- Email: [email protected]
Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer
Theriogenology 67 (2007) 341–345 www.theriojournal.com
Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer S. Bhojwani 1, H. Alm 2, H. Torner 3, W. Kanitz 4, R. Poehland * Research Unit Reproductive Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Received 5 April 2006; received in revised form 21 August 2006; accepted 21 August 2006
Abstract The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90 min before culture. Treated oocytes were then divided into BCB (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB oocytes (4%). These results show that the staining of bovine cumulus–oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future. # 2006 Elsevier Inc. All rights reserved. Keywords: Bovine nuclear transfer; BCB; Blastocysts; G6PDH
1. Introduction * Corresponding author. Tel.: +49 38208 68761; fax: +49 38208 68752. E-mail addresses: [email protected] (S. Bhojwani), [email protected] (H. Alm), [email protected] (H. Torner), [email protected] (W. Kanitz), [email protected] (R. Poehland). 1 Tel.: +49 38208 68768; fax: +49 38208 68752. 2 Tel.: +49 38208 68754; fax: +49 38208 68752. 3 Tel.: +49 38208 68759; fax: +49 38208 68752. 4 Tel.: +49 38208 68750; fax: +49 38208 68752. 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.08.006
Somatic cell nuclear transfer (SCNT) technique has succeeded in yielding bovine blastocysts but at a rather lower (averaging 20%) and inconsistent blastocyst rate as compared to in vitro-maturation (IVM) and fertilization (IVF). A host of factors have been shown to contribute to this low level of SCNT efficiency such as donor cell type, methods of embryo culture, imprinting defects, reprogramming failures, the rather inefficient artificial methods of activation, lab to lab
342
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
variation as well as the oocyte source and quality at the beginning of maturation [1]. The follicular oocytes are commonly recovered from ovaries of slaughtered cattle (often cows with reduced reproductive performance or which are slaughtered at the end of their economic period) and are thereby heterogeneous in quality and developmental competence [2]. Immature oocytes are known to synthesize a variety of proteins [3], among them, glucose-6phosphate dehydrogenase (G6PDH). This enzyme is known to be a component of the pentose phosphate cycle which provides ribose phosphate for nucleotide synthesis and much of the NADPH utilized as a hydrogen or electron donor in reductive biosynthetic reactions such as the formation of fatty acids. The G6PDH enzyme is active in the growing oocyte [4], but has decreased activity in oocytes that have finished their growth phase [3]. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colourless [5]. Studies with prepuberal goat oocytes showed that more competent oocytes could be selected using the BCB stain [6–8]. Both the rate of maturation to metaphase-II and the rate of normal fertilization of oocytes retaining the BCB stain were higher than in those metabolising the stain and in control oocytes. Activity of G6PDH has also been found to be inversely associated with maturation and fertilization of pig oocytes [5]. There have been limited reports on the use of BCB staining in the bovine: heifer oocytes classified as BCB+ had a significantly higher rate of blastocyst development than BCB or control heifer oocytes (12.3, 1.6, and 5.2%, respectively), but this rate was still lower than that of unstained cow oocytes (30.0%; P < 0.05) [9]. In yet another study in bovine [1], BCB staining was found to be effective in enhancing the blastocyst development rate after IVF where cow oocytes classified as BCB+ had a significantly higher rate of blastocyst development than BCB or control oocytes (34, 4, and 18%, respectively). The aim of the present study was, therefore, to determine whether a BCB staining as an indicator for G6PDH activity could be used to select developmentally competent bovine oocytes before IVM and thereby increase the efficiency of blastocyst development after bovine SCNT. 2. Materials and methods Unless otherwise indicated, all plastic ware, i.e. culture vessels and dishes used in our experiments were
obtained from Nunc, Wiesbaden, Germany, while all chemicals and medium, etc. were purchased from Sigma, Deisenhofen, Germany. 2.1. Oocyte collection Oocytes were collected by the method of ovarian slicing [10] from abattoir-derived ovaries 4–6 h after the slaughter. Prior to slicing, each ovary was placed in a Petri dish and covered with Tissue culture medium TCM-199 with Earle’s salts and 25 mM HEPES (M 7528) containing 0.5 IU/ml heparin and supplemented with 10% (v/v) heat-inactivated FCS (F 7524). All follicles present on the surface of the ovary were sliced with a scalpel and the oocytes were located with a stereo microscope. The medium used for recovery was TCM-199 with Earle’s salts (see above). Only oocytes with a compact cumulus investment were selected for further culture and were designated as COCs. 2.2. Brilliant cresyl blue staining To carry out the BCB test the compact COCs were washed three times in Dulbecco’s PBS modified by the addition of 0.4% BSA (A-7888; mDPBS). Then the COCs were exposed to 26 mM of BCB (B-5388) diluted in mDPBS for 90 min at 38.5 8C in humidified air [1]. The concentration of 26 mM had earlier been found to be effective for goat oocytes [7] as it was supportive of a high rate of selected oocytes without apparent loss of viability. It was also emphasized [11] that BCB could be used effectively in the study of embryo metabolism without being lethal after exposure with 26 mM BCB. COCs of the control group were incubated directly after selection without exposure to BCB (control). Following BCB exposure, the COCs were transferred to mDPBS and washed twice. After washing, the COCs were examined under a stereomicroscope and divided into two groups according to their cytoplasm colouration: oocytes with any degree of blue colouration to the cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB) (Fig. 1). 2.3. In vitro maturation (IVM) The collected COCs were washed twice in maturation medium (TCM-199) supplemented with 20% (v/v) heat-inactivated FCS (Sigma F 7425) and 10 mg/ml FSH (Ovagen, icp, New Zealand) and then incubated in maturation medium for 21 h at 38.5 8C in 5% CO2 in air [10,12].
