The zona pellucida of the koala (Phascolarctos cinereus): its morphogenesis and thickness

Abstract

In this study the ultrastructural organization of the koala oocyte and the thickness of the surrounding extracellular coat, the zona pellucida, has been determined to ascertain whether there is coevolution of the morphology of the female gamete with that of the highly divergent male gamete that is found in this marsupial species. Ovaries from several adult koalas were obtained and prepared for transmission electron microscopy. Oocytes in large tertiary follicles were somewhat smaller than those of most other marsupials, although their ultrastructural organization appeared similar and included many yolk vesicles. The zona pellucida surrounding the oocytes in tertiary follicles was approximately 8 microm thick and thus is of similar thickness to that of some eutherian mammals but at least twice as thick as that of most marsupial species so far studied. The results indicate that the koala oocyte is unusually small for a marsupial species whereas the zona pellucida is, by contrast, much thicker. How this relates to sperm-egg interaction at the time of fertilization has yet to be determined.

Fig 1

Transmission electron micrographs of primordial (a,b) and primary (c,d) ovarian follicles of a koala. Note that the primordial follicle, surrounded by ovarian interstitial tissue (I), consists of a simple squamous layer of follicular epithelium (FE) and, within the oocyte, a prominent lipid droplet (LD) occurs adjacent to the nucleus (N) (a). Mitochondria (M) are oval in shape, while cell junctions (arrowhead) occur between the interdigitating membranes of the oocyte and follicle cells (b). The primary follicle, enclosed by a basement membrane (B), contains a single layer of cuboidal granulosa cells (Gc) around a primary oocyte (Oo); within the ooplasm, electron-dense cortical granules (arrowheads) are present and pockets in which zona matrix (arrow) occurs are present between the oocyte and granulosa cells, as well as close to the basement membrane (c). Matrix of the zona pellucida (ZP) is present between oocyte microvilli (Mv) and cytoplasmic extenstions of the granulosa cells (arrow) (d); a golgi complex (G) lies adjacent to the oocyte–granulosa intercellular junction. Scale bars: a = 5 µm; b = 2 µm, c = 2 µm; d = 1 µm.

Fig 4

Thick plastic (a) and transmission electron micrographs (b,c) of an oocyte of a large tertiary ovarian follicle. The oocyte (Oo) is invested by two or three layers of cumulus cells and a broad zona pellucida (ZP); a nucleus (N) is evident around which unstained yolk vesicles and numerous lipid yolk droplets are present (a). The zona pellucida is less dense and less compact than in earlier follicles (b), while Golgi complexes (G) are still closely associated with the cumulus cell – oocyte intercellular junctions (arrows) (c). PVS, perivitelline space. Scale bars: a = 25 µm; b = 5 µm; c = 1 µm.

Fig 2

Transmission electron micrographs of primary (a,b) and secondary (c,d) ovarian follicles. In late primary follicles, a uniform zona pellucida (ZP) surrounds the oocyte (Oo); it is less regular adjacent to the granulosa cells (Gc) (a). At higher magnification, the zona pellucida appears dense and granular, whilst electron-lucent yolk vesicles (Yv) and a Golgi complex (arrowhead), adjacent to an intercellular junction (arrow), occur within the ooplasm (b). The oocytes of secondary follicles are surrounded by a multilaminar granulosa cell epithelium (c), while at higher magnification Golgi complexes (arrowheads) are found immediately beneath the microvilli of the oolemma, whereas the zona pellucida is dense and compact (d); N (nucleus). Scale bars: a = 5 µm; b = 1 µm; c = 10 µm; d = 2 µm.

Fig 3

Transmission electron micrographs of early tertiary ovarian follicles. The oocyte (Oo) is surrounded by a broad and dense zona pellucida (ZP) and two or three layers of cumulus cells (Cc) (a). Multiple cytoplasmic extensions (arrows) of the surrounding cumulus cells traverse the zona pellucida (b). A, follicular antrum. Scale bars: a = 10 µm; b = 2 µm.