The estrous cycle of captive woodchucks (Marmota monax)

Changes in vaginal cytology were assessed and correlated with temporal changes in circulating concentrations of progesterone (P) and estradiol-17 beta (E2) during the breeding season (February to March) in a seasonally breeding rodent, the woodchuck (Marmota monax). Ten individually caged adult females, maintained under laboratory conditions for 3-11 mo, were studied. Vaginal smears were taken each morning for 2 consecutive months beginning 1 February 1990. Seven of 10 females exhibited readily identifiable estrus, characterized by a clear predominance (83%) of cornified cells. The earliest estrous smear was recorded on 3 February and the latest on 12 March. These animals were monoestrous and remained in a prolonged estrous period during their brief breeding season. The average duration of estrus was 18.1 +/- 2.1 days, ranging from 12-27 days. Levels of P and E2 were determined in serum samples taken before, during, and after estrus from 7 females who exhibited estrus. No changes in the circulating levels of P were apparent during the estrous cycle. However, there was a consistent pattern of estradiol secretion characterized by elevated levels of E2 before and during estrus, followed by a significant (p less than 0.05) decline in E2 levels one week after the end of estrus. Elevated levels of E2 preceded and coincided with maximal degree of vaginal cornification. Thus, the termination, but not the onset, of estrus in woodchucks reflected closely the temporal pattern of changes in serum E2 levels during the breeding season.     

Diurnal and seasonal profiles of melatonin in the liver and their correlates of ovarian functions under natural photo-thermal conditions in adult carp Catla catla

Present study aimed to demonstrate daily rhythm features of hepatic melatonin concentrations in relation to ovarian functions during four reproductive phases of an annual cycle by measuring the levels of melatonin, 17-β estradiol (E2), vitellogenin (Vg) and maturation inducing hormone (MIH) in the liver and/or serum of adult carp Catla catla. Melatonin titres in liver, irrespective of reproductive phase, underwent daily variations with a peak in early dark phase and nadir at midday. However, the acrophase (Ø) of serum melatonin varied from late night in preparatory phase to midnight in the remaining parts of annual cycle. Their amplitude was highest during post-spawning phase and lowest during spawning phase. Hepatic E2 levels showed daily peak at midday and seasonal peak during pre-spawning phase. Though levels of serum Vg proteins and MIH did not exhibit daily variations, underwent seasonal changes with the highest and lowest values during spawning and post-spawning phases respectively. Hepatic melatonin titres always displayed significant negative correlation with the levels of both E2 and Vg. In essence, our study presented the first data on the daily and seasonal rhythm features of hepatic melatonin in carp and underlined their temporal relationship with the functions of ovary in any fish species.     

Reinitiation of spermatogenesis by exogenous gonadotropins in a seasonal breeder, the woodchuck (Marmota monax), during gonadal inactivity.

The present study was undertaken (1) to document structural and functional changes in the testes of seasonally breeding woodchuck during active and inactive states of spermatogenesis and (2) to evaluate the ability of exogenous gonadotropins to reinitiate spermatogenesis outside the breeding season. During seasonal gonadal inactivity, there were significant (P less than 0.05) reductions in volumes of several testicular features (testis, seminiferous tubules, tubular lumen, interstitial tissue, individual Leydig cells, Leydig cell nuclei, and Leydig cell cytoplasm) as compared with gonadally active animals. The diameter of the seminiferous tubules was decreased by 26%, and Leydig cell numbers also declined in the regressed testes. These changes were accompanied by a decline in testosterone (T) levels in both plasma and testis, and reduction in epithelial height of accessory reproductive organs. A hormonal regimen was developed that would reinitiate spermatogenesis in captive, sexually quiescent woodchucks. A combination of PMSG and hCG markedly stimulated testicular growth and function and restored spermatogenesis qualitatively. Quantitatively normal spermatogenesis was restored in 2 of 6 treated males. Morphometric analyses revealed substantial increases in seminiferous tubular diameter and in the volume of seminiferous tubules, tubular lumen, total Leydig cells, and individual Leydig cells in the hormone-treated animals. These increased values corresponded to 99, 75, 68, 51, and 200%, respectively, of the values measured in naturally active woodchucks. Leydig cell numbers, however, remained unchanged and approximated only 31% of the number found in naturally active testes. Hormonal stimulation also resulted in a significant rise in serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating spermatogenesis was treatment with 12.5 IU of PMSG twice a week for 4 weeks followed by 12.5 IU of PMSG + 25 IU of hCG twice a week for 4 weeks.     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.     

