Number Of Cortical Granules Research Articles

Vincristine exposure impairs mouse oocyte quality by inducing spindle defects and early apoptosis.

Vincristine (VCR) is a microtubule-destabilizing chemotherapeutic agent commonly administered for the treatment of cancers in patients, which can induce severe side effects including neurotoxicity. In context of the effects on female fertility, ovarian toxicity has been found in patients and mice model after VCR exposure. However, the influence of VCR exposure on oocyte quality has not been elucidated. We established VCR exposure in vitro and in vivo model. The results indicated in vitro VCR exposure contributed to failure of oocyte maturation through inducing defects in spindle assembly, activation of SAC, oxidative stress, mitochondrial dysfunction, and early apoptosis, which were confirmed by using in vivo exposure model. Moreover, in vivo VCR exposure caused aneuploidy, reduced oocyte-sperm binding ability, and the number of cortical granules in mouse oocyte cortex. Taken together, this study demonstrated that VCR could cause meiotic arrest and poor quality of mouse oocyte.

Effects of programmed cell death protein 10 on the Schistosoma japonicum female reproductive system

This study aimed to investigate the effects of programmed cell death protein 10 (PCDP10) on the female reproductive system of Schistosoma japonicum, one of the major infectious agents of schistosomiasis. We found that PCDP10 was widely distributed in the integument, the worm parenchymal area, and the vitellarium of the female worm, but was localized to a lesser extent in the ovary and testicles. RNAi experiments successfully achieved gene knockdown, and the ultrastructural morphology of the adult reproductive organs was observed. The results demonstrated that, compared with those of the negative control group, the number of cortical granules around oocytes decreased and the number of immature oocyte cells increased. Fusion of yolk globules occurred, and the number and the diameter of yolk droplets decreased significantly. Real-time PCR showed that the expression of yolk glands reached its peak before ovulation and then decreased. The TUNEL assay results showed that apoptosis in the RNAi group was significantly higher than that in the negative control group. These results suggested that SjPCDP10 plays an important role in the female reproductive system. In conclusion, PCD10 is involved in oocyte growth and development, especially in eggshell formation, which may provide a reference for further elucidating the molecular mechanism of PCDP10 involved in egg formation and embryo development in Schistosoma japonicum.

Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation.

The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.

Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes

Before human MII oocytes are vitrified they are usually denuded from their cumulus cells. In this study we wanted to investigate the effects of an intact corona radiata on the vitrification and fertilization of human oocytes. The study comprised two different parts. In Part 1, 36 MII stage oocytes, from 6 patients, were randomly assigned into a control group, a group of vitrified-warmed oocytes without a corona radiata and a group of vitrified-warmed oocytes with an intact corona radiata. In each group of 12, 6 oocytes were used for evaluation of the zona pellucida solubility (hardening) and another 6 oocytes were used for the analysis of their ultrastructure. In addition, six polyspermically fertilized oocytes were used as positive controls for zona pellucida hardening. In Part 2, 16 patients in total produced 107 fresh and 98 vitrified-warmed oocytes, with or without an intact corona radiata. All oocytes were fertilized via conventional IVF and embryos were transferred according to our standard ET routines. The oocyte survival and fertilization rates, embryo quality and pregnancy and implantation rates were evaluated. There were no differences in oocyte survival, zona pellucida solubility (hardening) or the number of cortical granules between the vitrified-warmed and fresh oocytes. There were also no differences in the zona pellucida solubility and the number of cortical granules between vitrified-warmed oocytes with or without an intact corona radiata. However, the oocytes with an intact corona radiata had a higher fertilization rate after conventional IVF insemination. No differences were seen in the survival and cleavage rates, the percentage of high-quality embryos or the clinical outcome. Zona hardening and ultrastructural damage do not seem to occur in vitrified human oocytes. An intact corona radiata in vitrified-warmed oocytes retains their fertilization capacity in conventional IVF, but does not improve the embryo quality. Poor fertilizing capacities of vitrified-warmed oocytes without an intact corona radiata seem to have been due to the complete removal of the cumulus cells.

Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.

Cortical granule exocytosis in hamster eggs requires microfilaments

Earlier work has demonstrated that hamster eggs that do not release a second polar body after fertilization in vitro lack a block to polyspermy (Stewart-Savage and Bavister, 1987: Gamete Res 18:333-338). Since polar body release requires microfilaments, the involvement of microfilaments in cortical granule exocytosis was examined. When hamster eggs were treated with cytochalsin B (CB) for 1 hr and then coincubated with sperm for 90 min, there was a dose-dependent increase in both the percentage of eggs with more than one sperm penetrating the zona pellucida and the mean number of sperm that penetrated the zona, with a maximum effect at 20 micrograms CB/ml (100% polypenetration, 3.0 +/- 0.3 sperm/egg). Cytochalasin-treated eggs retained 85% of their cortical granules 55 min after insemination, as compared to unfertilized eggs. Longer time periods did not result in any further reduction. As seen with the scanning confocal microscope, an extensive microfilament network was present in the cortex of untreated eggs, with the cortical granules located within the cortical network. The cortical microfilament network was highly reduced in CB-treated eggs. When viewed with the electron microscope, the same number of cortical granules were located next to the plasma membrane in both cytochalasin-treated and untreated, unfertilized eggs. These data indicate that intact microfilaments are required for normal cortical granule exocytosis in the hamster egg, but the role of the microfilaments in exocytosis is unresolved.

Structural and endocrine aspects of equine oocyte maturation in vivo

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.

Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle ( T. Ducibella, E. Anderson, D. F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184–197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2 f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.

