International Journal of Morphology
versión On-line ISSN 0717-9502
Int. J. Morphol. v.26 n.1 Temuco mar. 2008
Int. J. Morphol., 26(1):113-120, 2008.
Structural and Ultrastructural Aspects of Folliculogenesis in Didelphis albiventris, the South-American Opossum
Aspectos Estructurales y Ultraestructurales de la Foliculogénesis de Didelphis albiventris, la Zarigüeña Sudamericana
*Maria Dalva Cesario & **Selma M. Michelin Matheus
* Departamento de Morfologia, Instituto de Biociências, UNESP, São Paulo, Campus de Botucatu, Brasil.
SUMMARY: The ovarian histology, the structural and the ultrastructural characteristics of the folliculogenesis in Didelphis albiventris were described in detail. Recent studies suggest that methatherian mammals have unusual reproductive cycle but there are few informations regarding the marsupials reproductive life. Despite of the opossum folliculogenesis pattern resembles methatherian and eutherian pattern in many aspects, the analysis shows some peculiar features of the oocyte structure and ultrastructure that make available new data on the reproductive biology of marsupials.
KEY WORDS: Folliculogenesis, Oocytes; Opossum; Marsupial.
RESUMEN: Fueron descritas con detalles la histología ovárica, las características estructurales y ultra-estructurales de la foliculogénesis del Didelphisalbiventris. Estudios recientes sugieren que mamíferos metaterios tienen un ciclo reproductivo inusual, pero existen pocas informaciones en relación a la vida reproductiva de los marsupiales. A pesar de que el modelo de foliculogénesis de la zarigüeya se parece al modelo metaterio y euterio en muchos aspectos, el análisis muestra algunos rasgos peculiares de las estructura y ultra-estructura del oocito, que colocan a disposición nuevos datos en la biología reproductiva de los marsupiales.
PALABRAS CLAVE: Foliculogénesis; Oocitos; Zarigüeya; Marsupial.
Marsupials differ from eutherian mammals in their anatomical and physiological reproductive aspects although Mackay et al. (2004) have studied the postnatal reproductive tract development and have found that the mesonephric and paramesonephric ducts differentiation takes place after gonadal differentiation, according to the normal eutherian pattern. The opossum is a polyoestrous seasonal breeder (Fleming, 1973) and is sexually mature when in its 6 or 8 month-old and the females have two years of reproductive life. According to Collins (1973), Didelphis in Brazil has its first litter in August and September.
Earlier studies on marsupial oogenesis had suggested that it follows the biphasic growth pattern, typical from eutherian mammals (Lintern-Moore et al., 1976; Lyne & Hollis, 1983; Kress et al. 2001). Because of the unusual reproductive cycle among the marsupials, data regarding oocyte organelles and nucleus, polyovular follicles, oocyte development, folliculogenesis control, embryonic membranes and implantation have been published (Harrison & Weir, 1977; Hughes, 1977). Lyne & Hollis described the Graafian follicles in Isoodon macrourus and Perameles nasuta. The grown follicles are usually very large but the oocyte size does not increase concomitantly.
Among the marsupials, researches were undertaken in bandicoot ovary (Ullmann, 1979, 1981, 1989) and in Monodelphis opossum oocytes (Baggott et al., 1987, Mate et al., 1992). Oocytes maturation and in vitro fertilization studies in marsupials, especially Dasyuridae, were described by Selwood, (1982), Breed & Leigh, (1990), Breed, (1996) and Hickford et al. (2001). Kress et al. described the oogenesis pattern in Sminthopsis macroura and found that the timetable of oogenesis is accelerated as in other marsupials showing relatively early female maturation.
The available literature suggests that researches have been developed related to eutherian mammals and few of them are related to methatherian ones (Wolgemuth et al., 1984). Especially in Didelphis albiventris, the South-American opossum, no description of the folliculogenesis pattern was found. The goal of this paper is the description of some structural and ultrastructural characteristics of the south-american opossum ovary, with special attention to the ovarian follicles. These findings may contribute to the better understanding of the folliculogenesis and the reproductive behavior of this animal.
MATERIAL AND METHOD
Adult females specimens of 400500g weight were trapped in Botucatu City, São Paulo State, Brazil (IBAMA license N 002252/97-24). The experiments were conducted in accordance with the ethical guidelines for animal use, determined by the Brazilian College of Animal Experimentation (COBEA). The animals were anaesthetized by ether inhalation and perfused by Karnovsky´s solution (2% glutaraldehyde - 4% paraformaldehyde in Sorensen´s phosphate buffer 0.1 M, pH 7.3); through the left ventricle. The ovaries were removed and fixed overnight in Karnovsky´s solution.
For light microscopy, tissues were prepared to embed in Historesin (Historesin Jung 70-2218-500), according to standard methods and semithin sections were cut on Leica mod. RM 2065 microtome. These sections were stained with haematoxilyn and eosin and viewed under Zeiss Axiophot microscope.
For TEM, tissues were fixed for 24 hours in Karnovsky´s solution, washed in 0.1 M pH 7,3 PBS, post-fixed in 1% osmium tetroxide in PBS, for 2 hours, at the darkness, washed in distilled water (three changes of 5 minutes), stained in the block with 0.5% aqueous solution of uranyl acetate for 2 hours, dehydrated through a graded series of acetone, embedded in 1:1 Araldite - 100% acetone solution (12 hours) and embedded in Araldite. Ultrathin sections were double stained with uranyl acetate and lead citrate. The sections were examined and photographed under a PHILIPS EM 301.
