ACTA

ZOOLOGICA

SINICA

ACTA ZOOLOGICA SINICA

Vol. 47 No.4 Aug. 2001

ABSTRACT

Phylogenetic relationships among Crotalinae based on mitochondrial

cytochrome b gene sequence variations

ZHOU Ji-Liang^①② ZHANG Ya-Ping^② HUANG Mei-Hua^①CHEN Yong-Jiu^②

CHEN Xiao-Qing^① YAO Geng-Dong^①

(① College of Medicine, Zhejiang University, Hangzhou 310031, China)

(② Open Laboratory of Cellular Evolution, Kunming Institute of Zoology,

The Chinese Academy of Sciences, Kunming 650223, China)

Abstract Crotalinae are biomedically important, venomous snakes that are distributed across Asia and western Hemishere. Phylegenetic relationships and taxonomy of these pit-vipers belonged to genus Gloydius remains controversial. This paper analyzed the phylogenetic relationships and classification of pit-vipersx (Viperidae, Crotalinae, Gloydius) which are distributed in China based on mitochondrial cytochrome b gene sequence variations. We have sequenced mtDNA cytochrome b gene 789 bp or 744 bp fragment from Gloydius saxatilis (Emelianov), Gloydius shedaoensis (Zhao), Gloydius strauchii (Bedriaga), Gloydius strauchii monticola (Werner), Gloydius ussurriensis (Emelianov), Trimeresurus stejnegeri Schmidt and Gloydius brevicaudus (Stejneger) from two different localities. Sequence compositions and variations were analyzed with MEGA1.02 software. Comparison of aligned sequences revealed 157 variable and 154 phylogenetically informative sites. Combined with the sequence of Dinodon semicarinatus from Genbank, one most-parsimonious tree was drived from heuristic search using the Maximum-parsimony analysis in PAUP4.02b software. Our molecular data show T.stejnegeri is the most basal taxon in our pit-viper samples, and the clade consist of G.strauchii and G.strauchii monticolawhich are distributed in Hengduan montain area is sister group with another clade consist of G.brevicaudus, G.ussurriensis, G. shedaoensis, G.saxatilis which are distributed in northeast China.

Our results support the following conclusions: (1) Gloydius origicnoted from two areas: northeast China and hengduan Montain area; (2) we discussed the origin of G.shedaoensis, and showed that is a subspecies of G. Saxatilis; (3) G. ussurriensis and G. brevicaudus are classified as two species. [Acta Zoologica Sinica 47(4):361-366, 2001]

Key words Crotalinae, Cytochrome b gene, Sequence analysis, Molecular phylogenetic tree

E-mail: zhoujl@cmm.zju.edu.cn

Comparison of RAPD patterns variations in 11 species of Catantopidae*

ZHENG Zhe-Min^① WANG Gui-Ling^② HUANG Yuan^①

①College of Life Science, Shaanxi Normal University, Xi’an 710062, Shaanxi, China)

( ② Fisheries College, Shanghai Fisheries Uinversity , Shanghai 200090, China)

Abstract Catantopidae is a dominant family of Acridodea and has about over 3 000 species. In order to investigate genetic relationships, random amplified polymorphic DNA (RAPD) technique was applied to assess RAPD patterns variations in 11 species belonging to 8 genera of Catantopidae. Among 23 arbitrary primers (10 bp) under predetermined optimal reaction conditions, 8 primers were informative and yielded a total of 148 clear and reproducible bands. The patterns showed polymorphic variations among and within different genera and species. RAPD data were used to calculate Nei's similarity index. Molecular phylogentic tree were constructed by un- weighted pair-group method with arithmetical averages (UPGMA). The results showed the species of each genus first clustered respectively and 8 genera of Catantopidae were divided into two branches, whose conclusion coincided with morpholgical (eg. wingmorphs). In one branch, 2 genera ( Yunnanacris Chang and Pedopodisma Zheng) had short fore-wings, whereas 6 genera (Calliptamus Serville, Choroedocus I. Bolivar, Shirakiacris Dirsh, Apalacris Walker, Xenocatantops Dirsh and Eucoptacra I. Bolivar) in the other branch had long fore-wings. From these results, we found that the species with similar morphological characters shared a higher similarity on RAPD bands. [Acta Zoologica Sinica 47(4):367-370, 2001]

Key words Catantopidae, RAPD, Patterns, Phylogeny

E-mail: zhmzheng@yahoo.com.cn

Dynamics of material and energy during incubation

in the soft-shelled turtle (Pelodiscus sinensis )

DU Wei-Guo JI Xiang XU Wei-Qing

(Department of Biology, School of Life Sciences, Hangzhou Normal College, Hangzhou 310036, China)

Abstract We incubated a total of 132 Pelodiscus sinensis eggs at 30℃ using wet vermiculite as the incubation substrate, of which the moisture was kept constant at - 220 kPa water potential. We paid particular attention to the growth trajectory of embryo and embryonic use of material and energy during incubation. Eggs were weighed at 7-day intervals to test for temporal changes in egg mass. From the tenth day of incubation, we opened 15 eggs at 5-day intervals and separated them into shell, embryo and yolk. The three egg components were oven dried to constant mass at 65℃, weighed and preserved frozen for later determination of composition. Upon emergence, size (carapace length and width) and mass were measured on each hatchling. Hatchlings (n = 17) were then killed by freezing to - 15℃ for later study. Upon thawing, we separated each hatchling into carcass, residual yolk and fat bodies. The three components were oven dried to constant mass at 65℃, weighed and preserved frozen for later determination of composition. We extracted non-polar lipids from dried samples in a Soxhlet apparatus for a minimum of 5.5 h using absolute ether as solvent. The amount of lipids in a sample was determined by subtracting the lipid-free dry mass from the total sample dry mass. The total lipid in each hatchling was calculated as the sum of the lipids in its carcass, residual yolk and fat bodies. We determined energy density of dried samples using an adiabatic bomb calorimeter and ash (inorganic material) content in each sample using a muffle furnace at 800℃ for a minimum of 8 h and then weighing the remaining ash. The incubating eggs did not show significant temporal changes in mass over the course of incubation, suggesting that mass gain or loss due to exchanges of water between incubating eggs and their surroundings was negligible in this study. The incubation length averaged 45.5 days. At the stage of 0～20, 20～30, 30～40 and 40～45 days of incubation, the developing embryos mobilized approximately 6%, 24%, 59% and 4% of the total egg energy in the yolk of the freshly laid egg, respectively. Embryos grew slowly during the first 30 days and last 5 days of incubation, as indicated by the small increase in embryo mass and the low rate of embryonic mobilization of energy. The maximum embryonic growth occurred at the stage of 30～40 days of incubation. The pattern that embryos grew slowly in the last days of incubation was much similar to that seen in some other turtles, presumably resulting from the synchronized emergence of hatchlings. Embryos used both yolk and eggshell as the sources of inorganic material for development, because total ash in newly emerged hatchling exceeded that in the yolk of the freshly laid egg. This claim could be further substantiated by the fact that shells from hatched eggs were lighter in mass and contained less quantities of ash than those from freshly laid eggs. During incubation, approximately 79.6 % dry material, 58.7 % non-polar lipids and 66.5 % energy in egg contents of the freshly laid egg were transferred into the hatchling. Of all energy in the newly emerged hatchling, 71.8 % was in the carcass, 19.4 % in fat bodies and 8.8 % in the residual yolk. [Acta Zoologica Sinica 47(4):371-375, 2001]