Oogenesis in the free proglottis ofTrilocularia acanthiaevulgaris (Cestoda, Tetraphyllidea)

The early (ovarian) stages of oogenesis in Trilocularia acanthiaevulgaris have been studied by light microscope histochemistry and transmission electron microscopy. The process proceeds as far as meiotic prophase in the primary oocyte. The oogonia and early immature primary oocytes occupy the anterior and outer edges of the ovary and are typically undifferentiated cells, showing a high nucleo-cytoplasmic ratio. The scant cytoplasm is packed with free ribosomes and contains a small number of mitochondria and a few short strands of granular endoplasmic reticulum (GER). The oogonia undergo a number of mitotic divisions, marked by the presence of centrioles. The immature primary oocytes enter meiotic prophase, as evidenced by the appearance of nuclear synaptonemal complexes. The maturing primary oocytes, which occupy the inner, central region of the ovary, undergo a growth phase that is accompanied by an increased nucleolar volume and export of RNA to the cytoplasm via nuclear pores, and by an increase in the number of mitochondria. A GER network develops and, together with the Golgi complexes, is involved in the production of a small number of cortical granules. The GER often takes the form of concentric cisternae. Numerous lipid droplets are also present in the cytoplasm. The mature primary oocytes predominate in the posterior region of the ovary, near the oviduct. They represent a resting phase in development, in which cellular activity is minimal. Lipid droplets are abundant and the cortical granules remain in more central regions of the cell and do not migrate to the periphery of the cell. The ovary contains a second, non-germinal type of cell, the follicle cell. The cell body is smaller in size than the oogonia, and cytoplasmic processes from it ramify around the periphery of the ovary. The different cell types within the ovary are embedded in a cytoplasmic matrix that contains a number of organelles.

Human nonovulatory oocyte‐cumulus complexes: Ultrastructure, macromolecular synthesis, and developmental potential

AbstractHuman nonovulatory oocyte‐cumulus complexes were obtained from large antral follicles 34 h after the injection of hCG. Their ultrastructure, RNA and protein synthesis, and the ability of oocyte maturation and subsequent fertilization in vitro were the subject of this study. The oocyte nucleus was situated in the cell periphery, possessing one or two dense nucleoli. Intranucleolar vacuoles were sometimes present. Mitochondria and smooth endoplasmic reticulum were the most abundant cytoplasmic structures. The number of cortical granules was rather low. Cumulus cells showed responsiveness to exogeneous hCG/endogeneous LH stimulation as morphologic signs of the oocyte‐cumulus uncoupling and of the early luteinization. Furthermore, the oocytes were capable of meiotic maturation in vitro, and following subsequent in vitro insemination the majority of them were penetrated by spermatozoa. However, polyspermy was detected in most cases, and cleavage was not achieved. A developmental defect of nonovulatory oocytes was suggested by an extremely low level of RNA synthesis while continuing active protein synthesis by nonovulatory oocytes was demonstrated. In contrast, the associated cumulus cells synthesized both RNA and protein, suggesting that the majority of granulosa cells of the nonovulatory follicles was probably unaltered. It is hypothesized that the development of nonovulatory follicles after induction of ovulation might reveal the developmental pattern which would lead, in some cases, to ovulation of nonviable human oocytes both in stimulated and natural cycles.

Effect of age and of follicular aging on the preovulatory oocyte.

The ultrastructure and ability of oocytes to mature in vitro were examined in oocytes collected from preovulatory follicles of mature normal-cycling proestrous rats (Day 3 of the cycle) and of aged proestrous rats (Day 3) and during a spontaneous period of ovulatory delay (Days 3–5). These studies demonstrated that aging resulted in: 1) the germinal vesicle (nuclear) membrane becoming undulated and the nucleoplasm becoming denser; 2) a deterioration of the stacked arrangement of the cytoplasmic rays with the formation of single stranded rays; 3) a reduced ability to form a polar body; 4) an increased tendency for the nucleolus to remain intact after germinal vesicle breakdown was completed; and 5) an increased percentage of oocytes that fragment and/or degenerate in vitro. These combined ultrastructural and physiological studies indicate that aging alters the oocyte such that it is unable to complete meiosis. Thus, fertilization of these aged oocytes could result in chromosomal and developmental errors within the developing embryos. Additional structural alterations were observed in the aged oocyte. These changes include a decrease in the number of cortical granules and microvilli, and a separation of the zona pellucida from the oocyte. The fibrils which compose the zona pellucida also become organized into continuous bands. These changes are similar to changes which render the zona pellucida impenetrable to spermatozoa. Thus, the aged oocytes would have a reduced fertilizability and this could partially account for the lower fertility of older irregularly cycling rats.

An electron microscopic study of mouse eggs matured in vivo and in vitro.

AbstractMouse oocytes obtained at various times after administration of HCG or after culture were studied with the electron microscope. Gradual changes were observed in the perivitelline space, plasma membrane and cortical cytoplasm. Disappearance of the regular microvilli, the formation of lamellae‐like processes and vesicular bodies were observed. A progressive increase in the number of cortical granules and multivesicular complexes was observed simultaneously with the disappearance of the rough endoplasmic reticulum. In general, eggs which were matured either in vivo or in vitro showed similar patterns of morphological differentiation. Degenerative changes were seen in most eggs cultured for 17 hours, but not for nine hours or less. In aged tubal eggs recovered at 26 and 36 hours after injection of HCG, the number of cortical granules was decreased relative to previous stages and their extrusion from the egg was apparent. The fertilizability of the eggs matured in vitro and the role of the cortical granules are discussed in terms of the reaction of the zona pellucida.