Zona-intact Oocytes Research Articles

90 Guinea pig sperm is capable of fertilising bovine zona-intact oocytes in vitro.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Effects of fusion-activation interval and embryo aggregation on in vitro and in vivo development of bovine cloned embryos

Nuclear reprogramming in somatic cell cloning is one of the key factors for proper development, with variations in the protocol appearing to improve cloning efficiency. This study aimed to determine the effects of two fusion-activation intervals and the aggregation of bovine cloned embryos on subsequent in vitro and in vivo development. Zygotes produced by handmade cloning were exposed to two fusion-activation intervals (2 h or 4 h), and then cultured in microwells either individually (1 × 100%) or after aggregation of two structures (2 × 100%). Zona-intact oocytes and zona-free oocytes and hemi-oocytes were used as parthenote controls under the same fusion-activation intervals. Day-7 cloned blastocysts were transferred to synchronous recipients. Cleavage (Day 2), blastocyst (Day 7) and pregnancy (Day 30) rates were compared by the χ2 test (P < .05). Extending fusion-activation interval from 2 to 4 h reduced cleavage (91.0 vs. 74.4%) but not blastocyst (34.8 vs. 42.0%) rates. On a microwell basis, cloned embryo aggregation (2 × 100%) increased cleavage (91.5% vs. 74.4%) and blastocyst (46.0% vs. 31.3%) rates compared to controls (1 × 100%), but did not improve the overall embryo production efficiency on Day 7 (23.0% vs. 31.3%), on a per reconstructed embryo basis, respectively. Treatments had no effects on in vitro developmental kinetics, embryo quality, and in vivo development. In summary, the fusion-activation interval and/or the aggregation of cloned bovine embryos did not affect cloning efficiency based on the in vitro development to the blastocyst stage and on pregnancy outcome.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

Developmental potential of zona pellucida–free oocytes obtained following mild in vitro fertilization

To determine the developmental potential of oocytes in which the zona pellucida was damaged and subsequently removed, producing "zona-free" (ZF) oocytes that were cultured until the blastocyst stage. ZF eggs from cycles with more than one oocytes retrieved (n = 97) were compared with zona-intact (ZI) oocytes originating from the same patient. Private infertility clinic. Infertile patients (n = 135) undergoing minimal ovarian stimulation or natural-cycle in vitro fertilization treatment during 2010-2012. ZF oocytes undergoing intracytoplasmic sperm injection (ICSI) fertilization, blastocyst culture, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT). Rate of fertilization, cleavage, and blastocyst development. Live birth rate and neonatal outcome in subsequent SVBT cycles. There were no significant differences in fertilization (77% vs. 77%), cleavage (75% vs. 75%), or blastocyst development rates (39% vs. 32%) between the internally controlled ZF and ZI groups, respectively. Survival after thawing (90% vs. 100%) and live birth rates (37% vs. 36%) per thawed blastocyst were also similar. Newborns originating from all ZF and ZI oocytes had a similar gestational age at delivery (38.3 ± 3.7 wk vs. 39.5 ± 1.5 wk) and birth weight (3,115 ± 946 g vs. 3,010 ± 441 g). Our retrospective comparative study suggests that ZF eggs could be as successfully fertilized and cultured until the blastocyst stage as ZI control eggs without adversely affecting subsequent pregnancy rates and basic neonatal outcome.

Laser assisted IVF: A simplified technique to improve zygote production rates without the need for ICSI

Background/Aim: Spermatozoa from inbred mice provide a useful model to study male infertility as they produce a low number of zygotes with conventional IVF. This study aimed to assess whether the use of zona laser dissection might improve the number of zygotes developing after conventional IVF with inbred spermatozoa. Materials & Methods: Mouse oocytes from an inbred C57BL/6J strain and a hybrid B6D2F1 strain were allocated to either conventional IVF or laser assisted IVF, using spermatozoa from the inbred strain C57BL/6J. Laser assisted IVF involved use of a 1.48 diode microsurgical laser device to ablate three holes into each zona immediately prior to insemination. Results: Use of a laser significantly improved the zygote production rate compared with zona intact oocytes (88.4% vs. 18.3% for inbred oocytes, and 92.3% vs. 1.6% for hybrid oocytes, P Conclusion: Zona laser dissection is a simple technique that avoids the need for ICSI, significantly reduces operator time and allows numerous oocytes to be treated in a short period. Since high numbers of zygotes can develop using laser assisted IVF for this animal model where the spermatozoa are of poor quality, one might speculate that laser assisted IVF may have a possible role in the treatment male infertility in a clinical setting.