Key words Soft-shelled turtle (Pelodiscus sinensis ), Egg, Incubation, Hatchling, Material and energy dynamic

E-mail: duweiguo@mail.hz.zj.cn

Movement and brooding behaviour of blood pheasant

( Ithaginis cruentus ) in brooding period*

JIA Chen-Xi^①② ZHENG Guang-Mei^② ZHOU Xiao-Ping^③ ZHANG He-Min^③

(①Beijing Normal University, Beijing 100875, China)

(②Institute of Zoology , The Chinese Acaderny of Sciences , Beijing 100080)

(③ Wolong Nature Reserve , Sichuan 623006, China)

Abstract Blood pheasant (Ithaginis cruentus) is endemic to Qinghai-Tibetan Plateau and mainly distributes in the high mountains of China. The hatched chicks have to depend mainly on heat from the brooding behaviour of the adult female to survive because of their limited thermoregulatory capacity. Most studies on the daily movement and brooding behaviour of Galliformes centered on the captive birds, but the information on the birds in the field remains unclear. We studied the effects of the age of chicks and ambient temperature on the duration and frequency of brooding bout, and the relationship between the daily movement and the age of chichs of broods form November 1994 to September 1995 in Wuyipeng of Wolong Nature Reserve, Sichuan Province, China. Radiotelemetry was used for following the daily movement and focal sampling method was used for observing brooding behaviour of Blood pheasant. The main results are showed as follows.

Totally 110 brooding bouts of two females, 96 from brood No. 4 and 14 from brood No. 3, were observed and analyzed in this study. The duration of brooding bouts lasted 7.87 ± 0.45 min. Regardless of the age of the chicks ( r = 0.063, P = 0.540), and tended to increase as the ambient temperature decreased but not significant (r = -0.245, P=0.016). The interval between two brooding bouts was 13.60 ± 1.15 min. Brooding bouts tended to decrease in frequency both as the chicks grew older (r = -0. 878, P < 0.001), exceptions occurred on cloudy, raining and cold days, and, although not significant (r = -0.439, P = 0.053), as the ambient temperature increased. Brooding behaviour disappeared from the behavioral repertoire at about 40 days of age. Based on our results, we concluded that the brooding frequency of Blood pheasant was influenced by both the ambient temperature and the thermoregulatory capacity of chicks, and the brooding duration was not related with the age of chicks.

The average distance per hour moved by broods in one day tended to increase as the chicks grew older ( r = 0. 680, P < 0.001). The size of brood' s home range in July (0.17 km²) was larger than that in June (0.15 km²). It is suggested that the Blood pheasant with broods appeared more mobile as the chicks grow older. [Acta Zoologica Sinica 47(4):376-380, 2001]

Key words Blood pheasant (Ithaginis cruentus), Brooding period, Brooding behaviour

E-mail: jiacx@263.net

Developmental traits of captive sichuan snub-nosed monkeys

( Rhinopithecus roxellana ) at different age stages

LIANG Bing^① QI Han-Jun^② ZHANG Shu-Yi^①* REN Bao-Ping^①

(① lnstitute of Zoology , The Chinese Academy of Sciences , Beijing 100080, China)

(② Beijing Raising and Breeding Center of Endangered Animals, Beijing 102602, China)

Abstract Morphological and physiological characteristics, and behavior of 46 captive Sichuan snub-nosed monkeys(Rhinopithecus roxellana ) were studied during 1989～1998 at the Beijing Center for Breeding Endangered Animals. 28 of these animals were born at this center, with their birth dates and parents being recorded. Body length, weight, canine teeth length and upper-lip wart size of these animals were measured from time to time respectively. Reproductive behavior of all individuals over the age of 2 was observed, and the occurrence of the first menarche and solicitation of the females and the first mating and ejaculation of the males was recorded.

Based on these characteristics, the life span of Sichuan snub-nosed monkeys was classified into 5 developmental stages: infant, juvenile, adolescent, sub-adult and adult. The ages of these five stages are as follows: infant, 0～1 year; juvenile, 1～3 years for females and 1～4 years for males; adolescent, 3～4 years for females and 4-6 years for males; sub-adult, 4～6 years for females and 6～8 years for males; adult, older than 6 years for females and older than 8 years for males.

Our study revealed that from infancy to the juvenile stage, the increase in body size and weight in both sexes was comparable. However, beginning at the adolescent stage, the rate of development was greater in males than in females. In the adult stage, sitting height, tail length, head circumference, canine teeth length and hair length were each greater in adult males than in adult females, and the average weight of adult females was 54 % of that in adult males. The adult male and the adult female displayed remarkable sexual dimorphic.

Menarche in females was observed at the age of 3.2～4.5 (3.6 ± 0.5, n = 5) years, and females started to give birth at the age of 4～6 (5.0 ± 0.7, n = 5) years, 80 % of those started to give birth before 5 years old. Males displayed noteworthy ejaculation at the age of 6.5 years, and the earliest age of successful reproduction in this sex was 7～8 years. Additionally, observation of the upper-lip warts revealed that this unique structure, existing only in males, began to emerge at the age of 5.4～6.1 years, and then grew larger and larger until the age of 6.3～7.0 years when the upper-lip warts were about 2.4×0.9×0.9 cm. Thus, the upper-lip warts could be considered a sign of males' sexual maturity, or a secondary sexual trait of adult males. [Acta Zoologica Sinica 47(4):381-387, 2001]

Key words Sichuan snub-nosed monkeys (Rhinopithecus roxellana), Developmental age stages, Morphological traits, Reproductive behavior

E-mail: liangb@panda.ioz.ac.cn

Thermogenic capacity and expression of uncoupling protein gene of brown adipose

tissue from mongolian gerbils(meriones unguiculatus) during cold acclimation

LI Qing-Fen^* LIU Xiao-Tuan HUANG Chen-Xi SUN Ru-Yong

(Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering,

College of Life Sciences, Beijing Normal University, Beijing 100875, China )

LIN Qi-Shui

(Shanghai Institute of Biochemistry , The Chinese Academy of Sciences , Shanghai 200031, China)

