Biol Res 37: 767-775, 2004

Gametogenesis and nucleotypic effects in the tetraploid red vizcacha rat, Tympanoctomys barrerae (Rodentia, Octodontidae)

MILTON H. GALLARDO¹, ORLANDO GARRIDO², RAÚL BAHAMONDE¹ and MARCELO GONZÁLEZ¹

¹ Instituto de Ecología y Evolución and ² Instituto de Embriología, Universidad Austral de Chile, Valdivia, Chile

Dirección para Correspondencia

ABSTRACT

Nucleotypic effects link DNA content with nuclear size and cell dimensions of reproductive cells in polyploid organisms. We studied the gametogenesis of the allotetraploid rodent Tympanoctomys barrerae, aiming to determine these effects in reproductive cells. The species' cofamily members, Octodon degus and Spalacopus cyanus were used as control. Spermatogenesis and oogenesis in T. barrerae follows the pattern of differentiation and sequence of events of the control species, but varied nucleotypic effects were observed. Exceedingly large, spatulated spermatozoa with a submedially attached flagellum are characteristic of male T. barrerae. The diameter of the nuclei of primordial and growing follicles as well as those of the Graaff follicles, of the granulose, and of luteal cells are significantly larger and heavily heterochromatic. Moreover, the width of the pellucid zone is 108% thicker in T. barrerae than in S. cyanus. Binucleation was recorded in 26% of luteal bodies examined whereas no binucleated cells are detected in the diploid control. Likewise, large heterochromatic nucleoli were observed in the follicle cells but not in S. cyanus. This finding and the high heterochromatin content of reproductive cells in the red vizcacha rat is probably associated with its genome complexity so that redundant genetic information is silenced through heterochromatinization.

Key terms: nucleotypic effects, oogenesis, spermatogenesis, , Octodontidae, Tympanoctomys barrerae, tetraploidy.

INTRODUCTION

Gametogenesis is the inclusive process by which diploid cells undergo meiosis to produce highly differentiated and specialized haploid gametes (Hale, 1996). Although gamete formation differs in each sex, the sperm and the ovum are homologous and have evolved in morphophysiological concert. Indeed, the sperm must recognize and bind tightly to specific components of the egg's zona pellucida for fertilization to occur (Wassarmann & Litscher, 1995).

Unlike ovogenesis, spermatogenesis is a most dramatic modification that reflects the potential of evolutionary adaptations to cope with functional needs at the cellular level (Bedford & Hoskins, 1990). Associated with these requirements, the complex morphology and extensive variation in head and flagellar dimensions of mammalian spermatozoa appears associated with adaptive roles for optimal motility, gamete number, and size (Gomendio & Roldán, 1991, 1993; Roldán et al., 1992). But the role of the sperm is relevant also in connection with development since its entry position determines the plane of initial cleavage of the egg, and therefore defines the early antero-posterior axis of mouse embryos (Piotrowska & Zernicka-Goetz, 2001). The direct relationship between DNA content and cell dimension is well known and results in larger reproductive structures (flowers and seeds) of polyploid plants (Stebbins, 1971). These nucleotypic effects are claimed to result from increased genome size (Price et al., 1973; Gregory & Hebert, 1999; Gregory, 2000).

The large genome size described in the red vizcacha rat, Tympanoctomys barrerae (16.8 pg DNA) has overturned previous ideas about the constraints imposed by a mammalian genetic system (Gallardo et al., 1999). This genome size, ostensibly resulting from tetraploidization, more than doubles the grand mean of rodents (7.8 pg) and of mammals (6.3 pg) as a whole. Associated with its DNA content, a significant increase in the somatic cell dimension has been reported in T. barrerae (Gallardo et al., 2003). Aiming to assess the kind and degree of nucleotypic effects in the reproductive cells of T. barrerae, its spermatogenesis and oogenesis is studied.

MATERIALS AND METHODS

Four male and five female T. barrerae collected in Nihuil, Mendoza province, Argentina were used in this study. Two male Octodon degus collected in Peñuelas National Park (Valparaíso province, Chile) and three female Spalacopus cyanus (Octodontidae) collected in Quirihue (Chillán province, Chile) were included for comparative analyses. Animals were sacrificed with an overdose of ether anesthesia. Gametogenesis and sperm morphology were investigated by scanning (SEM) and transmission electron microscopy (TEM). Tissues for TEM observations were fixed according to Rodríguez (1969) and embedded according to Richardson et al. (1960). Ultrathin sections were mounted in copper grids and stained with uranyle acetate and lead citrate (Glauert, 1965). Smears of mature spermatozoa were fixed as described above and prepared for SEM. The smears were dehydrated in acetone, critical-point dried and coated with gold. SEM observations were made using a Leo 420 microscope. TEM observations were conducted on a Philips EM 300 and on a Hitachi H 700 microscope. The significance of cell size differences was tested by the Student's t-test.

