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The metabolic mechanisms to circannual changes in body mass of bears have yet to be elucidated. We hypothesized that the Japanese black bear (Ursus thibetanus japonicus) has a metabolic mechanism that efficiently converts carbohydrates into body fat by altering insulin sensitivity during the hyperphagic stage before hibernation. To test this hypothesis, we investigated the changes in blood biochemical values and glucose and insulin responses to intravenous glucose tolerance tests (IVGTT) during the active season (August, early and late November). Four, adult, female bears (5-17 years old) were anesthetized with 6 mg/kg TZ (tiletamine HCl and zolazepam HCl) in combination with 0.1 mg/kg acepromazine maleate. The bears were injected intravenously with glucose (0.5 g/kg of body mass), and blood samples were obtained before, at, and intermittently after glucose injection. The basal triglycerides concentration decreased significantly with increase in body mass from August to November. Basal levels of plasma glucose and serum insulin concentrations were not significantly different among groups. The results of IVGTT demonstrated the increased peripheral insulin sensitivity and glucose tolerance in early November. In contrast, peripheral insulin resistance was indicated by the exaggerated insulin response in late November. Our findings suggest that bears shift their glucose and lipid metabolism from the stage of normal activity to the hyperphagic stage in which they show lipogenic-predominant metabolism and accelerate glucose uptake by increasing the peripheral insulin sensitivity.

Feral raccoons (Procyon lotor) have been increasing in number since 1979 and are currently subject to pest control in Hokkaido. One of the reasons for the increase in numbers is thought to be the high reproductive potential of raccoons, but little is known about their reproduction. The main aim of this study was to clarify seasonal changes in spermatogenesis and peripheral testosterone concentration of raccoons in Hokkaido. In the present study, external characteristics and histology of the testis and epididymis and the plasma testosterone concentration were investigated in 68 feral, male raccoons culled for pest control and once a month in one live, captive male. The feral males exhibited seasonal changes in spermatogenesis, showing active spermatogenesis in autumn, winter and spring (October-June) with noted spermatogenesis and inactive spermatogenesis in summer (July-September) with lower mean levels of spermatozoa in the cauda epididymis. Even in the inactive period, spermatozoa were observed in about half of the individuals (14/26); therefore, individuals producing spermatozoa existed every month throughout the year. Testosterone concentrations were significantly high in the winter mating season. In the captive male, the testosterone concentrations were low from June to August, and spermatozoa could not be observed from July to September. These results suggest that raccoons exhibit seasonality of reproduction, but the time and duration of spermatogenetic decline varies widely among individuals. This individual variation in the inactive period is a feature of male raccoon reproduction and is unique among seasonally breeding mammals.

Testes of Japanese black bears (Selenarctos thibetanus japonicus) were collected from 27 bears shot by hunters and biopsied from 13 bears captured by traps. The size and weight of the testis, and the diameter of seminiferous tubules increased in 2 or 3 year-old bears. The percentage of sexually matured bears in each age group was 0% (1 year-old), 50% (2 year-old) and 100% (over 3 year-old), respectively. Therefore, puberty was assumed to be at 2 or 3 year of age. Only Sertoli and large round cells were the cell population in the seminiferous epithelium of prepuberal testis. The morphological characteristics of the large round cells were identified with that of gonocyte as reported in other species and were judged to be the stem cells in the testis of the prepuberal bear. The large cells resembling gonocyte were present in the seminiferous tubules of the puberal testis. In the present study, these cells were referred to as gonocyte-like cell. These cells increased in number during the non-breeding season. The gonocyte-like cells were morphologically similar to those of undifferentiated type A-spermatogonia. Therefore, these cells may be equivalent to undifferentiated type A-spermatogonia, that is, the stem cells in the testis of the puberal bear for resumption of spermatogenesis.

In order to develop a method for estimating the success/failure rates of reproductive processes, especially those of ovulation and neonate nurturing, in the Japanese black bear (Ursus thibetanus japonicus), we examined offspring status, corpora lutea (CLs), placental scars (PSs) and corpora albicantia (CAs) in 159 females (0-23 years old) killed as nuisances on Honshu Island of Japan during 2001-2009. PSs were found to remain in the uterus at least until November of the year of parturition. CA detectability began to decline after September of the year of parturition. Monthly and age-specific proportions of CL-present females revealed that the post-mating season starts in August, and that the age of first ovulation is 4 years. These results indicate that the success rate of ovulation (SRO: the probability that solitary/non-lactating mature females actually succeed in ovulation) can be estimated by calculating the proportion of CL-present females amongor = 4-year-old females without PSs captured from August to November; the early litter loss rate (ELLR: the probability that parenting females lose all of their cubs [0-year-old offspring] before mating season) can be estimated by calculating the proportion of CL-present females among those with PSs and CAs captured in August or later. The estimated values of SRO and ELLR were 0.93 (62/67) and 0.27 (6/22), respectively.

Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was

It was investigated in the domestic fowl, whether a mechanism for a synchronous start of the proliferation of the differentiating spermatogonia in a transversely cut seminiferous tubule (called the "co-ordination" between germ cell association) operates effectively in this species. A trapezoid-shaped cellular association demarcated by two perpendiculars from the tubular lumen to the basement membrane was the unit of observation (provisionally called a "column" Each column is composed of a bundle of elongate spermatids and the neighbouring, various types of germ cells). The extent of closeness of the co-ordination was examined in a total of 2,650 columns, based on the continuity of the spermiogenic step-number of the youngest generation of spermatids within adjacent columns. In no case did all columns in a tubular cross section show the same-step spermatids. In most cases (50.2% of 2,650 columns), the same number was kept in only 2 to 6 adjacent columns. The arrangement of different but consecutive numbers (e.g., 1-2-3 or 1-2-1) was observed in a total of 882 columns. The co-ordination mechanism, therefore, is considered to work, albeit incomplete, among a limited number of adjacent columns in this species.

Ovaries having corpora lutea and uteri of 6 female Hokkaido brown bears (Ursus arctos yesoensis), which were legally killed by hunters from October to December in Hokkaido, Japan, were examined macroscopically and histologically. Four of the 6 female bears had unimplanted embryos in the uterine lumen during the period of delayed implantation between October 19 and November 20. Ovaries of bears which had unimplanted embryos contained the corpora lutea with extensive vacuolation or numerous granules in the cytoplasm of luteal cells. These bears had well-developed uterine glands which had tall epithelial cells with vacuoles in the cytoplasm of glandular cells and coagula in the glandular lumina. It is concluded that Hokkaido brown bears exhibit obligate delayed implantation and that this period of delayed implantation continues at least until late November.

Male Japanese black bears (Ursus thibetanus japonicus) have an explicit reproductive cycle. The objective of this study was to clarify the variation of plasma testosterone, FSH, inhibin, LH levels and testicular gonadotropin receptor mRNA expression of male bears associated with their testicular activity. Notably, this study investigated peripheral FSH concentration and localization of gonadotropin receptor mRNAs for the first time in male bears. Blood and testicular tissue samples were taken from captive, mature, male Japanese black bears during testicular active, regressive and recrudescent phases. Plasma hormone concentrations were measured by immunoassays, and gonadotropin receptor mRNA expression in the testis was investigated by in situ hybridization technique and also by real-time PCR. There were significant variations in plasma testosterone and inhibin concentrations. Changes in FSH concentration preceded these hormones with a similar tendency. Hormones started to increase during denning, and achieved the highest values at the end of the recrudescent phase for FSH and in the active phase for testosterone and inhibin. These changes in hormone concentrations were accompanied by testicular growth. In situ hybridization analysis revealed that FSH and LH receptor mRNA was possibly expressed in Sertoli cells and Leydig cells, respectively, as they are in other mammals. However, neither plasma LH concentration nor testicular gonadotropin receptor mRNA expression level varied significantly among the sampling months. These results suggest that FSH, inhibin and testosterone have roles in testicular activity in male bears. This study provides important endocrine information for comprehending seasonal reproductivity in male Japanese black bears.

Serum progesterone (P) concentrations using P-EIA kit (Ovucheck, Cambridge Life Science Co., Ltd.) were examined in 8 captive and 7 free-ranging female Hokkaido brown bears (Ursus arctos yesoensis). The intra- and inter-assay coefficients of variation were 8.9%, 12.6% and 16.6%, 22.7%, respectively, based on 2 serum samples. There was a significant correlation between EIA and radioimmunoassay results based on 64 serum samples (r = 0.725; p less than 0.01). Serum P concentrations were examined in 5 pregnant, 2 solitary non-pregnant bears and a lactating non-pregnant bear in captivity. Annual changes of P levels in pregnant bears were observed as a small elevation during the mating season (May-June), a re-elevation in September-October and a sharp elevation in November-December. The sharp elevation was suspected to reflect changes when implantation occurred. Annual changes of P levels in solitary non-pregnant bears were similar to those in pregnant bears. An annual change of P levels in a lactating non-pregnant bear maintained levels under 5 ng/ml. Two of 7 free-ranging bears exhibited P levels over 1 ng/ml and the birth of cubs was confirmed in the following year in 1 of the 2 bears. P concentrations of other free-ranging bears exhibited less than 1 ng/ml, and these bears were considered to be non-pregnant. It was concluded that P-EIA kit was available for measuring P concentrations in Hokkaido brown bears.