31 IN VITRO SURVIVAL AND PREGNANCY OUTCOME OF ZONA-FREE TRANSGENIC GOAT CLONED EMBRYOS AFTER OVIDUCTAL TRANSFER TO FEMALE RECIPIENTS ON DAY 1 OF DEVELOPMENT

Survival to term following embryo transfer is usually low after cloning by somatic cell nuclear transfer (SCNT). The aims of this study were to evaluate the effects of the zona pellucida (presence or absence) and the cell nucleus transfer method (cell fusion or cell injection) on the in vitro survival and pregnancy outcome of Day-1 goat cloned embryos transferred into the oviduct of recipient females. In vitro-matured goat oocytes from slaughterhouse ovaries were polar body selected, with a group of oocytes subjected to enzymatic zona pellucida removal. Zona-free (ZF) and zona-intact (ZI) oocytes were enucleated by micromanipulation procedures (Oback and Wells 2003 Cloning Stem Cells 5, 3–12; Keefer et al. 2000 Biol. Reprod. 66, 199–203). Somatic nucleus donor cells from 3 transgenic females for mammary gland expressing human lysozyme (Maga et al. 2003 Trans. Res. 12, 485–496) were either fused (CF) with enucleated ZI and ZF oocytes or injected (CI) into enucleated ZI oocytes, with the assessment of fusion or injection survival rates performed after 60 min. Two direct-current (DC) pulses were used to induce fusion in the ZI group (2 kV cm–1 each for 10 µs) and in the ZF group (1.0 kV cm–1 for 20 µs). Embryo reconstruction using ZI oocytes was done by micromanipulation. Zona-free (CF) or ZI (CF or CI) reconstructed cloned embryos were chemically activated in ionomycin/DMAP, followed by in vitro culture for 12 h prior to the surgical embryo transfer into the oviducts of synchronous female recipients (Day 1). Pregnancy diagnosis was carried out on Day 30 of gestation by ultrasonography. Survival after cell fusion, cell injection, and embryo transfer were compared by the χ2 test, for P &lt; 0.05. After 16 replications, 1047 in vitro-matured oocytes obtained from 208 does were used for embryo reconstruction (Table 1). In vitro survival was higher in ZI oocytes that were injected with somatic cells than in ZI oocytes or ZF subjected to cell fusion. Pregnancy rates were similar between groups, irrespective of the cell nucleus transfer method or the presence or not of the zona pellucida, but the overall efficiency (fetal survival/recipients) was higher in the ZI-CF group. Currently, 2 ongoing pregnancies carrying 3 cloned concepti from ZI oocytes fused to somatic cells are in late gestation (&gt;110 days). In conclusion, the cell injection method promoted higher survival and, consequently, better efficiency than cell fusion for the reconstruction of goat cloned embryos. However, the zona removal did not affect subsequent in vivo embryo development, as the transfer of zona-free embryos into the oviducts of synchronous recipients resulted in similar pregnancy rates than with zona-intact embryos. Table 1.In vitro survival and pregnancy outcome of goat cloned embryos after embryo reconstruction and transfer to female recipients on Day-1 of development

