Effect of co-culture canine cumulus and oviduct cells with porcine oocytes during maturation and subsequent embryo development of parthenotes in vitro.

In the estrus stage, canine oocytes are surrounded by cumulus cells and undergo maturation in the oviduct for 2-3 days after ovulation. We hypothesized that canine oviduct cells (cOC) and canine cumulus cells (cCC) during this stage might affect the maturation of oocytes and thereby improve subsequent embryo development. Therefore, the objective of this study was to compare the effects of a cOC and cCC co-culture on oocyte in vitro maturation (IVM) and subsequent embryo development, and to analyze the gene expressions in a molecular fashion what co-culture actually gives the specific pathways in which the co-culture cells act to improve maturation and embryo development. The effect of co-culture using cOC and cCC on porcine oocyte IVM was investigated. Thereafter, oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to oocyte maturation, embryo development and apoptosis were analyzed. Also, reactive oxygen species (ROS) levels after IVM was analyzed. The IVM rate and embryo development including cleavage, blastocyst formation rates, and total blastocyst cell numbers from cOC group were significantly higher than other groups (P < 0.05). The expression of SMAD2/3 and growth differentiation factor 9 (GDF9) was significantly increased in cOC and oocytes from the cOC group compared with other groups. Moreover, the levels of GDF9, prostaglandin-endoperoxide synthase 2 (PTGS2), WNT3A and matrix metalloproteinase 2 (MMP2) were significantly up-regulated in blastocysts from the cOC group. The concentration of ROS was significantly lower in the supernatant of cOC groups compared with other groups. Also, the expression of BCL2 was significantly increased in porcine cumulus cells and oocytes from cOC group. The present study demonstrated that co-culture with cOC improved in vitro porcine oocyte maturation and subsequent embryo development competence. Also, co-culture with cOC during IVM induces a suitable environment for oocyte maturation by enhancing the mRNA level of SMAD2/3 and GDF9, and for embryo development by elevating the expression level of PTGS2, WNT3A and MMP2. In addition, the decreased ROS level in cOC co-culture could have a beneficial influence on oocyte maturation.

Timing of fertile period for successful pregnancy in American Bully dogs.

Determination of the timing of the estrus cycle is essential for fertile mating. There are physiological variations among breeds, between bitches, and between cycles of the same bitch. If serial monitoring and many tools are applied, the exact moment of ovulation could be pin-pointed. However, it leads to time and costly difficulties. Progesterone concentrations during estrus cycles follow a specific pattern, and it is largely used in timing of fertile period. Although it has similar pattern in general, it is likely that breed-specific differences exist. The aim of this study was to investigate the way of timing the fertile period for successful pregnancy in American Bully dogs based on vaginal cytology and progesterone assay with minimized cost. To identify the empirical relations among reproductive characteristics, we performed statistical analyses on data from proestrus-to-estrus 27 American Bully dogs referred for 7 months. We found the significant correlations between the cyclic changes of vaginal cytology and progesterone assay. The relationship of serum progesterone concentrations with the days from vaginal discharge onset was analyzed through linear regression assay. In conclusion, we addressed two standards in the timing of fertile period with a minimal number of progesterone assays in the breeding management of American Bully dogs.

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.

Coincidence of Persistent Müllerian duct syndrome and testicular tumors in dogs.

BACKGROUND: Persistent Müllerian duct syndrome (PMDS), a rare form of male pseudohermaphroditism in dogs, is an abnormal sexual phenotype in males that is characterized by the existence of a hypoplastic oviduct, uterus, and cranial part of the vagina. Dogs suffering from PMDS are often accompanied by cryptorchidism. To date, it has been mainly found in the Miniature Schnauzer breed. CASE PRESENTATION: In this report, two cases of PMDS with a malignant testicular tumor originating from cryptorchidism in breeds other than the Miniature Schnauzer breed are described. The patients were a seven-year-old male Maltese dog and a 17-year-old male mixed-breed dog weighing 3.8 kg. They also exhibited an enlarged prostate with or without abscess and an elevated serum estradiol level and were surgically treated to remove the testicular tumor and Müllerian duct derivatives. CONCLUSIONS: It is recommended that PMDS should be differentially diagnosed by ultrasonography and that orchiectomy be performed at an early age in patients suspected to have cryptorchidism to prevent the ectopic testes from becoming tumorous.

Oocyte maturation-related gene expression in the canine oviduct, cumulus cells, and oocytes and effect of co-culture with oviduct cells on in vitro maturation of oocytes.