RESULTS

Spermiogenesis: During the Golgi phase, chromatin is observed across the nuclear periphery although small granules of heterochromatin are also seen in the central area. Euchromatin is observed as small granules dispersed in the central area of the nucleus. The Golgi complex consists of an array of flat vessels, vacuoles, and scattered granules in the perinuclear cytoplasm (Fig. 1A). The proacrosomal granule, formed from the Golgi complex, corresponds to the apical point of the spermatid. Several mitochondria are visible throughout the cytoplasm (Fig. 1A). The proximal and distal centrioles are seen in the inner central depression of the implantation fossa (Fig. 1B).

While the spermatid's nucleus is still spherical, heterochromatin granules migrate to the central area of the nucleus. At the initiation of this compacting phase, the nucleus evolves from spherical to a flat, elongated structure (Fig. 1C). As constitutive chromatin is compacted, the nucleus gets its electrodense appearance. Simultaneously, the basal cytoplasm for the manchette pulls the cytoplasm to the posterior end. The distal centriole disappears into the implantation fossa and the proximal centriole originates a typical axoneme that corresponds to the initial flagellum (9 + 2 microtubules). A profusion of mitochondria migrate to the proximal end of the flagellum and originates the intermediate piece of the spermatid (Fig. 1D). The subsequent events occurring during the spermatid differentiation are similar to those of other rodents (Breed, 1995). In fact, the spermgenesis of T. barrerae involves the formation of the acrosome, transformation of the nucleus shape, elimination of the cytoplasm, and configuration of the flagellum. During the cap phase, the acrosomal vesicle forms a distinctive structure in broad contact with the nucleus. The acrosomal granule is characterized by its electrodense content (Fig. 1C). By the time the spermatozoa reach the proximal cauda epididymes, their acrosomes are condensed.

Figure 1A. Initial spermatid in Golgi stage. Gc, Golgi complex; N, nucleus; Nu, nucleolus. 7500X TEM. Figure 1B. Spermatid at the beginning of flagellum formation. Dc, distal centriole; N, nucleus; F, spermatid at start of flagellum formation; Pc, proximal centriole. 7500X TEM. Figure 1C. Intermediate spermatid with oval nucleus in phase of acrosomal vesicle, Av; Ch, chromatin; N, nucleus. 7500X TEM. Figure 1D. Later spermatid with compacted nucleus, N; Pc, proximal centriole; M, mitochondria. 15000X TEM.

The basic design of the spermatozoon of`T. barrerae is similar to that of eutherian mammals (Jones, 1974; Bedford & Hoskins, 1990; Gage, 1998) but its total length (86 mm) exceeds significantly those of most rodent species so far reported (Cummins & Woodall, 1985, Gallardo et al., 2002). The sperm head has a large, flat, spatulated head, with a broad lateral phase (Fig. 2A). An intense nuclear reaction is obtained with the DAPI stain (not shown). Head dimensions are 14.2 mm in length, 13.7 in width, and 0.4 mm in height. The acrosomal vesicle is 8.2 mm in length and extends two thirds of the anterior pole of the nucleus (Fig. 2A). The nucleus shape is also flat and spatulated (0.25 mm in lateral view). Nuclear chromatin is moderately electrodense and homogeneously distributed. The implantation fossa containing the connecting piece has an excentrical position such that the flagellum attaches submedially to the truncated end of the head (Figs. 2A, 2B). The intermediate piece (7.42 mm in length and 1.34 mm in diameter) has the characteristic spiral mitochondrial sheath surrounding the axoneme and the outer longitudinal fiber (Figs. 2B, 2D). The nine striated columns of the connecting piece fuse with a corresponding number of outer longitudinal fibers like in other rodents (Breed, 1995). The principal piece (55.51 mm in length; 0.58 mm in width) looses its outer longitudinal fiber in position 3-8 while the formation of the columns and transversal ribs uniting them occurs like in other mammals (Fig. 2C, 2E). The longitudinal components have the typical microtubule arrangement (9+2; Fig. 2E). The end piece is formed by the axoneme (0.37 mm in width), surrounded by the plasmatic membrane (Fig. 2F). The diameter of the end piece (6.55 mm) gradually decreases throughout its length as the microtubules disappear.

