spermatogenesis and spermiogenesis in ascaris lumbricoides var. suum

Spermatogenesis and Spermiogenesis in Ascaris lumbricoides Var. suum

PAUL GOLDSTEIN I

Department of B i o l w , York University, Downsview, Ontario, Canada M3J IP3

ABSTRACT Reorganization of the prophase I nucleus marks the beginning of the first meiotic division. A pair of centrioles is present a t each pole a t metaphase I and mitochondria are not observed in the spindle area. A chromosomal pellicle, which resembles a kinetochore plate but has no apparent association with microtubules, surrounds each autosome a t metaphase I and 11. The sex body lags behind the autosomes at anaphase I and segregates differentially to one daughter cell. Mitochondria and a pair of centrioles are present in the spindle during the second meiotic division. Localized condensation of chromatin and fusion of the condensed chromatin of the secondary spermatocyte telophase nucleus results in a compact spermatid nucleus. Loss of spermatid cytoplasm is effected by the ejection of a cytophore vesicle.

The cytology of Ascaris has been of interest since i t was discovered (Boveri, 1887) that some species lose chromosomal material during the early embryonic cell divisions (Boveri, 1887; Lin, ’54; Goldstein, ’77). In an effort to elucidate the morphology of the chromosomes of Ascaris suum, I have undertaken an electron microscopic analysis of male and female reproductive cells. In the process, I have be- come acquainted with details of spermatogenesis and spermiogenesis which are of interest in comparisons with these processes in other organisms. In particular, this study reports the spindle organization, microtubule attachment, centrioles, chromosome movement at anaphase I and 11, nuclear condensation in secondary spermatocytes and the ejection of cytophores by the amoeboid sperm.

MATERIALS AND METHODS

Ascaris lumbricoides var. suum were collected from a local abattoir and immediately fixed in 4% glutaraldehyde, 0.05 M phosphate buffer, pH 6.3. In the lab the testes were re- moved, cut into 5-mm portions in consecutive series beginning from the distal germinal end, and placed into fresh fixative for two hours, Post-fixation was in Dalton’s Osmium-Chro- mic Acid (Zickler and Olson, ’75) for two hours at room temperature followed by dehydration through an alcohol and propylene oxide series, embedded in Epon, and stained with uranyl

J. MORPH.. 154: 317-338.

acetate and Fiske lead citrate (Fiske, ’66). Serial sections were cut on a Porter-Blum ultramicrotome and collected as described by Moens (’70) on formvar coated single-slot grids. The sections were examined on a Philips EM 200.

OBSERVATIONS

First spermatocyte division Reorganization of the A. mum spermato-

cyte nucleus marks the beginning of prometaphase of the first meiotic division. At late prophase and early prometaphase the heterochromatic ends of the bivalents lie close to the nuclear envelope (figs. lb,c), which is in contrast to the organization of the nucleus at pachytene where the synaptonemal complexes have no apparent association with the nuclear envelope (Goldstein and Moens, ’76). At prometaphase I spindle microtubules are present in the nuclear envelope gaps (fig. lc), The mitochondria are not present in the spindle (fig. la) which differs from meiosis 11 where the mitochondria are packed around the spindle (fig. 11).

The metaphase bivalents are minute, ap- proximately 1 pm, and have irregular shapes (figs. 2-6). Most of the chromosomes have a peculiar surface s t ructure called a “chromosomal pellicle” here. It is assumed ‘ Present address: Department of Genetics, North Carolina State

University. Raleigh. North Camlina 27607.

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318 PAUL GOLDSTEIN

that these are the autosomes and that the few chromatin bodies without a pellicle are the sex chromatin (Goldstein, '77a). The pellicle has a tripartite appearance (figs. 3-5) : an electron dense outer layer, 8-10 nm, a less dense middle layer, 55 nm, and an inner layer, 5 nm, that is continuous with the chromatin. Oogonial chromosomes of Ascaris have a well-defined kinetochore plate that is apparently associated with microtubules.

The bivalents a t metaphase do not appear to have a specialized region for microtubule attachment. In their extremely contracted s ta te the bivalents appear to have a t - tachments over an extended region (figs. 2-6). The microtubules may continue through or terminate in the autosome. The orientation of the chromosomes a t anaphase I is parallel to the equatorial plate (figs. 7-9). Anaphase bridges, or coated microtubules, are apparent in the interzonal region and are similar to those reported inHeteropeza (Fux, '74) (figs. 7, 17). The poles are devoid of mitochondria (fig. 7). Light microscopy of the first spermatocyte division indicates the large heterochromatic body that lags behind the autosomes (figs. 8, 9) and this is verifed by electron microscopy (fig. 7). This large heterochromatic mass segregates to only one daughter cell and behaves as a sex body.

