SciELO - Scientific Electronic Library Online

 
vol.19 número2ESTUDIO AL MICROSCOPIO ELECTRÓNICO DE BARRIDO DEL EPIDÍDIMO EN EL PERRO (Canis familiaris, L.)TIPOS DE FIBRAS MUSCULARES EN CERDAS DETERMINADOS POR COMBINACIÓN DE TÉCNICAS HISTOQUÍMICAS E INMUNOHISTOQUÍMICAS índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

Compartir


Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.19 n.2 Temuco ago. 2001

http://dx.doi.org/10.4067/S0716-98682001000200007 

DESCRIPTION OF THE CHARACTERISTICS OF OOCYTES AND ZYGOTES OF COYPU (Myocastor coypus)

DESCRIPCIÓN DE LAS CARACTERÍSTICAS DE OVOCITOS Y CIGOTOS DE COIPO
(Myocastor coypus)

* Felipe, Antonio; * Teruel, Miriam; **Callejas, Santiago & **Cabodevila, Jorge.

** Área de Ciencias Morfológicas, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Buenos Aires, Argentina.
** Área de Reproducción, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Buenos Aires, Argentina.

SUMMARY: The goal of the present work was to characterise morphologically the oocytes and zygotes of Myocastor coypus. In order to obtain the samples, a daily colpocytological follow-up and programmed mating of females were done. By flushing of the oviducts, oocytes (17) and zygotes (24) were collected. In the analysis of the oocytes and zygotes, morphological aspects, including analysis of cellular polarity and of the envelopments were considered. Oocytes presented spherical shape with a cellular diameter of 85.7 ± 3.1 µm. The nucleus presented a diameter of 28 ± 3.1 µm. The occupied perivitelline space was 98%, with a zona pellucida thickness of 15.9 ± 1.9 µm. The existence of ovular polarity was determined. Zygotes presented an external diameter, measured to the zona pellucida, of 125 ± 8.6 mm and an embryonic diameter of 80.6 ± 8.1 µm. In 91.7% of the cases, they were in the stage of 2-nucleus zygote. A homogenous distribution of cytoplasmic granulations was observed in all the zygotes. As the only and complete envelopment, zygotes presented the zona pellucida, obtaining few specimens with associated granulose cells.

  KEY WORDS: 1. Oocytes; 2. Zygotes; 3. Myocastor coypus; 4. Morphology.

INTRODUCTION

The mammalian oocyte possesses most of the material and protective envelopments inherited by the zygote at the beginning of the development of a new individual (Wassarman, 1988). In many mammalian species, the morphological characterisation of the post-ovulatory oocytes and zygotes from the analysis of specimens collected after controlled mating, has been performed (Enders et al., 1987; Tsutsui, 1989; Xie et al., 1990; Renton et al., 1991; Farstad et al., 1993; Wakayama et al., 1994) These studies have allowed the development of basic knowledge on domestic and laboratory animals (Hunter et al., 1969; Maraldi & Monesi, 1970; Breed & Leigh, 1990) and its biomedical applications (Palma & Brem, 1993). For Myocastor coypus, the current information on early embryonic development aspects is scarce. Hence, the objective of the present work is to morphologically characterise post-ovulatory oocytes and zygotes of Myocastor coypus.

MATERIAL AND METHOD

Oocytes and zygotes were obtained from 7 females, weighing 4.6 ± 0.7 kg and ageing 9.4 ± 0.5 months. The mating programme included a daily colpocytological follow-up and controlled mating. The confirmation of mating by colpocytology allowed assuming as time 0 the day on which it happened. The total number of oocytes and zygotes was 17 and 24, respectively, collected at 24-hour intervals form day 1 to 3 post-coitus, by flushing of the oviducts. The specimens were studied in fresh utilising an Olympus CO11 binocular microscope. For their morphological evaluation, they were transferred with capillary pipettes onto a smear glass with a drop of flushing liquid. A complete observation of each specimen was achieved rotating them by producing the movement of the surrounding liquid with a capillary pipette.

