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ACTA

ZOOLOGICA

SINICA

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CHINESE

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ACTA ZOOLOGICA SINICA

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Vol. 48 No.4 Aug. 2002

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ABSTRACT

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The Induction of Meiotic Resumption in Mammalian Oocytes

You-Qiang Su and John J. Eppig^*

(The Jackson Laboratory, Bar Harbor, Maine 04609, USA)

Abstract The mechanisms that promote the resumption of meiosis in mammalian oocytes remain poorly understood. Fully-grown oocytes within antral follicles are maintained at the germinal vesicle (GV) stage by meiosis-arresting factors. Cyclic AMP (cAMP) is the best-characterized meiosis-arresting factor; yet, other factors almost certainly also participate. The induction of oocyte meiotic resumption by the preovulatory luteinizing hormone (LH) surge is well established, however the processes involved are complex and inadequately defined. Two hypotheses prevail, though they are not mutually exclusive. The first hypothesis is that LH stimulation of granulosa cells terminates the flow of meiosis-arresting factors to the oocyte, thus depriving the oocyte of these factors and promoting the resumption of meiosis. The second hypothesis is that LH stimulates the generation of a meiosis-inducing signal by the granulosa cells that over comes or destroys the meiosis-arresting factor(s). On balance, current evidence favors the positive signal hypothesis. Moreover, recent studies suggest that this signal is probably generated downstream of LH-induced elevation of granulosa cell cAMP and the activation of mitogen-activated kinases (MAPK) in the granulosa cells. [Acta Zoologica Sinica 48(4):435-444, 2002, in English]

Key Words: Oocyte, Meiotic arrest, Meiotic resumption, Gonadotropin

*E-mail: yqs@jax.org

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Relationships between metabolic rates and body composition in the Mongolian gerbils (Meriones unguiculatus)

SONG Zhi-Gang WANG De-Hua

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

Abstract A central problem of evolutionary physiology concerns the physiological origin and functional significance of the intra- and inter-specific variation in basal metabolic rate (BMR). Previous studies on laboratory mice suggest that individuals with relatively high basal metabolic rates have relatively large masses of metabolically active tissues; energetically expensive organs that contribute to cold resistance. We measured the BMR and cold induced maximum metabolic rate (MMR) in 20 wild Mongolian gerbils (Meriones unguiculatus). Then the gerbils were dissected and 11 body parts were weighed. The BMR was 118.10 ml O₂/h and the MMR was 659.83 ml O₂/h (Mean body mass was 65.2 g). We analyzed the relationship between residual variations of BMR, MMR and 11 body parts, and found that none of the variations in body components could be linked to the variation in BMR, however the small intestine wet mass and total digestive tract length were highly correlated with MMR (n=20, r=-0.478, P=0.033 and n=20, r=-0.487, P=0.030, separately). We used principal component analysis to describe the variation in the morphology and found no components contributed significantly to variations in BMR or MMR. We also pooled body parts into three groups and found that none of them were correlated with variation in BMR or MMR. These data indicate that in vivo variation in BMR is not caused by the variation in the mass of the body parts that we examined. A metabolic mechanism to generate high MMR exists in which the the small intestine plays the major part, but the size of this metabolic machinery is not reflected by BMR. The correlation between variation in BMR and variation in MMR was not significant (n=20, r=0.059, P=0.806). Therefore, our results do not support the hypothesis that BMR variation reflects the evolution of metabolic machinery to generate high nonbasal metabolic rates during energetically demanding periods such as cold exposure.[Acta Zoologica Sinica 48(4):445-451, 2002]

Key words Mongolian gerbil(Meriones unguiculatus), Basal metabolic rate, Maximum metabolic rate, Body composition

*E-mail: wangdh@panda.ioz.ac.cn

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Kin recognition and mate choice in estrous females root voles(Microtus oeconomus)

ZHAO Ya-Jun^ＫＬ*牧ZHAO Xin-Quan^Ｌ LI Bao-Ming^ＫTAI Fa-Dao^Ｍ WANG Ting-Zheng^Ｍ

(Ｋ Ministry of Agriculture Key Laboratory for Agro-Biological Environmental Engineering,

China Agricultural University, Beijing 100083, China)

(Ｌ Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, China)

(Ｍ College of Life Sciences, Shannxi Normal University, Xi'an 710062,China)

Abstract The two most likely mechanisms for recognizing kin are recognition by phenotype maching, identifying individuals as kin because of their genetic similarity,and recognition by association, classifying individuals as kin because of previous direct familiarity with them. Effects of kin recognition on maternal mate choice and inbreeding avoidance were examined in a laboratory colony of root voles(Microtus oeconomus), a generally polygynous species. At weaning age (20days), sibling pups from each of 20 litters were removed from their parents' cage and reared together in a new cage for 5 days, then fostered apart until they were at least 60 days of age at which age females first come into estrus. Between 26 and 65 days of age, i.e., two days before mate choice experiments began, females were assigned to one of 3 treatment groups: (1) females kept familiar with their male siblings by being allowed to sniff them for 1 hour every other day; (2) females kept apart from their male siblings; (3) females kept familiar with their male siblings and also familiarized with nonsibling males by being able to sniff them for 1 hour every other day. At 67 days of age, the mating preferences of estrus females with respect to sibling and nonsibling males were examined using three different experiments in a Y-shaped maze, each of which involved 30 animals divided into 10 groups. Female behaviours measured included the number of visits to different males and their duration, frequency and duration of sniffing/licking, duration of aggressive behavior, number of mountings, and the duration of lordosis, allogrooming, and self-grooming. The Wilcoxon Matched-pairs Test was used to compare behavioral differences in estrous females presented with male siblings and familiar/non-familiar nonsiblings. In experiment 1, estrus females spent notably more time visiting, mating and associating with novel nonsiblings than familiar siblings. In experiment 2, estrus females showed significantly preferred novel nonsiblings to unfamiliar siblings. In experiment 3, estrus females displayed showed a marked preference for familiar nonsiblings compared to familiar siblings. Estrus females preferentially sniffed / licked and groomed nonsiblings over siblings in all three experiments. These results suggest that: (1) kin recognition is primary mechanism of inbreeding avoidance in estrus female root voles; (2) memory of a sibling's scent may be a mechanism of sibling recognition by estrus females, and familiarity after weaning can reinforce permanence of this memory; (3) post-coital familiarity may be a mechanism of maintaining inbreeding avoidance in female root voles. [Acta Zoologica Sinica 48(4):452-458, 2002]