Centrioles The minute centriole of A. suum is present

throughout spermatogenesis and is 0.18 Frn in diameter, 0.27 pm in length, with a central lumen of 79 nm in diameter. The centriole has a 9-fold symmetry composed of single microtubules and a central cartwheel is absent (fig. 18a). The nine subunits are connected by a dense, continuous ring (fig. 18a).

During prophase of meiosis I a single centriole is associated with the outer membrane of the nuclear envelope (fig. 18c). Often a procentriole, the earliest event in centriole reproduction (Bernhard and deHarven, '60; Kalnins and Porter, '69; Stubblefield and Brinkley, '68) is observed a t right angles adjacent to the parent centriole during pachytene (fig. 18c) and a t anaphase I. Analysis of serial sections indicates that a pair of centrioles is present a t each pole a t anaphase I (fig. 18b) and metaphase I1 (fig. 11). A single centriole is observed embedded in the granular ribonucleoprotein layer sur- rounding the spermatid nucleus (figs. 19,19a).

Second spermatocyte division and spermiogenesis

A nuclear envelope reforms around the telophase nucleus of the secondary spermatocyte. The cell observed during the short interphase between the two meiotic divisions has a nucleus with a convolute nuclear envelope and numerous fibrillar, refringent vesicles in the cytoplasm. A granular nucleolus is present inside the nucleus (fig. 10).

A chromosomal pellicle is present on the chromosome a t metaphase I1 (fig. 5). There is a concentration of mitochondria a t the spindle poles (fig. 11). The synchrony of the earlier stages of spermatogenesis is lost as anaphase I, metaphase 11, telophase I1 and spermatids may be observed in the same region of the seminiferous tubule.

The telophase nucleus of the early spermatid does not have a nuclear envelope. Rather, localized condensation of chromatin and fusion of the condensed chromatin during the remainder of spermiogenesis, results in a compact spermatid nucleus (figs. 12a-f). A granular ribonucleoprotein layer surrounds the nucleus (fig. 12f) (Clark et al., '72). In the mature spermatozoan a nucleus of similar structure is present (fig. 16).

Elimination of cytoplasm of early spermatids occurs during the condensation of the telophase I1 nucleus. The cytoplasm that will be injected as a cytophore is differentiated in the spermatid (figs. 13,13a). There is no apparent polarization of cytophore formation on the spermatid and more than one cytophore may be ejected (fig. 14). The cytophore vesicle contains rough endoplasmic reticulum and dictyosomes (fig. 15).

DISCUSSION

Unusual aspects of spermatogenesis in Ascaris were initially described by Van Beneden and Julin (1884). Light microscopic studies have revealed aberrant behavior of heterochromosomes during meiotic divisions; condensation of the secondary spermatocyte nucleus; and elimination of cytoplasm, as a cytophore, during spermiogenesis. A description of the fine structure of the spermatocyte divisions in Ascaris megalocephala (Favard, '61) verified the extrusion of a cytophore, but did not detail the ultrastructural morphology of other phenomena observed in earlier studies.

SPERMATOGENESIS IN ASCARIS 319

In A. s u m kinetochore-like structures (chromosomal pellicles) are present on the spermatocyte autosomes during the meiotic divisions. The function of the pellicle is unknown. Apparently, microtubules are not directly associated with it, therefore, the pellicle may not be involved in chromosome movement.

Kinetochores The kinetochore region of chromosomes has

been suggested to play a critical role in the movement and orientation of chromosomes during meiosis (Luykx, '65). Kinetochore plates are present during mitosis but absent during meiosis in Oncopeltus (Buck, '67); Rhodnius (Buck, '67); Philaenus (Ris and Kubai, '70); Bombyx (Friedlander and Wahr- man, '70) and Ascaris suum.