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae

The recognition and binding of a free-swimming spermatozoon to an ovulated oocyte is one of the most important cellular interactions in biology. While traditionally viewed as a simple lock and key mechanism, emerging evidence suggests that this event may require the concerted action of several sperm proteins. In this study we examine the hypothesis that the activity of such proteins may be coordinated by their assembly into multimeric recognition complexes on the sperm surface. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we tender the first direct evidence that human spermatozoa do indeed express a number of high molecular weight protein complexes on their surface. Furthermore, we demonstrate that a subset of these complexes displays affinity for homologous zonae pellucidae. Proteomic analysis of two such complexes using electrospray ionization mass spectrometry identified several of the components of the multimeric 20S proteasome and chaperonin-containing TCP-1 (CCT) complexes. The latter complex was also shown to harbor at least one putative zona pellucida binding protein, ZPBP2. Consistent with a role in the mediation of sperm–zona pellucida interaction we demonstrated that antibodies directed against individual subunits of these complexes were able to inhibit sperm binding to zona-intact oocytes. Similarly, these results were able to be recapitulated using native sperm lysates, the zona affinity of which was dramatically reduced by antibody labeling of the complex receptors, or in the case of the 20S proteasome the ubiquitinated zonae ligands. Overall, the strategies employed in this study have provided novel, causal insights into the molecular mechanisms that govern sperm–egg interaction.

151 COMPARISON OF CELL NUMBERS OF ZONA-INTACT AND ZONA-FREE PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS CULTURED IN VITRO

Application of an artificial stimulus to activate oocytes and induce development is essential for the successful animal cloning by nuclear transfer (Zhu et al. 2002 Biol. Reprod. 66, 635-641). The embryo’s developmental competence could be further improved with optimal in vitro culture conditions (Du et al. 2007 Theriogenology 68, 1104-1110). Cell number determination is a commonly used and simple criterion to assess developmental quality of pre-implantation stage mammalian embryos (Lagutina et al. 2007 Theriogenology 67, 90-98). Our aim of the study was to investigate porcine embryos activated and cultured in different ways using total cell numbers as the only quality measure. After 43-44 h of in vitro maturation and cumulus cell removal, zona-intact (PAZI) or zona-free oocytes (PAZF; after pronase treatment) were subjected to parthenogenetic activation (Day 0) with a single 80-μs DC pulse of 1.26 kV cm-1 or 0.86 kV cm-1 (Kragh et al. 2005 Theriogenology 64, 1536-1545), followed by a 4-h treatment with 5 μg mL-1 of cytochalasin B and 10 μg mL-1 of cycloheximide. Subsequently, the well of the well system (Vajta in vitro 2000 Mol. Reprod. Dev. 55, 256-264) was used for culture of all PAZF and half of the PAZI embryos (PAZF-WOW and PAZI-WOW groups, respectively), whereas the remaining PAZI embryos were cultured in groups of 25-30 (PAZI group). All cultures were performed in porcine zygote medium 3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119). On Day 6, all these in vitro cultured embryos were fixed and stained with Hoechst 33342 and cell numbers were counted on the microscopic pictures taken using fluorescent light. Data analysis was performed using ANOVA. Four replicates were performed with a total of 462 PAZF-WOW, 484 PAZI-WOW, and 467 PAZI group embryos. Embryos of each group were then divided into 5 groups based on their cell number (&lt;5 cells, 5-8 cells, 9-16 cells, 17-32 cells, &gt;32 cells). Percentages of embryos in each group are shown in Table 1. The average cell numbers of zona-intact embryos from PAZI-WOW and the PAZI group were similar to each other (P &gt; 0.05), whereas the cell numbers of PAZF-WOW embryos were significantly different from both PAZI-WOW and PAZI group embryos (P &lt; 0.05), with more embryos having higher cell numbers.The results demonstrate that zona-free parthenogenetically activated embryos cultured in WOW have higher cell numbers than embryos with intact zona pellucida. Accordingly, the presence of zona pellucida may compromise embryo development under certain in vitro culture situations. Table 1.Distribution (in %) of parthenogenetically activated porcine embryos according to their cell number on Day 6 after activation

54 IN VITRO DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY HANDMADE CLONING FROM DISTINCT CELL CULTURE CONFLUENCES AND AGGREGATION SCHEMES

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitro development of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) &gt;90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1 than cycling cells at the log phase (&gt;91% v. 59%; Sun et al. 2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1 proportion in that cell population. Table 1.In vitro development of bovine cloned embryos from distinct cell culture confluences and aggregation scheme This study was supported by FAPESP and CAPES/Brazil.