The Japanese black bear (Ursus thibetanus japonicus) is a typical seasonal breeder that has a mating season in early summer. Spermatogenesis and testicular steroidogenesis are known to develop and regress annually; however, its molecular mechanism has not yet been investigated. In the present study, we clarified the mRNA sequence of 5 steroidogenic enzymes (P450scc, 3betaHSD, P450c17, 17betaHSD3 and P450arom) using RT-PCR and RACE methods and the localization of these gene expressions in the bear testis using an in situ hybridization technique. The amino acid sequence deduced from each mRNA sequence had high homology with the corresponding sequences of other species and possessed a motif typical of the P450 family or short chain alcohol dehydrogenase family. Expression of P450scc, 3betaHSD and P450c17 mRNA in interstitial tissue indicated that conversion from cholesterol to androstenedione occurs in Leydig cells. On the other hand, the mRNA of 17betaHSD3, which plays a central role in synthesizing testosterone, was detected not only in the interstitium but also inside the seminiferous tubules, along the basement membrane. P450arom mRNAs were distributed in the seminiferous tubules. These results suggest the possibility of testosterone and estradiol-17beta synthesis inside the seminiferous tubules in the bear testis. We expect that the results of this study will be useful for further investigation of the molecular mechanism of steroidogenic seasonality in the bear testis.

We examined morphological growth variations in skull features between the Kuril harbor seal and the spotted seal in Hokkaido, Japan. Skulls from 80 Kuril harbor seals and 41 spotted seals were collected, and we measured 29 metric and 6 non-metric cranial characteristics. Three growth classes were defined according to the postnatal developmental stage: pups (0 year), subadults (1-4 years old) and adults (more than 5 years old). We detected sexual dimorphism in Kuril harbor seal pups, subadults, and adults. Although interspecies differences were detected in each growth class, Kuril harbor seals were larger and more massive than spotted seals; this feature was already detectable in pups. We did not detect certain cranial characteristics with which to identify the two species, but it was possible to identify any unknown specimens to their species, sex, and growth class using the cranial data generated in this study. Using 6 non-metric cranial characteristics, we identified significant interspecies differences with regard to the shape of the temporozygomatic suture and the extent of the nasal-incisive suture; the shape of the temporozygomatic suture and the shape of the nares were indicators of growth class in Kuril harbor seals. Although non-metric cranial characteristics have a lower discriminating power than metric characteristics, they are easy to use in the field even by inexperienced researchers.

Testicular steroidogenesis and spermatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P450c17, 3betaHSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3betaHSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.

Japanese black bears, Ursus thibetanus japonicus, have been classified as a vulnerable species so that data on reproduction are needed to maintain and/or extend their population. They are known to have a peculiar style of reproduction, giving birth to their neonates and raising them during denning, a period of complete fasting. In this study, we investigated the metabolic rate and milk composition of mother bears raising neonates, and the changes in body weight of the neonates under captive conditions. Seven female bears kept in dens were weighed once a month, and the amount of energy they used was calculated. From birth, cubs were also weighed and their growth rate was determined. In addition, the milk composition was analyzed to investigate its characteristics. As a result, it was found that mother bears used 34% more energy than did solitary females. There was no significant difference in the energy used for nursing whether they had single or twin cubs. On the other hand, the body weight gain of single cubs was significantly higher than that of twin cubs, suggesting that the growth of the cubs was highly affected by the suppression of mother's energy consumption during the fasting period. The milk had high fat and low sugar concentrations. This indicates that mother bears used the fat accumulated prior to denning for their main energy source when raising cubs. Considering all results together, Japanese black bears showed remarkable efficiency in the use of energy for reproduction during the fasting period.