Key words Root vole (Microtus oeconomus), Estrus female, Mate choice, Kin recognition, Familiarization, Avoidance of inbreeding, Polygyny

*Email:yajunzhao@263.net

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Volatile compounds in the anal sac secretion of the Siberian weasel (Mustela sibirica)物狀玟

ZHANG Jian-Xu ZHANG Zhi-Bin^* WANG Rui CHEN Yi WANG Zu-Wang

(State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture,

Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China)

Abstract The volatile constituents of the anal-sac secretion of the Siberian weasel(Mustela sibirica) were sampled using the headspace technique and solvent desorption and then examined by gas chromatography-mass spectrometry. Six compounds detected were all sulfur-containing: (1) 2,2-dimethylthietane; (2) Cis or trans-2,4-dimethylthietane; (3) Trans-2,3-dimethylthietane; (4) 2-ethylthietane; (5) 2-propylthietane; (6) 3,3-dimethyl-1,2-dithiacyclopentane. Although the chemical constituents of the anal sac secretion of siberian weasels had great similarity to those of other Mustela species, inter-species differences between them and other Mustela species existed. Moreover, 2-ethylthietane was present only in females. These differences in the chemical composition of anal gland secretions make it possible to communicate information about species and sex. Many studies have documented that synthetic 2,2-dimethyl thietane and 2-propylthietane were effective rodent repellents, and since both 2,2-dimethylthietane and 2-propylthietane occurred in the Siberian weasel, the anal sac secretions of Siberian weasels may be highly repellent to rodents. [Acta Zoologica Sinica 48(4):459-464, 2002]

Key words Siberian weasel (Mustelia sibirica), Anal sac secretion, Volatile components, Chemical communication

*E-mail: zhangzb@panda.ioz.ac.cn

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Effects of tetrachlorobipenyls on maternal behavior in laboratory rats

WANG Xiong-Qing

(Department of Biology, Leshan Teachers College, Leshan 614004, Sichuan, China)

Abstract Polychlorinated biphenyls (PCB) are widespread environmental contaminants that are possible health hazards for humans. To evaluate the developmental effects of PCBs on human beings, rats were used as a model to study the effects of PCBs on maternal behavior. Five groups of time-mated Long-Evans rats (Rattus norvegicus) were injected daily with either non-coplanar 2,2',4,4'-tetrachlorobiphenyl (PCB 47) at the dosage of 1 or 20 mg/kg body weight, or with coplanar 3,3',4,4'- tetrachlorobiphenyl (PCB 77) at the dosage of 0.25 or 1 mg/kg body weight or sesame oil (control group). Treatment extended from gestational day 7 to 18. All the behavioral tests were done between 0900 to 1400 h under red illumination. A thirty-minute maternal behavior test and an induced retrieving test were performed once every three days with the former from postnatal day (PND) 0 to 15 and the latter from PND 0 to 12. If the tests were given on the same day the maternal behavior test was done first. Results show that control mothers spent less time with pups in the nest as they got older. However, PCB-treated females did not respond in this way. PCB 47-trerated mothers showed significant differences in the duration of crouching, licking, spontaneous retrieving and nest building on PND 0 and 6, whereas PCB 77-treated mothers showed significant differences in the duration of crouching and time in the nest only on PND 15. During tests for induced retrieving of pups, PCB 77-treated mothers showed an increase in the duration of retrieving on PND 12. The results of this study suggestion that the fact PCBs disturb maternal behavior of rats would have potentially negative effects on humans. [Acta Zoologica Sinica 48(4):464-470, 2002]

Key Words Long-Evans rat, Maternal behavior, Polychlorinated biphenyl, Tetrachlorobiphenyl

*E-mail: wangxio@ls-public.sc.cninfo.net

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Habitat distribution of snow finches (MONTIFRINGILLA) in China

QU Yan-Hua LEI Fu-Min^* YIN Zuo-Hua

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

Abstract Due to the inaccessibility of the Tibetan Plateau there has been insufficient information to determine the distribution of snow finches (Montifringilla), which mainly occur on this high plateau and adjacent areas in the alpine zone. Based on specimen collection records from museums, published references and field investigations, we used GIS (Geographic Information System) software and the WHR (Wildlife- Habitat Relationship) model to predict suitable snow finch habitat. The primary information used to predict suitable habitat included range, elevation and vegetation maps, and data on species/habitat associations. A method for predicting the likelihood of occurrence of different snowfinch species according to vegetation type was developed but only actual presence/absence data were used with respect to altitude. Potentially suitable habitats were identified by overlaying the suitable vegetation map layer with the suitable elevation map layer. Because most data from the specimen collections records contained only county names instead of detailed geographic coordinates, we overlaid potentially suitable habitats with a map of snow finch occurance by county to predict suitable habitats. Comparison of the predicted distribution with the current distribution map shows that most parts of the Tibetan plateau and adjacent areas that presently have no snow finches records contain potentially suitable habitats. The confirmed range of this group comprises only 1/3 to 1/5 of the area of the predicted potential distribution. Overlaying the predicted distribution of six Montifringilla species suggests that inner areas of the Tibetan Plateau contain habitat suitable for five or six species and could be the center of snow finches distribution. ﹛﹛Compared with the traditional dot-distribution maps, WHR-generated maps were more accurate, especially for poorly surveyed regions. Previous work (Cheng, 1981, 1983; Fu, 1998), suggested that Montifringilla species were not uncommon and locally distributed. However, our models suggest that Montifringilla species are widely distributed in most alpine meadow and grasslands of the Tibetan Plateau and adjacent areas. Such models can be an effective tool for species conservation management and wildlife fieldwork, especially in poorly surveyed regions. [Acta Zoologica Sinica 48(4):471-479, 2002]