Fertilization of C57BL/6 mouse sperm collected from cauda epididymides after preservation or transportation at 4 °C using laser-microdissected oocytes

The C57BL/6 mouse is commonly used to produce transgenic and knockout strains for biomedical research. However, the motility and fertility of its sperm decrease markedly with freezing. Short-term preservation of sperm without freezing can avoid this. Furthermore, such samples can be transported safety without the special skills or equipment needed for the transportation of live animals or frozen products. We evaluated the motility and fertility of sperm collected from cauda epididymides after preservation or transportation at 4 °C. Oocytes with the zona pellucida subjected to laser-microdissection were used to assist fertilization in vitro. Although the motility of sperm gradually decreased with storage ( P < 0.05), no disruption of the sperm plasma membrane was seen. The proportion of zona-intact oocytes fertilized with sperm preserved for 0, 24, 48 and 72 h were 70, 14, 5 and 1%, respectively. On the other hand, 45, 20 and 14% of laser-microdissected oocytes were fertilized by sperm preserved for 24, 48 and 72 h, respectively ( P < 0.05). The fertility of sperm collected from cauda epididymides of two transgenic strains after transportation at 4 °C were also significantly increased using laser-microdissected oocytes rather than zona-intact oocytes (57 and 68% vs. 5%, P < 0.05). Efficient production of offspring from sperm preserved or transported at 4 °C was achieved using laser-microdissected oocytes. Thus the fertility of sperm preserved or transported at 4 °C could be maintained, although motility gradually decreased with storage. Laser-microdissected oocytes will contribute to the efficient production of embryos and offspring using such preserved sperm samples.

Imaging the Zona Pellucida of Canine and Feline Oocytes Using Scanning Electron Microscopy

Ultrastructure of the zona pellucida (ZP) of canine and feline oocytes has not been fully investigated. The objective of the study was to evaluate the potential use of the low vacuum scanning electron microscope (LVSEM) with oocytes. This required development of a method to prepare canine and feline cumulus-oocyte complexes (COCs) for LVSEM to provide ultrastructural information on the ZP. COCs were collected from ovaries, and cumulus cells were either partially or completely removed to reveal the ZP. COCs and zona-intact oocytes were fixed at 4 degrees C for 1 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently viewed wet or further processed by critical point drying, and viewed uncoated or sputter coated with gold. Although the spongy surface of the ZP was visible at low vacuum in uncoated oocytes, coated oocytes had more details at high vacuum. The ZP surface of canine and feline oocytes contained numerous various-sized, spherical or elliptical pores that narrowed centripetally splitting into several smaller, deep pores. The round to oblong cumulus cells tightly surrounded the ZP. Each corona radiata cumulus cell tapered into a thin projection that entered the ZP. Our detailed techniques will enable future studies connecting ultrastructural and molecular aspects of oocyte maturation and development in mammals.

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.

47 EFFECT OF THE TYPE OF CYTOPLASTS, SOURCE OF KARYOPLASTS, AND ACTIVATION PROCESS ON IN VITRO DEVELOPMENT OF BOVINE CLONE EMBRYOS

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

Role of SPRASA in Sperm-Zona and Sperm-Oolemmal Binding.

