Sow Oocytes Research Articles

IN VITRO BISPHENOL A EFFECT ON TFAM AND SIRT1 GENE EXPRESSION IN PORCINE OOCYTE MITOCHONDRIA

"Mitochondria are the main cellular organelle responsible for energy production, having an essential role in maintaining cellular homeostasis. In this study, the gene expression of TFAM (Transcription Factor A Mitochondrial) and SIRT1 (Silent Information Regulator) in sow oocytes cultured in vitro was assessed using the RT-qPCR reaction. The primers were tailored after our own design. The groups were: V1 (matured oocytes, control group), V2 (matured oocytes with hormones), V3 (medium supplemented with Bisphenol A), V4 (medium supplemented with Bisphenol A and hormones). Our findings reveal a reduction in SIRT1 activity following maturation in all experimental groups, while TFAM activity displayed more elevated levels, seemingly independent of the SIRT1 gene expression. The highest values of gene expression for TFAM and SIRT1 were obtained in V2 (supplemented with FSH and LH, - 0.277 and 0.010) and V4 (FSH, LH and bisphenol A - 0.272 and 0.015) without significant differences (p=0.941). Bisphenol A alone generated low values, presumably due to its endocrine disruptor action. We concluded that FSH/LH addition might rescue some of the TFAM expression during bisphenol treatment, but the mechanism might be independent of SIRT1."

Cumulus-oocyte complexes from sows show differences in lipid metabolism compared to cumulus-oocyte complexes from prepubertal gilts during in vitro maturation.

This study aimed to evaluate the effects of donor age on lipid metabolism during in vitro maturation (IVM) of pigs cumulus-oocyte complexes (COCs). We evaluated transcript levels of genes, the percentage of ooplasm occupied by lipid droplets (LD) and evaluated DNA methylation in COCs from sows and prepubertal gilts. Transcript levels of six genes (ACACA, ACSS2, FASN, FABP3, SLC27A4, PLIN2), which were analyzed in cumulus cells (CCs), increased after 44 h of IVM in the sow group. In the gilt group, only FASN expression increased, while NR3C1 expression decreased after IVM. The measurement of LD in oocytes showed an accumulation of lipids in sow oocytes during IVM, while gilt oocytes showed a decrease in LD. FABP3 and NR3C1 methylation patterns exhibited a demethylation pattern in CCs and oocytes from gilts and sows and showed statistical differences between groups. CCs from sows had a better capacity to change transcription levels of the major genes involved in lipid metabolism during IVM than CCs from gilts. This difference may be involved in accumulation of lipids, acquisition of competence, and maturation of enclosed oocytes. Our results contribute to a better understanding of mechanisms involved in lipid metabolism and acquisition of competence in porcine COCs.

Quality Evaluation of Gilts and Sow’s Oocytes During In Vitro Maturation Based on Mitochondria Distribution


 Mitochondria is the most frequently studied cell organelle in relation to development competence of the oocytes both in vivo and in vitro. Developmental competence of oocyte is crucial for embryonic development, the molecular activity until the activation of the embryonic genome being based on the maternal reserves stored in the oocyte. Study was designed to qualitatively evaluate 133 COC class C1 and class C2 gilts and sow’s oocytes according to the distribution of mitochondria after IVM, based on Rhodamine 123 stain solution. Regarding mitochondrial distribution, the percentage of oocytes that had a homogeneous distribution around the germinal vesicle was higher in group C1 than C2 and also higher in sow oocytes than in gilts oocytes. For class 1, the difference was 10.58% in favor of sows, the same trend is maintained for C2, the difference being 4.37%. The results are confirmed by morphological examination, where C1 sow’s oocytes matured 26.09% more, compared to gilts oocytes, a difference maintained for C2 oocytes, being 27.4% more sow’s oocytes IVM compared to gilts. Based on mitochondrial distribution we observed that the stage of sexual development of females influences the IVM of oocytes. For pig’s IVF it’s recommended to use C1 sow oocytes.

Utilization of Rosmarinic and Ascorbic Acids for Maturation Culture Media in Order to Increase Sow Oocyte Quality Prior to IVF.