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
Fig. 1. Differentially stained bovine COCs after exposure to BCB stain (BCB+, blue-coloured; BCB, unstained).
2.4. Culture of nuclear donor cells Bovine fibroblasts from the sixth passage of a primary male cell line were utilised as nuclei donors. For this purpose, a cell line was established using mechanically isolated pieces of ear skin isolated from an adult bull using a method described earlier [12]. The small tissue pieces (approx. 1 mm diameter) were cultivated for 1–2 weeks in TCM-199 containing 20% FCS, 1 mM sodium pyruvate and antibiotics (mixture of a working solution of 100 IU/ml penicillin and 100 mg/ ml streptomycin). The cells growing in monolayer were cultivated up to confluence. Cells in G0/G1 phase of cell cycle were enriched by serum deprivation in culture medium (0.5% FCS, 2 days) for subsequent use in SCNT experiments [12]. 2.5. Somatic cell nuclear transfer (SCNT) For the production of nuclear transfer embryos, the zona-free method of Handmade Cloning (HMCTM) was utilised [12–14]. Approximately 100 COCs (per experiment) were vortexed for 3 min in 0.5 mg/ml hyaluronidase to remove the cumulus. Oocytes were then transferred for 8 min into 1.5 mg/ml pronase (Sigma Protease P 8811) to remove the zona pellucida. After this they were manually bisected under stereomicroscopic control with Ultra Sharp Splitting Blades (AB Technology, Pullman, WA) in TCM-199 medium containing 20% FCS, 2.5 mg/ml of cytochalasin B and 10 mg/ml fluorochrome Hoechst 33342 (Sigma B 2261). All stained demi-oocytes were arranged in drops of TCM199 (three per drop) and positions of half-oocytes without chromatin staining (cytoplasts) were registered under fluorescence microscope. Meanwhile, the somatic cells
343
were prepared by repeated washing of the monolayer with Ca++- and Mg++-free PBS and subsequent trypsination (39 8C, 5 min in 100 ml of 0.05% trypsin dissolved in PBS). The somatic cells were separated by vigorous pipetting and stored in 2 ml Eppendorf tubes in HEPES-buffered TCM-199 medium supplemented with 20% calf serum (T20) at room temperature until fusion. For the first fusion, half of the total quantity of prepared cytoplasts were individually transferred to 500 mg/ml phytohemagglutinin (PHA) for 3 s and then quickly dropped onto a single somatic cell for attachment. Following attachment, the cytoplast-somatic cell pair was transferred to a fusion chamber with 0.5 mm separation of wires (CFA 500 Kru¨ss, Hamburg, Germany). Wires were covered with 1 ml of fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2) at 26–27 8C. The electrical alignment was carried out using 15 V ac at 700 kHz and the first fusion was performed with a double dc pulse of 65 V, each pulse for 20 ms and 0.1 s apart. For the second fusion, all remaining cytoplasts and fused pairs were transferred to the fusion chamber—one fused pair was then attached to each cytoplast. A double fusion pulse with the same parameters but at 45 V dc was applied and the reconstructed embryos were then transferred into culture medium, covered with oil, and incubated for 4 h in 5% CO2 in air at 39 8C. Two-step activation was subsequently carried out—first with 2 mM Ca ionophore (A 23187, 5 min, RT) and second with 2 mM 6-dimethylaminopurine (6-DMAP, Sigma D 2629, 5% CO2 in air, 39 8C for 6 h). Embryos were then washed twice in culture medium and cultured individually in well-of-thewells (WOWs) [15] in Synthetic Oviductal Fluid (SOF) medium (Minitu¨b, Germany) supplemented with 10% oestrous cow serum and covered with mineral oil. Embryo culture was performed at 39 8C in 5% CO2, 5% O2, and 90% N2. The number of blastocysts were recorded on day 8 of culture. 