The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 +/- 0.4, 4.5 +/- 0.3, 10.4 +/- 0.7 and 69.6 +/- 3.1 mum (mean +/- SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate pH, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 +/- 0.36 ml, 7.3 +/- 0.4, 659 +/- 644 x 10(6)/ml, 214 +/- 208 x 10(6), 82.9 +/- 9.6%, 89.3 +/- 9.5% and 97.0 +/- 3.2% (mean +/- SD; n=21, in ejaculate pH n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean +/- SD%; n=23) were simply bent tail (19.9 +/- 22.6), distal droplets (13.5 +/- 11.7), proximal droplets (9.6 +/- 7.8), teratoid spermatozoa (6.7 +/- 10.7), knobbed acrosome (4.9 +/- 8.6), acrosome damage (3.7 +/- 2.8) and bent midpiece (3.7 +/- 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species.

The assay of serum leptin concentrations in the Japanese black bear (Ursus thibetanus japonicus) was tested by canine-leptin-specific sandwich enzymelinked immunosorbent assay (ELISA). A dose-response curve of the bear serum was linear and parallel to the canine leptin standard curve. In mated and unmated bears, the serum leptin concentration was stable at low levels from May to August or September and a gradual increase from September or October, followed by a remarkable increase in late November. We conclude that this method may be useful for measuring bear serum leptin concentrations and that serum leptin concentrations change annually with a peak in late November., イヌレプチン特異的酵素抗体測定法により、ニホンツキノワグマ（以下クマと略）の血清レプチン濃度測定を試みた。交尾群および非交尾群の血清レプチン濃度は、5～8 または9 月に低レベルで推移し、9 または10 月から徐々に増加し11 月下旬に顕著に上昇した。本測定法はクマ血清レプチン濃度測定に有効性が示唆され、レプチン濃度は11 月下旬にピークを有する明瞭な周年変化を示すことが明らかとなった。

To verify target organ(s) of leptin in the reproductive system of the Japanese black bear, we examined the expression of leptin receptor (Ob-R) protein in ovaries and uteri collected from July to December 2006 by immunohistochemical techniques. Eleven of 22 female Japanese black bears examined had corpora lutea (CLs) in their ovaries and were thought to have undergone or to have terminated delayed implantation in the early pregnancy stage. The CLs were classified into 3 types based on morphological features. The maximum diameters of Type 1 CLs ranged 3 to 7 mm, and the luteal cells contained numerous vacuoles in the cytoplasm, which suggests that this type of CL was functional. The maximum diameters of Type 2 CLs were approximately 7 mm, and the luteal cells contained fewer vacuoles in the cytoplasm, which suggests that this type of CL was in the early stage of regression. Finally, the maximum diameters of Type 3 CLs were approximately 1 mm, and these CLs contained collagen fibers among their luteal cells, which suggests that this type of CL had regressed. The 3 types of CLs showed different reactions to Ob-R, with positive staining in Type 1, much less positive staining in Type 2 and nearly negative staining in Type 3. In bears having CLs with functional and regressive features (Type 1 and 2 CLs), Ob-R was also immunolocalized in the developed glandular and ductal endometrial epithelium. In contrast, the Ob-R was absent in the undeveloped endometrial epithelium in bears with regressed (Type 3 CLs) or no CLs. These findings suggested that leptin directly targets CLs and the endometrium so as to develop and maintain them during the delayed implantation period in Japanese black bears.

Reproductive physiology of Hokkaido brown bears, Ursus arctos yesoensis was studied morphologically, ethologically and endocrinologically. Age of sexual maturity was confirmed to be 24 and 14 years from the histological examination of testes and ovaries in male and female bears, respectively. Sexual behavior was observed during the breeding season to obtain data such as copulation process, changes of mating number in a day and with day and estrous duration of each female. Annual changes of serum testosterone levels, testis sizes and cell constructions in seminiferous tubules exhibited that the level of breeding function (spermatogenesis) begun to increase from February when wild bears were hibernating. It was considered that implantation occurred in late Novemberearly December and then fetuses grew as sigmoid curve by examining the change of serum progesterone levels and the ultrasonographical fetus growth. Histological examinations of ovaries and uteri and flushing in uteri resulted in finding about morphology of corpora lutea, uterine glands and embryos during the delay period. It was suggested that embryo loss, abortion and/or infant death occurred from the comparison of numbers among corpora albicantia, placental scars and litters which wild mature female bears were accompanied by.