Abstract Cold-induced thermogenesis and gene expression of uncopling protein (UCP) of brown adipose tissu(BAT) in Mongolian gerbil, Meriones unguiculatus, were investigated. UCP specific for BAT in the gerbil was detected by using a [³²p] -labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectl determined by titration with its specific ligand [³H -labeled GTP. Type Ⅱ T4 5'-deiodinase in BAT was assayed concomitantly by a radioimmunoassay using a triiodothyronine RIK kit. We found that during cold exposure (4±2℃) from 1 day to 4 weeks, the GTP-binding capacity of BAT mitochondria increased gradually, an reached its maximum (0.94±0.07 nmol/mg mitochonrial protein, 1.7-fold of control) after three weeks. The trascriptional form of UCP mRNA in BAT showed only one species with a length of about 1.5 kb. The UC mRNA was significantly upregulated (about 2.6-fold vs control) after only 1 day in cold, and reached maximur (7-fold vs control) after I week, then remained at a relatively high level but tending to slowly decrease. The activity of T₄ 5'-deiodinase in BAT obviously increased by 517% after exposure to cold for 1 day, and increase much more with the increase of time in cold exposure. The results indicated that, even during acute cold exposure, the UCP mRNA had been upregulated. Such rapid upregulation of UCP mRNA may be necessary for BAT to synthesize new UCP and finally to acquire its optimal function of thermogenesis. The activation of T₄ 5'-deicdinase may be an important regulatory factor for the upregulation of UCP mRNA and the recruitment of BAT. The basal GTP-binding capacity of BAT mitochondria is higher in Mongolian gerbil than in rats kept in the same or similar warm environment temperature, indicating higher basal thermogenie activity of BAT in Mongolian gerbil. It may be an adaptive strategy of the Mongolian gerbil to large oscillations in daily temperature in its habitat. [Acta Zoologica Sinica 47(4):388-393, 2001]

Key words Mongolian gerbil, T₄ 5'-deiodinase(Meriones unguiculatus), Mitochondrial GTP-binding, Gene expression, Brown adipose tissue.

E-mail: lidu@bnu.edu.cn

Distribution of TrkA, TrkB, TrkC and TrkE neurotrophin receptors

in Chinese earthworm( Pheretima aspergillum )

LUO Zhen-Guo^* ZHANG Xiao-Yun

(Department of Biotechnology, Shenzhen University, Shenzhen 518060, Guangdong)

Abstract Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system. The high-affinity signal-transducing TrkA, TrkB, TrkC and TrkE receptors of neurotrophin are distributed widely in neural tissues and non-neural tissues of vertebrate. Trk receptors also are discovered in Arthropoda, Mollusca. The previous research results show that the number of subtype of Trk receptors decreases with the fall of evolutionary rank. Are there TrkA, TrkB, TrkC and TrkE receptors in Annelida ? Which subtype in nervous tissue? Which subtype in non-nervous tissue? All these questions remains unclear. The present study was undertaken to investigate the occurrence and distribution of TrkA, TrkB, TrkC and TrkE in the Chinese earthworm (Pheretima aspergillum ). The Pheretima aspergillum was used as the experimental animal. All animals were fed with paper pulp and gelatin in order to discharge earth and sand. They were deeply anesthetized with 10% ethanol and dissected. The samples were fixed in fixative containing 4% paraformaldehyde and 2% picric acid in 0.01 mol/L PBS (pH 7.2) at 4℃ for 6 hours, transferred to 30% sucrose-PBS until completely infiltrated, and then they were embedded in OCT compound (USA), frozen quickly in liquid nitrogen and sectioned in 20 btm thickness in Leica-ll00 cryostat (Pharmacia). Alternate serial sections were stained with polyclonal antibodys against TrkA, TrkB, TrkC and TrkE by employing ABC immunocytochemistry technique. The immunostained consecutive sections were observed and the positive cells and fibers were examined under light microscope. It was found that Trk was distributed in not only neural tissues but also non-neural tissues, TrkA receptor existed in subpharyngeal ganglion, ventral nerve cord, enteric neurons, nervous fibers between muscle cells, but not in cerebroganglion, TrkB only existed in intestinal epithelium, TrkC existed in eerebroganglion, subpharyngeal ganglion, ventral nerve cord, enteric neurons, nervous fibers between muscle cells, and TrkE neurons existed in cerebroganglion, while TrkE nerve fibers in subpharyngeal ganglion and ventral nerve cord. The results indicated that TrkA, TrkB, TrkC and TrkE receptors were distributed in Annelida with low evolutionary rank, and the evolution of neurotrophic faetors-Trk regulating system may be traced back with a long history. Each of the different Trk proteins binds and is responsive to different neurotrophic factors. TrkA, TrkB and TrkC served as the primary receptor for NGF, BDNF and NT-3 respectively. TrkE also was the primary ligand for NGF. The fact that TrkA, TrkB, TrkC and TrkE were distributed in neuron populations of different portions of the Earthworm suggested that these neurons were influenced by different neurotrophic factors. The Earthworm possesses stronger regeneration ability than vertebrates when injured. TrkA, TrkB, TrkC and TrkE may provide important insights into regeneration common to neural and non-neural tissues. [Acta Zoologica Sinica 47(4):394-397, 2001]

Key words Chinese earthworm (Pheretima aspergillum), TrkA, TrkB, TrkC and TrkE receptors, neurotrophic factor, immunocytochemistry

*E-mail: sms@szu.edu.cn

Distribution of sex steroid hormones and their receptors in the gonads and

nervous system of amphioxus (Branchiostoma belcheri) *

FANG Yong-Qiang^* WENG You-Zhu HU Xiao-Xia

(Third Institute of Oceanography, SOA, Xiamen 361005, Fujian, China )

Abstract Immunocytochemical localization were investigated for the first time in the gonads, brain vesicle and nerve tube of Amphioxus using polyclonal antibodies against 17β-estradiol, testosterone and progesterone receptors. The results indicated that the receptors of estrogen, androgen and progestogen existed in oogonia and oocytes of different developmental stages. At the small growth stage, three kinds of the sex steroid receptors usually were localized in the cytoplasm or nuclear membrane of oogonia and early primary oocyte, and localized in the cytoplasm and nucleoplasm of oocytes at the large growth stage and mature stage. In male, three kinds of sex steroid receptors were distributed in the spermatogonia, spermatocytes and spermatids (except estrogen receptor), and the positive reaction (substance) was localized in the cytoplasm and nucleus of these spermatogenic cells. In addition, the gonads at different stages and nervous system in amphioxus were also investigated by using immunocytoehemieal method and sex steroid hormones (17β-estradiol, testosterone and progesterone) antibodies. The results showed that immunopositive substance existed in the ovary and testis of different stages. At the early developmental stage of ovary, the cytoplasm and nucleolar membrane of oogonia and early primary oocyte showed immunopositive reaction with strong or medium intensity to 17β-estradiol, testosterone and progesterone antibodies. At the large growth stage and mature stage, immunopositive reaction to testosterone and progesterone antibodies in cytoplasm and nueleolar membrane of oocyte weakened obviously, but the immunopositive reaction to 17β-estradiol antibodies strenthened notably. In male, the immunopositive reaction in spermatogonia, spermatocyte and spermatid to testosterone antibodies strengthened gradually during the development and maturation of testis. At large growth stage and mature stage, the spermatogonia and spermatocyte showed strong immunopositive reaction to progesterone antibodies, but immunonegative reaction to 17β-estradiol antibodies at the mature stage. In sperm, the reaction was negative. On the other hand, we found for the first time that mammalian neurosteroids-like immunopositive nerve cell and its fiber existed in the brain vesicle and nerve tube of Amphioxus, and the positive substance distributed in the cytoplasm of nerve cell, while nucleus showed negative reaction. These results provide a new morphological proof that sex steroid hormones participate in the regulation of gonadal development and functional maturation of reproductive endocrine control axis in Amphioxus. [Acta Zoologica Sinica 47(4):398-403, 2001]

