Gautam Kumar Deb

Effect of cytoplasmic lipid content on in vitro developmental efficiency of bovine IVP embryos

The objective was to evaluate the effect of cytoplasmic lipid content on the embryonic developmental efficiency of bovine in vitro embryo production (IVP) embryos. Ovaries from Korean native cows (Bos taurus coreanae) were collected from a local abattoir, and cumulus-oocyte complexes (COCs) were recovered from follicles 2 to 8mm in diameter. The oocytes were divided into three groups, dependent on their cytoplasm color: pale color (PC), brown color (BC), and dark color (DC). The COCs were fertilized using frozen-thawed semen from a single Hanwoo bull. Based on measurement of the cytoplasmic color intensity of oocytes after 22h of in vitro maturation (IVM), the DC group had lower (P<0.05) color intensity than that in the BC and PC groups (56.3+/-2.7, 93.3+/-5.1, and 123.9+/-12.0, respectively). Based on MitoTracker Green FM staining, the number of mitochondria in the DC (170.1+/-31.2) group was significantly higher than that in the BC (137.5+/-30.8) and PC (105.5+/-25.3) groups. The cleavage rate in the DC (81.5%) group was also higher than that in the PC (50.4%) group (P<0.05), as was the development rate to blastocyst stage (18.9% vs. 9.8%). Finally, cell numbers of blastocysts in the DC (150.8+/-28.0) group were higher (P<0.05) than that in the BC (107.6+/-17.8) and PC (80.5+/-12.3) groups. In conclusion, cytoplasm color was a useful selection parameter for abattoir-derived oocytes destined for IVP.

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.

9- retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.

Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts

The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).

Proteomic analysis of placentas from cloned cat embryos identifies a set of differentially expressed proteins related to oxidative damage, senescence and apoptosis†

Production of cloned mammals by somatic cell nuclear transfer is associated with functional and structural abnormalities of placentation and with abnormal fetal development. A proteomic analysis was performed in domestic cats (Felis catus) to compare cloned term placentas (CTP) obtained from cesarean section (CS) to control placentas obtained from CS or vaginal delivery. The expression of 20 proteins was altered in CTP (p<0.05) compared to control placentas. The two control groups showed that the method of delivery, vaginal delivery or CS, did not affect protein expression (p>0.05). A total of 13 proteins were up‐regulated in CTP, including apoptosis‐related cathepsin D (CD), annexin A1 and heat shock protein 27 (HSP 27), and seven proteins were down‐regulated in CTP, including prohibitin (PHB). The expression of PHB and CD was confirmed by Western blotting and immunofluorescence staining. The abnormal expression of PHB and CD correlated with the generation of reactive oxygen species, leading to decreased mitochondrial membrane potential and telomeric DNA, which are associated with cellular senescence and apoptosis. In summary, a specific pattern of abnormal protein expression is associated with the impaired development and functions of cloned placentas and hence with decreased fetal viability. Strategies aimed at restoring normal placental protein expression may increase the efficiency of somatic cell nuclear transfer and transgenic cat production and help restore endangered species.

Effect of equine chorionic gonadotropin on the efficiency of superovulation induction for in vivo and in vitro embryo production in the cat

The effects of various dosages of equine chorionic gonadotropin (eCG) on superovulation induction for in vivo and in vitro embryo production were examined in stray cats (Felis catus). Cats (n=286) were allocated into five treatment groups with 0, 50, 100, 200, or 400 IU eCG, followed by 100 IU human chorionic gonadotropin (hCG). In vivo- and in vitro-produced blastocysts were obtained by artificial insemination (AI) and in vitro fertilization (IVF), somatic cell nucleus transfer (SCNT), or parthenogenetic activation (PA). The percentage of cats that developed mature follicles, the percentage of cats with collected embryos, and the mean number of in vivo blastocysts per cat were higher in the 200 IU treatment group (43.9%, 31.8%, and 1.53, respectively) compared with those of the other groups (P<0.05). The percentage of follicular developed cats, the percentage of cumulus-expanded oocytes, and the mean number of collected cumulus-oocyte complexes per cat in the 200 IU (56.7%, 67.8%, and 26.2, respectively) and 400 IU (53.3%, 64.2%, and 26.7, respectively) groups were higher than those in the other groups (P<0.05). Furthermore, the percentage of in vitro-produced blastocyst per cleaved embryos and the average cell number of the blastocysts from IVF (52.7% and 125.8, respectively) was higher than those of the blastocysts from PA (30.1% and 85.2) and higher than those of the blastocysts from SCNT (15.3% and 37.5; P<0.05). In conclusion, the current study demonstrated that in vivo and in vitro embryo production were affected by the dosage of eCG; the best results were obtained with 200 IU.

Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos

BackgroundThe in vitro culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality.MethodsCumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured in vitro, fertilized in vitro with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse in vitro embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR.ResultsThe coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (CD9, 0.4-fold; AKRAB1, 0.3-fold) and in cocultured zygotes (CD9, 0.3-fold; AKRAB1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (PGSH2, 5.0-fold; TXN, 4.3-fold; PLAU, 1.7-fold) and cocultured zygotes (PGSH2, 14.5-fold; TXN, 3.2-fold; PLAU, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However, the expression of PLAC8, TGF-β1, ODC1, ATP5A1 and CASP3 did not differ between the three culture groups.ConclusionsResults show that the agar chip-embedded helper embryo coculture system enhances developmental competence and embryo quality in cultures of limited numbers of high pedigree single cow OPU presumed zygotes.

9-cis Retinoic acid inhibits cumulus cell apoptosis during the maturation of bovine cumulus-oocyte-complexes1

Cumulus cell (CC) apoptosis is inversely correlated with embryonic development in vitro. Therefore, inhibition of CC apoptosis is important for proper embryonic development and quality. Retinoic acids (all-transRA and 9-cisRA) are natural components of retinoids, and 9-cisRA is the physiologically active metabolite of retinoic acid in vitro. During in vitro maturation, 9-cisRA enhances oocyte competence through multiple mechanisms affecting the oocyte and preimplantation embryo; however, the effect of 9-cisRA on CC apoptosis has yet to be elucidated. The aim of the present study was to evaluate the effect of 9-cisRA on CC apoptosis and to identify the molecular mechanism underlying that effect. Bovine slaughterhouse cumulus-oocyte complexes (COC) were matured in vitro in the absence or presence of 5 nM 9-cisRA. Cumulus cells were collected from immature and matured COC for the detection of apoptosis and gene expression analysis. Results showed that 9-cisRA reduced the number of apoptotic CC by about 2.7 fold (P < 0.023), compared with control. However, apoptosis is rare in CC of immature COC (0.01% ± 0.001). Transcripts involved in the caspase cascade were down-regulated upon exposure to 9-cisRA, including tumor necrosis factor alpha (TNF-α, 11.1 fold, P < 0.001), tumor necrosis factor alpha receptor 1 (TNFR1, 2.3 fold, P < 0.01), caspase 9 (CASP9, 2.0 fold, P < 0.031), caspase 8 (CASP8, 2.2 fold, P < 0.012), and caspase 3 (CASP3, 2.1 fold, P < 0.006), while antiapoptotic B-cell lymphoma 2 (BCL2) transcript was increased (3.1 fold, P < 0.004), compared with control. In addition, 9-cisRA inhibited mitogen activated protein kinase mRNA expression in CC, including extracellular signal-regulated kinase 1/2 (ERK1, 2.7 fold, P < 0.02; ERK2, 2.7 fold, P < 0.03), and c-Jun N-terminal kinase (JNK, 1.6 fold, P < 0.044), as well as the activator protein-1 (AP1) family members c-jun (1.6 fold, P < 0.041) and c-fos (2.0 fold, P < 0.06). The transcript abundances of TNF-α, TNFR1, CASP9, CASP8, CASP3, ERK1, ERK1, JNK, and BCL2 were increased, while c-fos and c-jun mRNA expression was decreased in the matured CC. On the basis of the data, we suggest that 9-cisRA inhibits CC apoptosis during in vitro maturation of bovine COC.

Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos

It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.