Circannual changes in serum testosterone concentrations of adult and yearling woodchucks (Marmota monax)

Testicular volumes and serum testosterone concentrations were determined biweekly in 5 adult and 4 yearling woodchucks maintained indoors from December through August. Food and water were provided ad libitum except for 2 mo beginning at the winter solstice when feeding and lighting (12L:12D) supplemental to available natural light were discontinued. Temperatures fluctuated with outdoor temperatures greater than 4 degrees C. No significant hibernation occurred. Testes in adults were small in December (0.3-1.8 cm3), largest in February and March (3.5-5.6 cm3), and smallest in late June (0.1-0.5 cm3). Testosterone was basal in December (less than 0.6 ng/ml), maximal (3.4-6.6 ng/ml) between early January and late March, and minimal from April through August (less than 0.8 ng/ml). In yearlings, maximum testes volumes (1.6 cm3) and serum testosterone (0.9 +/- 0.2 ng/ml) were less, and occurred later, than in adults. Testosterone levels and testis volumes measured in newly captured woodchucks in March and April and again 2-3 mo later were generally similar to those of their laboratory counterparts. Thus, in woodchucks: annual cycles of testosterone production and of testes recrudescence and regression parallel each other with maxima during the short, late-winter breeding season; those cycles are not altered significantly by the absence of hibernation or the present conditions of captivity; and yearling males apparently are not an important part of the breeding population.     

Further observations on estrus and ovulation in woodchucks (Marmota monax) in captivity.

The woodchuck is a seasonally breeding sciurid rodent. Female woodchucks are monoestrous and, when isolated from males, remain in a prolonged period of estrus characterized by a clear predominance of cornified cells in the vaginal smear. This study was designed to characterize relationships between the degree of vaginal cornification and sexual receptivity, and to study ovulation and related phenomena of this species in captivity. Fourteen individually caged adult females, maintained under standard laboratory conditions for 9-23 mo, were used in this investigation. Females exhibiting predominantly (67-97%) cornified smears were always receptive, regardless of the time interval from the onset of estrus, and mated within 24 h of pairing. Mated females allowed to complete pregnancy gave birth to live pups 30-32 days later. Litter size ranged from 3-7 pups. Serum progesterone (P) levels increased to approximately 2 ng/ml during the first week of pregnancy and greater than 5 ng/ml during the second and third weeks of pregnancy. Serum estradiol (E2) levels were elevated during the first week of pregnancy and began to decline thereafter. Examination of ovarian serial sections revealed that ovulation took place between 20 and 32 h after copulation. Serum levels increased significantly (4-fold) after ovulation (1.2 +/- 0.3 vs. 0.3 +/- 0.01 ng/ml). However, the circulating levels of E2 remained unchanged between the periods before (53 +/- 1 pg/ml) and after ovulation (60 +/- 3 pg/ml). Ovulation was not simultaneous in all mature follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory assessment of hepatic injury in the woodchuck (Marmota monax)

Normal reference values for total serum protein, albumin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), gamma glutamyl transferase (GGT), alkaline phosphatase (AP), and total bilirubin were established in 48 clinically healthy woodchucks. To validate the use of these biochemical tests in the woodchuck for assessment of liver injury, carbon tetrachloride was administered to produce hepatocellular necrosis and the common bile duct was surgically occluded to produce cholestasis. Biochemical tests were performed prior to experimental treatment and thereafter in surviving woodchucks for a period of 6 weeks. There were marked increases in the serum activities of AST, ALT, and SDH following carbon tetrachloride administration and all 3 enzymes appeared to be useful markers of acute hepatocellular injury. The predominate biochemical abnormalities in woodchucks with bile duct obstruction were hyperbilirubinemia, hypercholesterolemia and increased serum AP and GGT activities. The increase of GGT occurred earlier following bile duct obstruction and the magnitude of increase was greater than that of AP, suggesting that GGT would be the preferred serum enzyme test in the woodchuck for assessment of cholestatic liver injury.     