Figure 2A. Mature spermatozoon indicating the acrosome, A; end piece, Ep; head, H; intermediate piece, Ip; principal piece, Pp. TEM Figure 2B. Longitudinal section of the nucleus indicating its basal part, the intermediate piece, Ip, and the principal piece, Pp. The implantation fossa, If; the longitudinal fibers, Lf, the mitochrondria, M; the nucleus, N, and the residual dropped (Rd) are indicated. 8000X TEM. Figure 2C. Longitudinal section of the principal piece. The axoneme, Ax; the longitudinal fiber, Lf, and the ribs, R are indicated. 18000X TEM. Figure 2D. Cross section of the intermediate piece. Ax, axoneme; Lf, longitudinal fiber; and the ribs, R. 30000X TEM. Figure 2E. Cross section of the principal piece indicating the axoneme, Ax; the longitudinal column, Lc; the longitudinal fiber, Lf, and the ribs, R. 44000X TEM. Figure 2F. Cross section of the end piece showing the axoneme, Ax, and the plasmatic membrane, Pm. 30000X TEM.

Follicular development: Macroscopically, the ovary is ovoid, has a smooth surface, and lacks the ovarian sac. The oocyte nuclei of T. barrerae are moderately electrodense, spherical in shape, slightly excentric in position, with large clumps of heterochromatin in the periphery. The mitochondria have an electrodense appearance and are homogeneously distributed in the cytoplasm (Fig. 3A). Large deposits of heterochromatin are observed in the central nuclear area corresponding to the nucleolus of the follicle cells; a feature not observed in S. cyanus (Figs. 3A, 3B). The oocytes of S. cyanus are also spherical and slightly excentric. The fine-grained, homogeneous distribution of heterochromatin gives the oocyte's nucleus a clear appearance (Fig. 3B). Mitochondria are moderately electrodense and homogeneously distributed. Primary oocytes do not differ significantly in size between T. barrerae and S. cyanus but nuclei are significantly larger in the former species (P < 0.05; Table I). Likewise, no difference in total diameter is observed in the granulose cells of both species, but nuclei diameter is significantly larger in T. barrerae (P < 0.05; Figs. 3C, 3D). Unlike the discrete distribution of heterochromatin in S. cyanus (Fig. 14), large clumps of heterochromatin covering up to 50% of the nuclei are observed in the luteal cells of T. barrerae (Fig. 3E). Significantly larger cell diameter compared with the control is observed in the Graaff follicles (48.8%) and in the mononucleated luteal cells (46%) of T. barrerae (Table I). The most extreme difference in size is noted in the width of the pellucid zone which is 108% thicker in T. barrerae relative to control species (Table I).

Figure 3A. Primary follicle of T. barrerae. Nf, nucleus of follicle cells; No, nucleus of oocyte. 3600X. Figure 3B. Primary follicle of S. cyanus. Nf, nucleus of follicle cells; No, nucleus of oocyte. 3600X. Figure 3C. Granulosa cells of T. barrerae. Ht, heterochromatin; N, nucleus; Oo, oocyte; Pz, pellucid zone. 6500X TEM. Figure 3D. Granulosa cells of Spalacopus cyanus. Nucleus, N; the oocytes, Oo; pellucid zone Pz. 6500X TEM. Figure 3E. Binucleated luteal cells of T. barrerae. Note the large amount of heterochromatin, Ht. Lipid accumulation, L; mitochondria, M. N1 and N2 refers to each of the two nuclei. 5500X TEM. Figure 3F. Luteal cell of S. cyanus depicting lipid deposits, L and the nucleolus, Nu. Note less heterochromatin, Ht, relative to T. barrerae. 5500X TEM.

Marked hypertrophy of the mononucleated luteal cells reaching 46% is noted in T. barrerae when compared with S. cyanus (P < 0.05; Table I). Moreover, binucleation is observed in 26.2% of its luteal bodies, but not in the control species (Fig. 3E). Interestingly enough, significantly smaller nuclei are observed in the binucleated cells of T. barrerae when compared to the mononucleated cells of S. cyanus (Fig. 3F; Table I).