For each specimen, it was determined: cellular polarity, based on the position of the nuclei or pronuclei and the presence and position of the cytoplasmic granulations; the total diameter, considering as the external diameter the zona pellucida (ZPED); the internal diameter of the zona pellucida (ZPID); the oocyte and zygote diameters (cellular diameter, CD); the perivitelline space (PS), considering the space present between the oocyte or zygote and the internal surface of the zona pellucida. For the ZPED, ZPID and CD, it was calculated the mean of three diameters in straight angle passing through the centre of the specimen. The PS was estimated by the mean of 5 measures taken from the internal surface of the zona pellucida to the cellular surface. The shape, mean size and position in the cell were determined for the nucleus of oocytes and pronuclei of zygotes. Measures were taken with a micrometric ocular fitted to the stereomicroscope.

The volumes of each very oocyte and zygote and the potential space of the vitelline cavity were obtained from the measures taken for determining CD and ZPID. As to the assumption of specimen sphericities, the volumes were estimated as the product of three diameters times 0.523

(Rowlands & Heap, 1974). Based on the volume values, the occupied perivitelline space was calculated, which was expressed as percentage.

Morphological characteristics of the envelopments were determined (Dumont & Brumet, 1985; Breed & Leigh). For the cumulus oophorus, the radiated crown and the very cumulus were distinguished (Talbot, 1985), the disposition and association of the granulose cells with the zona pellucida were observed and the mean of its size was estimated measuring 10 cells per oocyte. In the case of the polar bodies, it was registered their number, localization, shape and mean size. It was considered the presence of cellular extrusions and their position, in a similar way as that observed in other species (Nilsson, 1980). The morphological signs utilised for determining oocytes from zygotes were based on the criteria of Bedford (1982) and Bavister (1989), as indicators of fecundation. Those oocytes and zygotes presenting morphological alterations were considered as abnormal (Palma & Brem). The mean value and its standard deviation were determined for all the embryonic measures obtained.

Fig. 1. Oocyte and zygote of Myocastor coypus.
A: Diagram of the morpholo-gical structure of an oocyte; B: micropho-tograph of an oocyte, 20X; C: Micropho-tograph of a zygote, 20X.

ZPED: zona pellucida external diameter; ZPID: zona pellucida internal diameter; CD: cellular mass diameter; PS: perivitelline space; OPS: occupied perivitelline space.
Values are expressed as mean ± standard deviation of the mean.

RESULTS

Oocytes: The oocytes presented spherical shape (Figs. 1A and B), with a round nucleus of 28.0 ± 3.1 µm of diameter. Their morphometric characteristics are presented in Table I. It was possible to determine the existence of cellular polarity in oocytes, having observed their nuclei in an eccentric position, close to the polar corpuscle. This determined the area considered as animal pole. As to the cytoplasmic polarity, the cytoplasm of the oocytes showed a translucid appearance and an accumulation of granules or drops of refringent material localised eccentrically toward the area assumed as vegetative pole (Fig. 1 A). The oocytes presented a double envelopment: the cumulus oophorus and the zona pellucida. The cells of the most inner layers of the cumulus oophorus, adjacent to the zona pellucida, presented ovoidal shape and radial disposition. These cells showed cytoplasmic projections passing through the zona pellucida and taking contact with the surface of the gamete. This cellular arrangement was interpreted as corresponding to the radiated crown. The most outer layers, considered as the very cumulus oophorus, formed an area of spheroid cells with a dispersed general appearance. The size of the granulose cells was 10.9 ± 1.5 µm. The zona pellucida was as a translucid and very refringent envelopment, with a thickness of 15.9 ± 1.9 µm, whose superficial aspect could not be observed because the cells of the radiated crown masked it. Except for the sector occupied by the only polar corpuscle, the existence of a perivitelline space was not observed. The polar corpuscle, with flatten ovoidal shape, presented a mean diameter of 20.6 ± 3.1 µm.