Key words Amphioxus (Branchiostoma belcheri), Steroid hormones, Receptors, Immunocytochemistry, Gonads, Nervous system

*E-mail: fant98@public.xm.fj.cn

Immunomodulation function of Grass carp (Ctenopharyngodon idellus) interferon

SHAO Jian-Zhong^* XIANG Li-Xin

(College of Life Science, Zhejiang University, Hangzhou 310012, China)

Abstract Interferon is an important cytokine that has been shown to have antiviral and immunomodulation effects from previous studies carried out in human and higher vertebrates. However, the immunomodulation effects of interferon were not reported in fish before. In this paper, we studied the immunomodulation effects of Grass carp (Ctenopharyngodon idellus) interferon for the first time in order to investigate and understand the biological role of interferon in fish. Grass carp macrophages and lymphocytes were isolated from the peripheral blood and head kidney by density and gradient centrifugation of Percoll and Ficoll-Urografin. Activity of the microphages was determined by assaying their intra-cellular activation markers like superoxide radical (0^2-), hydrogen peroxide (H₂0₂), acid phosphatase (ACP) as well as their phygocytic and bactericidal action. The activity of lymphocytes was determined by monitoring the transformation index of ³H-TdR incorporation. It was discovered that the immunomodulation effect of Grass carp interferon on macrophages was concentration-dependent. At concentration above 50 U/ml, it was shown that the macrophages exhibited not only an increased activity of 0^2-, H₂0₂, ACP but also a significant rise in phagocytic and bactericidal action. The results of the work suggested that Grass carp interferon has apparent effects on macrophages. The activation time was shortened with the increasing of interferon doses. Our results also showed that unlike the case with macrophages, Grass carp interferon did not have direct regulatory effects on lymphocytes. However, it could regulate T lymphocyte transformation induced by mitogen PHA and Con A as well as B lymphocyte transformation induced by SPA and LPS. It was shown that Grass carp interferon has two effects for the regulation of lymphocytes. When the concentration of interferon was 250～500 U/ml and 50～500 U/ml, it had positive regulatory action on the transformation of T and B-lymphocytes respectively; when the concentration increased to 5 000 U/ml, it had negative regulatory or inhibitory effects. The overall results suggested that fish interferon has an important immunomodulation effect on fish macrophages and lymphocytes. It can be used as not only an antiviral medicine to cure diseases of fish, but also an immunomodifier to increase the immune level of fish. [Acta Zoologica Sinica 47(4):404-411, 2001]

Key words Grass carp (Ctenopharyngodon idellus), Interferon, Immunomodulation, Macrophage, Lymphocyte

E-mail: lscshaoj@mail.hz.zj.cn

Effect of Mifepristone on gonadotropin secretion and its

mechanism in cultured pituitary cells

ZHU Si-Jun^①②* ZHUANG Lin-Zhi^① XIE Zhong-Ming^②

XU Yi-Shu① ZHAO Bai-Ge②*

(① Institute of Zoology, The Chinese Academy of Sciences, Beijing100080, China)

(② Shanghai Institute of Planned Parenthood Research, Shanghai200032, China)

Abstract Clinical research has observed that Mifepristone (MP) could decrease gonadotropin secretion when it was administrated to the woman with normal menstrual cycle, whereas its underlying mechanisms are poorly understood. In this study, the action of MP on GnRH-, high extracelar K⁺ ([K⁺]_e)-, Phorbol 12-myristate-13-lcetate (PMA)-induced LH release in cultured rat anterior pituitary cells was investigated. The anterior pitutaries of the female mature Sprague Dawley rats were dissociated with 0.25％ collagenase, 0.1％ hyaluronadase md 10 ug DNase I according to the method from Vale. Isolated pituitary cells were cultured with Ham Fl2 and Dulbecco modified Eagle Medium 1:1 supplemented with charcoal treated fetal calf serum for three days and then exposed with MP or/and hormones. In short term treatment groups (4-hour treatment), cells were exposed in 3×10^-9 mol/L, estaradiol (E₂) for 52 hours first and then 10^-7 mol/L MP or 10^-7 mol/L progesterone (P) or 10^-7 mol/L MP+ 10^-7 mol/L P was added in the medium at the last 4 hours of culture. In long term treatment groups, cells were exposed in 3 ×10^{- 9} mol/L E₂ semitaneously with either MP or P or MP + P for 52 hours and then treated with 10^-9mol/L GnRH or 10^-8 mol/L PMA or 60 mmol/L K⁺ for another 3 hours. The collected media were stored at -20℃ for luteinizing hormone (LH) radioimmunoassay. The results demonstrated that MP could inhibit GnRH-induced LH secretion in a dose- and time-dependent manner and also antagonized the regulatory effect of P on GnRH-induced LH secretion. Further studies showed that 4 hours treatment of MP could decrease KCI and PMA stimulated LH release and in contrary, P augmented KCl and PMA stimulated LH release. In the long term treatment groups, neither KCl nor PMA induced LH secretion was influenced by P, but P could inhibit the KCI stimulated LH secretion. When MP was treated concomitant with P, both stimulatory and inhibitory effects of P on the high [K⁺]_e and PMA induced LH secretion were reversed by MP( P<0.05). These data indicate that the mechanisms of the MP action on GnRH-induced LH secretion probably through both the modulation of voltage sensitive Ca²⁺ channels (VSCC) and PKC activity. [Acta Zoologica Sinica 47(4):412-418, 2001]

Key words Rat, Pituitary cell, Mifepristone, Progesterone, Luteinizing hormone, Voltage sensitive calcium channel, K + , Protein kinase C

*E-mail: sijunzhu@students.uinc.edu

Expression of beta subunit of Rhesus monkey chorionic gonadotropin (rmCGp) DNA in HeLa cells and the immune response in BALB/C mouse inoculated by rmCG13 DNA vaccine

CHEN Yun LIU Zhe PENG Jing-Pian^*

ZHANG Fu-Chun CHEN You-Zhen WANG Bin

(State Key Laboratory of Reproductive Biology, Institute of Zoology,

The Chinese Academy of Sciences, Beijing 100080)