The opossum has two ovaries located in the pelvis cavity and histologically they are covered by a cuboidal epithelium, which is squamous in some extension. Immediately underneath this epithelium there is the tunica albuginea of irregular dense connective tissue. Each ovary is divided in two regions: a well-developed cortical region of dense stromal tissue and an internal medullary region of connective tissue with many blood vessels. The ovarian follicles with the oocytes are lying in the cortical region. The histological limit between both regions can readily be seen as the cortex is mainly acidophilic and the medulla is eosinophilic (Fig. 1).
When the oocyte begins its growth phase, the single layer of follicular cells around it becomes cuboidal epithelial cells that form the primary follicle (Figs. 1 and 5). As the oocyte develops the mitochondria take the elongated profile and are grouped under the plasmalemma. Electron-lucent vesicular bodies are common components in the ooplasm. In addition, the oocyte begins to be surrounded by a translucent noncellular material, the zona pellucida. A space develops between the oocyte and the surrounding follicular epithelium into which irregularly shaped microvilli project from the oolema and from the neighboring follicular cells, over the whole surface of the oocyte (Figs. 8 and 9).
The follicular growth is characterized by a steady increase in the number of follicular cells and the granulosa becomes a two-layer follicular cells (Fig. 6) and later, a multilayered envelope of follicular cells. The granulosa, than, acquires a theca tissue with two thecal layers: theca interna, which is of dense connective tissue with several blood vessels and theca externa of connective tissue (Figs. 6, 7 and 8). Numerous lipid-like droplets are present in the cytoplasm (Figs. 6 and 7).
The Graafian follicle exhibits an incipient antrum at the late stage and as the antrum expands, the oocyte takes up an eccentric position surrounded by two or more granulosa cell layers, the corona radiata and the cumulus cells with dilated intercellular spaces (Figs 10, 12 and 13). The oocyte ooplasm becomes full of electron-lucent vesicular bodies and elongated mitochondria are grouped at the cortical region (Fig. 11).
Light and electron-microscopic studies provided strong evidence that the ovarian histology and the folliculogenesis in opossum resemble that of the eutherian pattern in some respects. There is a well-developed cortex of dense stromal tissue covered peripherally by a cuboidal germinal epithelium, which is squamous in some extensions and a medullary region with connective tissue and many blood vessels. Among the methatheria Lyne & Hollis have observed the same structure in Isoodon macrourus and Perameles nasuta.
The ovarian follicles with the oocytes are in the cortical region and primordial follicles that have a monolayer of flattened follicular cells around the oocyte can be easily identified. Our data about oocytes ooplasm and nuclei are in agreement with Ullmann (1979) for bandicoot; Frankenberg et al. (1996) for brushtail possum; Lucci et al. (1999) for goat and Browder (1985) for mammals in general.
Numerous pore complexes perforate the nuclear envelope of the opossum oocyte. According to Browder these pore complexes are ubiquitous structures found in all eukaryotic cells and have a highly regular architecture. They are important gateways in the macromolecules translocation.
Hyttel et al. (1997), Lucci et al. and Kress et al. found that the communication between the oocyte and the granulosa cells is apparently mediated through an endocytotic pathway as signaled by the abundant coated pits and vesicles present in the oocyte. We have found some vesicles in the analyzed oocytes but they are not in the same pattern described by these authors. The electron-lucent vesicular bodies observed in the ooplasm as the oocytes increase, are probably yolk spheres, protein vesicles, lipid and carbohydrate, the egg nutritional substances constituents. The same structural pattern was found in dogs and pigs oocytes and in pre-implantation embryos (Landin e Alvareng & Bicudo, 1997). The authors discuss that they are essential during the pre-implantation period as a nutritional source for these embryos. The similar pattern of oocyte ooplasm opossum leads us to suppose that in opossum embryos the same nutritional source is required but a further study about the nature of this droplets must be conducted.
Electron-dense bodies are usually dispersed throughout the ooplasm and they could be RNA and proteins, (secreted granulations). During oocytes growth the rounded mitochondria became elongated and gradually underwent a peripheral dislocation. Cui et al. (2005) studied oogenesis in the common brushtail possum and found their oocytes with a cytoplasm rich in mitochondria. Hyttel et al. observed the same change in cattle and Lucci et al., in goat. Frankenberg & Selwood (2001) found that the brushtail possum ova are slightly larger on average than those of eutherian animals and the same electron-lucent vesicles and electron-dense granular bodies were found in growing opossum oocytes. Harder & Jackson (2003) found in Monodelphis domestica, that the antral follicles diameters were 393 ± 4 mm.
Previous studies indicate that the zona pellucida consists of glycoproteins synthesized by the oocyte and the granulosa cells during the onset of growth phase and the ZP glycoproteins appear only after multiple layers of these cells are present (Selwood, 2000). Jewgenow & Rudolph (2001) concluded that the zona pellucida in cats is exclusively produced by granulosa cells and its synthesis takes place at every stage of follicular development. Our results demonstrated that the opossum zona pellucida construction occurs in the same way as in those animals. Sharman, (1961) found that marsupial's follicular envelope is typically thinner than that of other mammals. According to Hughes the zona pellucida, a protein- and carbohydrate-rich viteline coat, is of ovarian cell origin and is fully formed at the ovulation time.
Our results provided the first description of some structural and ultrastructural features of folliculogenesis in Didelphis albiventris, the South-American opossum. This description makes available data that are fundamental to the studies of its reproductive biology.
ACKNOWLEDGEMENTS. We would like to thank the Centro de Microscopia Eletrônica, IB, UNESP, Botucatu, SP, Brasil, for the facilities in use.
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Dr. Maria Dalva Cesario