The beneficial effect of antioxidant supplementation in maturation culture media of sow oocytes was evaluated by the expression quantification of apoptotic genes and the genes that ensure stability of germ cells during fertilization. The oocytes were cultivated for 44 h in conventional medium (C) or in medium supplemented with 105 µM rosmarinic acid (R) and 0.5 mM ascorbic acid (A) and classified into three quality classes by morphological observation from which the total RNA was isolated. The gene expression of Ptx3 and the apoptotic regulator p53, Bax and BCL-2 were evaluated by quantitative PCR technique. The decreased expression of the Bax gene in the A and R groups, compared to the control, indicates a protective role of antioxidants in the cells. Cell homeostasis was maintained, as reflected in the ratio of Bax/Bcl-2 in class I COCs (cumulus-oocyte complex) regardless of the experimental group, indicating minimum cellular stress. The expression of p53 genes was higher in all class III COC, but in A1 and R1 the expression was lower than in C1, and a similar Ptx-3 gene decreased significantly in groups A1, A2, A3 and R1 compared with control groups. Antioxidant supplementation showed beneficial effects on all morphological classes of pig COCs.

Cytological features of pig oocytes in the process of spontaneous maturation

Aim. The study was aimed at determining the cytological changes, related to the germinal vesicle (GV) of porcine oocyte during the follicular maturation. Methods. The ovaries were classifi ed according to the estrous cycle phase, judging by the status of yellow bodies and follicles. The oocytes were isolated from each animal using dominating and atresiated follicles separately. The oocytes were used to prepare whole mounts by a specially designed express-method, which is thermal fi xation (about 95 °С) and their further transfer into the medium for clarifi cation. The whole procedure of preparing the mount from one oocyte or their group lasted for about 10 min. The microscopic studies were conducted using the phase contrast. A total of 831 oocytes were studied. Results. It was established that sow oocytes had eccentric location of GV which may be found in oocytes from early antral follicles. This condition is kept until the beginning of meiosis restoration and GV destruction. The nucleolus is also located eccentrically. Another relevant cytological change was gradual condensation of chromatin around the nucleolus, starting from separate clots on its surface and further formation of circular structures. Conclusions. Contrary to laboratory rodents, GV in porcine oocytes has eccentric localization, which is set even in early antral follicles and remains until the beginning of meiosis restoration. The process of chromatin transformation was found to be its gradual condensation around the nucleolus which may be defi ned as a nucleolus complex.

86 IN VITRO-MATURED GILT OOCYTES CAN HAVE EQUAL OR BETTER DEVELOPMENTAL COMPETENCE THAN SOW OOCYTES WITH NEW MATURATION MEDIA

It has long been thought that oocytes obtained from sows yielded a higher level of developmental competence compared with oocytes obtained from prepubertal gilts. Because gilt-derived oocytes are more readily available to our laboratory and they are less developmentally competent, we hypothesised that by making alterations to our maturation system we could improve the developmental competence of the gilt-derived oocytes to that of their sow-derived counterparts. We performed 2 experiments that evaluated the ability of each source of oocyte to develop to the blastocyst stage, using altered maturation media. The first experiment focused on the developmental ability of each source of oocytes, through IVF and culture. The second experiment again focused on the developmental competence of each oocyte source but through somatic cell NT. For both experiments, the sow-derived oocytes were obtained from Desoto Biosciences and the gilt ovaries were collected from Smithfield Inc. in Milan, Missouri. Both sets of oocytes were in vitro matured in M199 supplemented with 0.57 mM cysteine, 5 μg mL−1 LH and FSH, and 10 ng mL−1 epidermal growth factor; however, the gilt derived media was altered to contain 40 ng mL−1 fibroblast growth factor 2 and 20 ng mL−1 insulin-like growth factor and leukemia inhibitory factor. Additionally, the maturation media for the sow-derived oocytes contained the addition of 5 μg mL−1 insulin and 10% follicular fluid. In the first experiment we performed IVF on oocytes from the 2 sources as per our laboratory standard IVF procedure, co-incubating the oocytes with 0.25 × 106 porcine semen for 4 h, followed by washing and moving the oocytes to MU2 culture media at 38.50°C in 5% CO2, humidified air overnight. After overnight culture the presumptive zygotes were transferred to the same conditions with 5% CO2, 5% O2, and 90% N2. After an additional 5 days, blastocyst development was assessed. The gilt oocytes yielded 39.3a ± 7.2% blastocyst, and the sow oocytes had a blastocyst rate of 24.9b ± 6.9%, with an n of 389 and 313, respectfully. Statistical analysis was performed by using Genmod in SAS 9.4. In the second experiment, using standard laboratory protocol for somatic cell NT, we activated both sets of oocytes with 200 μM thimerosal for 10 min followed by 30-min incubation with 4 mM dithiothreitol. The embryos were co-incubated for 15 h with 500 nM Scriptaid in the MU2 culture media in 5% CO2, humidified air; then these embryos were also moved to 5% CO2, 5% O2, and 90% N2 and cultured to Day 6. The sow oocytes produced a blastocyst percentage of 38.6%, and the gilt oocyte group had a blastocyst percentage of 43.5%, with an n of 290 and 285, respectfully. There was no difference statistically between these treatments. Both gilt and the sow oocyte sources have yielded live piglets at this time. We concluded that the maturation system used for our gilt-derived oocytes resulted in equal or better development in vitro compared with the sow-derived oocytes. Follow-up experiments evaluating in vivo development are needed for a complete comparison. This work was funded by Food for the 21st Century University of MO, and the NIH U42OD011140.