Abstract The human chorionic gonadotropin (hCG) as one of glycoprotein hormones family shares a common alp but differs in the hormone-specific beta subunit. Because of the physiological and temporal specific it Gl3 is a good target molecular to develop immuno-contraceptives. To study the feasibility of human DNA immuno-contraceptive by using hCG gene, the Rhesus monkey was chosen as an in vitro and in vivo model to understand the immune response, anti-fertility effect and safety of β-CG DNA immuno-contraceptive. pCMV4- rmCGβ, a kind of DNA vaccine inserted with full-coding cDNA sequence of beta subunit of rhesus monkey (Macaca mulatta) chorionic gonadotropin (rmCGβ), was constructed to understand the feasibility of developing DNA immuno-contraceptive. In this work, the full-coding eDNA sequence of rmCGβ was inserted into eukaryotic expression vector pCMV4 to construct recombinant expression vector pCMV4-rmCGβ. Using HeLa cells transient expression system, the capability of expression of pCMV4-rmCGβ was detected in vitro, in which the mRNA and the protein of rmCG expressed in HeLa cells were determined by RT-PCR and ELISA, respectively. The pCMV4-rmCGβ DNA were transfected into HeLa cells with lipid, and the culture sera and cells were collected at 24 h, 48 h, and 72 h after transfection. The results showed that there was the highest level expression of rmCGβ at 24 h after transfection at mRNA level, and the expression was gradually decreased from 48 h to 72 h. The mRNA expression of treatment assay showed significant difference compare to untreated HeLa cells and lipofectamine Lipid control assay (P < 0.05). At protein level, the highest level expression of rmCGβ reached 0.5 μg (equivalent to hCGβ) /10⁶ cells, and the rmCGβ protein was mainly with intracellular or membrane associated state. To detect the ability of biological function of pCMV4-rmCGβ plasmid DNA in vivo, the female BALB/c mice were inoculated with pCMV4-rmCGβ plasmid DNA from 10 μg to 100 μg with intramuscular inoculation method. The sera samples were collected at 2 week, 4 week, 6 week, and 10 week post-immunization. The results of ELISA showed that the intensive humoral immune responses in female BALB/c mice were elicited by inoculating pCMV4-rmCGβ DNA vaccine. The highest level of antibody titer was above 6 000. These intensive immune responses were no dose-dependent on the quantity of pCMV4-rmCGβ DNA from 10μg to 100 μg, and could last at least 10 weeks. The results indicate that the recombinant pCMV4-rmCGβ eukaryotic expression vector has biological function, which can express rmCGβ-subunit in vitro cells and in vivo tissue. Moreover, the rmCGβ protein could be expressed by the muscle of BALB/c mice and presented by the Antigen Present Cells to induce intensive humoral immune responses. In brief, This work establishes the base for further anti-fertility and safety detection of pCMV4-rmCGβ DNA vaccine, and should be valuable in developing CGβ DNA immuno-contraceptive. [Acta Zoologica Sinica 47(4):419-424, 2001]

Key words: Rhesus monkey (Macaca rnalatta ), rmCGβ, DNA vaccine, HeLa cells, Expression, Immuniltion

E-mail: pengjp@panda.ioz.ac.cn

Analysis of the polymorphism of mtDNA D-Loop in three

breeds of experimental miniature pig in China

LIU Zhong-Lu^① WEI Hong^①* ZENG Yang-Zhi^②

WANG Ai-De③ GAN Shi-Xiang④

(①Laboratory Animal Center, Third Military Medical University, Chongqing 400038, China)

(②College of Animal Resources, Yunnan Agriculture University, Kunming 650201, China)

(③Faculty of Animal Science, Guangxi University, Nanning 530005, China)

( ④ Laboratory Animal Center, Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China)

Abstract The present study is to analyze the polymorphism of the mitochondrial DNA (mtDNA) D-loop in Guizhou miniature Xiang pig, Guangxi Bama miniature pig and Xishuangbanna Small-ear inbred pig, all of those are used laboratory miniature pigs in China, and Landrace, and to look for its cytoplasmic DNA markers to identify them.

Pure and intact total DNA was obtained with leukocyte sediment added to the digestive solution STE (10 immol/L Tris-HC1 pH 8.0, 0.25 mmol/L EDTANa₂, 100 mmol/L NaCl), SDS (sodium dodecyl sulphate) and f pr0tease K, and was purified by Phenol/Chloroform extraction and ethanol precipitation. A pairs of primers were designed to amplify mtDNA D-loop of the pigs for PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism), the amplification products were digested by 23 restriction enzymes and detecte with 2 % agarose gel. Clear amplification band were obtained, and the fragment length was the same as that c early reports. Among the 36 pigs examined, there was no variation among or within each of the breeds showin monomorphism. Another pairs of primers were used to amplify mtDNA D-loop 5'-end 227 bp high variable regions, the amplification products were detected by PCR-SSCP (single string conformation polymorphism) an PCR-direct sequencing. There was no variation among or within three breeds of Chinese laboratory miniatm pigs besides Landrace. By comparison of the sequences of the porcine mtDNA D-loop 5'-end, no difference was detected among the three breeds of Chinese laboratory miniature pigs, while 4 base variation with Landrace.

It was concluded that the polymorphism of mtDNA D-loop within the three breeds of Chinese laboratory miniature pigs was poor, useful cytoplasmic DNA markers for distinguishing among them could not be obtaine by the methods of PCR-RFLP, PCR-SSCP and PCR-directing sequence, also it could be used to distinguish them from Landrace, more attention should be paid to the study of the nDNA. [Acta Zoologica Sinica 47(4):425-430, 2001]

Key words Miniature pig, PCR, mtDNA, D-loop, Polymorphism

E-mail: weihong@mail.tmmu.com.cn

Ultrastructure of the gametogenesis of the chick Coccidiae (Eimeria tenella)

AN Jian

(Department of Animal Science and Technology, Beijing Agricultural College, Beijing 102206)

WANG Ming KONG Fan-Yao YIN Pei-Yun

(College of Veterinary Medicine , Zhongguo Agricultural University, Beijing 100094, China)

Abstract The ultrastructure was described to interpret the life cycle of Coccidiae. The chick (Gaullus domesticus) was chosen as object of study and reared with coccidian-free food in the coccidian-free coop and house. At 14 days old, each of the chickens was inoculated with 20 000 sporulated-oocyst of E. tenella by mouth. At 132^nd hour and 144^th hour after being inoculated, the tissue sample of experimental chicken was taken from the caecum and was treated according to the inquiring of the electron microscope cutting section, which was cut by microtome. The ultrastructure of the period of gameogenesis of coccidia was observed through an electron microscope and described.

The microgametocyte (MI) and macrogametocyte (MA) is developed side by side in the neighbour host cell during the gametogenesis, and the microgamete (MIG) and macrogamete (MAG) are also developed in it. After the last generation merozoite (ME) enters the host cell, the ME turns into round by swelling and growing, and then it transforms into the MI and the MA. After that the MIG is constructed. At the beginning of the MIG being constructed, the number of nucleus increases in the cytoplasma by nucleus fission. There is a lot of nucleus in the MI. After the number of the nucleus stops increasing, the nucleus began to move toward the limiting membrane. And then two centrioles above the nucleus appear. And the nucleus and the mitochondria are included in the limiting membrane and project into the parasitophorous vacuole (PV), the limiting membrane of the MI near to the protruding begins to sink. The shape of the MIG, which is amateur, can be seen and linked with the residium of the MI. The symbol of the mature MIG is done away from the residium. The mature MIG had a banana-shaped body, which consists of two slender flagella, and a prominent mitochondria and a big nucleus. The big nucleus, whose anterior end is tipper than posterior end, occupies most of the mature MIG. There is quite little cytoplasma in mature MIG between the limiting membrane and the nucleus. The flagella consists of 9 groups of 3-microtubules and 1 group of 2-microtubules. There are more than 6 adherous microtubules, which run longitudinally from the basal body region to the posterior end.