Serum sex steroid and prolactin profiles were examined in the male American black bear, Ursus americanus during denning. Sera collected in December and the following March from 8 denning male black bears in Minnesota, U.S.A. were assayed for testosterone, estradiol-17 beta and prolactin. Eight bears were confirmed to be the denning mode based on a serum urea to creatinine ratio less than 10. Serum testosterone concentrations tended to increase from December to the subsequent March whereas serum estradiol-17 beta concentrations tended to decrease during this period. There were few changes in serum prolactin concentrations between December and March. These findings suggest that spermatogenesis and testicular steroidogenesis initiated during denning may be influenced by changes in serum sex steroid concentrations in the American black bear.

A free-flying Great Egret (Ardea alba) captured in Gifu, central Japan, in May 2006 had a large mass on the right carpal joint. The tumor was diagnosed as chondrosarcoma by histopathologic examination.

The effect of anesthetizing with a 1:1 combination of tiletamine hydrochloride and zolazepam hydrochloride (TZ) was evaluated in 75 Japanese black bears. TZ was administered to 43 captive and 11 wild, 8 captives and 13 hibernating captive bears at the doses of approximately 9.0 mg/kg (usual dosage), 18.0 mg/kg (high dosage) and 5.0 mg/kg (low dosage), respectively. Sufficient anesthetic effects were achieved in all bears, and rectal temperatures, heart rates and respiratory rates did not change significantly during an hour handling. Complete blood cell examinations showed no abnormal data. A combination of TZ would be an efficient and safe drug for chemical immobilization of Japanese black bears.

We investigated the distribution of 3 types of steroidogenic enzymes, P450scc, 3betaHSD, and P450c17, in wild raccoon dog ovaries by immunohistochemistry. Six pairs of ovaries were obtained from wild raccoon dogs between 2001 and 2003, with 3 of the 6 pairs of ovaries containing corpora lutea. P450scc, 3betaHSD, and P450c17 were localized in the granulosa and theca cells of these raccoon dogs. Furthermore, lutein cells were stained positively for P450scc and 3betaHSD in the pregnant and non-pregnant raccoon dogs. These results suggest that granulosa and theca cells may synthesize progesterone and androgens, which may play an important role in follicular development, and that lutein cells are a major source of progesterone in wild raccoon dogs.

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits during the breeding season in the wild male Japanese black bear. Histological observations of testes were performed and seminiferous tubule diameters were measured. The sections of the testes were immunostained by the avidin- biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/ activin beta(A), and inhibin/activin beta(B) during the breeding season. Serum concentrations of immunoreactive (ir-)inhibin, testosterone, and luteinizing hormone (LH) were measured by radioimmunoassay. Higher values of seminiferous tubule diameters and all types of spermatogenic cells including mature-phase spermatozoa were found during the breeding season. There were seasonal changes in serum concentrations of ir-inhibin, testosterone, and LH. Ir-inhibin was positively correlated with testosterone, and LH. In addition, immunoreactivity of inhibin alpha, beta(A), and beta(B) subunits were also detected in Sertoli and Leydig cells during the breeding season. These results suggest that Japanese black bear testes may secrete bioactive inhibins during the breeding season and that the circulating inhibin may be a useful indicator of the testicular function in wild male Japanese black bears.

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Gottingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.

The present study determined the contamination levels and congener-specific accumulation features of dioxins and related compounds (DRCs) in wild terrestrial mammals such as large Japanese field mice (LJFM), lesser Japanese moles (LJMs), and raccoon dogs (RDs) collected from Kanto region in Japan during 2001. The toxic equivalent quantity (TEQ) levels in the carcasses or adipose tissues were in the order of RDs > or = LJMs > LJFM. Comparison of DRC congener profiles in the three species and principal component analysis (PCA) demonstrated a higher contribution of OCDD, T4CB77, and P5CB118 in LJMs. Analysis of liver-adipose distribution of DRC congeners in RDs showed that livers contained significantly higher TEQs than adipose tissues, indicating that liver is a depository organ and critical for determining the toxicokinetics of DRCs. As for most T4, P5, H6CDD/DFs and for P5CB126, H6CB169 and mono-ortho PCB congeners, their liver/adipose concentration ratios in RDs revealed a tendency to increase with hepatic TEQ levels, suggesting TEQ-dependent hepatic sequestration.

Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17 degrees C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 microM A23187 (Ca2+/A23187) at 37 degrees C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.

Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by (1)H-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following: Neu5Ac(alpha2-3)Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]Gal(beta1-4)Glc. Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-6)Gal(beta1-4)Glc.

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.

Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.

It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.