中国石龙子繁殖季节血浆性类固醇激素水平的变化

本文研究了中国石龙子繁殖季节中性腺变化与血浆性类固醇激素水平波动间的相互关系。结果表明：雌性中国石龙子卵黄生成需要雌二醇(17β-estradiol,E2)的刺激。体现在卵巢重量于4月底至5月中旬间达峰值,血浆E2水平于3月底至最高值,此后显著回落,并于排卵前再次回升;血浆孕酮(progesterone,P)的生理功能在于输卵管卵的维持、促进胚胎发育和卵壳的形成。从3月中旬至4月底间,血浆P水平在基础值附近波动。5月中旬,随动物的排卵,血浆P水平迅速上升,至该月底达最高值,于产卵后迅速下降;E2随P的上升而下降,两者呈反向变动。雄性血浆睾酮(testosterone,T)水平与生精作用、维持攻击、求偶及交配行为密切相关。血浆T水平于动物出眠后呈现显著的上升趋势,并于4月中旬达峰值。5月中旬后,随睾丸的萎缩,血浆T水平也缓慢下降,至6月底达最低值。     

Seasonal changes in serum leptin, food intake, and body weight in photoentrained woodchucks

Male woodchucks (Marmota monax) were maintained in northern vs. southern hemisphere photoperiods, provided feed and water ad libitum, and evaluated every 2 wk for 23 mo for body weight, absolute and relative food intake, body temperature, serum testosterone, and serum concentrations of leptin measured using an anti-mouse leptin enzyme-linked immunoassay. During late spring and summer, body weight increased 56 +/- 4% above winter nadirs, and during the autumn and early winter weights decreased 27 to 43% below midsummer maxima. Serum leptin initially increased during increases in body weight, in the late spring, reached peak values (490 +/- 32 pg/ml) in summer during the initial decline in body weight, and later decreased along with body weight to reach basal values (20 +/- 5 pg/ml) in late winter. Spontaneous declines in food intakes in summer began 2-6 wk before resulting declines in body weight and occurred during increases in leptin >100 pg/ml. The rate of decline in food intakes was greatest when serum leptin was at or near peak values. Food intake increased in late winter when leptin was low and 7-10 wk before resulting increases in body weight. Testis recrudescence occurred when leptin was declining to near basal levels. The results suggest that leptin is involved in the hormonal regulation of the circannual cycle in the drive for voluntary food intake in this species.     

Seasonal changes in angiotensin converting enzyme activity in male and female frogs (Rana esculenta)

Gonad, lung, kidney and serum angiotensin converting enzyme (ACE) activities were determined by specific substrate hydrolysis in male and female Rana esculenta over 1 year. Ovary ACE activity showed the highest values among the different tissues, with a significant peak (223+/-52 nmol min(-1) mg protein(-1)) in late winter-early spring. Testis ACE activity followed a significant seasonal cycle, increasing from September to peak in April (2.5+/-0.8 nmol min(-1) mg protein(-1)) and then decreased in the post-reproductive period. Lung and kidney ACE activities were not correlated with the annual reproductive cycle phases. In serum a peak of activity was present in the post-reproductive period both in male and female frogs. The present data show a correlation between ACE and the annual reproductive cycle of R. esculenta.     

Persistent free-running circannual reproductive cycles during prolonged exposure to a constant 12L:12D photoperiod in laboratory woodchucks (Marmota monax).

