SciELO - Scientific Electronic Library Online

 
vol.37 issue4  suppl.ATime in physics and biologyAnalysis of rhythmic variance - ANORVA. A new simple method for detecting rhythms in biological time series author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 suppl.A Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000500006 

 

Biol Res 37: 767-775, 2004

ARTICLE

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).

 

TABLE I

Cell dimensions (mm) and standard deviations of different reproductive cell types of female T. barrerae and Spalacopus cyanus. N = sample size; S = significant; NS = non-significant


         

CELL TYPE

n

Spalacopus cyanus

Tympanoctomys barrerae


Oocyte diameter

20
28.42 ± 1.45
28.51 ± 0.92
NS
Nuclei of primordial follicle
20

17.98 ± 0.81

19.73 ± 1.21
S

Graaf follicle

25
74.21 ± 7.3
110.43 ± 5.1
S

Width of the pellucid zone

20
3.82 ± 0.68
7.97 ± 0.59
S

Nuclei of mononucleated luteal cells

200
2.99 ± 0.35
4.37 ± 0.39
S

Nuclei of binucleated luteal cells

1000
2.90 ± 0.27
Nuclei of granulosa cells

25

2.98 ± 0.44
3.36 ± 0.45
S

 

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.

REFERENCES

BACHMANN K (1972) Genome size in mammals. Chromosoma 37: 85-93         [ Links ]

BEATTY RA, FECHHEIMER S (1972) Diploid spermatozoa in rabbit semen and their experimental separation from haploid spermatozoa. Biol Reprod 7: 267-270         [ Links ]

BEDFORD JM, HOSKINS D (1990) The mammalian spermatozoon: morphology, biochemistry and physiology. In: LAMMING GE (ed) Marshall's Physiology of Reproduction. 2. The Male Reproductive System. London: Churchill Livingstone. pp: 379-568         [ Links ]BERRÍOS M, FLECHON JE, BARROS C (1978) Ultrastructure of Octodon degus spermatozoon with special reference to the acrosome. Amer J Anat 151: 39-54         [ Links ]

BLANCHETTE EJ (1966) Ovarian steroid cells. II. The lutein cell. J Cell Biol 31:517-523         [ Links ]

BREED WG (1995) Spermatozoa of murid rodents from Africa: morphological diversity and evolutionary trends. J. Zool 237: 625-651         [ Links ]

COMAI L (2000) Genetic and epigenetic interactions in allopolyploid plants. Plant Mol Biol 43: 387-399         [ Links ]

CUMMINS JM, WOODALL PD (1985) On mammalian sperm dimensions. J Reprod Fertility 75: 153-175         [ Links ]

FERRARI MR, SPIRITO SE, GIULIANO SM, FERNÁNDEZ HA (1998) Chromatin cytophotometric analysis of abnormal bovine spermatozoa. Andrologia 30: 85-89         [ Links ]

GAGE MJG (1998) Mammalian Sperm morphometry. Proc Royal Soc London B 258 97-103         [ Links ]

GALITSKI T, SALDANHA AJ, STYLES CA, LANDER ES, FINK GR (1999) Ploidy regulation of gene expression. Sci 285: 251-254.         [ Links ]

GALLARDO MH, BICKHAM JW, HONEYCUTT RL, OJEDA RA, KÖHLER N (1999) Discovery of tetraploidy in a mammal Nature 401: 341         [ Links ]

GALLARDO M, MONDACA FC, OJEDA RA, KÖHLER N, GARRIDO O (2002). Morphological diversity in the sperms of caviomorph rodents. Mastozool Neotropical 9: 159-170         [ Links ]

GALLARDO MH, BICKHAM JW, KAUSEL G, KÖHLER N, HONEYCUTT RL (2003) Gradual and quantum genome size shifts in the hystricognath rodents. J Evol Biol 16: 163-169         [ Links ]

GALLARDO, MH, KAUSEL G, JIMÉNEZ A, BACQUET CN, GONZÁLEZ CA, FIGUEROA J, KÖHLER N, OJEDA RA (2004). Whole-genome duplications in South american desert rodents (Octodontidae). Biol J Linn Soc (in press)         [ Links ]

GLAUERT AM (1965) Section staining, cytology, autoradiography and immunochemistry for biological specimens. In: KAY B (ed) Techniques for Electron Microscopy. Oxford and Edinburgh: Blackwell Scientific. pp: 254-310         [ Links ]

GOMENDIO M, ROLDÁN ERS (1991) Sperm competition influences sperm size in mammals. Proc R Soc London B 252: 7-12         [ Links ]