205 HOLDING PIG OOCYTES AT 24°C PRIOR TO IN VITRO MATURATION ALTERS THE DEVELOPMENTAL CAPACITY AFTER IN VITRO FERTILISATION BUT NOT PARTHENOGENETIC ACTIVATION

The in vitro production of porcine embryos is of great interest because of the increasing importance of the swine as an animal model and a tissue donor for biomedical or biotechnological applications. Availability of ovaries at selected time of the day can be a limitation; therefore, the possibility to maintain immature oocytes for some hours can be very useful. The aim of this study is to determine whether holding recovered oocytes at 24°C for 24 h alters the maturation process and/or the developmental capacity. Immature sow oocytes were either matured in vitro for 42 h at 38.5°C (control group; CTR) or kept in 2 mL of HEPES-SOF in the dark at 24°C for 24 h before maturation (experimental group; +24 h). After maturation, cumulus cells were removed, and the number at metaphase II were recorded. For parthenogenetic activation (PGA), oocytes with a visible polar body were activated at 48 h of maturation as previously described (Lagutina et al. 2006). For IVF experiments frozen-thawed boar semen was prepared through a discontinuous density gradient, washed in TALP Ca2+-free, diluted in TALP : SOF = 1 : 1 supplemented with 6 mg mL–1 of fatty acid-free BSA, hypotaurine and epinephrine, mixed with oocytes after partial removal of the cumulus cells, at 43 h of maturation and cultured in 5% CO2 in humidified air at 38.5°C. After 24 h of IVF, oocytes were denuded and cultured in mSOF-1 in atmosphere of 5% O2 and 5% CO2. The same culture conditions were used after parthenogenetic activation. Half of the medium was changed with mSOF-1 at Day 3 and with mSOF-2 at Day 5. The cleavage and the cumulative Day 7 blastocyst (BLD7) rates and cell number of IVF BLD6 were recorded. For each group, blastocysts on Day 6 were fixed and cell number counted, whereas the other embryos were left in culture until Day 7 (cumulative D7 = BLD6 fixed + BLD7). All experiments were done in 3 replicates. The data were compared by Student’s t-test and chi-square test. Maturation rates as recorded for the presence of the polar body did not differ (CTR: 255/312, 82%; +24 h: 208/256, 81%). There was no significant difference (P < 0.05, chi-squared test) between CTR and +24 h group cleavage (144/165: 87% and 127/138: 92%, respectively) and BLD7 rate (47/165: 28% and 34/138: 25%, respectively) in the PGA. Whereas no difference (P < 0.05, chi-squared test) was observed between CTR and +24 h group cleavage (111/180: 62% and 99/186: 53%, respectively) in the IVF, but the BLD7 rate in +24 h group was significantly lower (48/180: 27% in the CTR group, 27/186: 15% in the +24 h group). However, the cell number of IVF BLD6 was not altered by holding at 24°C (n = 22: 25 ± 10 cells in the CTR, n = 8: 22 ± 13 cells in the +24 h) (P < 0.05, 2-tailed Student t-test). These experiments show that holding at 24°C for 24 h before maturation can alter the developmental capacity of IVF-produced embryos but not that of parthenogenetically activated ones. More replicates are needed to study the kinetics of maturation and to confirm our results. MitCare project (ERC n 322424) is acknowledged for support of this project.

Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer

In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

Effect of Two Methods of Sperm Cells Processing on Achieving Fecundity "In Vitro" in Sow Oocytes

SUMMARY Considerable progress has been made the vitro production of pig embryos using improved methods for vitro maturation (IVM) and fertilization (IVF). Variatio n among boars, ejaculates and IVF protocols used differe nt laboratories appears to influence the incidence of polyspermy ( Revel F. et al., 1995) , after the first division that occurred the zygote content within 48 hours of cultivation th e thermostat ( Xu, X. et al. , 1996a). To highlight the effect of the two methods of sperm processing achieving fecundity in vitro on sow oocytes we have conducted two separate experiments: one consisted of placing together a group of cumulus-oocyte complex with processed sperm cells Percoll concentration gradient, and the other consisted of a number of COC cultivated with processed sperm cells through swim-up procedure ( J.J. Parrish et al., 1989) On the case of spermatozoids processing Percoll concentration gradient, out if 92 COC used for vitro fecundation, 13.04% of it had the first cleavages. Only 6.90% of the zygotes had the first cleavages o ut of 58 COC used on vitro fecundation, there were used processed spermatozoids by the method „swim-up”. If we evaluate the results of vitro fecundation, we see that from the total number of cumulus-oocyte complexes cultured the fecundation medium only 10.7% suffer the first division. If we compare the results on the effectiveness of t he two methods of processing bull and boar sperm, we find that the use of Percoll gra dient for choosing the most vigorous sperm cells and for their capacitation has an distinctive ly superior effect compared to the swimming competition procedure (swim-up).

51 SCRIPTAID AND MG132 IMPROVE DEVELOPMENT OF PORCINE EMBRYOS RECONSTRUCTED BY SOMATIC CELL NUCLEAR TRANSFER

Nuclear transfer efficiency in pigs and other large animal species is low. Previous studies have shown that histone deacetylase inhibitor (Scriptaid) and proteasomal inhibitor (MG132) treatment of somatic cell nuclear transfer (SCNT) pig embryos enhances blastocyst formation and pregnancy. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early development of cloned pig embryos reconstituted by SCNT. A total of 328 sow oocytes procured from ART (Madison, WI, USA) were reconstructed using α-1,3-Galactosyltransferase knockout hDAF transgenic pig fetal-derived fibroblast cells. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h and then treated with 500 nM Scriptaid for another 16 h. The SCNT embryos were washed and cultured in Porcine Zygote Medium 3 for 7 days. Percent cleavage was determined on Day 2, and blastocyst formation and cell number were determined on Day 7. The experiment was repreated 8 times. There was no difference (P > 0.05) in percent cleavage (57.9 to 66.7%), or cell number (25.5 to 30.6) among the 3 groups. Interestingly, while there was no difference in the percent blastocyst between the 1 μM and 0 μM MG132 treatment groups, more oocytes from the 1 μM MG132 group developed into blastocysts than in the 10 μM MG132 group (25.1 ± 4.6% v. 12.9 ± 3.3%; P = 0.045). Further research will be conducted to transfer these embryos to surrogate gilts to determine the true developmental competence of these embryos. Supported by the National Institutes of Health National Center for Research Resources (RR018877 and RR013438), and Food for the 21st Century.

48 DEVELOPMENTAL COMPETENCE OF CLONED OR PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS: EFFECT OF DIAMETER OF PREPUBERTAL GILT OOCYTES

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al. 1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor's age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120 kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitro matured for 42 to 44 h as described for sow oocytes (Li et al. 2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al. 2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al. 2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium was applied for 4 h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110 μm, from 105 to 116 μm), S group (mean diameter 101 μm, from 93 to 106 μm) and C group (mean diameter 107 μm, from 93 to 116 μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results

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.

Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes

Using reverse transcription and real-time quantitative PCR analysis we evaluated the transcript levels of integrins (alphaL, alphaM, beta1, and beta6), CD9 and CD18 antigens as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes isolated from puberal gilts (n=20) and multiparous sows (n=20). We found significantly (p<0.05) higher transcript contents of alphaL, alphaM, beta1, and beta integrins, CD9 antigen, and pZP2 and pZP3 in puberal gilt oocytes compared to multiparous sow oocytes. Our results suggest that a decrease in the level of oocyte transcripts encoding essential proteins involved in oocyte fertilization may be associated with increased porcine female age.