The seasonal changes of the cytoskeletal protein expressions were immunohistochemically investigated in the testes of Japanese black bear, Ursus thibetanus japonicus. A strong immunoreaction for alpha-smooth muscle actin is restricted to the vascular smooth muscle cells and the peritubular cells which surround the seminiferous tubules by several layers throughout the year. Weak immunoreactions for B4 antigen and desmin were observed in the vascular smooth muscle cells and in a part of peritubular cells throughout the year. A strong immunoreaction for vimentin was also detected in the fibroblasts and Leydig cells, in addition to the vascular smooth muscle and epithelial cells and the peritubular cells throughout the year. A strong alpha-tubulin immunoreaction was detected in the elongating spermatids during the acrosome phase of spermiogenesis in May and June. The cytoplasm of several Sertoli cells was faintly immunoreacted for vimentin in the basal and lateral region, while an intense alpha-tubulin reaction was seen in the entire cytoplasm in May, April and June. In November, January and March, the immunoreactions for vimentin and alpha-tubulin strongly accumulate in a perinuclear region of Sertoli cells when developmental spermatids are not seen in the seminiferous tubules. These accumulations in the immunoreactions for vimentin and alpha-tubulin seem to be caused by the reduction in size of Sertoli cells cytoplasm with season. However, the seasonal changes of distributions in the cytoskeletal proteins are obscure in the bear testes. These results suggest that the contents of cytoskeletal proteins may not change in relation to the morphological differences with season in the testes of the seasonal breeders.

Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c 17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c 17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.

Spermatogenic cycle in the testis of the japanese black bear (Selenarctos thibetanus japonicus) was studied by light and transmission electron microscopy. By light microscopy, spermatids were allocated into eleven steps based on morphological changes in the nucleus and the acrosome of spermatids. Cellular associations of the seminiferous epithelium were allocated into eight stages based on the changes in the nucleus and acrosome of spermatids, appearance of meiotic figures and time of spermiation. Cross-sections of the seminiferous tubule seldom contained more than one type of stage. Spermatids at steps 1-2 had the well-developed Golgi complex. The crescent-shaped Golgi complex was accompanied by the acrosome extending over the nucleus at steps 3-5. At step 6, spermatids faced the base, and the outer membrane of the acrosome converged upon the plasma membrane of spermatids. The acrosome projected into the cytoplasm of Sertoli cells at step 9. At step 11, most of the cytoplasm was phagocytosed by Sertoli cells, and spermatids were released in the lumen to become spermatozoa.

Corpora lutea of the sika deer (Cervus nippon, Cervidae) were histologically examined in order to elucidate their retrograding processes after parturition. They showed a remarkable regression within one month as luteal cells decreased in number and, by nine months after parturition, turned into masses of blood vessels containing many arterioles with a diameter of 30 microns or less. In retrograde corpora lutea (RCL), older than 1.5 years after parturition, arterioles of these dimensions decreased markedly, while those measuring 50 microns or more became dominant. These findings indicate that an examination of RCL can distinguish females less than nine months after parturition from those more than 1.5 years after parturition. Based on the findings of RCL from the ovaries of a 10.5-year-old female, it is suggested that RCL remain in the ovaries for at least 8.5 years after parturition. It was, however, difficult to distinguish RCL of pregnancy from retrograde accessory corpora lutea (RACL). In order to arrive at a complete count of RCL, the ovaries should be examined at thicknesses of 0.5 mm. To check the presence/absence of RCL of pregnancy within nine months or less after parturition, sectioning at thicknesses of 2 mm is sufficient.

Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig 125I-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4-14.8 ng ml-1; t = 15-30 min, 18.4-28.7 ng ml-1). The sensitivity of the assay was 0.08 ng per tube. Intra- and interassay coefficients of variation were 5.5% (n = 6) and 5.7% (n = 6), respectively. Serum concentrations of prolactin changed seasonally, with the lowest concentrations in December (mean +/- SD = 1.1 +/- 0.1 ng ml-1); this was followed by a gradual increase between January (2.6 +/- 0.6 ng ml-1) and April (6.4 +/- 1.2 ng ml-1) and the highest concentrations in May (17.6 +/- 4.7 ng ml-1), preceding peak testosterone concentrations in June. The observation that prolactin secretion increased with increasing daylength suggests that photoperiod may be an external regulator. The presence of high concentrations of prolactin before peak testosterone concentrations suggests that prolactin may play a role in regulating seasonal changes in the testes.

Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.