Anatomia Histologia Embryologia
ORIGINAL ARTICLE
Oocyte Ultrastructural Characteristics in Camel (Camelus dromedarius) Primordial to Large Antral Follicles N. Davoodian1, F. Mesbah2 and M. Kafi1* Addresses of authors: 1 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, 71345 Shiraz, Iran; 2 Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, 1731 Shiraz, Iran
*Correspondence: Tel.: +98 71 16138700; fax: +98 71 12286940; e-mail:
[email protected] With 14 figures and 1 table Received September 2010; accepted for publication October 2010 doi: 10.1111/j.1439-0264.2010.01049.x
Summary This study was conducted to describe in detail the ultrastructural features and morphological characteristics of camel oocytes from preantral follicles in relation to the sequential stages of follicular development and also for oocytes from antral follicles in relation to their diameter. Camel oocytes from primordial, primary, secondary and also early to late antral follicles were processed and examined by light and transmission electron microscopy. Primordial follicular oocytes were characterized by a layer of flattened granulosa cells around and also eccentric nucleus and few cytoplasmic organelles in the peripheral region. Up to the secondary follicle stage, flat cells were replaced by cuboidal granulosa cells and their number increased and also an increase in the number of organelles such as vesicles, mitochondria and endoplasmic reticulum was observed. In the early antral stage, the formation of zona pellucida, appearance of microvilli and pleomorphic mitochondria was seen and the nucleus was dislocated to the peripheral region. During final growth phase, the extent of endoplasmic reticulum, vesicles and mitochondria increased, the number of lipid droplets decreased and cumulus cell process endings (CCPE) were observed. In conclusion, the growth of camel oocyte is associated with progressive increase in the number of mitochondria, endoplasmic reticulum, Golgi complexes and cytoplasmic vesicles as well as decrease in the number of lipid droplets and the nucleus migration from an eccentric in preantral to a peripheral location in antral follicles.
Introduction Various attempts have been made to improve the reproductive efficiency and genetic potential of dromedary camel through the application of advanced reproductive technologies including embryo transfer (Skidmore et al., 2002), in vitro fertilization (Khatir et al., 2007) and very recently embryo cloning (Wani et al., 2010). Although these studies have resulted in production of the dromedary camel embryos, however, a convincing success rate has not been obtained yet. Therefore, a better understanding of the biology of the camel follicular and oocyte developments is of vital importance to devise an optimal system for in vitro culture of the follicle and oocyte. 120
Ultrastructural changes in the oocyte during folliculogenesis have been studied in different farm animals such as cattle (De-Loos et al., 1992; Fair et al., 1997), ewes (Brand and Jong, 1973; Cran et al., 1980) and Goats (Lucci et al., 2001). Investigations of camel oocyte ultrastructure have largely concentrated on the oocytes from antral follicles (Nili et al., 2004; Kafi et al., 2005). One aspect which appears critical but has not been examined is the ultrastructural changes of the oocyte during follicular growth. Therefore, the purpose of this study was to investigate systematically the ultrastructural features and morphological characteristics of camel oocytes from different size follicles at each stage of follicle growth using both light and transmission electron microscopy (TEM). ª 2010 Blackwell Verlag GmbH • Anat. Histol. Embryol. 40 (2011) 120–127
N. Davoodian, F. Mesbah and M. Kafi
Oocyte Ultrastructural Characteristics in Camel
Materials and Methods Sample collection Camel ovaries were collected at slaughter and were kept at 32–35C in normal saline (0.9%) until arrival at the laboratory, which was reached within 3 h. Processing of camel ovarian tissues with preantral follicles for TEM Ovaries were segmented (1 mm3) and then were fixed in Karnovsky solution (2.5% glutaraldehyde and 4% paraformaldehyde in 0.2 m cacodylate buffer) and post-fixed in 1% OsO4 in 0.1 m cacodylate buffer for 2 h. The segments of ovarian tissues were then dehydrated by passing through ascending graded ethanol, embedded in resin (Agar 100: Agar scientific Ltd, Cambridge, Essex, UK) and finally sectioned into semi-thin sections (ultra microtome OMU3, C. Reichert, Vienna, Austria). Semi-thin sections (0.5–1 lm) were stained with toluidine blue and examined at the LM level for the presence of preantral and early antral follicles containing an intact oocyte with nucleus and then ultra-sectioned. Ultra-thin sections (60– 90 nm) were collected on copper grids, stained with uranyl acetate for 15 min, washed repeatedly with distilled water and dried. Subsequently, they were stained with lead citrate for 12 min, washed, dried and examined under Philips, CM10 TEM (Philips Analytical, Eindhoven, the Netherlands). The follicles were classified into three categories: (1) resting primordial (Fig. 1), with a single layer of flattened granulosa cells (n = 10); (2) primary (Fig. 2), with a single layer of cuboidal granulosa cells (n = 10); (3) secondary (Fig. 3), with an incomplete or complete two layers of cuboidal cells (n = 6).
Fig. 1. Light micrograph showing a resting primordial follicle. Note the flattened granulosa cells (FG) and the oocyte containing an eccentrically located nucleus (N) (·720).
ª 2010 Blackwell Verlag GmbH • Anat. Histol. Embryol. 40 (2011) 120–127
Fig. 2. Light micrograph showing a primary follicle. Note the cuboidal granulosa cells (CG) and the oocyte containing an eccentrically located nucleus (N) (·720).
Fig. 3 Light micrograph showing a secondary follicle. Note the bilayer of cuboidal granulosa cells (CG) and the oocyte containing an eccentrically located nucleus (N) (·720).
Processing of isolated oocytes from antral follicles for TEM Camel ovaries collected at slaughter, held at 32–35C in normal saline (0.9%), were transported to the laboratory within 3 h of collection. Antral follicles were categorized into four groups based on their diameter as
