Geon A Kim

Generation of transgenic dogs that conditionally express green fluorescent protein

We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet‐on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP‐positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies. genesis 49:472–478, 2011.

Melatonin regulates lipid metabolism in porcine oocytes

It is being increasingly recognized that the processes of lipogenesis and lipolysis are important for providing an essential energy source during oocyte maturation and embryo development. Recent studies demonstrated that melatonin has a role in lipid metabolism regulation, including lipogenesis, lipolysis, and mitochondrial biogenesis. In this study, we attempted to investigate the effects of melatonin on lipid metabolism during porcine oocyte in vitro maturation. Melatonin treatment significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related to lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI‐58, HSL, and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria, and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid β‐oxidation (CPT1a, CPT1b, CPT2, and ACADS) and mitochondrial biogenesis (PGC‐1α, TFAM, and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria, and ATP. In addition, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, β‐oxidation, and mitochondrial biogenesis‐related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development.

Unilateral cryptorchidism induces morphological changes of testes and hyperplasia of Sertoli cells in a dog

Cryptorchidism is one of the most common genital defects in dogs. This study investigated the effects of abdominal cryptorchidism on morphology, cell proliferation, and Sertoli cell condition in a dog with spontaneous unilateral cryptorchidism. Elective orchidectomy was performed on the abdominal right testis and the scrotal left testis. Significant reductions in numbers of spermatogonia, spermatocytes, and spermatids were observed in hematoxylin and eosin stained sections of the cryptorchid testis. The size of the epididymal duct was smaller than that of the control testis. Based on Ki67 immunohistochemistry, the proliferative activity of spermatogonia and spermatocytes was significantly decreased in the cryptorchid testis. However, proliferative activity was increased in the epididymal duct. Based on GATA-4 immunohistochemistry, Sertoli cells were relatively resistant to cryptorchidism, and the proliferative activity of Sertoli cells was markedly increased in the cryptorchid testis than in the control testis. These results suggest that spontaneous unilateral cryptorchidism causes morphological defects in spermatogonia and spermatocytes in the testis and changes the size of the efferent ductule of the epididymis. In addition, spontaneous unilateral cryptorchidism increases proliferative activity of Sertoli cells, which may be a predisposing factor for Sertoli cell cancer in cryptorchid testes.

Production of offspring from cloned transgenic RFP female dogs and stable generational transmission of the RFP gene

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild‐type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild‐type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations. genesis 49:835–840, 2011.

Effect of culture medium type on canine adipose-derived mesenchymal stem cells and developmental competence of interspecies cloned embryos

Canine adipose-derived mesenchymal stem cells (ASCs) are promising as donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that different cell cultures possess different capacities to support pre-implantation development of SCNT embryos. The aim of this study is to investigate whether two culture medium (RCMEP, Dulbeccos modified Eagles medium [DMEM]) affect gene expression of ASCs, subsequent development of interspecies SCNT (iSCNT) and gene expression of cloned embryos. The RCMEP-cultured cells contained significantly greater amounts of SOX2, NANOG, OCT4, DNMT1, and MeCP2 than DMEM-cultured cells (P < 0.05). In iSCNT, the use of DMEM medium for culturing cells resulted in similar development to the blastocyst stage than those derived from RCMEP cultured cells (4.5% and 3.2%, respectively; P > 0.05). The expression of all transcripts except for DNMT1 in cloned blastocysts from RCMEP cultured cells followed those of cloned blastocysts derived from DMEM cultured cells. The alteration of gene expression in ASCs by culture medium was not manifested in the iSCNT embryos derived from these cells. Although the culture medium can induce changes of gene expression by ASCs, such alterations in donor cells did not affect the developmental competence or gene expression patterns of iSCNT embryos.

Employing mated females as recipients for transfer of cloned dog embryos

It has been suggested that co-transferring parthenogenetic embryos could improve the pregnancy success rate with cloned embryos in mammals. As an alternative to co-transferring parthenotes, in dogs we employed recipient females that possessed in vivo-fertilised embryos as a result of mating to determine whether mated bitches could be suitable recipients for cloned embryos. The effect of using mated recipients on implantation and pregnancy rates of canine somatic cell nuclear transfer embryos was also determined. Cloned embryos were transferred into the oviducts of naturally synchronous females that had mated with male dogs before ovulation. The pregnancy rate appeared to be similar between mated recipients (50%) and non-mated recipients (28.57%; P>0.05). However, the delivery rate of cloned pups was significantly higher in mated recipients than non-mated recipients (10.53 vs 2.38%; P<0.05). A decrease in progesterone levels in the mated recipients before the due date induced natural delivery. However, cloned pups in non-mated recipients were delivered by Caesarean section because the fall in progesterone concentration in these females did not occur until the due date. The present study demonstrated for the first time that mated female dogs can be used as recipients for cloned embryos.

