Shingo Hatoya

Expression of estrogen receptor α and β genes in the mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle

Estrogen receptor α (ERα) and ERβ mRNA levels were measured in the mediobasal hypothalamus, the anterior pituitary and the ovary of beagle bitches at various stages of the estrous cycle. With polymerase chain reaction analysis we detected ERβ gene transcripts in all tissue samples. The levels of hypothalamic and pituitary ERα and β mRNAs increased from mid anestrus to proestrus and declined thereafter. In the ovary, ERα mRNA levels increased from proestrus to diestrus and were positively correlated with plasma progesterone levels (r=0.62, P<0.01), whereas ERβ mRNA levels increased from mid anestrus to proestrus and were positively correlated with plasma estradiol-17β levels (r=0.73, P<0.001). These results suggest that the rise in hypothalamic and pituitary ERα and β mRNAs is associated with termination of anestrus, and that increases in ovarian ERα and β mRNAs may be involved in initiating development of the follicle or corpora lutea.

Evaluation of transplantation of autologous bone marrow stromal cells into the cerebrospinal fluid for treatment of chronic spinal cord injury in dogs.

OBJECTIVE To evaluate effects of transplantation of bone marrow stromal cells (BMSCs) into the CSF for the treatment of chronic spinal cord injury in dogs that had not responded by 1 month after decompressive surgery. ANIMALS 23 dogs. PROCEDURES Dogs with paraplegia and loss of nociception in the pelvic limbs for at least 1 month after decompressive surgery were assigned to transplantation or control groups. Dogs in the transplantation group received BMSCs injected into the CSF 1 to 3 months after decompressive surgery. Dogs in the control group did not receive additional treatments. Improvements in gait, proprioceptive positioning, and nociception were evaluated by use of the Texas Spinal Cord Injury Scale for ≥ 6 months after BMSC transplantation. RESULTS 6 of 10 dogs in the transplantation group regained the ability to walk, whereas only 2 of 13 dogs in the control group regained the ability to walk. Scores for the Texas Spinal Cord Injury Scale in the transplantation group were significantly higher than scores in the control group at the endpoint of the study (6 months after BMSC transplantation or after decompressive surgery for the transplantation and control groups, respectively). Only 1 dog (transplantation group) recovered nociception. All dogs from both groups had fecal and urinary incontinence. No complications were observed in relation to BMSC transplantation. CONCLUSIONS AND CLINICAL RELEVANCE Injection of BMSCs into the CSF caused no complications and could have beneficial effects on pelvic limb locomotion in dogs with chronic spinal cord injuries.

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway.

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0-100 ng/ml) for 18 h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100 ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100 ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100 ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100 ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18 h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

Safety of Autologous Bone Marrow Stromal Cell Transplantation in Dogs with Acute Spinal Cord Injury

OBJECTIVE To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). STUDY DESIGN An open-label single-arm trial. ANIMALS Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. METHODS Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dogs femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. RESULTS Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. CONCLUSIONS Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.

Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells

Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

IFNγ Markedly Cooperates with Intratumoral Dendritic Cell Vaccine in Dog Tumor Models

Dendritic cell (DC)-based immunotherapy can trigger effective immune responses against cancer in human patients. Although accompanied by little toxicity, further improvements are needed to optimize immune responses for fully satisfactory clinical outcomes. IFNγ, a potent inducer of T helper type 1 immune responses, is considered an important tool to realize improvements. In this study, we sought to clarify the effect of IFNγ on the maturation and activation of DCs and the clinical outcome of DC-based cancer therapy in dogs. In vitro experiments indicated that IFNγ significantly enhanced the expression of immune stimulatory molecules and interleukin-12 by DCs derived from canine monocytes. IFNγ also significantly strengthened DC-mediated growth suppression against tumor cell lines. DC inoculation with concomitant delivery of IFNγ into primary or recurrent tumors elicited significant clinical responses, including four complete responses and two partial responses against malignant tumors, also eliciting partial responses against benign but actively growing tumors. Together, our results indicate that combining IFNγ and DCs could induce strong immune responses against tumors, significantly improving clinical outcomes. The present study of dogs bearing common types of cancer in humans offers a unique line of support for the development of human cancer therapies.

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra.

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-alpha mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P<0.05). In bitches with pyometra, endometrial TGF-alpha and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P<0.05). In normal bitches, positive immunohistochemical staining for TGF-alpha, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-alpha and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-alpha by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: Changes with age and effects of cryptorchidism

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.

Evaluation of Serum Phosphorylated Neurofilament Subunit NF‐H as a Prognostic Biomarker in Dogs With Thoracolumbar Intervertebral Disc Herniation

OBJECTIVE To investigate whether pNF-H is a prognostic biomarker of spinal cord injury (SCI) in paraplegic dogs with thoracolumbar intervertebral disc herniation (IVDH). STUDY DESIGN Prospective, case-control clinical study ANIMALS Dogs (n = 60) with SCI from IVDH and 6 healthy dogs. METHODS Serum from 60 thoracolumbar IVDH dogs (Grade 4: 22 dogs; Grade 5: 38 dogs) collected 1-3 days after injury, and 6 control dogs, was analyzed using enzyme-linked immunosorbent assay (ELISA) against a phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H). Serum pNF-H levels were compared between different IVDH grades and their prognostic value was investigated. RESULTS pNF-H levels were significantly greater in Grade 5 than Grade 4 dogs. There were significant differences in pNF-H levels between dogs that regained voluntarily ambulation and those that did not. All 8 dogs that had high pNF-H levels 1-3 days after injury did not regain the ability to walk after surgery. CONCLUSIONS Serum pNF-H levels might be a biomarker for predicting prognosis of canine SCI.