2.6. Blastocyst evaluation At the end of the culture period, the blastocysts were fixed in buffered formal saline (containing 5% formaldehyde) and subsequently stained with Hoechst 33342 (Sigma B 2261). The number of nuclei in the blastocysts were then recorded under fluorescence microscope. 2.7. Statistical analysis The data were evaluated by non-parametric oneway analysis of variance (ANOVA). Because of the
344
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
Table 1 Enhancement of blastocyst rate after bovine SCNT upon selection of oocytes based on BCB staining and number of nuclei in bovine SCNT blastocysts recovered on day 8 of culture (mean S.D.) Groups
Embryos n
Cleavage day 2 p.SCNT n (percent of total embryos)
Blastocysts day 7 p.SCNT n (percent of cleaved embryos)
Number of nuclei in blastocysts (n blastocysts) ð¯x S:D:Þ
Control BCB+ BCB
148 218 149
108 (73) a 170 (78) a 76 (51) b
23 (21) a 67 (39) b 3 (4) c
102.7 6.8 a (23) 112.8 9.7 b (67) 58.3 8.1 c (3)
Within columns, values with different letters (a, b, c) differ significantly (P < 0.05).
non-normality of the data, the NPAR1WAY procedure of SAS/STAT software was used to calculate simple linear rank statistics for the Wilcoxon, median, Van der Waerden, and Savage scores to test for the differences in location. Group effect in the model was BCB+, BCB and control. Differences having P values of the test equal to or less than 0.05 (P 0.05) were considered statistically significant. 3. Results A total of 1108 bovine COCs were recovered and used for the investigation. Of these, 314 were used for the production of control embryos while 794 were subjected to BCB staining procedure out of which 472 (59.4%) were selected as BCB+ (blue stained oocytes) and 322 (40.6%) were selected as BCB (colourless oocytes) for the production of SCNT embryos. In terms of the proportion of cleaved embryos recorded two days after SCNT, significant differences (P < 0.05) were found among the BCB+ (78%) and the BCB (51%) groups, whereas no significant differences in terms of the cleavage rate were recorded between the control group (73%) and the BCB+ group (78%) (Table 1). Significant differences (P < 0.05) among groups were also observed on day 8 after SCNT, when the embryos reached the blastocyst stage (Table 1). The proportion of blastocysts from the BCB+ selected COCs was significantly higher (39%) than both the control (21%) and the BCB (4%) groups. Also, the rate of blastocyst development in the control group was significantly higher than that for BCB COCs (Table 1). Embryos in the blastocyst stage from all groups had normal morphology though they appeared to be comparatively small in the BCB group. When the cell numbers in the blastocysts were recorded, it was found that all the three groups (control, BCB+ and BCB) were significantly different from each other. The highest number of blastocyst nuclei were recorded in the BCB+ group and the lowest number in the BCB group (Table 1).