Effects of mineral supplements on ovulation and maturation of dog oocytes

The aim of this study was to assess the effects of trace mineral supplements near the time of ovulation on the number of ovulated oocytes, in vivo oocyte maturation and pregnancy for dog cloning. Sixteen oocyte donor dogs were used in each control and mineral supplement group, and 136 and 166 corpora lutea were counted from each group. No significant difference was observed between oocyte recovery rates in the control (91.2 ± 2.7%) and mineral (89.9 ± 2.7) groups. Proportions of mature (86.2 ± 7.2 and 88.4 ± 6.8%) and aged (13.8 ± 7.2 and 11.6 ± 6.8%) oocytes were not different in the control and mineral groups, respectively. Oocytes with fair (91.5 ± 3.6 and 93.6 ± 2.1%) and poor (8.5 ± 3.6 and 6.4 ± 2.1%) quality also showed no difference between the control and mineral groups. The concentrations of manganese and ferrous iron were higher and lower on the day of ovulation, respectively, in both groups, but trace element concentrations in peripheral blood were not affected by mineral treatment. Oocytes were used to make cloned embryos; after embryo transfer, four and two pups were delivered from the control and mineral group, respectively, but there was no difference in the delivery rate (4.6 and 2.7%). In conclusion, intravenous mineral supplements administered once close to the LH surge in oocyte donor dogs and recipients had no effect on the number of ovulated oocytes, in vivo oocyte maturation or pregnancy in dog cloning in this study.

Lanosterol influences cytoplasmic maturation of pig oocytes in vitro and improves preimplantation development of cloned embryos

Lanosterol is a precursor of meiosis-activating sterols in the cholesterol biosynthetic pathway and induces a physiological signal that instructs the oocyte to reinitiate meiosis. In this study, we examined the effect of lanosterol on IVM of porcine oocytes, specifically on nuclear maturation, cytoplasmic maturation by investigating intracellular glutathione (GSH) levels and lipid content, embryonic development after parthenogenetic activation and somatic cell nuclear transfer (SCNT), and on gene expression in cumulus cells, oocytes, and SCNT-derived blastocysts. There was no significant difference in nuclear maturation rates between the control and treatment groups (10, 50, and 100 μM of lanosterol added to IVM culture medium). Supplementation with 50-μM lanosterol significantly increased lipid content and GSH levels and decreased reactive oxygen species levels compared with the control. In addition, oocytes treated with 50 μM of lanosterol exhibited significantly increased blastocyst formation rates and total cell numbers after parthenogenetic activation (30.3% and 63.9 vs. 21.6% and 36.5, respectively) and SCNT (18.2% and 53.7 vs. 12.6% and 37.5, respectively), when compared with the control group. Cumulus cells treated with 50 μM of lanosterol showed significantly increased 14α-demethylase, Δ14-reductase, and Δ7-reductase mRNA transcript levels. Significantly increased PPARγ, SREBF1, GPX1, and Bcl-2 and decreased Bax transcript levels were observed in mature oocytes treated with 50 μM of lanosterol compared with the control. SCNT blastocysts derived from 50-μM lanosterol-treated oocytes had significantly higher POU5F1, FGFR2, and Bcl-2 transcript levels than control SCNT-derived blastocysts. In conclusion, supplementation with 50 μM of lanosterol during IVM improves preimplantation development of SCNT embryos by elevating lipid content of oocytes, increasing GSH levels, decreasing reactive oxygen species levels, and regulating genes related to the cholesterol biosynthetic pathway in cumulus cells, to lipid metabolism and apoptosis in oocytes, and their developmental potential and apoptosis in blastocysts.

Altering histone acetylation status in donor cells with suberoylanilide hydroxamic acid does not affect dog cloning efficiency

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs, and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on dog cloning efficiency. Relative messenger RNA expressions of the bax1/bcl2 ratio and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 hours) were compared. Treatment with 1 μM for 20 hours showed significantly lower bax1/bcl2 and Dnmt1 transcript abundance. Acetylation of H3K9 was significantly increased after SAHA treatment, but H4K5, H4K8 and H4K16 were not changed. After SCNT using control or donor cells treated with SAHA, a total of 76 and 64 cloned embryos were transferred to seven and five recipients, respectively. Three fetuses were diagnosed in both control and SAHA-treated groups by ultrasonography 29 days after the embryo transfer, but there was no significant difference in the pregnancy rate (4.2% vs. 4.3%). In conclusion, although SAHA treatment as used in this study significantly decreased bax1/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 acetylation, it was not enough to increase in vivo developmental competence of cloned dog embryos.

Duration of gestation in pregnant dogs carrying cloned fetuses

The aim of this study was to investigate gestation duration and the physiologic characteristics of pregnant dogs bearing cloned fetuses, especially in the prepartum period. A retrospective study was performed to compare gestation duration in females pregnant with cloned (somatic cell nuclear transfer) fetuses (cloned group) with those bearing noncloned fetuses (control group), and effects of litter size, birth weight, and breed of somatic cell donors on gestation duration in the cloned group were evaluated. Clinical delivery onset signs associated with serum progesterone concentration and rectal temperature were also compared in both groups. The gestation duration calculated from day of ovulation was significantly longer in the cloned (62.8 ± 0.3 days) versus the control group (60.9 ± 0.5 days; P < 0.001). There was a negative correlation between litter size and gestation duration including both groups (r = -0.59; P < 0.01), but there were no differences between birth weights or breed of cell donors and gestation duration in the cloned group. Even though the basal rectal temperature in the prepartum period was not different between control and cloned groups (36.9 ± 0.1 °C and 37.2 ± 0.1 °C, respectively), serum progesterone concentration on delivery day was significantly higher in the cloned group (2.2 ± 0.4 ng/ml) compared with the control group (0.5 ± 0.1 ng/ml; P < 0.05). The longer gestation duration of pregnant dogs bearing cloned fetuses might be because of the smaller litter size in this group. Also, the weaker drop in serum progesterone levels in the prepartum period in cloned dog pregnancies indicates that the parturition signaling process might be altered resulting in longer gestation periods.