Hong-Shu Sui

Chromatin configurations in the germinal vesicle of mammalian oocytes

In all the studied mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configuration). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configuration) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. Oocytes may also take on a SN/corresponding configuration during early atresia, but they undergo GVBD at the advanced stage of atresia. As not all the species show the SN configuration while in all the species, gene transcription always stops at the late stage of oocyte growth, it is suggested that not the formation of perinucleolar rings but a thorough condensation of GV chromatin is essential for transcriptional repression. The GV chromatin configuration is highly correlated with oocyte competence; oocytes must end the NSN configuration before they gain the full meiotic competence, and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. While factors inhibiting follicle atresia tend to synchronize oocytes in a chromatin configuration toward maturation, factors inducing follicle atresia tend to synchronize oocytes in a chromatin configuration reminiscent of early atresia. Furthermore, although condensation of GV chromatin is associated with transcriptional repression, both processes may not be associated with histone deacetylation during oocyte growth.

Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events

OBJECTIVE To study the mechanisms by which cumulus cells (CCs) promote oocyte maturation by observing the effect of removing the cumulus oophorus on nuclear and cytoplasmic events during in vitro maturation. DESIGN Experimental animal study. SETTING Academic institution. ANIMAL(S) Mice of the Kun-ming breed. INTERVENTION(S) Cumulus-free oocytes were cultured alone (DOs) or with a CC monolayer (coDOs), and the nuclear and cytoplasmic events were compared with those of oocytes matured in vivo or in vitro with the cumulus intact (COCs). MAIN OUTCOME MEASURE(S) Nuclear progression, spindle assembly, behavior of cortical granules (CGs) and mitochondria, levels of glutathione (GSH), and dynamics of maturation-promoting factor (MPF) activity during oocyte maturation under different conditions. RESULT(S) Cumulus removal increased MPF activity and accelerated the transition from the G2 to the M phase and the redistribution of CGs. Spindle assembly and mitochondrial congregation were impaired. In addition, removal of the cumulus caused a precocious exocytosis of CGs, leading to zona hardening and reduced penetrability of oocytes by sperm. After DOs were matured on the CC monolayer, however, these parameters were much improved, and the DOs acquired characteristics closer to those of cumulus-invested oocytes matured in vivo or in vitro. The level of intracellular GSH of DOs, on the other hand, did not differ from that of oocytes matured as COCs, suggesting that the mouse DOs can synthesize GSH on their own. CONCLUSION(S) While denuding mouse oocytes of CCs impaired in vitro cytoplasmic maturation, coculture with CCs promoted maturation, possibly through the regulation of MPF activity and meiotic progression.

Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes

The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 degrees C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 degrees C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 degrees C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 degrees C cytoplasts. While oocytes reconstructed between 37 degrees C ooplasts and 37 or 40 degrees C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 degrees C ooplasts and 37 degrees C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 degrees C compared with oocytes matured at 37 degrees C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 degrees C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 degrees C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 degrees C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Maternal Restraint Stress Diminishes the Developmental Potential of Oocytes

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.

Pyruvate prevents aging of mouse oocytes

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus-oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

Dynamic changes of germinal vesicle chromatin configuration and transcriptional activity during maturation of rabbit follicles

Dynamic changes in configuration of germinal vesicle (GV) chromatin and their effects on oocyte transcriptional activity and developmental competence were studied in rabbit oocytes. The results indicated that global condensation of GV chromatin, rather than the formation of a perinucleolar ring, would better reflect the global transcriptional repression and developmental competence of oocytes. Gonadotropins, by promoting large-scale chromatin remodeling, regulate oocyte transcriptional activity and developmental competence.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

Regulation of fusion of the nucleolar precursor bodies following activation of mouse oocytes: roles of the maturation-promoting factors and mitogen-activated protein kinases.

Fusion of nucleoli or nucleolus precursor bodies (NPBs) has been observed during somatic cell interphase and pronuclear development of human zygotes; however, the underlying mechanism is unknown. NPB fusion and its regulation by mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) were studied in activated mouse oocytes. Small NPBs appeared about 4 h after ethanol activation, and took about 1.5 h to fuse into a large NPB, which persisted for about 10 h before disappearance. Analysis of the temporal windows for kinase action indicated that a high MAPK activity during the first 2 h and a low MPF activity during the first 3-4 h after activation were essential for subsequent NPB fusion. A preactivation decline in MAPK activity was associated with decreased NPB fusion following activation of aged oocytes. While MAPK inactivation by regulator U0126 prevented NPB fusion in oocytes activated by ethanol or 5 min Sr2+ treatments, it had no effect on oocytes fertilized or activated by 6 h Sr2+ treatment. In most cases, while rates of pronuclear formation did not differ, rates of NPB fusion differed significantly between different treatments. Our results suggest that: (i) the MAPK and MPF activities at the initial stage of activation regulate NPB fusion after pronuclear formation; (ii) pronuclear assembly and NPB fusion are two separable events that might be controlled by different mechanisms; and (iii) high MAPK activity and low MPF activity at the initial stage of activation is essential for NPB fusion when only one calcium rise is induced by ethanol, while inhibition of MAPK activity does not affect NPB fusion when the repetitive intracellular Ca2+ rises are induced after fertilization.

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion‐enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion‐enabling factor (CEEF) in goat follicles. M‐199 medium was conditioned for 24 hr with cumulus‐denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG‐supplemented M‐199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2–4 mm) and small (0.8–1.5 mm) follicles, 71–83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5–6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80–90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half‐life than LH to expand mouse OOXs. Few meiosis‐incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF. Mol. Reprod. Dev. 75: 1387–1395, 2008.

Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self-made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk- and yolk-free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris-glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5 degrees C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non-centrifuged groups when semen was 11-fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11-fold diluted and stored at 5 degrees C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.