SPRASA (SPACA-3, SLLP-1) is a highly conserved protein located in the inner acrosome membrane of sperm. It was discovered based on its' reactivity with antisperm antibodies from some infertile men. SPRASA expression appears to be limited to the testis/sperm and others have shown that SPRASA-reactive antibodies inhibit human sperm binding to zona-free hamster oocytes. We have previously found that exposing gametes to SPRASA-reactive antibodies inhibits subsequent embryo development and have recently demonstrated that there are SPRASA binding sites on the oolemmal membrane of bovine oocytes. Using a bovine model, we investigated the effects of a SPRASA-reactive antiserum on sperm binding to oocytes at the zona pellucida and oolemmal membrane level in a bovine in vitro fertilisation model system. Bovine oocytes were obtained from Auckland Meat Processors and matured in vitro. To examine the effects of binding of sperm at the zona pellucida level, capacitated sperm and zona-intact oocytes were co-incubated with SPRASA-reactive antiserum, an irrelevant antiserum or in the absence of antiserum for 2 hours, followed by repeated pipetting with a Gilson pipette to remove loosely-bound sperm. Sperm and oocyte complexes were then stained with Hoechst and visualised on a fluorescent microscope. To examine the effects of binding of sperm and oocytes at the oolemmal membrane level sperm were capacitated then acrosome reacted using calcium ionophore then incubated with oocytes, from which the zona had been stripped using pronase, in the presence or absence of a SPRASA-reactive antiserum or an irrelevant rabbit antiserum for 2 hours. Following extensive washing, sperm/oocyte complexes were then stained with Hoechst and visualised on a fluorescent microscope. In both assays the number of sperm bound to each oocyte was counted. The significance of the difference between SPRASA-reactive antiserum and control groups was determined by the Student T-test. Analysis of sperm-zona binding showed that the number of sperm bound per zona-intact oocyte was not significantly different between the SPRASA-reactive antiserum treated group and the controls (P = 0.26). Similarly, SPRASA-reactive antiserum did not significantly alter the number of sperm binding to zona-free oocytes in comparison to the no antiserum control (P = 0.49). However, control rabbit antiserum appears to interfere with the binding assay, significantly increasing the number of sperm bound to the oolemmal membrane. In conclusion, this study shows that SPRASA-reactive antiserum had no effect on the binding of bovine sperm to bovine oocytes at either the level of the zona pellucida or the oolemmal membrane, and that the inhibitory effect of SPRASA-reactive antiserum on embryonic development that we have reported previously appears therefore to be downstream of sperm-oocyte binding. This study was supported by funding from the Marsden Fund of the Royal Society of New Zealand and the University of Auckland Staff Research Fund.

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo

Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.

227. Eggs with a surprise: the 'sperm-specific' protein SPRASA is also expressed in the oocyte

SPRASA is a newly identified protein which in silico analysis suggests is not expressed in other tissues. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm/oocyte recognition. The aim of this study was to investigate the spatial and temporal expression of SPRASA in reproductive and other tissues. Brain, thymus, heart, spleen, kidney, liver and the reproductive organs from duplicate female and male Balb/C mice were collected at several postnatal timepoints. RNA was extracted, reversed transcribed and analysed by quantitative real time PCR for SPRASA expression. Abattoir-derived, in vitro matured, bovine oocytes were examined for SPRASA expression by fluorescent immunochemistry. To examine SPRASA binding sites on oocytes, matured bovine oocytes were exposed to biotinylated recombinant human SPRASA or biotinylated α-lactalbumin (control), then visualised by confocal microscopy using DTAF-conjugated streptavidin. We found SPRASA mRNA was expressed in the reproductive organs of both females and male mice from postnatal day 10. Fluorescent immunochemistry indicated SPRASA was expressed on the oolemmal membrane and in the few cumulus cells remaining attached to zona-intact oocytes. Control preimmune serum did not stain the oocytes or cumulus cells. Recombinant human SPRASA bound to the oolemmal membrane of both zona intact and zona free bovine oocytes. To date the expression of SPRASA has only been reported in the testes/sperm with an additional single EST identified in brain. Our quantitative real-time PCR analysis demonstrated SPRASA is also expressed in the female reproductive organs. This was confirmed by our immunoassays which show oocytes and possibly cumulus cells express SRPASA while the oolemmal membrane has the ability to bind (sperm-derived) SPRASA. That SPRASA expression is restricted sperm and oocytes confirms the likely function of this protein in reproduction.