Serum levels of gonadal steroid were assayed at approximately 3-month intervals in groups of 5 to 8 male or female woodchucks which were exposed to a natural photoperiod for 1 year as yearlings or 3 years as adults (Study 1), or a constant photoperiod of 12L:12D from birth for 4.5 years (Study 2). After 4.5 years of 12L:12D, food intake was measured in November and compared with that in natural photoperiod animals (Study 3). Other groups of 11 males and 3 females were housed in 12L:12D for 2.5 years after capture at 2 months of age, and gonadal structure and serum steroid levels in November were compared with those of animals at selected times in the normal annual cycle (Study 4). All animals were provided food and water ad libitum and were not induced to hibernate. In Study 1, normal circannual breeding season elevations in testosterone in males and in progesterone in females were detected in most animals maintained in natural photoperiod. In Study 2, similar cycles persisted for 4.5 years in animals exposed to 12L:12D. However, based on quarterly blood samples, obvious asynchrony relative to natural light animals appeared to develop after 2, 3, or 4 years, with apparent free-running intervals of about 10 to 11 months. In Study 3, mean daily food consumption in late autumn for woodchucks in the 12L:12D group was 72% greater than animals in the natural photoperiod. In Study 4, some woodchucks exposed to 12L:12D for only 2.5 years had prematurely increased spermatogenic activity, Leydig tissue development, and elevated serum testosterone levels in November. They were similar in November to those in natural photoperiod animals in March, and significantly greater than those in natural photoperiod animals in November when normal regression and repair of the testis was complete. Likewise, females in the 12L:12D group had luteinized follicles and elevated progesterone in November which were not noted in natural photoperiod animals and which were similar to those observed during the spring in unbred females under normal conditions. The results suggest that circannual cycles of metabolic and reproductive activity in woodchucks persist in the absence of normal changes in photoperiod, are entrained to seasonal changes in the natural photoperiod, and can recede to a periodicity of less than 12 months within 2.5 to 4 years of laboratory maintenance in 12L:12D.     

Monitoring of early events of experimental woodchuck hepatitis infection: Studies of peripheral blood mononuclear cells by cytofluorometry and PCR

Abstract The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37%±25 and 17%±15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.     

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

Seasonal changes in serum dehydroepiandrosterone,androstenedione, and testosterone levels in the squirrel monkey (Saimiri boliviensis boliviensis)

The squirrel monkey (Saimiri boliviensis boliviensis) has a well-defined breeding season during which adult males undergo androgen-dependent morphological changes, with acquisition of active spermatogenesis. To assess the hormonal events of this annual cycle, blood samples were obtained weekly from ten adult males, and serum was assayed for testosterone (T), androstenedione (ΔA), and dehydroepiandrosterone (DHEA). A significant seasonal variation was noted in mean serum T (P < 0.02), ΔA (P < 0.02), and DHEA (P < 0.001) concentrations. Mean ΔA concentrations increased from a nonbreeding season nadir of 91.4 ± 12.9 ng/ml (mean ± standard error) to a prebreeding concentration of 139 ± 10.5 ng/ml and breeding season peak of 167.5 ± 15.4 ng/ml (P < 0.05). Mean DHEA concentrations increased from a nonbreeding season nadir of 8.3 ± 0.8 to a breeding season peak of 14.3 ± 1.2 (P < 0.001). Mean T levels in the nonbreeding (52.2 ± 11.6 ng/ ml) and prebreeding season (48.6 ± 7.4) were similar. However, T significantly increased during the breeding season to 103.5 ± 12.8 ng/ml (P < 0.05). Progressive changes in body weight and morphology paralleled the rise in serum ΔA levels. The pattern of peripheral serum androgen concentrations throughout the year would suggest annual activation of the hypothalamic-pituitary-adrenal and/or hypothalamic-pituitary-gonadal axes.     

Stimulation of testicular function during the nonbreeding season in the woodchuck (Marmota monax)

Treatment of male woodchucks with a series of s.c. injections of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) induced testicular growth, sperm production and marked increase in serum testosterone levels in the fall, approximately 4 mo before the expected spontaneous onset of testicular activity. These results suggest that it may be possible to induce and/or maintain reproductive competence in this species outside of its normally brief breeding season.     

