Daijiro Kumagai

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.

Enhancement of aromatase gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

The relationships among expression of cytochrome p450 aromatase (p450arom) mRNA in the mediobasal hypothalamus (MBH), ovarian aromatase activity, and estrogen secretion were examined throughout the estrous cycle in beagle bitches. Using polymerase chain reaction (PCR) analysis we were able to detect p450arom gene transcripts in the canine MBH. The level of hypothalamic p450arom mRNA increased during the progression of anestrus and declined thereafter. Ovarian p450arom activity, as measured by a (3)H2O assay, were low in anestrus, increased in proestrus, and declined thereafter. Ovarian p450arom activity and plasma estradiol-17beta levels were positively correlated (r=0.77, P<0.05). These results suggest that enhancement of hypothalamic p450arom gene expression is associated with termination of anestrus.

Immunohistochemical and morphometrical studies on myelin breakdown in the demyelination (dmy) mutant rat

The demyelination (dmy) rat is a unique mutant exhibiting severe myelin breakdown in the central nervous system (CNS). In this study, we conducted immunohistochemical and morphometrical investigations in the dmy rat. From around 6 weeks of age, the affected rats developed ataxia especially in the hindlimbs. Afterwards, ataxia worsened rapidly, resulting in complete paralysis of the hindlimbs and recumbency. Histopathology at 7 to 10 weeks of age revealed myelin destruction throughout the white matter of the CNS in the dmy rats. The most severely affected lesions were distributed in the corpus callosum, capsula interna, striatum, subcortical white matter, cerebellar peduncle, and ventral and lateral parts of the spinal cord. Immunohistochemistry demonstrated prominent astrogliosis and many ED-1 positive macrophages in the myelin-destructed areas. Until the 4th week, no significant differences in myelin thickness and fiber diameter were found between dmy and control rats. However, from 5 weeks of age, myelin thickness of residual myelinated fibers in dmy rats became significantly less than that in controls. These data indicated that the dmy phenotype shows a prolonged period of myelin destruction, suggesting that dmy mutation affects the adequate maintenance of myelin.

Immunophenotypical changes of neoplastic cells and tumor-associated macrophages in a rat dendritic cell sarcoma-derived transplantable tumor line (KB-D8)

Abstract. Basically, dendritic cell-derived sarcomas are characterized by expression of major histocompatibility complex class-II molecules, but the biological properties of the tumor cells remain to be elucidated. Recently, we established a novel transplantable cell line (KB-D8) from a dendritic cell sarcoma found in an F344 rat. In the present study, we investigated immunophenotypical changes of KB-D8 tumor cells and tumor-associated macrophages (TAMs) appearing in relation to tumor development in syngeneic F344 rats. A number of neoplastic cells in 0.5-cm-diameter KB-D8 tumors showed immunoreactions to OX6 (specific for rat antigen-presenting cells), ED1 (for rat exudate macrophages), and ED2 (for rat resident macrophages), and 72% and 11% of the OX6+ cells were double-immunostained with ED1 and ED2, respectively. Interestingly, the immunoreactions to these antibodies were gradually reduced with increasing size of KB-D8 tumors of 1-, 2-, and 3-cm diameter. These findings indicated that immunophenotypes of dendritic cell-derived sarcomas may be changeable depending on microenvironmental conditions in vivo. Many TAMs seen outside KB-D8 tumors reacted to OX6, ED1, and ED2; the numbers of TAMs immunopositive for these antibodies also decreased as the tumor grew. Similarly, the earlier temporary increase and subsequent gradual decrease in ED2+ and OX6+ cell numbers were observed in the spleen and liver of KB-D8-bearing rats. The reverse-transcription polymerase chain reaction showed mRNA expressions of granulocyte/macrophage-colony stimulating factor, monocyte-chemoattractant protein-1, and osteopontin in KB-D8 tumor tissues. Although the functional roles (biphasic roles: suppressing or promoting) of these factors should be investigated further in relation to tumor development, the factors might be partially responsible for the TAM reactions. KB-D8 would be a useful experimental model to investigate the biological characteristics of dendritic cell sarcomas and tumor immunology in the host.

Induction of fertile oestrus in bitches by an intranasal spray of gonadotrophin- releasing hormone agonist