The general TEM appearance of primary oocytes of both the primordial and the growing follicles of T. barrerae is also similar to those of other mammals. Although the oocytes of the Graaf follicles are significantly larger than in S. cyanus (Table I), their size is within the upper limit of cell dimension of eutherians (60-120 mm; Harrison & Weir, 1977). Characteristic electro-dense granules of heterochromatin are observed near the perinuclear membrane of granulosa and luteal cells of T. barrerae and S. cyanus (Figs. 3C-3F).

DISCUSSION

The sequence of differentiation events resulting in gamete production in male and female T. barrerae fits the general pattern described for mammals and particularly to those of the related octodontid, Octodon degus (Potocnjac & Bustos-Obregón, 1977; Montenegro et al., 1977; Berríos et al., 1978).

From varied positive correlations, the nucleotypic effect of genome size over cell dimensions has been advanced in diploid organisms (Bachmann, 1972; Price et al., 1973) and in cell lineages of a wide range of taxa (Gregory, 2000, 2001; Gregory & Hebert, 1999). This relationship, affecting the reproductive structures of polyploid plants is called the gigas effect (Stebbins, 1971). Enlarged sperm heads in abnormal, diploid gametes of rabbits and bovines are clear instances of this relationship in mammals (Beatty & Fechheimer, 1972; Ferrari et al., 1998). Moreover, larger cell size in isogenic strains of yeast results from ploidy-dependent repression of some G1 cyclins (Galitski et al., 1999). In Drosophila, cell size depends on some insulin-receptor signaling pathways associated to ploidy level (Stocker & Hafen, 2000).

Exceedingly large sperms compared to its relatives and to other South American caviomorphs are found in T. barrerae (Gallardo et al., 2002). Nevertheless, the sperm's head and tail increased at different rates since flagellum length is only 55% to 63% of its predicted value estimated from the isomorphic growth rate of the head (Gallardo et al., 2002). Whether this effect results from the different origin of these structures or from ploidy-dependent interactions cannot be ascertained at the moment. Unlike its relatives, head shape in T. barrerae is truncated whereas tail implantation gives the sperms an asymmetrical appearance. Nucleotypic effects in female T. barrerae are observed in the hypertrophy of the luteal cells and Graaf follicles relative to other mammals (Harrison & Weir, 1977; Cummings & Woodall, 1985). Although the oocyte diameter does not differ from the control species, nuclear size of the granulosa cells, of primordial follicles, and of mononucleated luteal cells is significantly larger (Table I) and is probably associated to genome size.

T. barrerae has been shown to have a duplicated genome size (Gallardo et al., 1999) and strict bivalent formation (Lanzone et al., 2001) as to support its hybrid origin (Gallardo et al., 2004). Allopolyploidization is often accompanied by rapid genetic and epigenetic changes including DNA methylation of ribosomal RNA and protein-coding genes and de-repression of dormant transposable elements (Comai, 2000; Wolfe, 2001; Liu & Wendel, 2002). For example, gene silencing of redundant ribosomal genes (rRNA) is achieved by heterochromatin condensation in allopolyploids (Pikaard, 2000; Viegas et al., 2002; Lawrence et al., 2004). In this context, the large heterochromatic content of follicle cells in T. barrerae (but not in the control species) may be associated to gene silencing of redundant genetic material (Mittelsten-Scheid et al., 1996). On the other hand, normal development in diploid mammals involves a two-fold increase in size of luteal cells but not in nucleus number (Blanchette, 1966; Pate, 1994; Niswender et al., 2000). Thus, our unexpected finding of luteal cell binucleation may result from incomplete diploidization in an organism with a complex genome of hybrid origin. The smaller size of binucleated luteal cells relative to those of the control species remains enigmatic since it departs from genome size expectations. Ongoing research will help us to shed light on this, and other aspects of the evolutionary dynamics of this peculiar species.

ACKNOWLEDGEMENTS

We thank F. Mondaca for fieldwork assistance. This research was partially supported by Fondecyt, Grant 1010727 to MHG and DID UACH S- 9232 to OG.

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Corresponding Author: Milton H. Gallardo. Instituto de Ecología y Evolución, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, Teléfono: (56-63) 221469, Fax: (56-63) 221344, E-mail: mgallard@uach.cl

Received: May 24, 2004. In revised form: September 20, 2004. Accepted: October 6, 2004