Key words Snow finches (Montifringilla), GIS, WHR model, Tibetan Plateau

*E-mail:leifm@panda.ioz.ac.cn

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Population genetic structure and geographic subdivision of the red panda (Ailurus fulgens)

LI Ming^ＫRAO Gang^ＬＭ牧WEI Fu-Wen^Ｋ玟FANG Sheng-Guo^ＬＭ TANG Chun-Xiang^ＮHidetoshi B. TAMATE^Ｏ

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

(ＬThe Key Laboratory of Conservation Genetics and Reproductive Biology for Endangered Wildlife, Ministry of Education, China) (ＭScholl of Life Sciences, Zhejiang University, Hangzhou 310012, China)

(ＮWolong National Natural Reserve, Wenchuan, Sichuan 623000,China)

(ＯDepartment of Biotechology, Ishinomaki Senshu University, Ishinomaki 986-8580, Japan)

Abstract Red panda (Ailurus fulgens) occurs only in Sichuan Province, Yunnan Province, and Tibet in China. We used a molecular method to reveal its present phylogeographic pattern. We defined four geographic regions:Xiangling (XL), Qionglai (QL), Yunnan (YN) and Tibet (TB) based on the geographic distributions of samples. A fragment of the left domain of the control region of mtDNA was amplified and sequenced from 26 hair samples in QL, XL and TB, and 6 skin samples in YN. Among the comparable sequences of 357 bp, 29 nucleotide sites were variable, including 8 transversions and 21 transitions. A total of 16 haplotypes were identified among the four geographic regions and the haplotype diversity was 0.57 in XL, 0.93 in QL, 1.0 in YN and 1.0 in TB, respectively, which indicated a high diversity within each region. The results indicated that the average genetic distance among all haplotypes was 1.6%, and the genetic variation between and within regions were 23.41% and 76.59%, respectively. The AMOVA and phylogenetic tree analysis demonstrated that no haplotype was shared among geographic regions, no geographic clustering was observed, there was distinctive geographic phylogeography between YN and XL, QL (F_ST was 0.336 and 0.351, respectively, P<0.001), but no obvious subdivision among other regions (P>0.05). We estimated the average split time of the haplotypes to be 20 million years ago (MYA) from genetic data and fossil records. Therefore, we suggest that the present distribution pattern of red panda in China results from recent population expansion based on Fu's test of neutrality and the above-mentioned results. [Acta Zoologica Sinica 48(4):480-486, 2002]

Key words Red panda (Ailurus fulgens), Phylogeography, Mitochondrial DNA control region, Population genetic structure, Geographic subdivision

*E-mail:weifw@panda.ioz.ac.cn

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Variability of the mitochondrial DNA control region of bottlenose dolphins (Genus:Tursiops) in Chinese waters

JI Guo-Qing YANG Guang^* LIU Shan ZHOU Kai-Ya

(Institute of Genetic Resources, College of Life Sciences, Nanjing Normal University, Nanjing 210097, China)

Abstract Two distinct morphotypes of bottlenose dolphins (Tursiops sp.) in Chinese waters have been recently referred to as T. truncatus and T. aduncus.物However, the genetic composition and divergence of bottlenose dolphins in Chinese waters is not clear. 424 base pairs (bp) of the mitochondrial control region collected from 30 bottle nose dolphins from the Yellow and East China Seas, the Taiwan Strait and the Gulf of Beibuwan were sequenced. Combined with previously published mtDNA control region sequences of bottlenose dolphins from Chinese waters, 54 variable sites were determined and 37 haplotypes were identified. No shared haplotypes were found between two morphotypes, and eight fixed diagnostic site differences separate the two morphotypes. Phylogenetic relationships among haplotypes were determined using the maximum likelihood method and the neighbor joining method. Maximum likelihood analysis was performed using the Phylogenetic Inference Package (PHYLIP) version 3.5c. The distance matrix for neighbor joining analysis was generated using the Tamura-Nei Gamma distance method of the Molecular Evolutionary Genetic Analysis (MEGA). Neighbor joining analysis was also performed using MEGA with 500 bootstraps. The average number of substitutions persite within groups (nucleotide diversity) and between groups (nucleotide divergence) were estimated using DnaSP. Haplotypic diversity for the two morphotypes of Chinese waters was also estimated. Reconstruction of the phylogenetic relationships among haplotypes divided the 37 haplotypes of bottlenose dolphins into monophyletic clades representing the two morphotypes. The average nucleotide divergence between the two morphotypes is 5.58%, which is about five times greater than between Delphinus species (1.09%). These support the conclusion that the two morphotypes of bottlenose dolphins in Chinese waters represent distinct species, i.e. T. truncatus and T. aduncus. Net nucleotide divergence between the two species was 3.79%. As the substitution rate for the cetacean mtDNA control region has been estimated to be 0.5% per million years, the two species probably diverged about 7.5 million years ago (Mya). Although the distribution of the two species overlap in the Taiwan Strait (and maybe

adjacent waters), there is no genetic interchange between them, indicating reproductive isolation. In order to conserve more genetic diversity of Tursiops in the Chinese waters, the two species should be treated as independent conservation management units in conservation planning. [Acta Zoologica Sinica 48(4):487-493, 2002]

Key words Bottlenose dolphin (Tursiops), Chinese waters, mtDNA control region

*E-mail: gyang@njnu.edu.cn

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Phylogenetic relationships of 20 snake species based on partial sequences of 12S rRNA gene