4. Discussion While suboptimal culture conditions undoubtedly contribute to the poor development after IVM/IVF and SCNT, the quality of the oocytes is probably the major limiting factor [1]. While follicles in different stages of growth and atresia contribute to the heterogeneous nature of the oocytes recovered from ovaries of slaughtered animals [16], the method of COC recovery may further influence the degree of heterogeneity of the recovered oocytes. When ovaries are sliced, COCs may be recovered not only from antral follicles on the surface [10] but also from smaller antral follicles from the inside of the ovary, which may be in earlier stages of follicular development after antrum formation. High fertilization and cleavage rates were observed after IVF in all groups of bovine oocytes irrespective of their G6PDH activity [1] thereby ruling out any possible adverse influence of BCB stain. In contrast, we recorded a significant decrease in the cleavage rates of the cloned bovine embryos from BCB oocytes which might owe its cause to the rather poor developmental competence of these BCB oocytes. It has been reported that the overall nuclear maturation rate of the oocytes remains unaffected by exposure to BCB [1]. However, the proportion of oocytes reaching metaphase-II after IVM in the BCB COCs was found to be significantly lower than in the control or BCB+ COCs in case of bovines [1] and goats [6,8] which probably might be the result of the unfinished growth of these oocytes [1]. Though the biochemical basis of BCB metabolism in COCs is not fully understood, some evidence for the ability of BCB to play a role as electron acceptor and thereby become colourless during the electron flow induced by the G6PDH-catalyzed oxidation of G6P and reduction of NADP+ has been reported [1]. The present bovine SCNT experiments provided a significantly increased blastocyst development rate for the BCB+ group (39%) as compared to the control (21%) and the BCB (4%) groups (Table 1) thereby highlighting the ability of the BCB stain to be able to differentially select the developmentally competent
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
oocytes. Similar findings regarding the accuracy of BCB stain for selecting against incompetent heifer oocytes [9] had earlier been reported though with a much lower blastocyst development rate (only 12%) in the competent (BCB+) group. In addition, in our SCNT experiments, the number of nuclei in the blastocysts was found to be the highest in the BCB+ group followed by the control group, whereas blastocysts from the BCB group had a significantly lower cell number than both the BCB+ and control groups (Table 1). These findings are consistent with results of other IVF studies [1,17] reporting a higher cell number for the developmentally competent (BCB+) group. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and subsequent blastocyst development in the bovine after SCNT. The BCB stain by itself does not improve the blastocyst development rate after SCNT, rather it helps in the selection of the developmentally competent oocytes (before in vitro maturation) which when subjected to the process of SCNT lead to an improvement of the overall blastocyst rate. Our results clearly indicate the influence of oocyte quality on SCNT results. In conclusion, the classification of bovine cumulus– oocyte complexes on the basis of BCB staining (or G6PDH activity) could be effectively used to select bovine oocytes in terms of their further developmental competence, and could, thereby, positively influence the blastocyst development rate after bovine SCNT. References [1] Alm H, Torner H, Loehrke B, Viergutz T, Ghoneim IM, Kanitz W. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology 2005;63:2194–205. [2] Gordon I. Recovering the bovine oocyte. In: Laboratory production of cattle embryos (biotechnology in agriculture no. 27)2nd ed., Cambridge, UK: CAB International/Cambridge University Press; 2003. pp. 79–111. [3] Wassarman M. The mammalian ovum. In: Knobil E, Neil D, editors. The physiology of reproduction, vol. 1. NY, USA: Raven Press; 1988. p. 69–102.
345
[4] Mangia F, Epstein CJ. Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities. Dev Biol 1975;45:211–20. [5] Ericsson SA, Boice ML, Funahashi H, Day BN. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology 1993;39:214 [Abstract]. [6] Rodriguez-Gonzalez E, Lopez-Bejar M, Velilla E, Paramio MT. Selection of prepubertal goat oocytes using the brilliant cresyl blue test. Theriogenology 2002;57:1397–409. [7] Rodriguez-Gonzalez E, Lopez-Bejar M, Izquierdo D, Paramio MT. Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation. Reprod Nutr Dev 2003;43:179–87. [8] Urdaneta A, Jimenez AR, Izquierdo D, Paramio MT. Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM–IVF prepubertal goat oocytes. Zygote 2003;11:131–8. [9] Pujol M, Lopez-Bejar M, Paramio MT. Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test. Theriogenology 2004;61:735–44. [10] Alm H, Torner H, Tiemann U, Kanitz W. Influence of organochlorine pesticides on maturation and postfertilization development of bovine oocytes in vitro. Reprod Toxicol 1998;12: 559–63. [11] Tiffin GJ, Rieger D, Betteridge KJ, Yadav BR, King WA. Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development. J Reprod Fertil 1991;93:125–32. [12] Bhojwani S, Vajta G, Callesen H, Roschlau K, Kuwer A, Becker F, et al. Developmental competence of HMCTM derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells. J Reprod Dev 2005;51: 465–75. [13] Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO. Somatic cell cloning without micromanipulators. Cloning 2001;3:89–95. [14] Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P, Schmidt M, et al. Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro. Biol Reprod 2003;68:571–8. [15] Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, et al. New method for culture of zona-included or zona-free embryos: the well-of-the-wells (WOW) system. Mol Reprod Dev 2000;55: 256–64. [16] Carolan C, Monaghan P, Gallagher M, Gordon I. Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro. Theriogenology 1994;41:1061–8. [17] Lonergan P, Khatir H, Piumi F, Rieger D, Humblot P, Boland MP. Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J Reprod Fertil 1999;117:159–67.
Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer S. Bhojwani 1, H. Alm 2, H. Torner 3, W. Kanitz 4, R. Poehland * Research Unit Reproductive Biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Received 5 April 2006; received in revised form 21 August 2006; accepted 21 August 2006
Abstract The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90 min before culture. Treated oocytes were then divided into BCB (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB oocytes (4%). These results show that the staining of bovine cumulus–oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future. # 2006 Elsevier Inc. All rights reserved. Keywords: Bovine nuclear transfer; BCB; Blastocysts; G6PDH
1. Introduction * Corresponding author. Tel.: +49 38208 68761; fax: +49 38208 68752. E-mail addresses: [email protected] (S. Bhojwani), [email protected] (H. Alm), [email protected] (H. Torner), [email protected] (W. Kanitz), [email protected] (R. Poehland). 1 Tel.: +49 38208 68768; fax: +49 38208 68752. 2 Tel.: +49 38208 68754; fax: +49 38208 68752. 3 Tel.: +49 38208 68759; fax: +49 38208 68752. 4 Tel.: +49 38208 68750; fax: +49 38208 68752. 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.08.006
Somatic cell nuclear transfer (SCNT) technique has succeeded in yielding bovine blastocysts but at a rather lower (averaging 20%) and inconsistent blastocyst rate as compared to in vitro-maturation (IVM) and fertilization (IVF). A host of factors have been shown to contribute to this low level of SCNT efficiency such as donor cell type, methods of embryo culture, imprinting defects, reprogramming failures, the rather inefficient artificial methods of activation, lab to lab
342
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
variation as well as the oocyte source and quality at the beginning of maturation [1]. The follicular oocytes are commonly recovered from ovaries of slaughtered cattle (often cows with reduced reproductive performance or which are slaughtered at the end of their economic period) and are thereby heterogeneous in quality and developmental competence [2]. Immature oocytes are known to synthesize a variety of proteins [3], among them, glucose-6phosphate dehydrogenase (G6PDH). This enzyme is known to be a component of the pentose phosphate cycle which provides ribose phosphate for nucleotide synthesis and much of the NADPH utilized as a hydrogen or electron donor in reductive biosynthetic reactions such as the formation of fatty acids. The G6PDH enzyme is active in the growing oocyte [4], but has decreased activity in oocytes that have finished their growth phase [3]. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colourless [5]. Studies with prepuberal goat oocytes showed that more competent oocytes could be selected using the BCB stain [6–8]. Both the rate of maturation to metaphase-II and the rate of normal fertilization of oocytes retaining the BCB stain were higher than in those metabolising the stain and in control oocytes. Activity of G6PDH has also been found to be inversely associated with maturation and fertilization of pig oocytes [5]. There have been limited reports on the use of BCB staining in the bovine: heifer oocytes classified as BCB+ had a significantly higher rate of blastocyst development than BCB or control heifer oocytes (12.3, 1.6, and 5.2%, respectively), but this rate was still lower than that of unstained cow oocytes (30.0%; P < 0.05) [9]. In yet another study in bovine [1], BCB staining was found to be effective in enhancing the blastocyst development rate after IVF where cow oocytes classified as BCB+ had a significantly higher rate of blastocyst development than BCB or control oocytes (34, 4, and 18%, respectively). The aim of the present study was, therefore, to determine whether a BCB staining as an indicator for G6PDH activity could be used to select developmentally competent bovine oocytes before IVM and thereby increase the efficiency of blastocyst development after bovine SCNT. 2. Materials and methods Unless otherwise indicated, all plastic ware, i.e. culture vessels and dishes used in our experiments were
obtained from Nunc, Wiesbaden, Germany, while all chemicals and medium, etc. were purchased from Sigma, Deisenhofen, Germany. 2.1. Oocyte collection Oocytes were collected by the method of ovarian slicing [10] from abattoir-derived ovaries 4–6 h after the slaughter. Prior to slicing, each ovary was placed in a Petri dish and covered with Tissue culture medium TCM-199 with Earle’s salts and 25 mM HEPES (M 7528) containing 0.5 IU/ml heparin and supplemented with 10% (v/v) heat-inactivated FCS (F 7524). All follicles present on the surface of the ovary were sliced with a scalpel and the oocytes were located with a stereo microscope. The medium used for recovery was TCM-199 with Earle’s salts (see above). Only oocytes with a compact cumulus investment were selected for further culture and were designated as COCs. 2.2. Brilliant cresyl blue staining To carry out the BCB test the compact COCs were washed three times in Dulbecco’s PBS modified by the addition of 0.4% BSA (A-7888; mDPBS). Then the COCs were exposed to 26 mM of BCB (B-5388) diluted in mDPBS for 90 min at 38.5 8C in humidified air [1]. The concentration of 26 mM had earlier been found to be effective for goat oocytes [7] as it was supportive of a high rate of selected oocytes without apparent loss of viability. It was also emphasized [11] that BCB could be used effectively in the study of embryo metabolism without being lethal after exposure with 26 mM BCB. COCs of the control group were incubated directly after selection without exposure to BCB (control). Following BCB exposure, the COCs were transferred to mDPBS and washed twice. After washing, the COCs were examined under a stereomicroscope and divided into two groups according to their cytoplasm colouration: oocytes with any degree of blue colouration to the cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB) (Fig. 1). 2.3. In vitro maturation (IVM) The collected COCs were washed twice in maturation medium (TCM-199) supplemented with 20% (v/v) heat-inactivated FCS (Sigma F 7425) and 10 mg/ml FSH (Ovagen, icp, New Zealand) and then incubated in maturation medium for 21 h at 38.5 8C in 5% CO2 in air [10,12].
