Rat Oocytes Research Articles

Efficient Activation of Reconstructed Rat Embryos by Cyclin-Dependent Kinase Inhibitors

BackgroundOver the last decade a number of species, from farm animals to rodents, have been cloned using somatic cell nuclear transfer technology (SCNT). This technique has the potential to revolutionize the way that genetically modified animals are made. In its current state, the process of SCNT is very inefficient (<5% success rate), with several technical and biological hurdles hindering development. Yet, SCNT provides investigators with powerful advantages over other approaches, such as allowing for prescreening for the desired level of transgene expression and eliminating the excess production of undesirable wild-type animals. The rat plays a significant role in biomedical research, but SCNT has been problematic for this species. In this study, we address one aspect of the problem by evaluating methods of activation in artificially constructed rat embryos.Principal FindingsWe demonstrate that treatment with a calcium ionophore (ionomycin) combined with a variety of cyclin-dependent kinase inhibitors is an effective way to activate rat embryos. This is in contrast to methods developed for the mouse embryo, which tolerates much less specific chemical treatments. Methods developed to activate mouse embryos do not translate well to rat embryos.ConclusionsActivation methods developed for one species will not necessarily translate to another species, even if it is closely related. Further, the parthenogenic response to chemical activators is not always a reliable indicator of how reconstructed embryos will react to the same activation method. A better understanding of rat oocyte physiology, although essential for developing better models of disease, may also provide insights that will be useful for making the SCNT process more efficient.

Analysis of different cell death processes of prepubertal rat oocytes in vitro

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

The reverse logic of multivesicular endosomes

Fifty years after the first description of curious “multivesicular bodies” in micrographs of rat oocytes (Sotelo & Porter, 1959), it might seem odd that the precise mechanistic basis for the formation of these ubiquitous intracellular organelles remains as perplexing as their morphology. The biogenesis of multivesicular bodies (MVBs)—also known as late multivesicular endosomes—represents a subset of the remarkable endosomal transformations that occur in eukaryotic cells. Situated between early peripheral rab5 GTPase‐positive endosomes and the terminal lysosome compartment, MVBs are characterized by the presence of intralumenal vesicles (ILVs) and sometimes also onion‐like whorls of internal membrane (Fig 1). The internal packaging of the limiting membrane to generate ILVs is widely accepted to partition certain transmembrane molecules that were present originally at the cell surface into the MVB lumen. The spatial remodelling isolates receptors from the surrounding cytosol and can terminate signal propagation as a prelude to preparing receptors for destruction by the lysosomal compartment. In fact, stimulation by epidermal growth factor (EGF) increases the number and size of MVBs (White et al , 2006), whereas incapacitating the core MVB biogenesis machinery leads to excessive and prolonged signal propagation (Vaccari & Bilder, 2005). However, before becoming fully committed to ILV production, nascent MVBs sort transmembrane proteins and lipids within emanating tubules to other intracellular destinations, such as the trans ‐Golgi network, recycling endosomes, or back to the plasma membrane (Fig 1). Figure 1. Endocytic sorting structures and pathways. Schematic of the main types of sorting endosome and the trans ‐Golgi network. The arrows indicate the temporal relationship of the different sorting trajectories. ESCRTs, endosomal complexes required for transport; ILV, intralumenal vesicle; LBPA, lysobisphosphatidic acid; MVB, multovesicular body. At the recent EMBO Conference on Endocytic Machineries in Control of Cell Signalling and Tissue Morphogenesis, held in Chania, Crete and organized by Harald Stenmark and Gillian …