The MAG and the MA are also covered with a unit membrane and located at the PV. After the ME turns into MA, the wall-forming bodies of the first type and wall-forming bodies of the second type begin to appear in it. As the MA developing, there is an increase in the number of amylopectin and in their size. The amylopectin can be observed throughout the MA to MAG. There are a lot of wall-forming bodies of the first type and wall-forming bodies of the second type, amylopectin and lipochondria in the mature MA. The wall-forming bodies of the first type are denser in the electron-density and lately appear than that of the wall-forming bodies of the second type. The nucleus, whose shape is not normal and that contains amorphous nucleoplasma, is a distinguished feature of developing MA, locates at the center of the MAG and MA. The electron density of the nucleolus is clear and compact. There are a lot of intravacuolar microtubules in the PV. All of the intravacuolar microtubules link with the limiting membrane. The difference can't be found between the mature MAG and the zygote. The formation of the oocyst is initiated after the MAG fertilized with a MIG. After fertilization, the wall-forming bodies begin to move to the limiting membrane, the moving of the wall-forming bodies of the first type is earlier than that of the wall-forming bodies of the second type, and then the wall is constructed with them. The wall of the oocyst consists of 5 layers. A large nucleus locates at the oocyst center. There is a lot of amylopectin and lipochondria in the oocyst.

In summary, the MI and the MA are developed side by side in the neighbour host cells during the gametogenesis. In the period of the MIG growth, the number of nucleus increases in the cytoplasms by nucleus fission, after that, the nucleus move toward the cell membrane. And the limiting membrane, which includes nucleu and mitochondria, project into the PV, and the MIG are directly developed. This proceeding belongs to directly development type. The MIG consists of two slender flagella, a mere big mitochondria, a big nucleus and quite little cytoplasms. More than 6 adherous microtubules run longitudinally from the basal body region to the posterior. The adherous microtubule can help the flagella sway, and help the MIG move and enter into the PV to fertilize with the MAG. A lot of intravacuolar tubules, which locate at the PV and connect with the limiting membrane of the MA, can transport the nutrition to the parasite and drain off the metabolism waste out of the parasite. The wall of the mature oocyst consists of 5 layers. The nucleus, which contained amorphous nucleoplasma, locates at the oocyst center. A lot of amylopectin and lipochondria, which exist in the cell plasma, can supply the energy and material to the oocyst for the sporulation. [Acta Zoologica Sinica 47(4):431-435, 2001]

Key words Eimeria tenella, Ultrastructure, Gametogeny

Structure of oral apparatus in Stylonychia mytilus

SUI Shu-Guang CHEN Ying QIU Zi-Jian SHI Xin-Bai*

(Department of Biology, Harbin Normal University, Harbin 150080, China )

Abstract The microstructure and ultrastructure of oral apparatus in Stylonychia mytilus were observed by means of continuous semi-ultrathin and ultrathin transverse sections. In contrast with light microscopic observation on protargol preparation of the whole oral apparatus, the stereoscopic positions of each buccal organelle were clarified. Both paroral mebarane (PM) and endoral membrane (EM) were made of cilia. PM originated from the edge of inner lip (IL) and contained two files of cilia. The EM originates from the right wall of peristome (PST) and stretched to the left side in a way of closely nearing the top of PST. It was formed by a file of cilia. A nonciliary thicker membrane tentatively named phago-assistant membrane (PAM) was found in the ventral side of the EM. It was formed by two layers of membrane and a longitudinal microtubule bundle layer between them. In posterior region of the peristome where the adoral zone of membranelles (AZM) had turned to the mid ventral surface, the phago-assistant membrane extended to the ventral side into a second membrane, they formed together a bracket-shaped membrane. At the cytostomal level, the phago-assistant membrane separateed itself from the cytostomal wall, and the cilia of endoral membrane and paroral membrane assumed a lengthened growth, three membranes tightening into a bundle and entering the cytopharynx (CPH). Sometimes, Stylonychia mytilus could engulf a food even as large as itself. For a long time, how could so large a food pass through such a narrow cytopharynx and the dynamic mechanism of this process perplexed researchers. The discovery of the phago-assistant membrane and the understanding of the stereoscopic structure of the oral apparatus make it possible to explain the possible mechanism of engulfing larger food in Stylonychia mytilus. Phago-assistant membrane may play an important role in this process. We argue that the phago-assistant membrane looks like a palisade that is made of microtubule. To some extent, it possibly expanded itself when food particle or larger food body was flushed into the peristome by the rapids from the AZM, and then it enclosed the food. The cooperative gliding of the microtubules might give rise to vermicular motion of the palisade that push the food pass the cytopharynx through. Meanwhile, the undulation of PM and EM participated in all the ingestion process.. [Acta Zoologica Sinica 47(4):436-441, 2001]

Key words Stylonychia mytilus, oral structure, Injecting function

Ultrastructure of Steinernema carpocapsae BJ in anhydroous dormancy

CHEN Song-Bi^①* YANG Huai-Wen^① JIANG Shu-Nan^② JIAN Heng^① YANG Xiu-Fen^①

(① Institute of Biological Control, Chinese Academy of Agricultural Sciences, Beijing 100081 China )

(② Department of Plant Protection, Southwest Agricultural University, Chongqing 400716, China )

Abstract SB high osmotic solution is a new type of dehydration agent, which administrated to entomopathogenic nematode Steinernema carpocapsae BJ for 10 hours to make them to be anhydrous and in dormancy. It is the easy, quick and safe way to make nematodes anhydrous and in dormancy. It will be a main proceeding of dehydration to make nematode into dormancy in the future. There are some special morphological characters of anhydrous dormant S. carpocapsae BJ reported in this paper, which are obtained by the observation under the scanning electron microscope, clairvoyant electron microscope and optical microscope. These special morphological characters are the most important parameters of anhydrobiotic nematode for S. carpocapsae, which are helpful to know whether nematodes have already been into dormancy or not, and which can tell the difference from the fresh nematodes. In fact, these morphological characters of anhydrous dormant nematode in the condition of fast dehydration were described for the first time.