TONG Zong-Zhong WANG Yi-Quan^* ZHOU Kai-Ya

(Institute of Genetic Resources, Nanjing Normal University, Nanjing 210097, China)

Abstract The conservative morphological characters of snakes complicate the analysis of phylogenetic relationships among these organisms. Recently, some authors have employed DNA sequence analysis to examine phylogenetic relationships among snakes but little work on China突s snakes has been published to date. The evolutionary history of snakes still remains controversial. In the current investigation, we try to shed light on these relationships via DNA sequence analysis of the mitochondrial 12S rRNA gene in 20 species of snake representing 13 genera from 4 families. About 800 bp of gene fragments from these species were sequenced, of which 494 variable sites were identified after combining and aligning the data with the homologous sequence of Sphenodon punctatus.物A phylogenetic tree constructed using the neighbor-joining method with Sphenodon punctatus as the outgroup indicates that the snakes examined in this study are clustered in four major clades corresponding to their four families. The first clade includes the 2 Boidae species, Erys tataricus and Python molurus biritattatus, the second is comprised of the 3 Viperidae species, Deinagkistrodon acutus, Trimeresurus stejneger and Vipera ursinii, the third contains the two Elapidae species, Naja naja and Bungarus multicinctus, and finally, the remaining 13 species of Colubridae form a large lineage in the phylogenetic tree. Consistent with the widely accepted opinion that it is the most primitive family among all current snakes, the Biodae branch diverges from the base of the tree. The Colubridae and Elapidae cluster together with a bootstrap value of 93 before they combine with the Viperidae lineage, strongly supporting the assumption that the Elapidae share an ancient ancestor with the Colubridae, rather than the Viperidae. Within the 13 species of the Colubridae, there are two major clades, the Rhabdophis and Sinonatrix clade, and theZaocys, Ptyas, Dinodon and Elaphe clade, with bootstrap values of 99 and 98 respectively. In the second clade, seven species of Elaphe form a common branch before they cluster with other genera, contrary to Dessauer's assumption of the polyphyletic origin of this genus. [Acta Zoologica Sinica 48(4):494-500, 2002]

Key words Snake,12S rRNA gene,DNA sequence,phylogeny

*E-mail: wangyqnj@jlonline.com

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Factors affecting the parthenogenetic activation of mouse oocytes with electrical stimulation

LI Jin-Song^ＫＬ牧HAN Zhi-Ming^ＫＭ牧ZHU Zi-Yu^ＫＮ牧WANG Min-Kang^Ｋ LIAN Li^ＫCHEN Da-Yuan^Ｋ*

(Ｋ State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences,Bejing 100080, China)

(Ｌ College of Animal Science and Veterinary, Yangzhou University, Yangzhou 225009, Jiangsu, China)

(Ｍ College of Life Science, Beijing Normal University, Beijing 100875,China)

(Ｎ College of Life Science, Suzhou University, Suzhou 215006, Jiangsu, China)

Abstract This study was conducted to determine the best conditions of parthenogenetic activation of mouse oocytes for nuclear transfer. Four factors〞field strength of the electric pulse (direct current), electrical fusion solutions (EFS), nuclear transfer manipulation medium pre-treatment and different mouse strains relating to the parthenogenetic activation of mouse oocytes by electric stimulation were studied. Oocytes were recovered from mice superovulated with PMSG and hCG and were denuded of cumulus with hyaluronidase. The denuded oocytes were stimulated in different EFS with three electroporations 30 min apart (two DC pulses of the same duration and different strength, 1 s apart). After cultured in CZB for 24‵30 hours, the parthenogenetically activated oocytes developed to the two-cell stage. Results were as follows. (1) With the field strength of 0.5 kv/cm‵1.25 kv/cm, the activation rates of mouse oocytes were not different (71.40%‵80.39%, P>0.05). However, when the field strength increased to 1.5 kv/cm, the rate of activation decreased markedly to 48.15%(P<0.05), whereas the rate of lysed oocytes increased markedly (29.63%, P<0.05). (2) EFS1 with sorbitol and EFS2 with mannitol had no effect on the activation rate, but it was easier to manipulate oocytes in EFS1 than in EFS2. So EFS2 could be replaced by EFS1 in mouse somatic nuclear transfers. (3) Compared with the control group, the pre-treatment of oocytes with nuclear transfer manipulation medium had no effect on activation rate under a field strength of 0.75 kv/cm. However, when the field strength increased to 1.0 kv/cm, the activation rate of oocytes in experimental group decreased significantly (P<0.05) and the rate of lysis increased significantly (P<0.05). (4) The activation rates of oocytes from Kunming and C57BL/6 mice were not different under field strengths of 0.75‵1.0 kv/cm. (P>0.05). In conclusion, the present study demonstrates that the higher activation rate of mouse oocytes was obtained in EFS1 stimulated with the field strength of 0.75 kV/cm after the treatment of oocyteds with nuclear transfer manipulation medium. [Acta Zoologica Sinica 48(4):501-505, 2002]

Key words Mice oocyte, Electrical stimulation, Field strength, Parthenogenetic activation, Electrical fusion solution, Nuclear transfer

*E-mail: chendy@panda.ioz.ac.cn

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Role of angiotensinｊon decidualization in human endometrium

WANG Qing-Bin JIAO Li-Hong YAN Jian-She LIU Jiang WANG Hong^*

(State Key Laboratory of Reprocuctive Biology, Institute of Zoology, Chinese Academy of Sciences,Beijing 100080, China)