175. EFFECTS OF SPECIES DIFFERENCES ON OOCYTE REGULATION OF GRANULOSA CELL PROLIFERATION

The role of oocytes in regulating ovulation quota between species is not fully understood. In sheep, the oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have profound effects on ovarian follicular development and ovulation-quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled thymidine uptake by granulosa cells (GC) both within and between the two species. For these experiments, 32 oocytes denuded of cumulus-cells were co-incubated with 20 000 GC from either species. Rat or sheep oocytes stimulated thymidine uptake by GC from the same species (P &lt; 0.005). Sheep oocytes also stimulated thymidine uptake by rat GC (P &lt; 0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with 3.2 µg/mL or 7.6 mg/mL monoclonal antibodies specific to GDF9 or BMP15 respectively and to a hydatids antigen (control). Both sheep and rat oocyte stimulation of thymidine uptake by GC was inhibited with the GDF9 antibody (P &lt; 0.05) but not control, irrespective of species of GC. Sheep oocyte stimulation of rat GC was also inhibited using an antibody to BMP15 (P &lt; 0.05). However, when using the BMP15 antibody to block the effects of rat oocytes on rat GC, the inhibition of thymidine uptake was modest (i.e. ~10%) albeit significant (P &lt; 0.05). The molecular forms of GDF9 and BMP15 in spent media were examined by Western blotting under reducing conditions. For both species, oocyte-secreted GDF9 was present in the mature form. For sheep oocytes, secreted BMP15 was present as promature and monomeric mature forms whereas from rats, trace amounts of mature form was sometimes, but not always, detected. Thus, in sheep both BMP15 and GDF9 are essential for regulating GC proliferation whereas in rats, although responsive to BMP15, GC proliferation is likely regulated primarily by GDF9.

Ultrastructural changes in rat oocytes after treatment with testosterone enanthate

Ultrastructural changes in rat oocytes after treatment with testosterone enanthate

Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells

Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

234 EXPRESSION OF NUCLEAR AND MEMBRANE MELATONIN RECEPTORS GENES AND THE CLOCK GENES IN RAT OOCYTES: PRELIMINARY RESULTS

There is evidence that melatonin acts directly on the regulation of ovary function. This action is probably attributed in part to melatonin receptors, which are known to be present in granulosa and cumulus cells (Kang J-T et al. 2009 J. Pineal Res. 46, 22–28). Melatonin is also known to be associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function (Arendt J 1998 Rev. Reprod. 3, 13–22). Circadian rhythms and clock genes appear to be involved in reproductive processes (Dolatshad H et al. 2009 Repro. Fertil. Dev. 21, 1–9). However, the presence of melatonin receptor genes and the clock genes has not been so widely studied or has never been reported to exist in mammalian oocytes.The aim of this study was to investigate the presence of nuclear (Rorα) and membrane (Mt1 and Mt2) melatonin receptors genes and the clock genes (Clock, Bmal1, Cry1, Cry21, Per1, Per2) in rat oocytes by reverse RT-PCR. Twenty-seven-day-old Wistar female rats were treated with 20 UI of pregnant mares serum gonadotropin for induction of follicular development and slaughtered 48 h later. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. The ovaries were removed and placed in TCM-199 supplemented with 100 UI mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.1% polyvinyl alcohol (H-199 medium). Germinal vesicle-intact oocytes, isolated from the ovarian follicles, were denuded from cumulus cells by vortexing for 5 to 8 min. The denuded oocytes were incubated for 5 min in H-199 medium with 0.1% pronase for removal of the zona pellucida. Pools of 80 oocytes per cDNA sample were used.As an internal control for the sample integrity, additional primers for RPL37a were included in each PCR reaction. All the 3 control PCR replicates showed a repeatable amplification. Polymerase chain reaction amplifications of cDNA yielded Rorα, Clock, Bmal1, and Cry1 products in 2 of 3 replicates. No expression of the MT1 and MT2 mRNA was observed. The preliminary results suggest the presence of a nuclear melatonin receptor gene and some clock genes in rat oocytes. However, additional studies are necessary to confirm this hypothesis. This research was supported by FAPESP.

Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro

Meiosis activating sterol, produced directly by lanosterol 14-alpha-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins' induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs' meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.

Evaluation ofIn VitroCultured Rat Oocytes, from Different Strains, by Spindle Morphology and Maturation-Promoting-Factor Activity Combined with Nuclear-Transfer Experiments

Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus.

The effect of the time interval between injection and parthenogenetic activation on the spindle formation and the in vitro developmental potential of somatic cell nuclear-transferred rat oocytes

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes continued to elongate with an increase in the culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.