Zygotes: The collected zygotes presented spherical shape (Figure 1 C), cytoplasmic refrigency and homogenous distribution of cytoplasmic granulations. In 91.7% of the cases, it was the 2-pronucleus zygote stage (Fig. 1C). It was no possible to differentiate the masculine from the feminine pronuclei, owing to the similarities in shape and size (22 ± 3.1 µm). In most of the cases, both pronuclei were observed close to each other and in a central position. In 8.3% of the zygotes, it was observed a diffuse nuclear area, in central position, with a diameter of 30 ± 1.7 µm. The 87.5% of the zygotes presented as the only and complete envelopment the zona pellucida, observing associated granulose cells (range 1 to 6) only in the 12.5%. The zona pellucida, with a thickness of 17 ± 1.5 µm, was of transparent, refringent, homogenous and without grooves. The internal surface was soft and the external surface, for all the cases, was irregular or with little waves. A remarkable characteristic of the zygotes was the presence of a well-defined perivitelline space (Table I), of which 67.4% was occupied by the embryonic mass (ranging from 40.4% to 88.9%). In 91.7% of the zygotes, the presence of two polar bodies was observed in the perivitelline space. These had spherical shape, lightly flatten, with diameters of 16.6 ± 5 mm and 16.4 ± 4 µm. The remaining 8.3% presented a third polar body, with a mean diameter of 10 ± 1.8 µm. No signs of fragmentation were present in the polar bodies. Spermatozoa were observed in 91.6% of the zygotes, most of them (71.4%) localised within the zona pellucida, and the rest of them in the perivitelline space. The mean of spermatozoa per zygote was 1.9 ± 1, ranging from 0 to 4.

Tabla I. Morphometric characteristics of oocytes and zygotes of Myocastor coypus.
Characteristic
Oocyte
Zygote

ZPED

113,6 ± 2,9 µm
125,1 ± 8,6 µm

ZPID

86,2 ± 2,9 µm
92,2 ± 6,7 µm
CD
85,7 ± 3,1 µm
80,6 ± 8,1 µm
PS
absent
7,1 ± 3,5 µm
OPS
98,0 %
67,4 %

DISCUSSION

The general morphology of Myocastor coypus oocytes was similar to that of other eutherian mammals studied by Wassarman. Their mean size was similar to that of mouse oocytes (Snell, 1969) and of hysthricomorphs such as the plains viscacha (Weir, 1971a), chinchilla, casiragua and degu (Roberts, 1971). Roberts & Perry (1974) indicated that in most of the hysthricomorphs, the distribution of granulations in the ovular cytoplasm is regular, not being evidences of polarity. In the coypu, however, the cytoplasmic granulations were accumulated close to the vegetative pole, similarly to that reported by Baviste for oocytes of myomorphic rodents, such as the mouse and rat, and of a lagomorph, such as the rabbit, in which a vacuolated area is observed in the vegetative pole.

As to the ovular envelopments, the oocytes of the coypu were similar to that of eutherian mammals, which are surrounded by covers (zona pellucida and radiated crown) (Dumont & Brummet, 1985). In this aspect, as result of the morphologic analyses done on the ovular envelopments of the coypu, they were differentiated, at the cumulus, in a cellular area, adjacent to the zona pellucida, corresponding to the radiated crown, and a peripheral area, of a more or less diffuse cells, corresponding with the cumulus properly defined, in agreement with the differentiation done by Talbot and Harper (1988), in other mammals. Similarly to that described for other rodents of the suborder Hysthricomorpha, the oocytes of the coypu were surrounded by a zona pellucida with superficial irregularities, as also with a frequent presence of follicular cells. Both characteristics were reported for the chinchilla (Weir, 1967), the aguti (Dasiprocta agouti) (Weir, 1971b) and the viscacha (Weir, 1971a). In most of the eutherian mammals, the zona pellucida is surrounded by the radiated crown during the in vivo fertilisation. Interspecies variations have been observed on its presence in the ovulation and its post-ovulatory persistence. In some ungulates, as the cow and sheep, the cumulus is quickly detached after ovulation (Blandau, 1961; Rodger & Bedford, 1982), and in monotrems and marsupials it is absent in the ovulation (Bedford). In other species, as the rabbit (Chang & Bedford, 1962), the Rhesus monkey (Harper), the papion (Hendrickx), the chinchilla (Weir) the agouti (Weir, 1971b), the viscacha (Weir, 1971a) and the casiragua (Roberts), the cells of the radiated crown do not disperse and stay associated with the oocyte and the zygote. Conversely to these, in the coypu, the cells of the radiated crown were almost absent in the zygote, in agreement to the observations of Hunter et al. in guinea pigs.

In a similar way to that observed in the coypu, Roberts & Perry reported that in hysthricomorphs, at the liberation of the ovule, the first polar body is present, and remarked that the perivitelline space is little prominent in fresh oocytes. Similar observations were done in laboratory rodents (Yanagimachi, 1988). Bedford indicated that in the rabbit, hamster and guinea pig, the first polar body remains as a stable structure in the smallperivitelline space until the first cleavage division has occurred. The retraction of the ovular cytoplasm, the appearance or spreading of the perivitelline space and the observation of two pronuclei in the ovular cytoplasm constitute the most convincing cytological evidences of fecundation (Yanagimachi) and were observed in the coypu. The amplification of the perivitelline space has been informed in other mammalian species such as the guinea pig and the mouse (Snell; Bedford; Yanagimachi and) and marsupials (Selwood, 1987).