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
Fig. 1. Differentially stained bovine COCs after exposure to BCB stain (BCB+, blue-coloured; BCB, unstained).
2.4. Culture of nuclear donor cells Bovine fibroblasts from the sixth passage of a primary male cell line were utilised as nuclei donors. For this purpose, a cell line was established using mechanically isolated pieces of ear skin isolated from an adult bull using a method described earlier [12]. The small tissue pieces (approx. 1 mm diameter) were cultivated for 1–2 weeks in TCM-199 containing 20% FCS, 1 mM sodium pyruvate and antibiotics (mixture of a working solution of 100 IU/ml penicillin and 100 mg/ ml streptomycin). The cells growing in monolayer were cultivated up to confluence. Cells in G0/G1 phase of cell cycle were enriched by serum deprivation in culture medium (0.5% FCS, 2 days) for subsequent use in SCNT experiments [12]. 2.5. Somatic cell nuclear transfer (SCNT) For the production of nuclear transfer embryos, the zona-free method of Handmade Cloning (HMCTM) was utilised [12–14]. Approximately 100 COCs (per experiment) were vortexed for 3 min in 0.5 mg/ml hyaluronidase to remove the cumulus. Oocytes were then transferred for 8 min into 1.5 mg/ml pronase (Sigma Protease P 8811) to remove the zona pellucida. After this they were manually bisected under stereomicroscopic control with Ultra Sharp Splitting Blades (AB Technology, Pullman, WA) in TCM-199 medium containing 20% FCS, 2.5 mg/ml of cytochalasin B and 10 mg/ml fluorochrome Hoechst 33342 (Sigma B 2261). All stained demi-oocytes were arranged in drops of TCM199 (three per drop) and positions of half-oocytes without chromatin staining (cytoplasts) were registered under fluorescence microscope. Meanwhile, the somatic cells
343
were prepared by repeated washing of the monolayer with Ca++- and Mg++-free PBS and subsequent trypsination (39 8C, 5 min in 100 ml of 0.05% trypsin dissolved in PBS). The somatic cells were separated by vigorous pipetting and stored in 2 ml Eppendorf tubes in HEPES-buffered TCM-199 medium supplemented with 20% calf serum (T20) at room temperature until fusion. For the first fusion, half of the total quantity of prepared cytoplasts were individually transferred to 500 mg/ml phytohemagglutinin (PHA) for 3 s and then quickly dropped onto a single somatic cell for attachment. Following attachment, the cytoplast-somatic cell pair was transferred to a fusion chamber with 0.5 mm separation of wires (CFA 500 Kru¨ss, Hamburg, Germany). Wires were covered with 1 ml of fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2) at 26–27 8C. The electrical alignment was carried out using 15 V ac at 700 kHz and the first fusion was performed with a double dc pulse of 65 V, each pulse for 20 ms and 0.1 s apart. For the second fusion, all remaining cytoplasts and fused pairs were transferred to the fusion chamber—one fused pair was then attached to each cytoplast. A double fusion pulse with the same parameters but at 45 V dc was applied and the reconstructed embryos were then transferred into culture medium, covered with oil, and incubated for 4 h in 5% CO2 in air at 39 8C. Two-step activation was subsequently carried out—first with 2 mM Ca ionophore (A 23187, 5 min, RT) and second with 2 mM 6-dimethylaminopurine (6-DMAP, Sigma D 2629, 5% CO2 in air, 39 8C for 6 h). Embryos were then washed twice in culture medium and cultured individually in well-of-thewells (WOWs) [15] in Synthetic Oviductal Fluid (SOF) medium (Minitu¨b, Germany) supplemented with 10% oestrous cow serum and covered with mineral oil. Embryo culture was performed at 39 8C in 5% CO2, 5% O2, and 90% N2. The number of blastocysts were recorded on day 8 of culture. 