A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis

The objective was to find out the functional roles of hydrogen peroxide (H2O2) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H2O2, total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H2O2 induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H2O2, reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H2O2 and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.

234 MECHANISMS OF SPONTANEOUS ACTIVATION IN RAT OOCYTES

MII-arrested rat oocytes spontaneously resume meiotic maturation soon after recovery from oviducts. Time elapsed post oocyte retrieval, environmental factors, certain genetic background have been implied to promote spontaneous release from MII arrest in rat oocytes. The precise mechanism behind this process is unknown. This study was undertaken to explore signaling pathways, which may be involved in spontaneous activation in rat oocytes. Using triple immuno-staining and epifluorescence microscopy we have described morphological changes in the meiotic spindle of rat oocytes during 6h of in vitro culture (immature CD female rats from Charles River Laboratories). An ELISA-based method was used to evaluate cdc2 activity in rat oocytes subjected to in vitro culture. SDS-PAGE and western blotting were performed to study levels of phosphorylated and total p42/p44 MAPK, phosphorylated on Tyr15 and total cdc2, cyclin B1 and β actin. Expression of Emi2 was analysed by RT-PCR. Data were analyzed using GLMM or ANOVA followed by t-test. Freshly collected oocytes contained well-preserved spindle with chromosomes aligned in metaphase plate. After being in culture for 2 h oocytes demonstrated signs of activation, such as spindle rotation, preparing to extrude second polar body, and some oocytes had entered anaphase. In the majority of oocytes cultured for 6 h spindles had disintegrated and chromosomes were scattered in the oocyte cytoplasm; microtubules were found around condensed chromosomes. Significant drop in cdc2 activity was detected in oocytes after 2 h of in vitro culture. In oocytes cultured for additional 4h of cdc2 activity returned to the level observed in freshly collected oocytes. Inhibitory phosphorylation of cdc2 on Tyr 15 was not associated with cdc2 inactivation. We were not able to detect changes in the level of cyclin B1 during the MII to MIII transition. Phosphorylated forms of activated p42/p44 MAPK in eggs were present throughout in vitro culture. Emi2 is a novel candidate cytostatic factor in vertebrate eggs. Expression of Emi2 was detected at mRNA level in rat eggs and zygotes with no expression at later stages of preimplantation development. Taken together, the onset of spontaneous activation in rat oocytes is correlated with a sharp decline in cdc2 activity and stable level of phosphorylated p42/p44 MAPK. Dynamics of cdc2 and p42/p44 MAPK activity during spontaneous activation resembles that during the MI to MII transition, although spontaneously activated rat oocytes do not form MIII spindle. Molecular factors involved in spindle assembly may be missing during the MII to MIII transition. Persistence of p42/p44 MAPK in spontaneously activated rat oocytes could account for the absence of pronuclear formation. The role of Emi2 remains to be investigated. This work is supported by the CMVM, ORS, Mary Orr Paterson studentships and the Framework 6 activity EURATOOLS.