Abstract The Angｊ levels and changes in human decidia at week 5‵9 of gestation were measured by RIA and quantitative in vitro autoradiography was used to map the density of Angｊreceptors (AngｊR). The inhibition of␛¹²⁵I␝ Angｊbinding by subtype-specific antagonist was used to examine AngｊR subtypes. The effect of Angｊ on PRL release in human decidual cells at early pregnancy was also observed. The results showed that (1) Angｊcontent was 116.8㊣8.42 pg/g wet weight in decidual tissue collected week 5 of gestation. Angｊlevels gradually increased as gestation proceeded. The highest value occurred in week 7 groups (7th w vs 5th w group,P<0.01). (2) Considerable AngｊR was detected in human decidual tissue at week 6‵8 of gestation. The Ang ｊ subtypeｉ(AT₁) receptor antagonist Losartant (Dup 753) caused substantial displacement, whereas the Ang ｊ subtypeｊ(AT₂) receptor antagonist (PD 123319) did not cause detectable displacement. Thus the AT

1 receptor was the predominant receptor expressed in human decidua at the early stage of pregnancy. (3) Angｊsignificantly stimulated PRL release in human decidual cells in a dose-dependent mode. The inhibitor of Angｊconverting enzyme (ACEI) induced the decrease of PRL secretion slightly. All the results indicated that Angｊplays an important role in the deciduialization of human endometrium, and is involved in embryo implantation and the maintance of gestation. [Acta Zoologica Sinica 48(4):506-510, 2002]

Key words Angiotensin ｊ, Decidualization, Angiotensin receptors, Prolactin, Angiogenesis

*E-mail: mwangh@panda.ioz.ac.cn

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Development of peritoneal lymphatic stomata and diaphragm lymphatic vessels in mice

LI Hui LI Ji-Cheng*

(Institute of Cell Biology & Tissue Engineering, Zhejiang University Medical College, Hangzhou310031,China)

Abstract The development and occurrence of the peritoneal lymphatic stomata (PLS) and diaphragm lymphatic vessels in mice, at different ages from the embryonic to the postnatal period, were studied with TEM, SEM and enzyme histochemistry. The data were analyzed with a computer image processing system and elescope software. The results showed that the diaphragm mesothelium was only covered with the flattened mesothelial cells (FMC) on embryonic day (ED)13. On ED 15, some cuboidal mesothelial cells (CMC) and nonage lymphatic stomata (NLS) were found scattered on the diaphragm mesothelium. However, the sub-peritoneal lymphatic capillaries did not appear until ED18. During this stage, it was noted that there was no absorptive function in NLS when trypan blue granules were injected into the peritoneal cavity. On postnatal day (PND) 1, the sub-peritoneal connective tissue and basal membrane of the mesothelial cells were spanned by the endothelial cell processes of lymphatic cells to form a sub-peritoneal channel connected with the NLS. Therefore, an absorptive route of material in the peritoneal cavity was formed and the PLS were found to have absorptive function following the application of trypan blue. On PND 5, the cuboidal mesothelial cells ridge (CMCR) appeared with the CMC area increasing and the sub-peritoneal lymphatic capillaries more abundant in the diaphragm. On PND 10, CMCR fused to form the band-like CMC area with numerous MLS in the muscular portion of the diaphragm. With the increase of age, the area and number of PLS were to increase gradually as well as lymphatic absorption from the peritoneal cavity. [Acta Zoologica Sinica 48(4):511-518, 2002]

Key words Mouse,Peritoneal lymphatic stomata,Diaphragm lymphatics,Mesothelium,Development

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

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Cloning, structural analysis and construction of homologous recombination vector of p53 gene in medaka fish (Oryzias latipes)

CHEN Song-Lin^Ｋ*玟HONG Yun-Han^Ｌ Manfred SCHARTL^Ｌ

(Ｋ Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, Shandong, China)

(ＬPhysiological Chemistry I, Biocenter of the University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany)

Abstract The tumor suppressor p53 is critical for guarding the genome from the incorporation of damaged DNA. In mammals p53 functions in the regulation of the cell cycle, cell death and differentiation. Mutation of the p53 gene can lead to cell transformation and neoplasia. Similarly, cancer is also a frequent disease in fish. In analogy to the mammalian situation fish p53 was expected to play a similar crucial role in tumorigenesis. However, by inspecting p53 cDNA sequences from tumors in different fish species, surprisingly not a single mutation was found. We have therefore considered the possibility that p53, unlike many other molecules which appear to play the same role in tumor development in higher and lower vertebrates, might have a different function in fish.

The p53 gene was cloned from the medaka fish (Oryzias latipes) using the ※long PCR" technique. Genomic DNA was isolated from the medaka embryonic stem cell-like cell line MES1 according to the standard procedure. Six pairs of overlapping primers (N8-C2, N1-C8, N15-C9, N14-C11, N21-C10 and N28-C1) were used to amplify the medaka p53 gene were as follows: N1,5∩- CAT GGA TCC TGT ACC CGA CCT G-3∩;N8, 5∩-ATC GAA TTC TGC AGT CGC CGA TTC AAA ATA TT-3∩;N14, ATC GAA TTC AGT GTT ACA GTT CCT TAT GAG C-3∩; N15, 5∩- ATC GGA TCC CAT TCT CAG GGC CAC TGC GGT CTA-3∩; N21, 5∩- ATC GGA TCC TTC TTC TTT TCC CAG ATC AAC CTT-3∩; N28, 5∩-ATC GGA TCC GGA AAT TTC ATC AGC TCA GTA AC-3∩; C1,5∩- ATC TCT AGA CTT TAT TTA ACA AGG AAT TTG GTA C-3∩; C2, 5∩-CAG GTC GGG TAC AGG ATC CAT G-3∩; C8, 5∩-ATC TCT AGA CCA CGT GCT CCG TCT TCT TGT AGA-3∩; C9, 5∩-ATC TCT AGA GAG CAG GAT GGT GGT CAT CTC AGA-3∩; C10, 5∩-GGG TCT AGA TTG TTC CAT GCA TGT GTT CTT TAA T-3∩; C11, 5∩-CTA TCT AGA GCA CAG ATT CTG ACC TCG AA-3∩. PCR products were cloned into the pBluescriptKSｊ+ vector and sequenced with the automated Alfexpress DNA analysis system. Sequence analysis revealed that the medaka p53 gene is 8 745 bp in length and consists of 11 exons. Comparison of the genomic organization of the p53 gene uncovered several differences between medaka and mammalian p53 genes: (1) Intron 1 (0.85 kb) of the medaka p53 gene is much smaller than that from the mouse (6 kb) and human (10 kb) p53 gene; (2) exon 3 (86 bp) is significantly larger than that of mammals (22 bp); (3) intron 10 (3.5 kb) in medaka p53 is 4 to 5 times larger than that in mice and humans. These data suggest that the medaka p53 gene differs from that in mice and humans in gene structure, especially in intron size. As the introns of the p53 gene in mammals contain important regulation elements and define some species specificity, these structural differences in the sizes of introns might point to a functional difference in transcriptional control.