In two light microscopic studies Lin ('54) and Schrader ('35) suggest the kinetochores of A. rnegalocephala are polycentric, since portions of the compound chromosomes are apparently lacking kinetic activity and are eliminated during the early embryonic divisions. Favard's ('61) study did not reveal the fine structure of the kinetochore. A similar process of chromatin elimination occurs in A. m u m (Goldstein, '77). The description of the ultrastructural morphology of the embryonic chromosomes in Ascaris has been hin- dered by the embryo shell impermeability to chemical fixatives. A well-defined kinetochore, although present on oogonial chromosomes, is not present on male spermatocyte chromosomes, and, therefore, it is difficult to accurately determine those chromosomal re- gions lacking kinetic activity.

Centrioles Centrioles, of uncommon structure, are

present through all stages of spermatogenesis inA. suum. A centriolar pair is present a t each pole during the first and second meiotic divisions. In A. suum the centriole does not function as a basal body in the formation of a flagellum since the sperm is amoeboid (Nelson, 1852) and lacks a flagellum (Foor, '70). Pickett-Heaps ('69) suggests that the nine triplet tubules of the centriole are essen- tial for the initial assembly of the subunits that form the nine doublets of the flagellum and that in those organisms (i.e.,Ascaris) that lack a centriole, a flagellum may not be present. The structure of the centriole inA. suum

is similar to that reported inA. megalocephala (Favard, '61) and the nematode Capillaria (Wright, '76).

The spermatid nucleus ofA. suum has no apparent nuclear envelope and is surrounded by a layer of ribonucleoprotein (Clark et al., '72; Foor, '70) in which the single centriole associated with the nucleus is embedded. Simi- lar observations have been reported in A. megalocephala (Favard, '61). The centriole is contributed by the sperm to the cytoplasm of the egg a t fertilization (Boveri, '01; Favard, '61; Fulton, '71).

Cytophores Loss of sperm cytoplasm during spermio-

genesis occurs in mammals (for review see Clermont, '72). Reduction in cytoplasm has been reported in A. megalocephala (Favard, '61; Hertwig, 1890; Mayer, '08), A. mystax (Marcus, '061, and A. canis (Marcus, '06). A similar process also occurs inA. suum. Romeiu ('11) observed the refringent vesicles, or "karyochondria," were not eliminated in the cytophore of A. megalocephala. The cytoplasmic components eliminated include mitochondria, dictyosomes and rough endoplasmic reticulum and were described as "plasto- chondria" (Faure-Fremiet, '12; Mayer, '08; Meves, '11; Romeiu, '11; Wildman, '13). The contents of the cytophore are not lost from the organism as the cytophore is absorbed by the cells of the testicular wall, as was similarly reported in A. megalocephala (Favard, '61).

ACKNOWLEDGMENTS

I wish to thank Doctor P. B. Moens for his helpful discussions during the course of the study and for his review of the manuscript. I also wish to thank Ms. M. L. Ashton for her technical assistance. This study was funded by the National Research Council of Canada, Grant 1901 to P. B. Moens.

LITERATURE CITED

Bernhard, W., and E. de Harven 1960 L'ultrastructure du centriole et d'autres elements de l'appareil achromatique. Proc. Fourth Int. Conf. Electron Micr. Heidelberg: Springer-Verlag. 2: 217-227.

Boveri, T. 1887 Ueber Differenzierung der Zellkerne wiihrend der Furchung des Eies von Ascaris mega- lorephala. Anat. Am., 2: 688-696.

1901 Zellen IV. Uber die Natur der Centroso- men. Jena &it. Naturwiss., 28: 1-220.

Buck, R. 1967 Mitosis and meiosis in Rhodniw prolixus: The tine structure of the spindle and diffuse kinetochore. J. Ult. Res., 18: 489-501.

320 PAUL GOLDSTEIN

Clark, W., R. Moretti and W. Thomson 1972 Histochemical and ultracytochemical studies of the spermatida and sperm of Ascaris lumbricoides var. mum. Biol. Reprod., 7: 145-159.

Clermont, Y. 1972 Kinetics of spermatogenesis in mammals: Seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev., 52: 198-236.

Faure-Fremiet, E. 1912 Le cycle germinatif chez l’As- curia m e g t a h e p f i Arch. Anat. Micr. Morph. Exp., 15: 435-758.

Favard, P. 1961 Evolution des ultrastructures cellu- lairen au coura de la spermatogemh de I’ASCaria Ann. Sci. Nat. Zwl. Ser., 12: 53-152.

Fiske, 5. 1966 An adaptation of Reynold’s lead citrate stain for high resolution autoradiograhy. J. de Micros., 5: 355-360.