The anhydrous dormant entomopathogenic nematode S. carpocapsae BJ mainly had 5 aspect characters in morphology. (1) The bodies were bending into slightly curve, on which the transverse wrinkles were regularly in rows and the lateral lines were in the shape of waves. While the bodies of the control were smooth, on which the lateral lines were very straight. (2) The bodies shrank evidently both in length and in width. The percent-age of shrinking reached a steady value, which were 33.1% in length and 28.4% in width of the control, respectively. (3) The cortex and matrix got thinner, while the underlying muscles grew thicker. (4) The muscle fibers were in disorderly arrangement. (5) The esophagus and organelle dwindled, while the lipid droplets in- [Acta Zoologica Sinica 47(4):442-446, 2001]

Key words Steinernema carpocapsae, Anhydrous dormant, Morphological characters

E-mail: songbichen@hotmail.com

Morphology of egg-larvae of Swimming crab (Portunus trituberculatus)

during embryonic development'

XUE Jun-Zeng^*

( Department of Biology, Hangzhou Normal College, Hangzhou 310036, China)

DU Nan-Shan LAI Wei

(Department of Biology, East China Normal University, Shanghai 200062, China)

Abstract Scholars have their own views for egg-larval stages of crustacean. We studied the embryonic development in order to explain the accurate stages for the Swimming crab (Portunus trituberculutus) by histological method. The egg-nauplius stages and egg-zoea stages were discovered in the embryonic development of the crab. The egg-nauplius stages were divided into egg-nauplius I and egg-nauplius Ⅱ.

The optic disc rudiment, antenule rudiment, antennna rudiment and mandible rudiment increased and formed optic lobe, antenule, antenna and mandible with the cleaving cells during the egg-nauplius I. These appendages were unsegmented. The thoracoabdominal process formed abdomen with the proliferating cells. The labrum rudiment and labium rudiment formed in the tip of the stomodeum. The maxillule, maxilla, cavity abdomen, optic ganglia and antennule ganglia formed during the egg-nauplius Ⅱ. The segmentation emerged between the abdomen and thorax. The egg-zoea stages were divided into egg-zoea I , egg-zoea Ⅱ and egg-zoea Ⅲ. The carapace, stomach, hint gut, compound eye and thoracic ganglia formed in the egg-zoea I. The antenna ganglia keep in touch with each other. Each of the optic ganglia was independent. The abdomen was divided into 6 segments. The appendages segmented during the egg-zoea Ⅱ. The ends of the appendages were divergent with setae. The parts of the compound eyes have formed. The shape of the yolk sac was like butterfly. The pigment cells, maxillipede I , maxillipede Ⅱ, heart, gonad and brain have formed. The mesoderm formed muscle and arranged themselves as beads in the next stage. The digestive system has formed except hepatopancreas. Compared with the egg-zoea I and egg-zoea Ⅱ, the brain and carapace further developed in the egg-zoea Ⅲ. The muscle and the hepatopancreas formed at the same time.

Different crustaceans have their respective egg-larva. Some crustaceans have only egg-nauplius; some have egg-nauplius and egg-zoea larvae, while others have their egg-macrophthal larva besides egg-nauplius and egg-zoea larvae. The egg-nauplius I and II for the crab were identified respectively by the pair number of appendages. The egg-nauplius I and egg-zoea I were identified by the compound eyes, carapace, hind gut. The egg-zoea I and Ⅱ identified by the pair number of appendages too. The egg-zoea Ⅱ and Ⅲ were idendified by whether muscle's emergence or not. Therefore, the standard differentiating the larva stages for egg-larvae of the crab are different from those of freely living larvae. Though the stages for egg-larvae of crustaceans are not the same as indicated by individual scholars, there exist some common points. Generally speaking the embryonic development of crustaceans mostly experiences stages of nauplius. The development of embryo involves nauplius

and zoea except prawn in Decapoda. [Acta Zoologica Sinica 47(4):447-452, 2001]

Kev words Swimming crab (Portunus trituberculatus), Embryonic development, Egg-larvae

*E-mail: wuhuixue@mail.hz.zj.cn

Oxytocin and its receptor in mammalian reproduction

FAN Heng-Yu^* YANG Zeng-Ming

(Department of Biotechnology, Northeast Agricultural University, Harbin 150030, China)

Abstract The nonapeptide hormone oxytocin, synthesized mainly in the mammalian hypothalamus, plays an important role in mammalian reproduction through the neuroendocrine, autocrine or paracrine pathways. Oxytocin receptor is a member of the G-protein-coupled receptor superfamily, consisting of seven hydrophobie transmembrane a-helices, and acts through the activation of phospholipase C. Oxytocin released from neurohypophysis can stimulate the contraction of uterine smooth muscle at term and that of myoepithelial cells that surround the alveoli of the mammary gland during lactation. The secretion of oxytosin is a typical neuroendocrine response and can be stimulated by various physiological and environmental stimulation such as the suck ling of the young animals during lactation, light-dark rhythm and the suppression of fetus on the uterus and vagina. Oxytocin is involved in the regulation of maternal behavior in the central nervous system. OTR are expressed in the brain of many mammals, though its distribution in the central nervous system may be different among species. The OTR expression in brain is regulated by the steroid hormones. OT may play a role in the process of luteinization in some species. Oxytocin also cooperates with prostaglandin F2a to elicit the leuteolysis of ungulates. OT secreted from the large luteal cells binds the OTR expressed by uterine epithelium cells, so as to stimulated the production of prostaglandin, forming a positive feedback that responsible for the luteolysis. During pregnancy recognition, the trophoblast cells secret interferon-r, which inhibits the expression of OTR in the uterus. The block of OT-OTR interaction are necessary for the suevival of corpus luteum during pregnancy. But it is possible that other mechanism of luteolysis and maternal pregnancy recognition may be exist in other species. All of these processes depend on the temporal-spatial expression of OT and OTR genes. Several hormones are involved in the regulation of their expression. Some physiological functions of oxytocin can be substituted for by other redundant mechanisms, since in the mice whose OTR gene have been knocked out, some of the physiological processes, but not all, are interfered. [Acta Zoologica Sinica 47(4):453-458, 2001]

Key words Mammal, Oxytocin, Oxytocin receptor, Reproduction

*E-mail: oocyte@china.com

Preliminary study on inducing polyploid in

Japanese scallop (Patinopecten yessoensis) by cytochalasin B

YANG Hui -Ping①^* LI Li^① GUO Xi-Ming^②

(① Experimental Marine Biology Laboratory, Institute of Oceanology,

The Chinese Academy of Sciences, Qingdao 266071, Shandong)

(② Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences,

Rutgers University, USA )

Abstract Triploid shellfish are useful for aquaculture because of their sterility, superior growth and improved meat quality. Tetraploid are also valuable for 100 % producing triploids through mating with diploid. We tested polyploid induction in Japanese scallop, Patinopecten yessoensis, by inhibiting.polar body I (PB group) and both polar body I andlI (PPB group) in newly fertilized eggs. Cytochalasin B (0.6 mg/L) was applied at 11～22 rain post fertilization (PF), and terminated in PB group when polar body I was released about 70% in untreated eggs, in PPB group when polar lobe was observed in control group. The treatment and its control were repeated 5～7 times using different pairs of parents. The ploidy was determined by counting chromosome number at embryo stage, and then was detected by flow cytometry (FCM) at larvae stage and juvenile stage.