For studying the function of p53 in fish, homologous recombination vectors were constructed on the basis of the neo and tk cassettes and a genomic fragment of p53 in multiple steps. First, two fragments (2 kb and 4.8 kb) of medaka p53 gene were amplified by Long-PCR, and ligated into basic plasmid pBluescript KSｊ+, resulting in plasmid pM53N32C10 (9.8 kb). Secondly, the 1.2-kb neo cassette gene was inserted at the unique BamHｉ site between the 2 kb and the 4.8 kb fragments in pM53N32C10 as positive selection marker. The insertion disrupts the open reading frame of the p53 gene. The plasmid in which the orientation of neo was the same as that of the p53 gene was designated as pM53+neo (11 kb). Finally, the 2.6-kb tk cassette was inserted at the Xbaｉsite in pM53+neo as a negative selection marker, generating homologous recombination vector pM53+N-K(13.6 kb). For transfection experiments, the plasmids pM53+N-K were linearized with Notｉ and transfected by electroporation. MES1 cells were plated in 10 cm-dishes at a density of 80% confluence the day before electroporation. Cells of 350 米l at 1.8℅10.7 cells/ml were electroporated in the presence of 48 米阱 of plasmid DNA using a single pulse delivered from an Easyject electroporator set at 250 V, 600 米F. Immediately after electroporation, the cells were diluted in 15 ml growth medium and plated in three 10-cm dishes. For drug selection experiments, 48 h after electrotroporation, the cells were subjected to drug selection by G418 or G418 plus Ganc. Positive selection of pM53 +N-K transfected MES1 cells resulted in 398-650 G418^rclones, while dual selection with G418 plus Ganc resulted in 56-75 G418^r-Gc^rclones. A ratio of 7.1‵11.3 between G418^rand G418^r-Gc^r colonies was obtained, demonstrating the feasibility of the p53 gene homologous recombination vector in MES1 cells. [Acta Zoologica Sinica 48(4):519-526, 2002]

Key words Medaka (Oryzias latipes), p53 gene, Structure, Homologous recombination vector, ES cell

*E-mail:chensl@ysfri.ac.cn

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Age discrimination of Selincuo schizothoracini (Gymnocypris selincuoensis)in Selincuo Lake,Tibeten Plateau

CHEN Yi-Feng^* HE De-Kui CNEN Yi-Yu

(Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072,China)

Abstract The present study validates age estimates from a suite of calcified structures (anal scales, dorsal fin spines and otoliths) and assesses the consistency of age interpretations for Gymnocypris selincuoensis from Selincuo Lake, a plateau lake in North Tibet Plateau at 4 530 m above sea level. Marginal increment ratio analysis and edge type analysis were introduced for judging the period when annuli formed. From May to August in 1997 and 1998, and December in 1997, about 5 000 individuals were collected for study. Among them 258 specimens were selected to identify the age of fish using three calcified structures. Anal scales and sectioned dorsal fin spines provided respectively the lowest (n=256, 62.30%) and highest (n=256, 98.41%) number of interpretable preparations. In most cases anal scales were uninterpretable because some annuli did not display a clear pattern of alternating zones of widely and closely spaced circuli. For juveniles, especially those with a standard body length less than 60 mm, annuli on anal scales could not be seen because the anal scales were too small and thin to be separated from the fish's body. In other cases annuli anal scales could not be recognized due to partial resorption and erosion. Although sectioned dorsal fin spines showed the highest interpretability, most results from older individuals were not reliable. Observed ages ranged from age 1 to 29 using otoliths, 1 to 20 using dorsal fin spines and 1 to 21 using anal scales. There was the same amount of bias in sectioned dorsal fin spines and otoliths with under eight annuli, but the annulus counts of sectioned dorsal fin spines were fewer than those of sectioned otoliths when the latter had more than eight annuli. There was a good agreement between anal scales and sectioned otoliths of 4‵14 annuli, but anal scales had fewer counts than sectioned otolith at 1‵3 annuli and also when they were from older individuals. The percentage of sectioned otoliths with an opaque edge increased from May to August and decreased in December. The marginal increment ratio of sectioned otoliths increased from May to August and peaked in December. Edge type analysis and marginal increment ratio analysis revealed that the annuli on otoliths form annually. Annuli formation occurred from April to June. In contrast to other calcified structures, sectioned otoliths provided a more accurate record of age. [Acta Zoologica Sinica 48(4):527-533, 2002]

Key words Selincuo schizothoeacini (Gymnocypris selincuoensis), Age, Otolith, Dorsal fin spine, Anal scale, Tibet

*E-mail: chenyf@ihb.ac.cn

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Microstructure and ultrastructure of vitellogenesis in oocytes of the stream salamander

(Batrachuperus pinchonii)

ZHANG Yu-Hui JIA Lin-Zhi

(College of Life Science, Shaanxi Normal University, Xi'an 710062, China)

