Young Kwang Jo

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.

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.

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.

Cloning of the short-tailed Gyeongju Donggyeong dog via SCNT: conserving phenotypic inheritance

Somatic cell nuclear transfer is a useful tool to maintain genetic information of animals. The Gyeongju Donggyeong dog is a breed registered as natural monument in Korea. The unique feature of the Donggyeong dog is its tail, as the Donggyeong dog can be classified as either short tailed or tailless. The aim of this study was to preserve the Donggyeong dog’s unique feature by cloning. Fibroblasts were obtained from a short-tailed Donggyeong dog. In vivo matured oocytes were enucleated, microinjected with a donor cell and fused electrically. Reconstructed embryos were transferred to six recipient dogs. One surrogate became pregnant, and one short-tailed Donggyeong dog was delivered. This study demonstrated that the phenotype of the Donggyeong dog could be conserved by somatic cell nuclear transfer.

Differential expression of estrogen receptor α and progesterone receptor in the normal and cryptorchid testis of a dog

Descending of the testes is an important process for spermatogenesis and cryptorchidism is one of the most relevant genital defects in dogs. In a previous study, we observed abnormal morphology and proliferation of Sertoli cells in a cryptorchid testis. In the present study, we investigated the expression of estrogen and progesterone receptors in the normal and cryptorchid testis of a dog. Elective orchidectomy was performed on the dogs abdominal right testis (undescended, cryptorchid) and scrotal left testis (descended, normal). In the normal testis, estrogen receptor α immunoreactivity was detected in Leydig cells alone, while estrogen receptor α immunoreactivity in the cryptorchid testis was significantly prominent in the Sertoli cells as well. In addition, progesterone receptor immunoreactivity in the control testis was detected in the spermatids, but was not detected in the cryptorchid testis. This result suggests that unilateral cryptorchidism causes increases of estrogen receptor α expression in Sertoli cells.

Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective agents

Cryopreservation procedures cause osmotic stress to spermatozoa following cryoinjury and reduce their content of osmolytes. Conventional method for cryoprotectant loading and dilution on canine semen freezing which could be categorized in single step protocol, makes decreasing in sperm performance such as motility, morphology and viability. Therefore, the objective of the present study was to determine if a multistep protocol using glycerol or ethylene glycol can be used to overcome the osmotic sensitivity of canine spermatozoa, and to identify osmolytes that were involved in regulation of osmotic stress. A multistep protocol, comprising serial loading and dilution of cryoprotective agents by dividing the total volume of extender into 4 steps (14%, 19%, 27%, and 40%) every 30s, was compared to a single step method. Frozen-thawed spermatozoa in the multistep group showed superior quality (P<0.05) compared with the single step process in progressive motility (23.3 ± 1.3% vs. 12.5 ± 1.6%), intact membranes (66.5 ± 2.8% vs. 49.5 ± 2.6%) and bent tail (29.2 ± 3.2% vs. 46.2 ± 1.9%). Multistep also succeeded in minimizing loss of the osmolytes carnitine (20.6 ± 2.0 nmol/U G6PDH vs. 10.8 ± 2.1 nmol/U G6PDH) and glutamate (18.4 ± 1.6 nmol/U G6PDH vs. 14.4 ± 0.8 nmol/U G6PDH) compared to the single step group. Moreover, glycerol with multistep was more advantageous for maintaining sperm quality than ethylene glycol. In conclusion, the multistep protocol with glycerol can be used to improve the morphology, motility and osmolytes content of frozen-thawed canine spermatozoa.

Immunohistochemical localization of glucose transporter 1 and 3 in the scrotal and abdominal testes of a dog

Glucose is essential for testicular function; the uptake of carbohydrate-derived glucose by cells is mediated by glucose transporters (GLUTs). In the present study, we investigated the activity of GLUT1 and GLUT3, the two main isoforms of GLUTs found in testes, in the left scrotal and right abdominal testes of a German Shepherd dog. Immunohistochemical analysis showed that GLUT1 immunoreactivity was absent in the scrotal and abdominal testes. In contrast, weak to moderate GLUT3 immunoreactivity was observed in mature spermatocytes as well as spermatids in the scrotal testis. In the abdominal testis, relatively strong GLUT3 immunoreactivity was detected in Leydig cells only and was absent in mature spermatocytes and spermatids. GLUT3 immunoreactivity was significantly decreased in the tubular region of abdominal testis and significantly increased in the extra-tubular (interstitial) region of abdominal testis compared to observations in the each region of scrotal testis, respectively. These results suggest that GLUT3 is the major glucose transporter in the testes and that abdominal testes may increase the uptake of glucose into interstitial areas, leading to an increased risk of developing cancer.

Ectopic liver and gallbladder in a cloned dog: Possible nonheritable anomaly

Ectopic liver and gallbladder are rare anomalies usually not accompanied by any symptoms and are found during surgical exploration or autopsy. We aimed to find a cause of this anomaly using somatic cell nuclear transfer (SCNT) technology, which can produce genetically identical organisms. A cloned beagle having ectopic organs was produced and died on the day of birth. Major and ectopic organs were fixed and underwent histologic analysis. SCNT was performed using cells derived from the dead puppy to produce reclones. Normality of internal organs in the original donor dog and recloned dogs was evaluated by computed tomography. While a liver without the gallbladder was located in the abdominal cavity of the cloned dog, a well-defined, reddish brown mass with a small sac was also positioned outside of the thoracic cavity. Histologically, they presented as normal liver and gallbladder. Five reclones were produced, and computed tomography results revealed that the original donor dog and reclones had normal liver and gallbladder structure and location. This is the first report of both ectopic liver and gallbladder in an organism and investigation on the etiology of these abnormalities. Normal organ structure and position in the original donor dog and reclones suggests that the ectopic liver and gallbladder is a possible nonheritable anomaly.

Survival of skin graft between transgenic cloned dogs and non-transgenic cloned dogs.

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues.