Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Social organization and range use in the Yunnan snub-nosed monkey Rhinopithecus bieti

We studied social organization, behavior, and range use of the Yunnan snub-nosed monkey (Rhinopithecus bieti) at Wuyapiya (99 degrees 12'E, 28 degrees 30'N, the People's Republic of China) over 12 months between May 1992 and June 1994. The Wuyapiya band contained greater than or equal to 175 members and had two levels of social organization. At one level, the monkeys formed multifemale, one-male units (OMUs) similar to those of many other colobines. At another level, 15 to 18 OMUs traveled together in a cohesive band. Unlike the bands of other species of Rhinopithecus, the Wuyapiya band of R. bieti did not show seasonal fission-fusion, although some social behavior, such as male-male aggression, was seasonal. With regard to range use, the Wuyapiya band had a large home range and long daily travel distances compared with other colobines. Minimum range size in 1 year at Wuyapiya is 16.25 km(2), although there is no asymptote for range size as a function of observation time. Range size for the Wuyapiya band is 25.25 km(2) over the 2-year study and appeared to cover 100 km(2) between 1985 and 1994. The primary food of R. bieti at Wuyapiya is lichens, which are ubiquitous in fir frees. The multitiered social organization of R. bieti appears to result from the interaction of food resource characters with the forces of mate competition, with band sizes based on female responses to the spatial and temporal characteristics of lichens and subdivisions within bands based on male competition for mates.

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

Metrical dental analysis on golden monkey (Rhinopithecus roxellana)

Dental variation in the Chinese golden monkey (Rhinopithecus roxellana) is here evaluated by univariate, bivariate, and multivariate analyses. Allometric analyses indicate that canines and P3s are positively, but other dimensions negatively scaled to mandible and maxilla, and to body size. With the exception of the mesiodistal dimensions of I-1 and M-3, and the buccolingual dimension of Pq, mandibular dental variables show similar scaling relative to body size. Analysis of residuals shows that males have significantly larger canine, P-3 and buccolingual dimensions of the postcanine teeth (M-2 and M-3) than females. A significant difference in shape between the sexes is found in the buccolingual dimension of the upper teeth, but not in the mandible. Unlike the situation in some other species, Female golden monkeys do nor exhibit relatively larger postcanine teeth than males, in fact, the reverse is true, especially for M(2)s and M(3)s. The fact that most of the dental variables show low negative allometry to body size might be related a cold environment that has led to the development of larger body size with I-educed energy loss. When the raw data are examined by Discriminant Function Analysis the sexes are clearly distinguishable.

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4+ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.

恒河猴大脑前额叶cDNA 文库的构建

为筛选出与认知、记忆相关的脑部表达基因及基因家族,利用TRIzol试剂从恒河猴大脑前额叶组织抽提总RNA,再用纯化试剂盒从总RNA中成功纯化出mRNA。按照Stratagene公司的cDNASynthesisKit(200401)、ZAP-cDNASynthesisKit(200400)和ZAP-cDNAGigapackⅢGoldCloningKit(200450)三个试剂盒的操作说明,构建了恒河猴大脑前额叶组织的cDNA文库。文库总库容为2·0×106克隆;绝大多数的cDNA插入片段≥0·5kb,平均长度≈1·0kb;cDNA片段与噬菌体载体重组率为97·3%。文库各项指标均达要求,为克隆大脑PFC区表达基因、测定基因编码区序列、揭示具有多种剪切组合模式等提供了可靠资源,也为相关基因表达调控的研究提供了方便。

猕猴胚胎干细胞的分离培养和鉴定

目的　从体外培养成熟囊胚中分离并鉴定猕猴胚胎干细胞(embryonic stem cell , ES cell) 。方法　猕猴卵母细 胞经体外成熟培养、体外受精和早期胚胎体外成熟培养后,获得猕猴囊胚。当囊胚由透明带自然孵出后,用细玻璃针剥离 囊胚中的内细胞团(inner cell mass , ICM) 并与饲养细胞进行共培养。由ICM分离,培养并鉴定胚胎干细胞集落。结果　由 4 只FSH 超排猕猴中共取得92 个处于GV 期的猕猴卵母细胞,选取其中的22 个用HECM210 培养基培养后,获得6 个高质 量的囊胚,由此6 个囊胚中分离得到3 个内细胞团,并由此最终获得1 株猕猴ES 细胞,即RS5 细胞。RS5 细胞具高比例核P 质比,核仁多,其细胞集落边缘平整,其内各单个细胞清晰。经约5 个月的连续传代后,仍保持了正常二倍体的核型,其染 色体数目为42 条。碱性磷酸酶细胞组织化学染色为阳性,说明RS5 细胞为未分化态的胚胎干细胞。经高密度和长时间 培养后,RS5 细胞可进一步分化为多种类型细胞。结论　RS5 细胞株具有自我更新能力和多分化潜能,属于胚胎干细胞。

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from greater than or equal to 1000 mum diameter follicles

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.