decline. Its significance is obscure, but it is worth noting thatNicander, Abdel-Raouf & Crabo (1961) observed similar changes in the gono-cytes of bull calves. With advancing development, the mitochondria once againbecome more randomly distributed. Their temporary 'polarization' cannottherefore be assumed to indicate the onset of some irreversible process ofdifferentiation. Since gonocytes undergoing mitosis at 15-5 to 18-5 days p.c.apparently contain randomly distributed mitochondria, it is possible thatmitochondrial 'polarization' is a first step in the long period of interphasethrough which all gonocytes are known to pass before the resumption of mitoticactivity at 4 to 5 days p.p. (Beaumont & Mandl, 1963). In the female, mitoticactivity is abruptly halted when the germ cells enter the prophase of meiosis(Beaumont & Mandl, 1962; Franchi & Mandl, 1962). It is possible, therefore,that the increase in the population of gonocytes is checked in some manner bymitochondrial 'polarization'. At present there is no evidence, however, that


gonocytes with mitochondrial 'polarization' are incapable of dividing mitoti-cally before entering upon their long interphase.

Following cessation of mitotic activity and re-distribution of mitochondria,the male germ cells acquire specific, though heterogeneous groups of organelles(A- and B-bodies) in their cytoplasm. The incidence of these inclusions is relatedto foetal age and, after birth, to the presence of cytoplasmic extensions linkingthe germ cell to the basement membrane. The 'attached' germ cells are believedto correspond to 'transitional' cells (Beaumont & Mandl, 1963) until they giverise, by mitosis, to spermatogonia type-A. Individual gonocytes show signs ofdegeneration at 2 to 4 days p.p., at a time when only few cells begin to extendcytoplasmic processes to the periphery of the cord. Widespread degenerationdoes not occur until 5 to 6 days p.p. The majority of cells affected appear to be'unattached' gonocytes at interphase; but occasional abnormal mitoses havebeen observed in centrally situated germ cells. The significance of the specificorganelles, of cytoplasmic contacts with the basement membrane, and of thedegenerative changes observed, will be discussed in turn.

The nature and function of the granular cytoplasmic inclusions, here calledA-bodies, are entirely obscure. The inclusions were first observed in specimensat 18-5 to 19-5 days; their incidence decreases rapidly after the 2nd day p.p.,when their presence is largely restricted to centrally situated germ cells seeminglylacking contact with the basement membrane.

Three other types of organelle (Bl5 B2 and B3) have been identified. Althoughforms intermediate in structure between each of the three types are neverdifficult to detect, there is no direct evidence that Bj-bodies, which appear first,subsequently become transformed to give rise to B3-bodies. It is nonethelesspossible—though unproven—that B2-bodies, whose size is intermediate betweenthat of the other two, are formed at the expense of Brbodies, and that theysubsequently develop into the large, more complex B3-forms.

B-bodies are particularly characteristic of gonocytes in the late foetal andearly neonatal period, though it was not possible to determine with certaintythat every germ cell contained them. The rapid decline in their incidence at3 to 4 days/)./?, coincided with the reduction in the numbers of typical roundedgonocytes; at this time, the majority of germ cells show signs of migratingtowards the periphery of the seminiferous cords. Certain B-bodies, particularlythose of type B3, are prominent in germ cells whose nuclear envelope, endo-plasmic reticulum and mitochondria show presumed degenerative changes.Excessive numbers of B3-bodies of irregular size and form are sometimes foundin such cells as early as 1 • 5 or 2 days p.p. It would appear, therefore, that thedevelopment of large B3-bodies is in some way correlated with abnormalities ingonocytes of post-natal animals. During the later part of the developmentalperiod studied, widespread breakdown of the central regions of some cordstakes place (cf. Beaumont & Mandl, 1963). It is tempting to suggest, therefore,that B-bodies play an important part in the autolysis of some gonocytes,


especially those failing to make contact with the basement membrane. If thiswere so, the organelles might be considered to be lysosomal in nature, and theirpresence, coinciding with high radiosensitivity (see Beaumont, 1960, 1962;Hughes, 1962), of considerable significance.

In their reviews of morphological and biochemical properties of lysosomes,de Duve (1959a, b) and Novikoff (1961) point out that lysosomes are highlyvariable in form, and that biochemical evidence does not confirm the existenceof a homogeneous class of particles. Indeed, functions attributed to intra-cellular hydrolases (de Duve, 1959a) are so varied as to preclude the possibilityof a common form for the lysosome. As judged by their form and/or enzymicproperties, and the physiological conditions under which they become manifest,lysosomes apparently occur in many different types of cell; but in all sites inwhich they have been identified, they appear to play a role in either cellulardifferentiation, catabolism or involution (e.g. Novikoff, Beaufay & de Duve,1956; deDuve, 1963; Novikoff &Essner, 1960; Moe & Behnke, 1962; Rahman,1962; Nilsson, 1962; Brandes et al., 1962; Behnke, 1963; Bonneville, 1963;Napolitano, 1963).

