Oocyte Fragments Research Articles

On fragmentation and elimination of ovarian oocytes.

Examination of numerous fragmented oocytes in the ovary revealed an asynchronous relationship between nuclear and cell divisions. The asynchrony was observed in non-fragmented oocytes as well and was considered to be one of the common processes leading towards oocyte elimination of the ovary. The present study additionally demonstrates the morphology of follicle cell degeneration observed on the surface of fragmented oocytes.

On induced fragmentation of ovarian oocytes.

Fragmentation of oocytes was induced in the ovary, via ovulation suppression, by administration of dehydroepiandrosterone acetate (DHA-Ac) to mature cycling rats. The maximal fragmentation ratio, 15.0 +/- 3.2%, was obtained by 10 mg/100 g b.wt/day for 7-day administration of DHA-Ac. The relationship of fragmentation to the first stage of meiosis is also discussed.

SEM cryofracture study of ovarian follicles of immature rats.

Follicles were examined with the scanning electron microscope (SEM) following preparation of cryofractured specimens of rat ovaries. After perfusion fixation with 2.5% glutaraldehyde buffered at pH 7.4 with 0.1M cacodylate, ovaries were trimmed of fat and decapsulated, fixed for an additional 12 hours, washed in distilled H2O for 20 hours and postfixed in 1% OsO4 in distilled H2O for 30 minutes. Following a brief wash, ovaries were dehydrated in a linear gradient of dist. H2O/ethanol, frozen in Freon 22, cryofractured under liquid nitrogen, brought to 1 degree C in ethanol, and critical point dried from CO2. Follicles cleaved through their oocytes were examined with SEM to determine the ultrastructural characteristics of developing and atretic follicles revealed by cryofracture. Cytoplasmic arrays of parallel sheets, nucleoli and microvilli were prominent structures of cryofractured oocytes. Numerous cytoplasmic projections of granulosa cells and oocyte microvilli penetrated the zonae pellucida of preantral follicles. The numbers of oocyte microvilli were greatly diminished in large antral follicles. Other structures made prominent by cryofracture included cell nuclei, a microvillous border at the granulosa-theca boundary, and numerous holes in the cytoplasm of thecal cells which correspond in size and distribution to the liquid droplets in these cells. The soluble proteins and glycoproteins of the follicular fluid were visualized as a homogeneous granular precipitate. The disappearance of oocyte microvilli and granulosa cell projections from the zonae pellucida and the blebbing of granulosa cells at the surface of the antral cavity appeared characteristic of an early stage of follicular atresia. Increased numbers of holes in thecal cells, oocyte fragmentation, and extensive dissociation and fragmentation of granulosa cells were typical of more advanced stages of atresia.

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.

Influence of culture media on in vitro fertilization of ovine tubal oocytes.

The ability of Synthetic Oviductal Fluid (SOF) and Minimum Essential Medium (MEM) to support sperm binding, sperm penetration and subsequent cell division of ovine tubal oocytes in vitro was evaluated. These media contained no protein, 3% (w/w) bovine serum albumin (BSA) or 20% (v/v) lamb serum (LS). Midventral laparotomies were performed on 49 ewes synchronized with progesterone and then superovulated, and tubal oocytes were collected by flushing the oviducts. Oocytes were pooled and randomly added to 2 X 10(7) ejaculated spermatozoa in 1 ml of treatment media that had been preincubated in tubes for 3 hr at 37 C under 5% CO2 in air. After an additional 3 hr of culture in a rotating tissue culture drum, oocytes were further cultured in microdrops of the same medium for 48 hr and observed for sperm penetration, cell division and fragmentation. Oocytes were then stained with aceto-orcein and observed for penetration, multiple nuclei and bound sperm. Percentage of oocytes showing penetration in SOF, SOF+ BSA, SOF+LS, MEM, MEM+BSA and MEM+LS was 14, 3.6, 7.8, 11.1, 8.7 and 12.8, respectively; percentage of oocytes cleaving was 8, 3.6, 5.9, 4.4, 4.3 and 4.3; percentage of oocytes fragmenting was 40, 60, 60.8, 20, 60.9 and 29.8, and the average number of sperm cells bound per oocyte was 82, 177, 117, 41, 56 and 47. No cell division was observed for control oocytes (no sperm added), but 72.7, 80 and 76.9% fragmented in SOF, SOF+BSA and SOF+LS, respectively. No difference was found among the media in their ability to support sperm penetration and subsequent cell division in vitro. All media tested supported a high incidence of sperm binding and oocyte fragmentation.

The role of germinal vesicle karyoplasm in the development of male pronucleus in the mouse

Zona-free mouse ovarian oocytes from Swiss albino females were bisected into nucleate and anucleate halves at the germinal vesicle stage (1), shortly after germinal vesicle breakdown (GVBD) (2) and in metaphase I (3). Sister halves were cultured for 15–17 h (ser. 1, 2) or 6.5–7.5 h (ser. 3) and those in which the nucleate fragment extruded the first polar body, were fertilized with F 1(C57BL/10 × CBA/H) spermatozoa. After an additional period of culture lasting for 6–7 1 2 (1, 2) or 16–17 h (3) the cells were fixed and examined in whole-mount preparations. Intact oocytes recovered from follicles at the same stages as those used for bisection, and ovulated oocytes (both zona-free) served as controls. Nucleate fragments and intact ovarian oocytes were fertilized in 65% and in the majority of them sperm heads were transformed into pronuclei; no differences were observed between the three series in the rate of fertilization and development of pronuclei. In anucleate oocyte fragments development of male pronuclei took place only if the dissolution of germinal vesicle had occurred prior to bisection. It is concluded that in the mouse, the germinal vesicle material is essential for transformation of sperm nucleus into the functional male pronucleus.

Meiotic maturation of mouse oocytes in vitro: Protein synthesis in nucleate and anucleate oocyte fragments

Nucleate and anucleate fragments of mouse oocytes have been isolated following treatment of fully grown oocytes with cytochalasin B. The nucleate oocyte fragments resume meiosis in vitro, progressing from dictyate of the first meiotic prophase to metaphase II ('meiotic maturation'), and exhibit all of the changes in protein synthesis normally associated with meiotic maturation of mouse oocytes. The anucleate oocyte fragments also undergo certain of the changes in protein synthesis associated with meiotic maturation, despite the absence of nuclear progression. These results suggest that the acquisition of meiotic competence (i.e. the ability to undergo meiotic maturation) during growth of the mammalian oocyte is due to changes in the quality, rather than the quantity, of cytoplasm and that the reprogramming of protein synthesis during meiotic maturation is directed by RNA templates already present in the cytoplasm. The behaviour of anucleate oocyte fragments is discussed in terms of the proposed role for nucleoplasm in the initiation of changes in protein synthesis during meiotic maturation of mouse oocytes.