Fwr, W. E. 1970 Spermatozoan morphology and zygote formation in nematodes. Biol. Reprod. Suppl., 2: 177-202.

Friedlander, M., and J. Wahrman 1970 The spindle as a basal body distributor. A study in the meiosis of the male silkworm moth, Bombyx mori J. Cell Sci., 7: 65-89.

Fulton, C. 1971 In: Origin and Continuity of Cell Organ- elles (J. Reinert and H. Ursprung, &.). Heidelberg: Springer-Verlag.

Fux, T. 1974 Chromosome elimination in Heteropezapyg- m a 11. Ultrastructure of the spindle apparatus. Chro- mosoma, 49: 99-112.

Goldatein, P. 1977 Chromatin dimunition in early em- bryogeneeis of Ascaris lumbricoides L. var. suum. J.

(1977a, submitted for publication) Ultrastruc- tural analysie of sex determination in Amaria suum Chromosoma, in press.

Goldatein, P., and P. B. Moens 1976 Karyotype analysis of Asearis lumbricoides var. suum male and female pachytene nuclei by 3.D reconstruction from electron microscopy of serial sections. Chromosoma, 58: 101-111.

Hertwig, 0. 1890 Vergleich der Ei-und Samenbildung bei Nematoden. Arch. Mikr. Anat., 36: 1-137.

Kalnins, V., and K. Porter 1969 Centriole replication during ciliogenesis in the chick tracheal epithelium. 2. Zellforsch., 100: 1-30.

Morph., 152: 141-152.

Lin, T. P. 1964 The chromosomal cycle of Parancaris equorum (Ascaris megalocephnla): Oogenesis and diminu- tion. Chromosoma, 6: 175-198.

Luykx, P. 1965 The structure of the kinetochore in meiosis and mitosis in Urechis eggs. Exp. Cell Rea., 39:

Marcus, H. 1906 Ei und Samenreife bei Ascaris canis. Arch. Mikr. Anat., 68: 441-490.

Mayer, A. 1908 Zur Kenntnie der Samenbildung bei Ascaris megalocephula Zool. Jahrb. Abt. Anat., 25:

Mevee, F. 1911 iiber die Beteilung der Plastochondrian an der Befruchtung des Eies von Ascaris megalocephalu Arch. Zellf., 6: 254.325.

Moens, P. B. 1970 Serial sectioning in electron mimsco- py. Proc. Canad. Fed. Biol. Soc., 13: 160.

Nelson, H. 1852 The reproduction ofAscaris mystax. Phil Trans. Roy. Soe. London, 2: 563-594.

Pickett-Heaps, J . D. 1969 The evolution of the mitotic apparatus: an attempt a t comparative ultrastructural cy. tology in dividing plant cells. Cytobios, 1: 257-280.

Ria, H., and D. Kubai 1970 Chromosome structure. Ann. Rev. Genet., 4: 263-294.

Romeiu, M. 1911 La spermatogenese chez Ascaris mega- locephulu Arch. Zellf., 6: 254-325.

Schrader, F. 1935 Notes on the mitotic behavior of long chromosomes. Carylogia, 6: 422-430.

Stubblefield, E., and B. Brinkley 1968 Architecture and function of the mammalian centriole. Symp. Int. Soc. Cell Biol., 6: 175-218.

van Beneden, E., and C. Julin 1884 La spermatogenese chez I’ascaride megalmphale. Bull. Acad Roy. Belg., 7:

Wildman, E. 1913 The spermatogenesia ofAscark mega. locephnla with special reference to the two cytoplasmic inclusions. the refractive body and the mitochondria: their origin, nature and role in fertilization. J . Morph., 24: 421-457.

Wright, K. 1976 Somatic centrioles in the parasitic nematode Capillaria hepatica J. Nematol., 8: 92.93.

Zickler, D., and L. Olson 1975 The eynaptonemal complex and the spindle plaque during meiosis in yeast. Chromoso- ma.50: 1-23.

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PLATES

PLATE 1

EXPLANATION OF FIGURES

l a Spermatocyte a t prometaphase I. Mitochondria (M) are scattered in the cytoplasm and not located a t the poles. Remnants of nuclear envelope (NE). Spindles (S). Re- fringent vesicle (RV). Bar is 1 pm.

b Bivalenta aaaociated with the nuclear envelope (NE) at late prophase. Remnants of the synaptonemal complex (SC) are apparent in the chromoaomal substance (CH). Bar is 0.1 pm.

c Spindle microtubules (arrows) are observed in nuclear envelope gaps a t pro- metaphaae I. Nuclear envelope (NE). Chromosome (CHI. Bar is 0.1 pm.