BITCHES are monoestrous, and ovulate only once or twice a year at fiveto 12-month intervals. A reliable method to induce ovulation and control the timing of pregnancy would aid the clinical management of prolonged anoestrus or infertility. Several protocols using various gonadotrophins have been used in attempts to induce oestrus and ovulation in anoestrous bitches (Renton and others 1984, Nakao and others 1985, England and Allen 1991). These protocols were not very effective, and would require further improvement to enable practical application. Fertile oestrus in anoestrous dogs has been induced successfully by the pulsatile administration of gonadotrophin-releasing hormone (GnRH) via an indwelling infusion catheter (Vanderlip and others 1987). However, many pet owners are reluctant to subject their animals to this procedure. In 1998, the authors presented an alternative approach to the pulsatile infusion of GnRH, using a depot-injectable form of the potent GnRH agonist leuprolide acetate (Inaba and others 1998). In human beings, the intranasal administration of a GnRH agonist is used as a painless, simple and practical method for ovarian stimulation in in vitro fertilisation (Elgendy and others 1998), central precocious puberty (Sizonenko and others 1990) and endometriosis (Franssen and others 1989). This short communication describes a study to determine whether an intranasal spray of leuprolide acetate can induce fertile oestrus in mature, anoestrous bitches, and to investigate the effect of a combination of leuprolide acetate administered intranasally and oestrogen administered orally. Twenty laboratory-bred, anoestrous beagle bitches, two to six years of age, were housed and studied in accordance with National Institutes of Health guidelines, the regulations of the local Institutional Animal Care and Use Committee, and with accepted veterinary medical practice. They were fed a commercial dog food once daily and were given water ad libitum. The time of the most recent oestrus of each bitch was known. The bitches were divided into three treatment groups: the first group, of seven bitches, was treated daily with 3·6 μg of an intranasal spray of a sustained-release formulation of leuprolide acetate (Leupron Depot; Takeda Chemical Industries). The second group, also of seven bitches, received 0·3 mg/kg estradiol-17β orally, once daily for three days, and then a daily intranasal spray of leuprolide acetate as described above. Six untreated bitches were studied as controls. The treatment with intranasal leuprolide acetate was continued either until the onset of oestrus or for up to a maximum of 14 days. The bitches were observed daily for vulval swelling and oestrous behaviour, and a vaginal smear was taken every day. The interoestrous interval was calculated as the interval between the first day of each standing oestrus. The bitches were allowed to breed on the first day of oestrus and undergo pregnancy and parturition; the length of gestation was calculated from the day of mating. Blood samples were collected daily by jugular venepuncture from the start of the treatment until the end of oestrus, in order to detect ovulation: ovulation was considered to have occurred if the level of progesterone in the plasma was more than 2 ng/ml (Wanke and others 1997). The concentration of progesterone in the plasma was measured by radioimmunoassay as described by Inaba and others (1998). The sensitivity of the assay was 10 pg per tube, and the intra-assay variation was less than 11·5 per cent. All the samples were assayed in duplicate in the same assay. The data were evaluated by analysis of variance, followed by Fisher’s protected least significant difference post hoc analysis, using the Stat View computer program (Abacus Concepts). The significance level was set at P<0·05. Behavioural signs of oestrus were induced in three of the bitches treated with leuprolide acetate alone and five of the bitches treated with leuprolide acetate and oestrogen (Table 1). These three leuprolide acetate-treated bitches showed a strong pro-oestrous and oestrous response, including normal oestrous behaviour, and accepted natural mating. Their plasma progesterone concentrations rose above 2 ng/ml during an apparently normal oestrus, and all three became pregnant and delivered normal litters. In one bitch, the vulval swelling and discharge were slight and lasted only for two to three days. The five bitches treated successfully with the combination of estradiol-17β and leuprolide acetate showed evidence of oestrus, became pregnant and delivered normal litters. All the bitches in which oestrus was not induced returned to normal oestrus within three months. The interoestrous interval of the bitches in which oestrus was induced was significantly shorter than that of the control dogs (Table 1). The litter sizes did not differ significantly between bitches that underwent induced or spontaneous oestrus (Table 1). No sign of fetal resorption or abortion was observed. The length of pro-oestrus, oestrus and gestation did not differ significantly between the groups. To the authors’ knowledge, this is the first report of the successful induction of oestrus in bitches by treatment with an intranasal spray of a GnRH agonist. One bitch appeared to respond weakly to the spray treatment, and did not allow the male to mount or show an increase in plasma progesterone above 2 ng/ml. It was assumed that the GnRH agonist stimulation was not sufficient to induce follicular growth and ovulation in this bitch. Veterinary Record (2006) 158, 378-379

Establishment of a Transplantable Rat Pulmonary Carcinoma-Derived Cell Line (IP-B12) as a New Model of Humoral Hypercalcemia of Malignancy and Bone Metastasis

A cloned cell line (IP-B12) derived from a transplantable rat pulmonary carcinoma (IP), of which neoplastic cells produce parathyroid hormone-related protein (PTHrP), was established. Tumors induced in syngeneic F344 rats by intraperitoneal injection of IP-B12 cells had features of pulmonary adenocarcinomas, consisting of neoplastic cells immunopositive to PTHrP. The IP-B12 tumor-bearing rats developed severe emaciation and hypercalcemia, with a marked elevation of plasma PTHrP level; there was an increase in osteoclastic areas of the femur and calcium depositions in systemic organs, indicating progression to humoral hypercalcemia of malignancy (HHM) in the tumor-bearing rats. In addition, the injection of IP-B12 cells into the left cardiac ventricle of syngeneic rats resulted in osteolytic skeletal metastases in the long bones and vertebrae. In the metastatic lesions, histologically, neoplastic cells showed an immunopositive reaction to PTHrP, and a prominent osteoclastic activity was seen; bone lesions, including osteolysis, fracture, and nerve compression as well as replacement of bone marrow cells by proliferated tumor cells were similar to those reported in human cancer patients with bone metastases. IP-B12 is a new animal model for HHM and osteolytic bone metastases, and will become a useful tool for studies on the pathogenesis and therapeutic strategies for such conditions.