Brandes et al. (1962) studied the prostatic epithelium in normal and castratedanimals by both histochemical and electron microscopic procedures. Theyreport that acid phosphatase activity is correlated with the presence of dense,membrane-bound organelles resembling lysosomes, both features becomingmore prominent after castration. The organelles described and illustrated byBrandes et al. are strikingly similar to many of the B3-bodies observed ingonocytes. Both are of variable size and shape, sometimes occur in a complexmultiple form or in clusters, they are bounded by a limiting membrane andcontain variably developed internal membranous and flocculent electron densematerial. Nilsson (1962) similarly describes three types of dense cytoplasmicorganelles in the uterine epithelium, whose incidence can be varied by theadministration of oestrogen. Those of one type detected are similar not onlyto lysosomes, as described by Novikoff & Essner (1960), but also to some of theB3-bodies in our material.

Lysosomes are apparently capable of storing a host of different enzymes (seeNovikoff, 1961). Of those that have been identified, acid phosphatase is oftenprominent (de Duve, 1959a) and the simplest to detect by histochemical or bycytochemical methods (Holt & Hicks, 1961; Sabatini, Bensch & Barrnett, 1963).Preliminary attempts to establish the presence of acid phosphatase activity inthe cytoplasm of gonocytes have so far failed, though sections of liver, pro-cessed simultaneously, indicated the presence of lysosomes in the expectedmanner. Moreover, Leydig cells in the sections of foetal rat testes showed someactivity; Jirasek's (1962) histochemical study of interstitial cells in the humanembryonic testis yielded similar results. The apparent absence of detectablequantities of acid phosphatase in B3-bodies does not, however, invalidate thehypothesis that they are lysosomal in nature (see Novikoff, 1961).


It is of interest to note that a marked rise in lysosomal enzyme activities, insome instances known to be associated with enlargement of lysosomes, occursnot only in spontaneously regressing tissues (e.g. embryonic Miillerian duct;tail of tadpole) but also in those subjected to a variety of noxious procedures(e.g. ligation, anoxia; see Novikoff, 1961). The levels of a variety of lysosomalenzymes in thymus and spleen have also been shown to rise steeply followingirradiation (e.g. Roth & Eichel, 1959; Roth, Bukovsky & Eichel, 1962; Roth &Hilton, 1963). It would be premature to postulate that the presence of lysosome-like bodies in gonocytes is a major cause for their high radiosensitivity, ionizingradiations rupturing their membranes and 'liberating' their enzymes. First, it ispossible that irradiation induces surviving cells in the spleen and thymus toproduce more enzymes, rather than lysosomal breakdown and subsequentrelease of pre-existing 'bound' enzymes. Second, Rahman (1962) suggeststhat radiosensitivity and abundance of lysosomes in thymus cells may beinversely correlated. Third, if ionizing radiations kill gonocytes by rupturingthe membranes of lysosome-like bodies, it would be expected that shortly afterirradiation, there would be signs of widespread lysis. Beaumont's observations(personal communication) point to the contrary: gonocytes exposed to a dosesufficient to induce subsequent sterility persist for several days after irradiation,and though many enlarge somewhat, none show signs of disintegration. It is asyet uncertain how long after irradiation gonocytes, or their daughter-cells,involute. If the major loss occurs at the first post-irradiation mitosis, the con-tribution of latent chromosomal damage may well outweigh that of enzymicrelease during interphase. Finally, mammalian oocytes in primordial follicles,known to be highly radiosensitive though mitotically inactive, apparentlylack lysosome-like bodies. In his careful study of unirradiated and irradi-ated oocytes in young mice, Parsons (1962) makes no mention of suchorganelles.

The disappearance of B-bodies from germ cells which, after birth, becomeattached to the basement membrane poses further problems. If the lysosome-like bodies contain potentially autolytic enzymes, it would appear that eitherthe enzymes are 'resorbed' or that they remain within the cell in some 'bound'or 'inhibited' form. The persistence of B-bodies in 'unattached' germ cells, andtheir abundance in cells with swollen endoplasmic reticulum and abnormalmitochondria, suggest that they may be somehow involved in spontaneousdegeneration. The establishment of cytoplasmic contacts between the germ celland the basement membrane appears to be a pre-requisite for subsequentsurvival and mitotic activity. What 'advantages', if any, the cell derives fromsuch contacts are as yet unknown. It is difficult to decide, in the light of thepresent observations, whether B-bodies disappear from germ cells shortlybefore or after the first contact with the basement membrane has been estab-lished. All that can be said at present is that the two phenomena are related in


time; but whether there is any causal relationship between the two remains tobe determined.