Conversely to that informed by Hunter et al. for the guinea pig, in which the pronuclei had a central position, in the coypu zygotes they were always observed in an eccentric position. This characteristic is in agreement to that reported in the rat and mouse by Bavister. With reference to other discernible components of the cytoplasm of the zygotes, in the guinea pig, Hunter et al. observed granulations uniformly distributed. These observations are in agreement with those in the coypu and would correspond to a characteristic of the granulations in many mammals (Bedford).

Similarly to that described by Bedford and Hunter et al. for the guinea pig, two polar corpuscle were clearly observed in most of the coypu zygotes and, in few occasions, three. As in the guinea pig, they presented ovoidal shape and no differentiation among them (Hunter et al.).

RESUMEN: El objetivo del presente trabajo fue caracterizar morfológicamente los ovocitos y los cigotos de Myocastor coypus. Se utilizaron 17 ovocitos y 24 cigotos obtenidos por lavaje de los oviductos de hembras, a las que se practicó seguimiento colpocitológico diario y apareamiento dirigido. En el análisis de los ovocitos y los cigotos se consideraron aspectos morfológicos y de la polaridad celular y envolturas. Los ovocitos presentaron forma esférica con un diámetro celular de 85,7± 3,1 µm. El núcleo, redondeado, presentó un diámetro de 28 ± 3,1 µm. El espacio perivitelino ocupado, fue del 98 %, con un espesor de la zona pelúcida de 15,9 ± 1,9 µm. Se determinó la existencia de polaridad ovular. Los cigotos presentaron un diámetro externo a la zona pelúcida de 125 ± 8,6 µm y un diámetro embrionario de 80,6 ± 8,1 µm. En el 91,7 % de los casos, se trató del estadio de cigotos de 2 pronúcleos. En todos los cigotos se observó una distribución homogénea de las granulaciones citoplasmáticas. Los cigotos presentaron a la zona pelúcida como única envoltura completa, obteniéndose pocos especímenes con células granulosas asociadas.

  PALABRAS CLAVE: 1. Ovocito; 2. Cigotos; 3. Myocastor coypus; 4. Morfología.

Prof. Dr. Antonio Felipe
Depto. de Ciencias Biológicas
Facultad de Ciencias Veterinarias UNCPBA
Pinto 399 (7000)
Tandil
Buenos Aires
ARGENTINA

Recibido : 23-04-2001
Aceptado: 30-05-2001

REFERENCIAS BIBLIOGRÁFICAS

Bavister, B. D. A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Research, 23:139-58, 1989.         [ Links ]

Bedford, J. M. Fertilization. In: Germ Cells and Fertilization. C.R. Austin & R.V. Short (eds.), Cambridge, Cambridge University Press, 1982.          [ Links ]

Blandau, J. R. Biology of eggs and implantation. In: Sex and internal secretions, W.C. Young (ed.), Baltimore, Williams & Wilkins, 1961.                [ Links ]

Breed, W. G. & Leigh, C. M. Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata. J. Morphol., 204:177-96, 1990.         [ Links ]

Chang, M. C. & Bedford, J. M. Fertilizability of rabbit ova after removal of the corona radiata. Fertil. Steril., 13:421-25, 1962.         [ Links ]

Dumont, J.N. & Brummet, A.R. Egg envelopes in vertebrates. En: Developmental Biology. A comprehensive synthesis. V. I: Oogenesis. Ed. by L.W. Browder, New York, Plenum Press, 1985.         [ Links ]

Enders, A.C.; Liu, I. K.; Bowers, J.; Lantz, K.C.; Schlafke, S. & Suarez, S. The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova. Biol. Reprod., 37:453-66, 1987.         [ Links ]

Farstad, W.; Hyttel, P.; Grondahl, C.; Mondain-Monval, M. & Smith, A. J. Fertilization and early embryonic development in the blue fox (Alopex lagopus). Mol. Reprod. Dev., 36:331-7, 1993.         [ Links ]