2.6. Blastocyst evaluation At the end of the culture period, the blastocysts were fixed in buffered formal saline (containing 5% formaldehyde) and subsequently stained with Hoechst 33342 (Sigma B 2261). The number of nuclei in the blastocysts were then recorded under fluorescence microscope. 2.7. Statistical analysis The data were evaluated by non-parametric oneway analysis of variance (ANOVA). Because of the
344
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
Table 1 Enhancement of blastocyst rate after bovine SCNT upon selection of oocytes based on BCB staining and number of nuclei in bovine SCNT blastocysts recovered on day 8 of culture (mean S.D.) Groups
Embryos n
Cleavage day 2 p.SCNT n (percent of total embryos)
Blastocysts day 7 p.SCNT n (percent of cleaved embryos)
Number of nuclei in blastocysts (n blastocysts) ð¯x S:D:Þ
Control BCB+ BCB
148 218 149
108 (73) a 170 (78) a 76 (51) b
23 (21) a 67 (39) b 3 (4) c
102.7 6.8 a (23) 112.8 9.7 b (67) 58.3 8.1 c (3)
Within columns, values with different letters (a, b, c) differ significantly (P < 0.05).
non-normality of the data, the NPAR1WAY procedure of SAS/STAT software was used to calculate simple linear rank statistics for the Wilcoxon, median, Van der Waerden, and Savage scores to test for the differences in location. Group effect in the model was BCB+, BCB and control. Differences having P values of the test equal to or less than 0.05 (P 0.05) were considered statistically significant. 3. Results A total of 1108 bovine COCs were recovered and used for the investigation. Of these, 314 were used for the production of control embryos while 794 were subjected to BCB staining procedure out of which 472 (59.4%) were selected as BCB+ (blue stained oocytes) and 322 (40.6%) were selected as BCB (colourless oocytes) for the production of SCNT embryos. In terms of the proportion of cleaved embryos recorded two days after SCNT, significant differences (P < 0.05) were found among the BCB+ (78%) and the BCB (51%) groups, whereas no significant differences in terms of the cleavage rate were recorded between the control group (73%) and the BCB+ group (78%) (Table 1). Significant differences (P < 0.05) among groups were also observed on day 8 after SCNT, when the embryos reached the blastocyst stage (Table 1). The proportion of blastocysts from the BCB+ selected COCs was significantly higher (39%) than both the control (21%) and the BCB (4%) groups. Also, the rate of blastocyst development in the control group was significantly higher than that for BCB COCs (Table 1). Embryos in the blastocyst stage from all groups had normal morphology though they appeared to be comparatively small in the BCB group. When the cell numbers in the blastocysts were recorded, it was found that all the three groups (control, BCB+ and BCB) were significantly different from each other. The highest number of blastocyst nuclei were recorded in the BCB+ group and the lowest number in the BCB group (Table 1).