Migration of gonocytes towards the periphery of the seminiferous cord hasbeen observed in other species (e.g. Courot, 1962; Sapsford, 1962a, b). Thatthey should be capable of doing so is hardly surprising in view of Blandau, White &Rumery's (1963) recent demonstration that amoeboid movement in female germcells persists up to the onset of pachytene. Blandau et ah were unable to observeany amoeboid movement in gonocytes after their inclusion within medullarycords, but the oldest animals they examined were approaching full term, whengonocytes are known to be tightly packed within the cords. With the subsequentrapid proliferation of somatic tissue (see Beaumont & Mandl, 1963) and therelative decrease in the incidence of germ cells per cross section of seminiferouscord, all that separates the centrally situated germ cell from the basementmembrane is somatic cytoplasm. This would presumably offer no greaterresistance to the 'streaming' of germinal cytoplasm than the loose tissuesurrounding developing oocytes.

The process of spontaneous degeneration, which affects a relatively smallproportion of the total population of male germ cells (Beaumont & Mandl,1963; see also Courot, 1962) differs strikingly both in time and nature from thatobserved in the female (Beaumont & Mandl, 1962; Franchi & Mandl, 1962).Pyknosis is rare, and it was impossible to decide whether such pyknotic nucleias were recorded in the youngest specimens were germinal or somatic in origin.At the time that many female germ cells are eliminated from the ovary (18-5 daysp.c. to 2 days/?./?.), the gonocytes are at interphase, and none or few degenerate.Only as late as 4 to 5 days p.p., when the rate of spontaneous atresia in thefemale has begun to fall, does degeneration set in on a detectable scale in themale. When it does so, the cell membrane ruptures, the cytoplasm streams outand the nucleus disintegrates. Dying or dead cells in the foetal and neonatalfemale appear to be removed rapidly (Beaumont & Mandl, 1962) by the phago-cyte-like activity of neighbouring somatic cells which engulf the debris (Franchi& Mandl, 1962). The process by which lysing gonocytes are eliminated fromthe testis appears to differ. Though the peripheral cytoplasm of 'supporting'cells occasionally contains cellular debris—which may or may not be germinal inorigin—no clear-cut signs of rapid phagocytic removal have been observed. Thecytoplasm of the degenerating germ cell disperses over a wide area, and smallfragments may be absorbed or 'digested' by the cytoplasm of all the neighbour-ing somatic cells confluent with the lysing gonocyte. The ingestion of degenerat-ing germ cells by Sertoli cells, observed by Lacy (1964) in adult rats followingirradiation or treatment with oestrogen, does not appear to occur in theneonatal rat. Although the developing 'supporting' cells (precursors of Sertolicells) are seemingly capable of engulfing cellular debris, the process whereby thegerm cells degenerate clearly differs between the unirradiated neonatal andirradiated adult rat.


SUMMARY

1. Male germ cells of foetal and neonatal rats (14-5 days p.c. to 7 days p.p.)were examined with the electron microscope. Female germ cells of youngspecimens (14-5 to 16-5 days p.c.) were re-examined for comparison.

2. Shortly after sex differentiation, male and female germ cells have manyfeatures in common. The endoplasmic reticulum in oogonia, however, is themore highly developed, while the outline of gonocytes becomes markedly moreangular as development proceeds. Between 15-5 and 18-5 days p.c, mito-chondria in the male aggregate at one pole of the cell; those of the female remainrandomly distributed. The significance of mitochondrial 'polarization' isobscure, but it is possible that the process is in some way related to cessation ofmitotic activity.

3. From 18-5 days p.c. onwards, when mitochondria become re-distributedevenly, groups of specific organelles (A- and B-bodies) appear in the cytoplasm.Three types of B-bodies have been identified. Of these, one type resembleslysosomes. The incidence of B-bodies increases during late intra-uterine lifeand reaches its peak between birth and 2 days p.p. Thereafter, they becomerarer, as do germ cells situated in the centre of the seminiferous cord. At thistime, a high proportion of gonocytes grow cytoplasmic extensions establishingcontact with the basement membrane; their nucleus and nucleolus change theirshape, while their mitochondria contain more regularly arranged parallel cristaethan do those of 'unattached' gonocytes. Cells showing these signs of 'stream-ing' or migration are believed to correspond to the 'transitional' cells identifiedunder the light microscope, and to be the immediate precursors of the firstgeneration of spermatogonia type-A. After 2 days p.p., B-bodies appearto persist almost only in those gonocytes that have failed to establish contactwith the basement membrane. The possible significance of B-bodiesin spontaneous degeneration and high radiosensitivity of gonocytes isdiscussed.