PURPOSE: In contrast to most other mammals, canine oocytes are ovulated in an immature state and undergo oocyte maturation within the oviduct during the estrus stage. The aim of the study was to investigate whether oviduct cells from the estrus stage affect the maturation of oocytes and show gene expression patterns related to oocyte maturation. METHODS: We analyzed MAPK1/3, SMAD2/3, and BMP6/15 expression in oviduct cells, cumulus cells, and oocytes from anestrus, estrus, and diestrus stages. Next, we investigated the effect of co-culture with oviduct cells derived from the estrus stage upon in vitro maturation (IVM) of canine oocytes. RESULTS: There was significantly higher MAPK1/3 (1.42 ± 0.02 and 2.23 ± 0.06), SMAD2/3 (0.77 ± 0.03 and 2.39 ± 0.07), and BMP15 (2.21 ± 0.16) expression in oviduct cells at the estrus stage (P < 0.05). In cumulus cells, MAPK1 (1.26 ± 0.07), SMAD2/3 (0.82 ± 0.01, 1.04 ± 0.01), and BMP6 (13.09 ± 0.11) expression was significantly higher in the estrus stage (P < 0.05). In oocytes, significant upregulation of MAPK1/3 (14,960 ± 3121 and 1668 ± 253.4), SMAD3 (774.6 ± 79.62), and BMP6 (8500 ± 895.4) expression was found in the estrus stage (P < 0.05). After 72 h of IVM culture, a significantly higher maturation rate was observed in oocytes co-cultured with oviduct cells (10.0 ± 1.5%) than in the control group (3.2 ± 1.4%). CONCLUSIONS: We demonstrate that oviduct cells at the estrus stage highly expressed MAPK1/3, SMAD2/3, and BMP15. Furthermore, canine oviduct cells from the estrus stage enhance the culture environment for canine oocyte maturation.

Establishment of Transgenic Porcine Fibroblasts Expressing a Human klotho Gene and Its Effects on Gene Expression and Preimplantation Development of Cloned Embryos.

Even though the functions of the klotho gene in aging of small animals such as mice have been well investigated, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. Therefore, the objective of this study was to generate porcine cell lines overexpressing human klotho (hKlotho) and tetracycline (Tet)-inducible hKlotho and to produce cloned embryos from these cell lines. We designed vectors for hKlotho overexpression (CA-Klotho) under control of CMV enhancer/chicken ß-actin (CAG) promoter and Tet-inducible hKlotho overexpression (Tet-Klotho, under control of doxycycline-dependent promoter). The vectors were transfected into porcine fibroblasts then CA-Klotho and Tet-Klotho cell lines were established. The Tet-Klotho (+) cell line was cultured in the presence of doxycycline (2 µg/mL), whereas the Tet-Klotho (-) cell line was cultured without doxycycline. In polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) assays, integration and expression of the hKlotho gene were confirmed in CA-Klotho, Tet-Klotho (+), and Tet-Klotho (-) cell lines. The CA-Klotho cell line was subjected to real-time PCR and showed positively changed expression of genes related to aging and cell survival. Somatic cell nuclear transfer was performed to generate hKlotho overexpression cloned embryos by using CA-Klotho and Tet-Klotho (+) cell lines; blastocyst formation frequency was significantly higher in cloned embryos from CA-Klotho and Tet-Klotho (+) (21.5% and 20.2%, respectively) compared with the control (8.4%). In conclusion, we established hKlotho overexpression and Tet-inducible hKlotho overexpression cell lines and porcine embryos cloned from these cell lines.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation.

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.

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 dog's 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.

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.

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.

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.

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.

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.

Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P < 0.05). In vivo developmental potential of cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P < 0.05). The culture medium can induce changes in gene expression of donor cells and telomerase activity, and these alterations can also affect in vivo developmental competence of the cloned embryos.

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.

Reduced birth weight, cleft palate and preputial abnormalities in a cloned dog.

The aim of the present study was to report a novel developmental abnormality in a cloned dog. A fibroblast cell line was established from an 8-year-old male German shepherd dog. In vivo matured oocytes were retrieved from a large breed dog, and the nucleus was removed from each oocyte. A donor cell was injected into an enucleated oocyte, and the oocyte-cell couplet was fused electrically. After chemical activation, the resulting embryos were transferred into a naturally estrus-synchronized recipient dog, and two cloned pups were delivered by Cesarean section 60 days later. One cloned pup (Clone 1) was healthy, but the other (Clone 2) had a birth weight of only 320 g and cleft palate, failure of preputial closure at the ventral distal part, and persistent penile frenulum. Clone 2 was raised by stomach feeding until Day 40 after birth, where palatoplasty was performed. The abnormalities in external genitalia in Clone 2 resulted in persistent penile extrusion that was surgically corrected. This complex developmental abnormality has not been reported in dogs previously.

Behavioral analysis of cloned puppies derived from an elite drug-detection dog.

Since the first cloned dog "Snuppy" was born, many cloned dogs have been produced by somatic cell nuclear transfer (SCNT) technology. We reported the production of seven cloned drug detection dogs (named "Toppies") in 2009. Although their genetic identity was confirmed, similarities in behavior and the drug-detecting ability were not examined. Therefore, this study is the first attempt to examine their behavior. We conducted the Campbell test which is commonly used to evaluate the tendency of dominance. Data were analyzed by the general linear mixed model. The scores among seven cloned puppies and four naturally-bred controls were significantly different (P < 0.0001). After the test, cloned and control puppies were trained according to the Korea Customs Detector Dog Training Center's manual. The selection rate for detector dog in the cloned puppies was higher (86 %) than that of naturally-bred dogs (30 %). Therefore, it can be concluded that drug detection dogs with high performance can be propagated more efficiently using SCNT.

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.

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.