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SPERMATOGENESIS IN ASCARIS Paul Goldstein

PLATE 1

PLATE 2


2 Microtubules (arrows) associated with metaphase I bivalents occur along an extended region of the chromosome. Bar is 1 pm.

3-5 The pellicle (PI that surrounde metaphase I and I1 chromosomss isa tripartite structure and is not directly associated with microtubules. Figures 3,4 are chromosomes at metaphase I and figure 5 is a chromosome from metaphaee. 11. Bar is 1 pm.

6 Microtubular chromosomal aesociation at anaphase I. Microtubules (arrows) are aa- mciated along an extended region of the chromosome. Bar is 1 pm.

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PLATE 2

325

PLATE 3


Figs. 7-9 Anaphase of the first spermatocyte division.

'I Figure 7 shows the aex body (SB) lags behind the autoeomea. Coated microtubules (cMT) are observed in the interzonal region. The pol- are devoid of mitochondria (MI. Refringent vesicles (RV). Bar is 5 pm.

8 Figure 8 is a light micrograph of Feulgen stained spermatocytes showing two cells at anaphase I with sex body (SB) lagging. Bar is 5 pm.

9 Figure 9 is a light micrograph of a 0.5 pm thick Epon section from which corresponding thin sections were cut. Anaphaee I divisions are observed (A). Bar is 20 pm.

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PLATE 3

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PLATE 4


10 A nuclear envelope (NE) reforms around the primary spermatoeyte telophaae nucleus during the short interphase prior to beginning the eeeond meiotic division. Nu- cleolus (Nu). Refringent vesicles (RV). Mitochondria (M). Bar is 5 pm.

11 Mitochondria (M) and a pair of centrioleg (C) are present at the poles during metaphase 11. Chromosome (CH). Refringent vesicles (RV). Bar is 5 pm.

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SPERMATOGENESIS IN ASCARIS Paul Goldatein

PLATE 4

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PLATE 5


Figs. 12a-f Condensation of secondary spermatocyte nucleus resulting in the compact spermatid nucleus.

12a Secondary spermatocyte nucleus (N) after fusion of chromoeomea at telophase II. Refringent vesicles (RV). Bar is 3 pm.

b Beginning of condensation of chromatin (HK). Centrioles (C). Microtubules (arrows). Bar is 1 pm.

c-e Further condensation and fusion of condensed chromatin. Bar is 1 p a

f A nuclear envelope is absent around the spermatid nucleus (N). Granular ribonucleoprotein layer (GR). Mitochondria (M). Bar is 1 pm.

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PLATE 5

331

PLATE 6


13-15 Differentiation of secondary spermatocyte cytoplasm and extrusion of the cytophore. The cytoplasm is extruded and the vesicles pinched off (fig. 15). Rough endoplasmic reticulum (RER). Dictyosomes (D). Refringent vesicles (RV). Micro- filaments (MF). Cytophore (CP). Bar in figure 13 is 10 pm. Bar in figure 13a is 2 pm. Bar in figures 14 and 15 is 6 q.

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PLATE 6

333

PLATE I


16 Mature amoeboid sperm in utem. The refringent vesicles have fused in the formation of the refringent cone (RC). Membrane specializations (MS). Nucleus (N). Mitochon- dria (M). Bar is 5 pm.

17 Coated microtubules kMT) observed in the interzonal region during anaphase 1. Bar is 1 sm.

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PLATE 7

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PLATE 8


Figs. 18a-c Centrioles in A. 8uum are present through all stages of spermatogenesis.

18a The centriole has a 9-fold symmetry of single microtubules and a central cartwheel is absent. A dense, continuous ring connects the subunits. Microtubules (MT). Dense ring (arrow). Bar is 1 p.

b A pair of centrioles is present at each pole at anaphaae I. Bar is 2 Nm.

c During the prophaee I of meiosis a procentriole Uarge arrow) is observed. Micro- tubules (small arrows). Bar is 1 Nm.

19,19a A granular layer of ribonucleoprotein (RNP) surrounds the spermatid nucleus (N). A nuclear envelope is absent. The centriole (C) is associated with RNP material. Bar is 1 pm.

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PLATE 8

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spermatogenesis and spermiogenesis in ascaris lumbricoides var. suum

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