新生与成年土拨鼠肝组织中部分差异表达的基因比较分析

目的新生土拨鼠感染土拨鼠肝炎病毒后,大部分发展为慢性肝炎,而成年土拨鼠感染后则多发生急性自限性肝炎。本实验目的就是寻找其肝组织中可能导致这种预后差异的关键基因。方法采用全基因组表达谱芯片技术,对比新生与成年小鼠肝组织基因表达差异,选取目的基因,再通过多个物种序列比对,设计简并引物,在土拨鼠肝组织eDNA中扩增对应基因,测序,再次设计引物,进行实时荧光定量PCR。结果与新生土拨鼠相比,成年土拨鼠肝细胞中与钙离子重吸收相关基因DNMl（Dynamin1）、DNM3（Dynamin3）及Prkcc（proteinkinaseC,gamma）表达率明显升高,分别上升2．65±0．25倍、1．90±0．34倍、2．94±0．54倍。结论在钙离子重吸收通路中,在两组小鼠肝脏中表达差异最明显的上述三个基因,在新生组与成年组土拨鼠之间也有明显差异。此类基因造成肝细胞内钙离子浓度的差别,间接影响其中肝炎病毒的复制。这种表达差异很可能是导致两个年龄段动物感染土拨鼠肝炎病毒后转归不同的原因之一。     

Serum steroid hormone levels in relation to the reproductive cycle ofSebastes taczanowskii andS. schlegeli

Synopsis Changes in serum steroid hormones were studied during the reproductive cycle of two viviparous rockfishes of the genusSebastes. During the annual reproductive cycle of femaleS. taczanowskii, serum levels of estradiol-17β (E₂) gradually increased from their lowest in August to maximal levels towards the end of vitellogenesis in February, and rapidly returned to low levels in pregnant females in April and May. Serum progesterone (prog) fluctuated at low levels throughout the annual reproductive cycle, while 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog) levels were high during gestation in May. Serum levels of these steroids were also measured in femaleS. schlegeli at weekly intervals during late vitellogenesis, gestation and post-parturition. In pregnant females, E₂ was high during vitellogenesis, then decreased and remained low throughout gestation. The 17α, 20β-diOHprog levels were low during vitellogenesis, then rapidly increased and remained high during gestation. On the other hand, prog values remained low, with temporal peaks coinciding with the peak of 17α, 20β-diOHprog during gestation. Based on these results and the literature, it is suggested that E₂ is the most important steroid involved in vitellogenesis and that 17α, 20β-diOHprog may play an important role in final oocyte maturation and subsequent gestation.     

Mating-related estradiol fluctuations during the estrous cycle of the Bolivian squirrel monkey (Saimiri boliviensis boliviensis)

The female squirrel monkey, Saimiri boliviensis, a New World monkey, has 10-day estrous cycles during the annual breeding season. Measurements of serum estradiol (E) concentrations in females housed with males in breeding pens revealed markedly higher levels than previously reported. Additionally, females in breeding pens appeared to have two distinct patterns of serum E peaks relative to the LH surge. Serum estrogen peaks averaging 5-fold greater than levels on the preceding day were observed on the same day as the LH surge, whereas other females had only a small E rise on the day of the LH surge followed by a 6-fold E rise on the next day. The serum progesterone (P) levels in all animals were depressed for 1-2 days before the LH surge but frequently started to rise on the day of the LH surge. The effect of the presence of a breeding male was examined by studying females housed in a group pen without exposure to a breeding male. In contrast to breeding-pen patterns, relatively small E rises were found in the 10 cycles observed. To further elucidate estrus-related E rises, a limited male-access paradigm was used to isolate mating-related hormone fluctuations. Pre-mating E levels had no marked rises; however, 4 h after mating, whether on the day of the LH surge or the next day, large E rises were found. These studies indicate that the LH surge in cycling squirrel monkeys is consistently preceded by a marked P nadir and associated with relatively small E rises.(ABSTRACT TRUNCATED AT 250 WORDS)