240 EFFECTS OF TIMING AND DIPLOIDIZATION ON ACTIVATION AND DEVELOPMENT OF RAT OOCYTES

The objectives of these studies were (1) to compare the effects of Cytochalasin B (CB) or 6-dimethyl amino purine (DMAP) in combination with SrCl2 on activation and development of rat oocytes and (2) to determine the optimal timing for collection and activation of rat oocytes following superovulation. In Experiment 1, rat oocytes collected at 16–18 h after hCG injection were immediately cultured in 10 mm SrCl2 alone, 10 mm SrCl2 + 2 mm DMAP for 4 h and 10 mm SrCl2 + 5 μg mL–1 CB for 5–6 h, respectively, they were then cultured in KSOM for 20 h and finally cultured in R1ECM for an additional 4 days, all at 37°C, 5% CO2 in air. There were no significant differences in pronuclear formation (SrCl2 alone: 51/53; DMAP: 139/140; CB: 139/140) and cleavage rate (SrCl2 alone: 52/53; DMAP: 129/140; CB: 130/140) observed among three groups (χ2-test, P &gt; 0.05). On Day 5 the frequency of development to blastocyst was not statistically different (P &gt; 0.05) between DMAP (55.71%, 78/140) and CB (54.23%, 76/140) treated groups; however, no blastocysts were produced in 10 mm SrCl2 alone. In Experiment 2, oocytes were collected at 14, 16, 18, 20 and 22 hours after hCG injections, respectively, and they were immediately cultured in 10 mm SrCl2 + 2 mm DMAP for 4 h and then cultured in the same conditions described for Experiment 1. Although development to blastocyst was slightly lower in the 14-h group (43.50%, 47/108), no significant differences in the frequencies of pronuclear formation, cleavage on Day 1 or development to blastocyst on Day 5 were observed between groups (χ2-test, P &gt; 0.05) (Table 1). All experiments were repeated at least three times. In conclusion, our results showed that SrCl2 combined with either CB or DMAP could equally, effectively activate rat oocytes. In contrast rat haploid oocytes activated in the absence of CB or DMAP were not able to develop to the blastocyst stage. Furthermore, rat oocytes collected between 14 and 22 h after hCG injection had an equal potential to develop to the blastocyst stage after activation by 10 mm SrCl2 + 2 mm DMAP. Table 1.Development of rat oocytes activated at different time points following

The Developmental Potential of Parthenogenetic and Somatic Cell Nuclear-Transferred Rat Oocytes In Vitro

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in rat oocytes. First, we compared the effects of two types of inhibitors of spontaneous activation, MG132 and demecolcine, on the developmental potential of parthenogenetic oocytes. The potential of activated oocytes to develop into blastocysts significantly decreased 2 h after oocyte recovery (77% vs. 7%). The developmental potential of oocytes preserved in MG132-supplemented medium for 1 to 4 h was high (62% to 77%), but the potential of those preserved in demecolcine-supplemented medium for 3 and 4 h was low (77% vs. 41% and 37%, respectively). Second, the effect of the duration of parthenogenetic activation on the developmental potential was examined. When oocytes preserved in MG132 for 4 h were treated with 10 mM strontium for 5 or 6 h, the potential of activated oocytes to develop into blastocysts was high (78% and 70%, respectively). Using the optimal conditions for parthenogenetic activation, we examined the potential of rat enucleated oocytes receiving cumulus cells to develop into blastocysts. In contrast to parthenogenotes, the potential of SCNT rat oocytes to develop into blastocysts was low (2%) even if then oocytes were treated with the histone deacetylation inhibitor trichostatin A. The reason for the low developmental potential of rat SCNT oocytes is discussed.

Treatment with Proteasome Inhibitor MG132 during Cloning Improves Survival and Pronuclear Number of Reconstructed Rat Embryos

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.

Production of reconstructed two-cell rat embryos after chemical inactivation of chromosomes in MII oocytes by etoposide

The application of etoposide for chemical enucleation of rat oocytes was tested. The reconstruction efficiency after chemical and mechanical enucleation was comparatively analyzed. The obtained data indicate similar viability of reconstructed rat embryos irrespective of the enucleation technique.

Combined apoptosis and autophagy, the process that eliminates the oocytes of atretic follicles in immature rats

We studied the alterations of dying oocytes in 1-28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.

Reduction in Rat Oocyte Fertilizability Mediated by S-(1, 2-dichlorovinyl)-l-cysteine: A Trichloroethylene Metabolite Produced by the Glutathione Conjugation Pathway

Trichloroethylene (TCE), a commonly used industrial degreasing solvent and environmental toxicant, reduces rat oocyte fertilizability by an incompletely understood mechanism. Previous evidence implicated cytochrome P450 dependent oxidation of TCE. The current study investigated a second pathway, glutathione conjugation using S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a mutagenic and cytotoxic TCE-metabolite. In vitro exposure of oocytes and in vivo exposure of females to DCVC significantly reduced oocyte fertilizability (63% vs. 26%; p < 0.005 and 60% vs. 36%; p < 0.005, respectively). Reduced fertilizability of oocytes following in vivo TCE exposure may be mediated partially by the glutathione conjugation pathway.

Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.