Piezo-assisted In Vitro Fertilization of Mouse Oocytes with Spermatozoa Retrieved from Epididymides Stored at 4 Degree C

This study was performed to investigate the effect of partial zona pellucida incision by piezo micromanipulation (ZIP) on the in vitro fertilizing ability of stored mouse spermatozoa. The storage conditions were optimized by storing the mouse epididymides at 4 C in mineral oil or in the mouse body for up to 4 days after death, and the retrieved spermatozoa were used to fertilize fresh oocytes. No significant difference was observed in fertilization rates between the treatments when epididymides were stored for up to 2 days, but the fertilization rates in mineral oil were higher (P<0.05) than those in the mouse body at 3 (41.4 vs. 16.2%) and 4 days (26.0 vs. 15.8%). Spermatozoa retrieved from epididymides stored in mineral oil were then used to fertilize fresh and vitrified oocytes with or without ZIP treatment. The fertilization rates of the ZIP fresh oocytes were higher than those of the zona-intact oocytes at each time point (1 to 4 days). After ZIP, the fertilization rates of spermatozoa stored for 1 and 2 days (91.2 and 86.6%, respectively) were similar (P>0.05) to that of fresh spermatozoa (91.9%). In regard to vitrified oocytes, the fertilization rates of zona-intact and ZIP oocytes using fresh spermatozoa were 46.7 and 84.7%, while the fertilization rates of vitrified ZIP oocytes using spermatozoa stored for 1 to 4 days ranged from 49.3 to 79.6%. When 2-cell embryos derived from ZIP fresh and vitrified oocytes inseminated with 2 day-stored spermatozoa were transferred into recipient females, 47.9 and 15.0% of the embryos developed to term, respectively. These results indicate that storing mouse epididymides at 4 C in mineral oil is more suitable than storage in the mouse body and that the ZIP technique improves the in vitro fertilizing ability of stored mouse spermatozoa in fresh oocytes and significantly increases the fertilization rate of vitrified oocytes with fresh spermatozoa.

50 EFFECT OF AGGREGATION OR FUSION ON DEVELOPMENT AND CELL NUMBER OF BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING AND PARTHENOGENESIS

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (&gt;90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

81 INDUCED BLASTOCOEL COLLAPSE IMPROVES SURVIVAL RATES OF PORCINE BLASTOCYSTS AFTER VITRIFICATION

Cryopreservation of porcine blastocysts is regarded as a demanding task because of the high chilling sensitivity and low survival rates. Ultrarapid vitrification techniques combined with the previous artificial induction of collapse of the blastocoel have been successfully applied in mouse and human. The purpose of our work was to use the Cryotop (Kitasato Supply Co., Tokyo, Japan) vitrification method combined with induced blastocyst collapse for cryopreservation of porcine blastocysts produced with parthenogenetic activation of in vitro-matured oocytes. A total of 1200 slaughterhouse-derived porcine oocytes were matured for 41–42 h. Zona-intact or zona-free oocytes (after pronase digestion) were activated with a single electric impulse of 63 or 43 V cm–1, respectively, and incubated in 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide for 4 h (Du et al. 2007 Cryobiology 54, 181–187). Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2, and 90% N2 and maximum humidity. On Day 6, collapse was induced manually in approximately half of the blastocysts with the use of a fire-polished holding pipette and injection pipette (Cook Australia, Brisbane, Queensland, Australia). Ten min after the manipulation, collapsed and control (not manipulated) blastocysts were vitrified with the Cryotop, and then warmed and diluted in 1, 0.5, and 0 m sucrose, respectively (Kuwayama et al. 2005, Reprod. BioMed. Online 11, 300–308). Reexpansion of blastocysts was evaluated under a stereomicroscope after 16–18 h of culture under conditions described above. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical calculations; values with P &lt; 0.05 were regarded as significant. Reexpansion rates of blastocysts are shown in Table 1. Our results indicate that manually induced collapse of the blastocoel increases reexpansion rates of both zona-intact and zona-free porcine blastocysts after Cryotop vitrification. The combination of these simple techniques may help to improve the efficiency of cryopreservation of pig embryos for both experimental and practical applications. Table 1. Reexpansion rates after vitrification of blastocysts with or without induced blastocoel collapse The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen, and ClausWillemoes for their help and excellent technical assistance.