In PB group, aneuploid (31.13%), triploid (3.96%), tetraploid (17.46%) and pentaploid (46.65%) embryos were produced, and in PPB groups, pentaploid embryos became higher (56.2 % ), triploid and tetraploid were 2.42 % and 9.11%. At day 3 PF, the larvae showed tetraploid, pentaploid and aneuploid peaks through checking with FCM in PB group, and showed mainly higher pentaploidy peak in PPB groups. However, at day 14 PF diploids were mainly left, sometimes with small triploid peak. It suggested that most tetraploid, aneuploid and pentaploid larvae were died within the first two weeks PF. At three months PF, a few diploid juveniles were harvested in three control groups. Only 12 juvenile scallops were harvested in one of treated group (PB-7), and 11 of them died accidentally, the alive one in treated group was triploid through checking with FCM. [Acta Zoologica Sinica 47(4):459-464, 2001]

Key words Japanese scallop (Patinopecten yessoensis), Triploid Tetraploid, Cytochalasin B, Polar body

*E-mail: hyang@hsrl.rutgers.edu

Follicular development and ovulation inducement in sow

LIN Feng^* YUAN Xi-Fan ZHANG Ying-Han

(College of Animal Husbandry and Veterinary Medicine, Northwest Science and

Technology University of Agriculture and Forestry, Yangling 712100, Shaanxi, China)

Abstract PMSG was used to study the follicular development and ovulation regularity of the sows. The results showed that the earlier copulation caused better ovulation effect after sows were injected PMSG, and the ovulation time of estrous sows centralized 12～63.5 h after accepting copulation. The observation of early embryos' morphology showed that the embryos in the oviduct were in 2～4 cell stage, when entered womb, they were in 4～8 cell stage during 51～59.5 h and all of them entered womb during 71～83.5 h after the sows accepted copulation. [Acta Zoologica Sinica 47(4):465-467, 2001]

Key words Sow, Follicular development, Ovulation inducement

*E-mail: linfengx@263.net

Effect of photoperiod and conspecific odor on the plasma testosterone

level of male Brandt's vole (Microtus brandti )

ZHANG Li^* SUN Ru-Yong FANG Ji-Ming

(College of Life Sciences, Beijing Normal University, Beijing 100875, China)

Abstract Chemical signals provide important cues in social behavior of many mammalian speciesly, mediating sexual, aggressive, parental and spacing behavior, as well as influencing an animal's internal hormonal milieu. Conspecific novel male's substrate was given as individual odor stimulus to adult male Brandt's voles (Microtus brandti) that left in individual cases in long-photoperiod (LD) and short-photoperiod (SD) resfeetioely. Theplasma testosterone of these male actors was mensurated by radioimmunoassay. The results indicated that plasma testosterone increased with the duration that the male vole was exposed to the odor of a novel male. Plasma testosterone concentration in LD males increased sigrifieanily than those in SD males in 30 minutes. After 1 hour or 2 hours, exposed to the novel scents, the testosterone levels of LD males were higher than SD males, but didn't show significant difference. The results demonstrated that the chemical signals induced releasing of androgen and the increase of plasma testosterone of males voles exposed to novel conspeeific odors was also influenced by the photoperiod. It suggest that the change of testosterone concentration would underlie the odor producing and odor preferences of Brandt's vole. [Acta Zoologica Sinica 47(4):468-472, 2001]

Key words Brandt's vole (Microtus brandti), Photoperiod, Odor, Testosterone

*E-mail: asterzhang@sina.com

The new distribution areas of Sichuan wood owl(Strix davidi) in Gansu, China

SUN Yue-Hua^①* Wolfgang Scherzinger^② LIU Nai-Fa^③ Siegfried Klaus^④ FANG Yun^①

(① Institute of Zoology, The Chinese Academy of Sciences, Beijing 100080, China )

(② Nationalpark Bayerischer Wald , Guntherstraβe 8, D-94568 St.Oswald, Germany)

(③ Department of Biology , Lanzhou University, Lanzhou 730000, China)

(④ Thüringer Landesanstalt für Urnwelt, Prüssingstraβe 25, D-07749 Jena, Germany)

Abstract New distribution areas of the Sichuan wood owl (Strix davidi ) were recorded at Lianhuashan Natural Reserve (Kangle county), Zecha (Luqu county) and Kache (Zhuoni county) in Gansu, China. Now its distribution includes west Sichuan, southeastern Qinghai and southern Gansu. Combined with the Ural Owl (Strix uralensis), this distribution range corresponds to the distribution of the genera Bonasa (B. sewerzowi and B.bonasia) and Perisoreus (P. internigrans and P. infaustus). All are related to the endemic birds in Qinghai-Tibet Plateau. Based on our work during 1995～1999, we reported our field observations and preliminary habitat characters of the bird. The main habitat of Sichuan wood owl in Gansu was found in the conifer and conifer-deciduous mixing forest from 2 900 m to 3 300 m. At the Lianhuashan Natural Reserve, the birds were observed many times, with some breeding activities. The population of Sichuan wood owl at Lianhuashan Mountains was an isolated one, and the reserve only protected about 3 360 hm². of its habitat. [Acta Zoologica Sinica 47(4):473-475, 2001]

Key words Sichuan wood owl ( Strix davidi), New distribution areas, Gansu, Habitat

*E-mail: sunyh@midwest.com.cn

Description of Paa polunini Smith(Amphibia: Ranidae) from nyalam of Xizang, China

FEI Liang^* YE Chang-Yuan

(Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)

Abstract The specimens, originally named Rana yadongensis (Wu, 1977), from Yadong and Nyalam, Xizang (Tibet) were examined and compared with the specimens [ Specimens of Paa polunini (No. 1975. 1036 et al. ) and Paa blanfordii (No. 1975. 1266 et al. ) in Museum National d’Historie Naturelle, Paris were examined] of topotypes of Paa polunini and Paa blanfordii from Nepal kept in Paris Museum National d’ Historie naturelle. The results indicated that Rana yadongensis from Yadong is a synonym of Paa blanfordii and Paa yadongensis from Nyalam is identified as Paa polunini (Smith, 1951). Morphorlogieal characters of Paa polunini were not reported in literature of China, in this paper its characters are described.

Paa polunini (Smith, 1951)

Diagnosis. Paa polunini Smith is closely related to Paa blanfordii Boulenger, but differs from the latter as follows: (1) dorso-lateral folds present; (2) skin of back of body and four limbs covered with numerous granules; (3) eolour brown above, uniform, back without spots; (4) no cross-bars on dorsal surface of four limbs. Paa blanfordii has: (1) dorso-lateral folds absent; (2) skin of back covered with round warts and glandular ridges; (3) dorsum and lateral sides of body with distinct spots; (4) black crossbars on four limbs. [Acta Zoologica Sinica 47(4):476-478, 2001]

Key words Paa polunini, Anura, Amphibia, Taxonomy

*E-mail: xiefeng6@mail.sc.cninfo.net

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