A New Approach to the Study of Ovarian Follicles by Scanning Electron Microscopy and ODO Maceration.

Abstract

Summary. The ultrastructure of the follicle-oocyte complex in rodents and humans was revealed by high resolution scanning electron microscopy (SEM) following the Osmium-DMSO-Osmium maceration method (TA-NAKA and NAGURO, 1981).In primary follicles, the majority of oocyte organelles such as mitochondria and Golgi complex components are concentrated in a juxtanuclear area. In particular, many spherical mitochondria are oriented all around the nucleus. After maceration of the ooplasm matrix, most of these mitochondria appear intermingled with numerous microtubules (MT) and associated with many Golgi vesicles. Such a nuclear polarization of organelles, essential to the oocyte metabolism, might depend upon a MT activity. MT might guide mitochondria to gather in the perinuclear region and further maintain their close associated to the nuclear envelope. A similar relationship among microtubules, vesicular Golgi complex and mitochondria has been also observed when, in maturing oocytes, these organelles migrate and gather in other areas of the ooplasm.A pattern common only to human developing follicles appears in the occurrence of long microvilli projected from follicle cells deep into the oocyte. These unusual microvilli running within the ooplasm are surrounded by several vesicles of the Golgi complex and endoplasmic reticulum, and often end close to the nucleus.In the antral follicle, the microvilli of corona cells, directed toward the oocyte (after their full exposure through the chemical dissolution of the zona pellucida matrix) are extremely numerous (up to 70/cell), long (up to 7/10um) and tortuous. They resemble epidydimal stereocilia, may be ramified and possess bulbous tips. In contrast, oocyte microvilli are thin and short. The"curly hair-like microvilli" of corona follicle cells and especially those terminating deep in the oocyte cytoplasm, besides being the expression of active exchange of nutrients, may serve to finely modulate oocyte maturation.The study of intracellular organelles by scanning electron microscopy (SEM) began about 20 years ago with the use of various cracking methods, including cryofracture and frozen resins (TANAKA, 1972;HAGGIS and PHIPPS-TODD, 1977). At that time, the utility of these techniques was quite limited in that the low resolution power of SEM rendered it impossible to remove the cell matrix embedding and masking the cellular structures.Today, the Osmium-DMSO-Osmium (ODO) method, developed by TANAKA and NAGURO (1981) and considered to be the most effective technique for visualizing cell organelles, is used extensively. Using this method, the present SEM study offers original illustrations of the three-dimensional (3-D) submicroscopic details of intracellular structures of oocytes. Microvilli embedded in the zona pellucida of oocytes and corona radiata cells are similarly studied. MATERIALS AND METHODSOvaries taken from 14 to 22-week-old human fetuses, and from adult humans and rats were used in the present study.Human ovaries were fixed in toto by immersion with 2.5% glutaraldehy...

Oocyte Follicle Cells Association during Development of Human Ovarian Follicle. A Study by High Resolution Scanning and Transmission Electron Microscopy.

“…(MAKABE et al, 1992) was advantageous in fully revealing the true 3-D patterns of follicular cells and their extensions.…”

“…This method was recently adapted quite advantageously also to human biopsies including fetal tissues (MAKABE et al, 1992). In fact, this is one of the best maceration techniques since it is most effective in preserving the membranous cellular structures, while at the same time dissolving the cell matrix.…”

Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus–oocyte complexes during in vitro maturation and fertilization

“…The cumulus oophorus is a group of follicular somatic cells closely associated with the oocyte to form the cumulus-oocyte complex (COC; Makabe et al 1992, Motta et al 1995. Before ovulation, and at the same time as the progression of oocyte meiosis to metaphase II, the cumulus cell (CC) mass undergoes a process called 'mucification' or 'cumulus expansion'.…”

Ultrastructure des cellules de Sertoli humaines aux stades embryonnaires précoces de la différenciation gonadique

“…Utilisant la technique pr~c~demment d~crite [11,21], les ~chantillons ont ~t~ congel~s sur des plaques m~talliques refroidies par l'azote liquide, bris~s en deux parties avec une lame de rasoir et un marteau et imm~diatement places dans une solution aqueuse de DMSO ~ 50%. Apr~s rin~age dans la solution tampon, les ~chan-tillons ont ~t~ places dans un bain de t~troxy- A la 6~me semaine du d~veloppement, le blast,me gonadique est constitu~ de gonocytes primordiaux et de cellules m~senchy-mateuses.…”

“…Dans ces conditions, le contact entre les cellules pr4sertoliennes et |es gonocytes primordiaux jouerait un rSle cl4 dans la diff~-renciation sexuelle masculine en contr61ant la prolif@ration et le d@veloppement pr@-coces des cellules germinales [9,10,14,15]. Ainsi que nous ravons montr@ dans de pr4-c~dentes publications [11,12] la m@thode l'Osmium-DMSO-Osmium (ODO) est la technique la plus efficace pour la mise en 4vidence des organites cellulaires et en combinaison avec la microscopie 41ectro-nique ~ balayage (MEB) ~ haute r4solution, elle permet d'obtenir des images originales de la micro-architecture tridimensionnelle du testicule [12].…”