4. A few isolated germ cells show degenerative changes during the first 3 daysafter birth. These frequently show swollen endoplasmic reticulum, abnormalmitochondria and an abundance of B-bodies. Widespread degeneration doesnot set in until about 5 days, and largely affects ' unattached' gonocytes at inter-phase ; a few degenerating cells undergoing mitosis have been observed. Degene-ration takes the form of lysis: the cell membrane ruptures, the cytoplasmstreams out over a wide area, and the nucleus becomes distorted. The processby which lysing germ cells are eliminated from the testis has not been fullyestablished.

5. The ultrastructure of spermatogonia type-A is essentially similar to that oftheir precursors. Most of the small germ cells corresponding to spermatogoniatype-A seen under the light microscope lie with their long axis parallel to thebasement membrane.


RESUME

Uultrastructure des cellules germinales chez les foetus et les nouveau-nes malesde rat

1. On a examine au microscope electronique les gonocytes males de foetus etde nouveau-nes de rat (de 14 jours \ post coitum jusqu'a 7 jours post partum).Pour faire la comparaison, on a examine de nouveau des gonocytes femelles dejeunes specimens (14 jours \ a 16 jours %p.c).

2. Peu apres la differentiation sexuelle, les gonocytes males et femelles ont encommun beaucoup de caracteres. Neanmoins, l'ergastoplasme des oogonies estle plus developpe, tandis que les contours des gonocytes deviennent nettementplus anguleux pendant que le developpement progresse. Entre 15 jours \ et18 jours \p.c, chez le male, les mitochondries s'agregent a un pole de la cellule;chez la femelle, elles restent reparties au hasard. La signification de cette* polarisation' mitochondriale est obscure, mais il est possible que le processussoit en relation d'une maniere quelconque avec l'arret de l'activite mitotique.

3. A partir de 18 jours \p.c, quand les mitochondries se trouvent de nouveauegalement reparties, apparaissent dans le cytoplasme des groupes d'organitesspecifiques (corps A et B). On a identifie trois types de corps B, dont l'unressemble aux lysosomes. Le nombre des corps B augmente pendant la fin dela vie intra-uterine et atteint son maximum entre la naissance et le 2e jour p.p.Ensuite, ils deviennent plus rares, comme les gonocytes situes au centre ducordon seminifere. A ce moment, une forte proportion de gonocytes emettentdes expansions cytoplasmiques etablissant un contact avec la membrane basale;leur noyau et leur nucleole changent de forme, tandis que leurs mitochon-dries contiennent des cretes disposees plus regulierement que ne le sont cellesdes gonocytes 'non attaches'. On croit que les cellules presentant ces signes' d'ecoulement' ou de migration correspondent aux cellules de transition identi-fiees au microscope ordinaire, et sont les precurseurs immediats de la premieregeneration de spermatogonies de type A. Apres le 2e jour p.p., les corps Bpersistent presque uniquement dans ceux des gonocytes qui n'ont pas etabli decontact avec la membrane basale. On discute la signification possible des corpsB dans la degenerescence spontanee et la forte radiosensibilite des gonocytes.

4. Quelques gonocytes isoles montrent des modifications de degenerescencependant les trois premiers jours apres la naissance. Ils montrent frequemmentun ergastoplasme gonfle, des mitochondries anormales et une abondance decorps B. La degenerescence etendue ne s'instaure pas avant 5 jours environ etelle atteint largement les gonocytes 'non attaches' en interphase; on a observela mitose de quelques cellules en degenerescence. Celle-ci prend Pallure d'unelyse: la membrane cellulaire se rompt, le cytoplasme s'ecoule sur une largesurface et le noyau se deforme. On n'a pas pleinement etabli le processus parlequel les gonocytes en cytolyse sont elimines du testicule.

5. L'ultrastructure des spermatogonies du type A est essentiellement sembl-


able a celle de leurs precurseurs. La plupart des petits gonocytes correspondantaux spermatogonies de type A vues au microscope optique ont leur grand axeparallele a la membrane basale.

ACKNOWLEDGEMENTS

The expenses incurred in this study were defrayed from grants, made to Professor SirSolly Zuckerman, F.R.S., by the Population Council, Inc. and by the Medical ResearchCouncil. The Authors are grateful to Professor Sir Solly Zuckerman, F.R.S., for hisencouragement and valuable criticisms.

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(Manuscript received 9th December 1963)

Documents

The ultrastructure of germ cell in foetas l and neonatal male rats · The ultrastructure of germ cell in foetas l and neonatal male rats by L. L. FRANCHI and ANITA M. MANDL From the