4. Discussion While suboptimal culture conditions undoubtedly contribute to the poor development after IVM/IVF and SCNT, the quality of the oocytes is probably the major limiting factor [1]. While follicles in different stages of growth and atresia contribute to the heterogeneous nature of the oocytes recovered from ovaries of slaughtered animals [16], the method of COC recovery may further influence the degree of heterogeneity of the recovered oocytes. When ovaries are sliced, COCs may be recovered not only from antral follicles on the surface [10] but also from smaller antral follicles from the inside of the ovary, which may be in earlier stages of follicular development after antrum formation. High fertilization and cleavage rates were observed after IVF in all groups of bovine oocytes irrespective of their G6PDH activity [1] thereby ruling out any possible adverse influence of BCB stain. In contrast, we recorded a significant decrease in the cleavage rates of the cloned bovine embryos from BCB oocytes which might owe its cause to the rather poor developmental competence of these BCB oocytes. It has been reported that the overall nuclear maturation rate of the oocytes remains unaffected by exposure to BCB [1]. However, the proportion of oocytes reaching metaphase-II after IVM in the BCB COCs was found to be significantly lower than in the control or BCB+ COCs in case of bovines [1] and goats [6,8] which probably might be the result of the unfinished growth of these oocytes [1]. Though the biochemical basis of BCB metabolism in COCs is not fully understood, some evidence for the ability of BCB to play a role as electron acceptor and thereby become colourless during the electron flow induced by the G6PDH-catalyzed oxidation of G6P and reduction of NADP+ has been reported [1]. The present bovine SCNT experiments provided a significantly increased blastocyst development rate for the BCB+ group (39%) as compared to the control (21%) and the BCB (4%) groups (Table 1) thereby highlighting the ability of the BCB stain to be able to differentially select the developmentally competent
S. Bhojwani et al. / Theriogenology 67 (2007) 341–345
oocytes. Similar findings regarding the accuracy of BCB stain for selecting against incompetent heifer oocytes [9] had earlier been reported though with a much lower blastocyst development rate (only 12%) in the competent (BCB+) group. In addition, in our SCNT experiments, the number of nuclei in the blastocysts was found to be the highest in the BCB+ group followed by the control group, whereas blastocysts from the BCB group had a significantly lower cell number than both the BCB+ and control groups (Table 1). These findings are consistent with results of other IVF studies [1,17] reporting a higher cell number for the developmentally competent (BCB+) group. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and subsequent blastocyst development in the bovine after SCNT. The BCB stain by itself does not improve the blastocyst development rate after SCNT, rather it helps in the selection of the developmentally competent oocytes (before in vitro maturation) which when subjected to the process of SCNT lead to an improvement of the overall blastocyst rate. Our results clearly indicate the influence of oocyte quality on SCNT results. In conclusion, the classification of bovine cumulus– oocyte complexes on the basis of BCB staining (or G6PDH activity) could be effectively used to select bovine oocytes in terms of their further developmental competence, and could, thereby, positively influence the blastocyst development rate after bovine SCNT. References [1] Alm H, Torner H, Loehrke B, Viergutz T, Ghoneim IM, Kanitz W. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology 2005;63:2194–205. [2] Gordon I. Recovering the bovine oocyte. In: Laboratory production of cattle embryos (biotechnology in agriculture no. 27)2nd ed., Cambridge, UK: CAB International/Cambridge University Press; 2003. pp. 79–111. [3] Wassarman M. The mammalian ovum. In: Knobil E, Neil D, editors. The physiology of reproduction, vol. 1. NY, USA: Raven Press; 1988. p. 69–102.
345
[4] Mangia F, Epstein CJ. Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities. Dev Biol 1975;45:211–20. [5] Ericsson SA, Boice ML, Funahashi H, Day BN. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology 1993;39:214 [Abstract]. [6] Rodriguez-Gonzalez E, Lopez-Bejar M, Velilla E, Paramio MT. Selection of prepubertal goat oocytes using the brilliant cresyl blue test. Theriogenology 2002;57:1397–409. [7] Rodriguez-Gonzalez E, Lopez-Bejar M, Izquierdo D, Paramio MT. Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation. Reprod Nutr Dev 2003;43:179–87. [8] Urdaneta A, Jimenez AR, Izquierdo D, Paramio MT. Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM–IVF prepubertal goat oocytes. Zygote 2003;11:131–8. [9] Pujol M, Lopez-Bejar M, Paramio MT. Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test. Theriogenology 2004;61:735–44. [10] Alm H, Torner H, Tiemann U, Kanitz W. Influence of organochlorine pesticides on maturation and postfertilization development of bovine oocytes in vitro. Reprod Toxicol 1998;12: 559–63. [11] Tiffin GJ, Rieger D, Betteridge KJ, Yadav BR, King WA. Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development. J Reprod Fertil 1991;93:125–32. [12] Bhojwani S, Vajta G, Callesen H, Roschlau K, Kuwer A, Becker F, et al. Developmental competence of HMCTM derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells. J Reprod Dev 2005;51: 465–75. [13] Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO. Somatic cell cloning without micromanipulators. Cloning 2001;3:89–95. [14] Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P, Schmidt M, et al. Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro. Biol Reprod 2003;68:571–8. [15] Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, et al. New method for culture of zona-included or zona-free embryos: the well-of-the-wells (WOW) system. Mol Reprod Dev 2000;55: 256–64. [16] Carolan C, Monaghan P, Gallagher M, Gordon I. Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro. Theriogenology 1994;41:1061–8. [17] Lonergan P, Khatir H, Piumi F, Rieger D, Humblot P, Boland MP. Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J Reprod Fertil 1999;117:159–67.