Harper, M. J. K. Gamete and Zigote Transport. In: The Physiology of Reproduction, ed. by E. Knobil and J. Neill et al., New York, Raven Press, 1988.         [ Links ]

Hendrickx, A.G. Embryology of the baboon. 1th. ed., Chicago, Univ. Chicago Press, 1971.         [ Links ]

Hunter, R. R. F.; Hunt, D. M. & Chang, M.C. Temporal and cytological aspects of fertilization and early development in the guinea pig, Cavia porcellus. Anat. Rec., 165:411-29, 1969.         [ Links ]

Maraldi, N. M. & Monesi, V. Ultrastructural changes from fertilization to blastulation in the mouse. Arch. D´Anat. microscop., 59:361-82, 1970.         [ Links ]

Nilsson, B.O. Comparative ultrastructure of the yolk material in preimplantation stages of the hamster, mouse and rat embryos. Gamete Res., 3:369-77, 1980.         [ Links ]

Palma, G. A. & Brem, G. Transferencia de Embriones y Biotecnología de la Reproducción en la Especie Bovina, 1th. ed., Buenos Aires, Hemisferio Sur, 1993.         [ Links ]

Renton, J. P.; Boyd, J. S.; Eckersall, P. D.; Ferguson, J. M.; Harvey, M. J.; Mullaney, J. & Perry, B. Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris). J. Reprod. Fertil., 93:221-31, 1991.         [ Links ]

Roberts, C. M. The early development of some hystricomorph rodents with particular reference to Chinchilla laniger. J. Reprod. Fertil., 27:488-9, 1971.         [ Links ]

Roberts, C. M. & Perry, J. S. Hystricomorph embryology. Symp. Zool. Soc. Lond., 34:333-60, 1974.         [ Links ]

Rodger, J.C. & Bedford, J. M. Separation of sperm pairs and sperm-egg interaction in the opossum Didelphis virginiana. J. Reprod. Fertil., 64:171-9, 1982.         [ Links ]

Rowlands, I.W. & Heap, R.B. Histological observations on the ovary and progesterone levels in the coypu (Myocastor coypus). Symp. Zool. Soc. Lond., 15:335- 40, 1974.         [ Links ]

Selwood, L. Embryonic development in culture of two dasyurid marsupials, Sminthopsis crassicaudata (Gould) and Sminthopsis macroura (Spencer) during cleavage and blastocyst formation. Gamete Res., 16:355-70, 1987.         [ Links ]

Snell, G.D. The early embryology of the laboratory mouse. In Biology of the laboratory mouse, Philadelphia,  Blakiston, 1969.         [ Links ]

Talbot, P. Sperm penetration through oocyte investments in mammals. Am. J. Anat., 174:331-46, 1985.         [ Links ]

Tsutsui, T. Gamete physiology and timing of ovulation and fertilization in dogs. J. Reprod. Fertil. Suppl., 39:269-75, 1989.         [ Links ]

Wakayama, T.; Matsubara, Y.; Imamura, K.; Kurohmaru, M.; Hayashi, Y. & Fukuta, K. Development of early-stage embryos of the japanese field vole, Microtus montebelli, in vivo and in vitro. J. Reprod. Fertil., 101:663-6, 1994.         [ Links ]

Wassarman, P. M. The mammalian ovum. En: The Physiology of Reproduction. E. Knobil and J. Neil et al., Raven Press, New York, 1988.         [ Links ]

Weir, B. J. Aspects of reproduction in some hystricomorph rodents. Ph.D. Thesis, Cambridge, University of Cambridge Press, 1967.         [ Links ]

Weir, B. J. The reproductive organs of the plains viscacha, Lagostomus maximus. J. Reprod. Fert., 25:365-73, 1971a.         [ Links ]

Weir, B. J. Some observations on reproduction in the female agouti, Dasyprocta aguti. J. Reprod. Fert., 24:203-11, 1971b.         [ Links ]

Xie, S.; Broermann, D. M.; Nephew, K. P.; Bishop, M. D. & Pope, W. F. Relationship between oocyte maturation and fertilization on zygotic diversity in swine. J. Anim. Sci. 68:2027-33, 1990.         [ Links ]

Yanagimachi, R. Mammalian fertilization. In: The Physiology of Reproduction. Ed. by E. Knobil and J.Neill et al., New York, Raven Press, 1988.         [ Links ]

 

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons