Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Melatonin defends mouse oocyte quality from benzo[ghi]perylene-induced deterioration

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon widely found in haze. Long-term exposure to humans or animals can cause serious damage to the respiratory system. Melatonin is an endogenous natural hormone synthesized and released by the pineal gland. In this study, we investigated the effects of melatonin on in vitro cultured B[ghi]P-exposed mouse oocytes and the protective roles of melatonin. Our data indicate that B[ghi]P exposure leads to meiotic maturation arrest and reduced ability of sperm binding and parthenogenetic activation. Also, B[ghi]P exposure disrupts actin filament dynamics, spindle assembly, and kinetochore–microtubule attachment stability, which results in oocyte aneuploidy. Simultaneously, B[ghi]P exposure disturbs the distribution of mitochondria, increases the level of oxidative stress, and induces apoptosis of oocytes. Whereas all of these toxic effects of B[ghi]P can be restored after melatonin supplement. In conclusion, our findings validate that melatonin has a certain protective effect on preventing the reduced oocyte quality caused by B[ghi]P exposure during meiotic maturation in mouse oocytes.     

Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice

Currently, maternal aging in women, based on mouse models, is thought to raise oocyte aneuploidy rates, because chromosome cohesion deteriorates during prophase arrest, and Sgo2, a protector of centromeric cohesion, is lost. Here we show that the most common mouse strain, C57Bl6/J, is resistant to maternal aging, showing little increase in aneuploidy or Sgo2 loss. Instead it demonstrates significant kinetochore-associated loss in the spindle assembly checkpoint protein Mad2 and phosphorylated Aurora C, which is involved in microtubule–kinetochore error correction. Their loss affects the fidelity of bivalent segregation but only when spindle organization is impaired during oocyte maturation. These findings have an impact clinically regarding the handling of human oocytes ex vivo during assisted reproductive techniques and suggest there is a genetic basis to aneuploidy susceptibility.     

Donor-dependent developmental competence of oocytes from lambs subjected to repeated hormonal stimulation

An unpredictability of ovarian response still remains the major problem concerning ovine reproductive programs. The influence of several environmental, genetic, and ovarian cycle effects on oocyte/embryo yield from donor females has been previously reported. The present research has been designed to exclude aforementioned causes of variability, thus to verify embryogenic competence in homogenous groups of animals. For this purpose we used prepubertal ewes kept under identical conditions. Initially, we stimulated three groups of prepubertal ewes at various ages and used a number of gonadotropin treatments to assess differences in oocyte competence between individuals. The results revealed the repeatability of response within individual donor lambs throughout the study. Moreover, once the variability in both oocyte and embryo yield between homogenous groups of donors was revealed alongside the influence of age and type of gonadotropin treatment (P < 0.001), we investigated whether the individual donor effect persisted among genetically similar animals. Therefore, we compared oocyte and subsequent embryo output of sibling lambs derived from the most efficient donor. Here the genetic homogeneity of sisters kept under identical conditions substantially improved the uniformity of either follicular response or embryo production, suggesting that the genotype plays a primary role in establishing follicular recruitment and developmental capability of oocytes. This observation consents to predict the ovarian performance from a single ewe already in early prepuberty (i.e., to qualify the female to breeding programs).     

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.     

Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro

Follicular oocytes collected prior to the expected time of the LH surge from PMSG-treated immature rats were incubated cummulus-intact (with or without LH) or cumulus-free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm-related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm-related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.     

NAMPT reduction‐induced NAD+ insufficiency contributes to the compromised oocyte quality from obese mice

Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD⁺) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD⁺ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity‐associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice.     

Changes at the hamster oocyte surface from the germinal vesicle stage to ovulation

Immature and ovulated hamster oocytes were studied with the scanning electron microscope. Immature oocytes at the germinal vesicle stage have their surface uniformly covered by microvilli. When meiosis has progressed to the first meiotic metaphase the overlying surface shows the differentiation of a circular area 19 μm in diameter with a low density of microvilli. Later, from this region the first polar body emerges, and the oocyte surface at the point from which it was extruded shows a cluster of cytoplasmic, conical projections. When the zona-free oocytes are cultured at 37°C for 5 minutes these projections disappear and the oocyte surface at that point becomes smooth. However, when the oocytes remain in the oviduct for several hours after ovulation these projections remain unchanged. The in vitro interactions of capacitated hamster sperm with the immature oocyte was always seen at microvillus surfaces and never associated with the differentiated regions.     

Ultrastructure of canine oocytes during in vivo maturation

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).     

Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes

The effects of the pesticide carbendazim (MBC) on the in vitro meiotic maturation of mouse oocytes were evaluated using conventional and confocal fluorescence microscopy. The response of oocytes exposed to 0, 3, 10, or 30 μM MBC during meiotic maturation was analyzed with respect to chromosome organization, meiotic spindle microtubules, and cortical actin using fluorescent labels for each of these structures. Continuous exposure to MBC during the resumption of meiosis resulted in a dose-dependent inhibition of meiotic cell cycle progression at metaphase of meiosis-1. Drug exposure at the metaphase-anaphase transition of meiosis-1 did not interfere with cell cycle progression to metaphase-2 except at high concentrations (30 μM). At the level of spindle microtubule organization, MBC caused a loss of nonacetylated microtubules and a decrease in spindle size at 3 or 10 μM concentrations. Thirty μM MBC prevented spindle assembly when added at the beginning of meiotic maturation or caused spindle pole disruption and fragmentation when added to preformed spindles. Spindle disruption involved a loss of phosphoprotein epitopes, as monitored by MPM-2 staining, and resulted in the appearance of dispersed chromosomes that retained a metaphase-plate location on spindle fragments associated with the oocyte cortex. Polar body extrusion was impaired by MBC, and abnormal polar bodies were observed in most treated oocytes. The results suggest that MBC disrupts cell cycle progression in mouse oocytes by altering meiotic spindle microtubule stability and spindle pole integrity. Mol. Reprod. Dev. 46:351–362, 1997. © 1997 Wiley-Liss, Inc.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for superovulation. The zona-free oocytes were incubated separately with specific ligand of integrins,an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal microscope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reorganization of cytoskelal protein, which may be involved in transmembrane signaling in egg activation.     

The effects of superovulation on Brucella abortus infection in the bovine uterus

Uterine flushings were collected three times at predetermined intervals from 11 mixed-breed beef cows and cultured for Brucella abortus . Prior to sampling, all cows had aborted fetuses from which brucellae had been isolated. Initial collections were made between 21 and 34 days following abortion. The second flushing was conducted at the onset of injections used for inducing superovulation and the third flushing was conducted 6 to 8 days after the ensuing estrus. The latter two flushes were conducted between 60 and 120 days following abortion. Brucellae were isolated from uterine flushings collected from 6 of the 11 cows on the initial round of sampling. Cultures of all subsequent uterine flushings collected before and after injections for superovulation were negative. It was concluded that the superovulatory treatment is not likely to reactivate the release of brucellae into the uterine lumen during the period when embryos are normally collected.     

Factors affecting the efficiency and reversibility of roscovitine (ROS) block on the meiotic resumption of goat oocytes

Goat oocytes from 2 to 4 and 0.8 to 1.2-mm follicles were freed (DOs) or not (COCs) of cumulus cells and cultured for different times in an inhibition medium supplemented with different concentrations of roscovitine (ROS). At the end of culture, oocytes were either cultured in a maturation medium for 24 hr and activated chemically for embryo development, or examined for GV chromatin configurations. Nuclear status was checked at different time points during maturation culture. Although both 200 and 250 microM ROS maintained 78-85% of oocytes at the GV stage for 24 hr, only oocytes blocked with 200 microM ROS developed to MII stage at a high rate after maturation culture. While few oocytes blocked with 200 microM ROS for 24 hr developed into morulae and none into blastocysts after activation, percentages of oocytes developing into morulae and blastocysts increased to the level of the control oocytes when the block time was reduced to 8 hr. While the GV and pMI stages were shortened with MI, and A/TI unaffected after oocytes were blocked for 8 hr, all the stages but A/TI were shortened after 24 hr of block. The sizes of nucleoli diminished with time and the GV chromatin configuration changed during ROS block. Significantly more DOs than COCs were blocked with 200 microM ROS, but none of the blocked DOs matured after drug withdrawal. However, maturation of the DOs improved significantly when ROS concentration was reduced to 150 microM or DOs were co-inhibited with COCs. The GV intact percentages of DOs did not differ after ROS inhibition with or without eCG, but those of COCs decreased significantly after ROS inhibited in the presence of eCG. When MII-incompetent oocytes from 0.8 to 1.2-mm follicles were inhibited with ROS for 8 and 24 hr prior to maturation culture, nuclear maturation improved significantly, activation rates were as high as that of the control oocytes, and some of the activated developed to 4- or 8-cell stages. It is concluded that (i) the efficiency and reversibility of ROS block was both drug concentration and exposure-time dependent; (ii) cumulus cells alleviated the toxicity of ROS on goat oocytes; (iii) eCG released goat oocytes from ROS block through the mediation of cumulus cells; (iv) ROS block quickened the nuclear maturation of goat oocytes and improved the developmental competence of meiosis-incompetent oocytes, possibly due to a sustained nuclear activity during inhibition culture; (v) oocyte nuclear maturation and activation did not depend upon cumulus expansion, but the embryo development occurred in association with cumulus expansion.