Abstract The microstructure and ultrastructure of several different developmental stages of the oocytes of the salamander Batrachuperus pinchonii were observed. Special attention was paid to changes in the organelles related to vitellogenesis. The results suggest that the nuage forms a focus of aggregation for membraneous organelles. The mitochondria can develop into the precursors of yolk platelets. Both endocytic vesicles and Mylin's body play a key role in vitellogenin endocytosis. Vitellogenesis in nonmammalian verteberate species results from the harmonious activity of the ooplasm and the organelles. Vitellogenesis is the consequence of whole cell activity, therefore intracellular pathways involved in vitellogenesis are diverse rather than unitary. The development of oocytes in different species reveal different patterns, a result of differential expression of intracellular pathways during vitellogenesis. [Acta Zoologica Sinica 48(4):534-542, 2002]

Key words Stream salamander (Batrachuperus pinchonii), Vitellogenesis, Microstructure, Ultrastructure

*E-mail: yu-hui zhang@163.com

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Preparation of interspecies chicken(Gallus domesticus)/Duck (Anas domesticus) chimeras

LI Zan-Dong^ＫLIU Chun-Hai^ＫHUANG Jin-Song^ＫSHA Jin^ＫWEI Hua^ＫSUN Ming-Jun^ＫZHAO Chen^ＫJIANG Sheng-Hua^ＫHiroshi KAGAMI^Ｌ

(ＫCollege of Biological Sciences, China Agricultural University, Beijing 100094, China)

(ＬFaculty of Agriculture, Shinshu University, Japan)

Abstract Blood samples were collected from stage 14 chicken embryos, dispersed in MEM medium containing 10% FBS and 10% DMSO, frozen to -80⊥ in a BICELL freezing vessel, then stored in liquid nitrogen for three months. The survival rate of the thawed PGCs was confirmed by the Typan Blue exclusive method. Over 80% of hawed PGCs were viable. PGCs were isolated from the thawed and fresh blood of chicken embryos by Ficoll density gradient centrifugation. The concentration of PGCs at stage 14 was 0.0126%, and the concentrated fraction contained PGCs at about 27.5% purity. About 50 PGCs could be obtained from each embryo. After PGCs were thawed and isolated, about 100‵200 PGCs in one microliter were injected into the dorsal aorta of each stage 15 Maya duck embryo from which one microliter blood had been drawn prior to injection for the purpose of reducing recipient PGCs. The recipient embryos were incubated until hatching. Of 110 Maya duck embryos injected with chicken PGCs, 7.3%(8/110) survived to hatching. Duck PGCs were also isolated from the recipient embryo blood. About 30 PGCs could be obtained from each duck embryo. To identify the presence of chicken PGCs in the gonads of recipient duck embryos, in situ hybridization (ISH) was performed with a chicken W chromosome-specific DNA probe labeled with digoxigenin. The specificity of the chicken W chromosome repetitive DNA probe was determined by hybridization of the probe with female chicken blood and duck blood. Female chicken blood had a positive reaction while duck blood had negative results in all three samples. Seventy six percent of duck gonads (16/21) at different ages of development showed positive reaction at various levels after ISH. These results suggest chicken PGCs, could migrate, colonize and proliferate in duck gonads, together with duck PGCs, during development. Transferring PGCs could produce chicken/duck germline chimeras. [Acta Zoologica Sinica 48(4):543-548, 2002]

Key words Chicken, Duck, Primordial germ cells (PGCs), Inter-species chimeras

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Culture of chicken stage x blastoderm cells

DU Li-Xin^* YIN Chun-Guang

(College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong, China)

Abstract Blastoderm cells modified genetically from stage X embryos of chicken could still enter recipient embryos at the same stage of development and were the able to produce chimeras. Donor cells suspended from stage X blastoderms and recombinant plasmid P^GS1 containing the infectious bronchitis virus S1 gene were em bedded in liposome and co-cultured. The co-culture was then treated in three ways: 1. Microinjected into the receptors (n=140)at the same stage of development directly. 2. The co-culture was anti-screened with G418 before being microinjected. 3. In order to produce chimeras donor cells were cultured in a monolayer in vitro for 48 h then co-cultured with recombinant plasmid P^GS1 before being microinjected into the receptors(n=190). PCR and RAPD analyses of DNA from the blood of young chickens and embryonic tissue indicated that the hatchability of the first group(5.3%) was significantly higher than that of the second(1.4%) and the third (2.1%) (One-Way ANOVA, P<0.05). The positive rate of exogenous DNA amplification in embryonic organs from the second group was higher than that from the first or third group. These results indicate that donor cells cultured in vitro for 48 h and co-cultured with plasmid DNA might migrate into recipient embryonic tissue to produce chimeras. It is, therefore, potentially possible to produce transgenic chickens using lipofection-mediated exogenous gene transfer to blastoderm cells isolated from stage X embryos. Subsequent injection of these cells into receptors at the same stage of development could result in the production of germline chimeras. [Acta Zoologica Sinica 48(4):549-553, 2002]

Key words Chicken, Blastoderm cells, Culture in vitro, PCR, RAPD

*E-mail:lxdu@sdau.edu.cn

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Oviposition sites and their effects on the reproductive success in the Chinhai salamander

(Echinotriton chinhaiensis)

XIE Feng^Ｋ*FEI Liang^ＬYE Chang-Yuan^ＫWANG Zu-Wang^ＬCAI Chun-Mo^Ｍ

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

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

(ＭZhejiang Natural Museum, Hangzhou 310012, China)

Abstract We studied oviposition sites and their effects on the reproductive success of the Chinhai salamander物 (Echinotriton chinhaiensis). The results showed that: (1) the female had strong preferences for spawning sites and oviposition microhabitat; (2) the most suitable oviposition sites were 30 cm higher than water level and 45 cm from the water body; (3) oviposition sites usually overlapped;(4) embryos could develop and hatch normally in water as well as on land. The hatching rate on land was high (94.3%), but the number of hatchlings that successfully migrated to water was low (52%㊣11%, n=3 in the laboratory, and approximatly 20% in nature). Based on the situation in laboratory, the average hatchling survival rate at all oviposition sites was 36.7%. Owing to oviposition on land, the reproductive success of Chinhai salamander was very low;(5) protecting the spawning sites and increasing the hatchling survival rate are the keys to conserving this species. [Acta Zoologica Sinica 48(4):554-557, 2002]