GOMENDIO M, ROLDÁN ERS (1993) Coevolution between male ejaculates and female reproductive biology in eutherian mammals. Proc R Soc London B 252: 7-12         [ Links ]

GREGORY TR (2000) Nucleotypic effects without nuclei: genome size and erithrocyte size in mammals. Genome 43: 895-901         [ Links ]

GREGORY TR (2001) Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma. Biol Rev 76: 65-101         [ Links ]

GREGORY TR, HEBERT PDN (1999) The modulation of DNA content: proximate causes and ultimate consequences. Genome Res 9: 317-324.         [ Links ]

HALE DW (1996) Mammalian spermatogenesis. In: VILLAR, HO (ed) Advances in Genome Biology. New York: JAI Press Inc. pp: 249-304         [ Links ]

HARRISON RJ, WEIR BJ (1977) Structure of the mammalian ovary. In: ZUCKERMAN L,WEIR BJ (eds) The Ovary. New York: Academic Press. pp: 113-217         [ Links ]

JONES RC (1974) The ultrastructure of spermatozoa from some hystricomorph rodents. In: AFZELIUS BA (ed) The Functional Anatomy of the Spermatozoon. Oxford: Pergamon Press. pp: 251- 258         [ Links ]

        [ Links ]

LAWRENCE RJ, EARKEY K, PONTES O, SILVA M, CHEN ZJ, NEVES N, VIEGAS W, PIKAARD CS (2004). A concerted DNA methylation/histone methylation switch regulates rDNA gene dosage control and nuclear dominance. Mol Cell 13: 599-609         [ Links ]

LIU B, WENDEL JF (2002) Non-mendelian phenomena in allopolyploid genome evolution. Curr Genomics 3: 1-17         [ Links ]

MITTELSTEN-SCHEID O, JAKOVLEVA L, AFSAR K, MALUSZYNSKA J, PASZKOWSKI, J (1996) A change in ploidy can modify epigenetic silencing. Proc Natl Acad Sci USA 93: 7114-7119         [ Links ]

MONTENEGRO MA, BUSTOS S, BERRÍOS S, BARROS C (1977) Activación y reacción del acrosoma de espermatozoides de Octodon degus. Medio Ambiente 3: 120-126         [ Links ]

NISWENDER GD, JUENGEL JL, SILVA PJ, ROLLYSON MK, MCINTUSH EW (2000) Mechanisms controlling the function and life span of the corpus luteum. Physiol Rev 80: 1-29         [ Links ]

PATE JL (1994) Cellular components involved in luteolysis. J Anim Sci 72: 1884-1890         [ Links ]

PIKAARD C (2000) Nucleolar dominance: uniparental gene silencing on a multi-megabase scale in genetic hybrids. Plant Mol Biol 43: 163-177         [ Links ]

PIOTROWSKA K, ZERNICKA-GOETZ M (2001) Role of sperm in spatial patterning of the early moue embryo. Nature 409: 517-521.         [ Links ]POTOCNJAC P, BUSTOS-OBREGÓN E (1977) Espermatogénesis y duración del ciclo del epitelio seminíferos de Octodon degus Molina. Medio Ambiente 3: 100-111         [ Links ]

PRICE JJ, SPARROW AH, NAUMAN AF (1973) Correlations between nuclear volume, cell volume and DNA content in meristematic cells of herbaceous angiosperms. Experientia 29: 1028-1029         [ Links ]

RICHARDSON K, JARRETE L, FINKE E (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35: 313-323         [ Links ]

RODRÍGUEZ E (1969) Fixation of the central nervous system by perfusion of the cerebral ventricles with a threefold aldehyde mixture. Brain Res 15: 395-412         [ Links ]

ROLDÁN ERS, GOMENDIO M, VITULLO AD (1992) The evolution of eutherian spermatozoa and underlying selective forces: female selection and sperm competition. Biol Rev 67: 551-593         [ Links ]

STEBBINS L (1971) Chromosomal Evolution in Higher plants. London: Edward Arnold Ltd Publisher         [ Links ]

STOCKER H, HAFEN E (2000) Genetic control of cell size. Curr Op Genet Dev 10: 529-535         [ Links ]

VIEGAS W, NEVES N, SILVA M, CAPERTA A, MORAIS-CECILIO L (2002) Nucleolar dominance: a "David and Goliath" chromatin imprinting process. Curr Genomics 3: 563-576         [ Links ]

WASSARMAN PM, LITSCHER ES (1995) Sperm-egg recognition mechanisms in mammals. Curr Topics Dev Biol 30: 1-19         [ Links ]

WOLFE KH (2001) Yesterday's polyploids and the mystery of diploidization. Nature Rev Genet 2: 333-341         [ Links ]

 

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

 

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License