101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P &lt; 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.

An Epigenetic Modifier Results in Improved In Vitro Blastocyst Production after Somatic Cell Nuclear Transfer

The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation could significantly improve blastocyst yield compared to the control (46.4+/-4.6% vs 17.7+/-4.9% for treated and untreated embryos, respectively; p<0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production.

Sexual maturity and reproductive phase of oocyte donor influence the developmental ability and apoptosis of cloned and parthenogenetic porcine embryos

This study investigated the influence of the sexual maturity and reproductive phase of oocyte donor on the developmental ability and quality of porcine embryos produced by somatic cell nuclear transfer (SCNT) or parthenogenesis (PA). Blastocyst quality was evaluated in terms of hatching ability, total nuclei number and types of apoptosis. Results revealed that maturation rate was not influenced by the reproductive status of the oocyte donor. However, when subjected to PA or SCNT, embryos derived from sexually mature sow oocytes developed to blastocysts at higher rates and had higher cell number than those derived from immature gilt oocytes ( p < 0.05). Significant effect of reproductive phase, luteal versus follicular, was also noted with luteal stage oocytes yielding higher ( p < 0.05) rate of blastocyst formation (PA: 54.3 ± 1.3% versus 44.8 ± 0.3%; SCNT: 29.4 ± 0.2% versus 22.7 ± 0.1%). Blastocysts derived from luteal phase oocytes also had higher ( p < 0.05) hatching ability (PA: 44.2 ± 1.1%; SCNT: 39.6 ± 4.7%) and cell number (PA: 77.4 ± 4.9; SCNT: 54.9 ± 2.4) than those derived from follicular phase oocytes (PA: 34.9 ± 0.9%, 67.2 ± 3.9; SCNT: 34.6 ± 2.7%, 47.5 ± 2.9). TUNEL assay and Hoechst 33342 staining revealed that percentage of blastocysts showing total apoptosis did not differ among the groups. However, luteal phase oocyte-derived blastocysts had the highest incidence of nuclear fragmentation. Among cloned blastocysts that showed the signs of apoptosis, the highest index of total apoptosis was observed in prepubertal oocyte-derived blastocysts (5.2 ± 0.7). Blastocysts derived from luteal phase oocytes showed the lowest TUNEL index (2.0 ± 0.5). The present study therefore, indicates that the sexual maturity and reproductive phase of cytoplast donor significantly influences the developmental ability, apoptosis and quality of blastocysts produced by SCNT or PA. Oocytes from sexually mature sows in luteal phase of their reproductive cycle may be better cytoplast recipients for SCNT.

The Effect of Energy Substrate Concentration and Amino Acids on the In Vitro Development of Preimplantation Porcine Embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5–8 mm) and small (2–3 mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.

Improved survival of vitrified porcine embryos after partial delipation through chemically stimulated lipolysis and inhibition of apoptosis

Mechanical removal of intracellular lipids has been the most effective approach to increase the cryosurvival of porcine embryos. In this experiment, we tested the hypotheses that the cryosurvival of porcine embryos can be improved after partial delipation through chemically stimulated lipolysis and that the survival can be further improved by inhibition of apoptosis. Porcine embryos were produced in vitro using sow oocytes. On Day 5 of embryonic development, embryos were cultured in the presence of 10 μM forskolin for 24 h. On Day 6 blastocysts were vitrified using an open pulled straw (OPS) method and warmed blastocysts were cultured 18 h for them to recover. A caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used at 20 μM during vitrification and subsequent culture to inhibit apoptosis. A 2 × 2 × 2 factorial design experiment was conducted to examine the effect of chemical delipation, vitrification and apoptosis inhibition. We also measured the lipolytic activity of porcine embryos cultured with or without forskolin. Chemical delipation increased the cryosurvival of porcine embryos compared to the controls (71.2 ± 2.8% versus 37.1 ± 5.1%). Apoptosis inhibition increased the ability of blastocysts to fully recover (23.8 ± 3.1% versus 14.6 ± 4.3%). However, there was no interaction between chemical delipation and apoptosis inhibition. Lipolytic agent treatment increased the lipolytic activity of porcine blastocysts. In conclusion, cryosurvival of porcine embryos was improved by partial delipation through chemical stimulation of lipolysis or apoptosis inhibition.