Key words Chinhai salamander (Echinotriton chinhaiensi), Oviposition site, Reproductive success

*E-mail: xiefeng@cib.ac.cn

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Seasonal variation of pineal melatonin content in the toad-headed lizard (Phrynocephalus przewalskii)物

LI Ren-De^* LI Yun-Hong CHEN Qiang

(School of Life Science, Lanzhou University, Lanzhou 730000, China)

Abstract Seasonal variation in the pineal melatonin content of toad-headed lizards was determined by HPLC. The highest melatonin content (1 017㊣209 pg/pineal) occurred in spring and the lowest (335㊣68.5 pg/pineal) in winter. Melatonin levels in autumn (735㊣133 pg/pineal) were higher than in summer (597㊣94.9 pg/pineal). We conclude that pineal melatonin content has remarkable seasonal variation that coincides with photoperiod and reproductive rhythms. [Acta Zoologica Sinica 48(4):558-562, 2002]

Key words Toad-headed lizard(Phrynocephlus przewalskii),Agamidae,Pineal body,Melatonin,Seasonal variation

*E-mail: lird@lzu.edu.cn

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Toxic effects of polychlorinated biphenlys on chicken embryos

FANG Chang-Ge ZHANG Cai-Qiao^* XIA Guo-Liang QIAO Hui-Li

(College of Biological Sciences, China Agricultural University, Beijing 100094, China)

Abstract The toxic effects of polychlorinated biphenlys (PCB) on embryonic chickens, especially on liver morphology, were studied. PCB (Aroclor 1 254) was injected into yolk of Hyline fertilized eggs, with doses of 0, 1, 10 and 100 米g/egg, respectively. The survival rate of chicken embryos in each PCB treatment group was significantly lower than that of the control group. The hatching rate of 1 米g/egg group was only 40%. Subcutaneous edema and hydrocephally were found in dead embryos and newly-hatched chicks of all treatment groups. Livers of newly-hatched chickens were removed for examination of histological structures. After PCB treatment, severe liver injury was apparent. The hepatic structures were damaged and steatosis was observed, together with necrosis and cell vacuolation and pyknosis. There were more Kupffer突s cells in the hepatic sinusoid. No PAS-positive staining was found in the hepatic cytoplasm, indicating that glycogen was exhausted in the hepatic cells. The higher death rate in PCB-treated groups may be related to glycogen exhaustion. These findings indicate that PCBs reduced the hatching rate by causing severe subcutaneous edema and liver injury in chicken embryos. [Acta Zoologica Sinica 48(4):563-566, 2002]

Key words Chicken embryos,Polychlorinated biphenyls,Edema,Liver

*E-mail:cqzhang@mail.cau.edu.cn

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Expression of TGF-汐 and EGF-R mRNA in the early embryonic development of mice

YAN Yun-Qin^* TAN Jing-He ZHANG Li-Xia

(Northeast Agricultural University, Harbin 150030,China)

(Animal Science Institute, Guangdong Province Academy of Agricultural Science, Guangzhou 510001,China)

Abstract This study was conducted to examine the activity of TGF-汐 and EGF-R mRNA using the RT-PCR technique in the early embryonic development of the mouse. Expression of TGF-汐 mRNA was initially observed in the 8 cell stage embryo of the mouse. The level of expression gradually increased in the morula and blastula. Expression of EGF-R mRNA was initially observed in the 4 cell stage embryo of the mouse and 8-cell stage embryo;morula and blastula all expressed EGF-R mRNA and the levels of expression had not changed. These results confirm that epidermal growth factors play a key role in early embryonic development of the mouse, especially TGF-汐 which can stimulate and regulate embryonic development via the autocrine system. [Acta Zoologica Sinica 48(4):567-569, 2002]

Key words Mouse, RT玨PCR,TGF-汐,EGF-R,Embryo development

*E-mail:yanyq@neau.edu.cn

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Molecular cloning and sequence analysis of cystatin mature peptide cDNAs from two species of sturgeons

BAI Jun-Jie^* LAO Hai-Hua YE Xing LI Ying-Hua LUO Jian-Ren

(Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Tropical 物and Subtropical Fish Breeding and Cultivation, Ministry of Agriculture, Guangzhou510380,China)

Abstract Cystatin, a superfamily of cysteine proteinase inhibitors related to cathepsins and other cysteine proteinases, is widely distributed in animal tissues and body fluids. Although considerable attention has been given to mammalian and avian cystatins, little data exist on cystatins from other vertebrates. In order to isolate fish cystatin cDNA, total RNAs were isolated from liver tissues of the Chinese sturgeon (Acipenser sinensis) and Amur sturgeon (Acipenser schrenckii), respectively. The cDNAs encoding the mature peptides of cystatin and the 3∩untranslated region of the two species of sturgeon were amplified by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as a template. The amplified cDNA fragments were inserted into pGEM-T Easy vector and sequenced and the amino acid sequences deduced. Nucleotide sequence analysis showed that both cDNAs encode 112 amino acid residues of the mature cystatin peptide. The similarity of the two sturgeon nucleotide sequence coding regions and the deduced amino acid sequences were 99.4% and 100%, respectively. Analysis of the amino acid sequences indicate that the cloned cystatins were the homolog of the mammalian cystatin C. The amino acid residues of the functional regions are well conserved among different species, but there is considerable divergence in large portions of the coding region of two sturgeon cystatins in a variety of species. [Acta Zoologica Sinica 48(4):570-573, 2002]

Key words Chinese sturgeon (Acipenser sinensis),Amur sturgeon(Acipenser schrenckii), Cystatin, Molecular cloning

*E-mail: jjbai@163.net

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