HSP70在食蟹猴睾丸不同发育阶段的表达研究

目的 观察热休克蛋白70(HSP70)在食蟹猴睾丸发育成熟过程中的表达特征,探讨其在睾丸发育成熟中的作用.方法 用Eli VisionTM免疫组织化学技术检测食蟹猴从新生猴期至成年猴期不同发育阶段睾丸HSP70的表达.结果 30只食蟹猴睾丸均表达HSP70.在新生猴期、童猴前期、童猴后期的睾丸中,HSP70主要表达于精原细胞的胞质;在青春猴期,HSP70主要表达于精母细胞的胞质中,精原细胞只有少量表达;在成年猴期,HSP70主要表达于精母细胞和精子细胞的胞质中,精原细胞则无明显表达.各个发育阶段中,支持细胞和间质细胞未见明显HSP70表达.结论 HSP70在食蟹猴睾丸不同发育阶段的表达差异,提示HSP70对睾丸的形态发育及精子发生起调节作用.     

链脲佐菌素诱导的食蟹猴糖尿病模型中胰腺巢蛋白阳性细胞的变化

目的探讨链脲佐菌素（STZ）诱导的食蟹猴糖尿病模型中胰腺巢蛋白（nestin）阳性细胞分布及生物学特性的变化。方法1型糖尿病模型食蟹猴5只,长期血糖监测和静脉葡萄糖耐量实验评价该模型的可靠性及稳定性,细胞培养、免疫荧光染色对糖尿病猴和正常猴的胰腺组织进行分析。结果（1）nestin阳性细胞在正常猴胰腺组织中主要分布于胰腺导管,少量分布于胰岛周边;而在糖尿病猴则主要分布于残存的胰岛中。（2）糖尿病猴胰岛内的大部分nestin阳性细胞同时表达胰升血糖素。结论STZ在选择性地破坏胰岛β细胞的同时,可引起胰腺nestin阳性细胞的分布及表型的变化。     

感染柯氏疟原虫的两种不同种群食蟹猴的临床表现及免疫应答

食蟹猴较恒河猴不易感染疟原虫。本文报道来源于菲律宾和毛里求斯的两种不同种的食蟹猴感染柯氏疟原虫(P.coatneyi)后在临床表现、寄生虫学、生物学及免疫学等方面的差异。     

营养性肥胖对SD大鼠睾丸eNOS和PCNA表达的影响

目的探讨营养性肥胖对SD大鼠睾丸间质细胞内皮型一氧化氮合酶（eNOS）和精原细胞增殖细胞核抗原（PCNA）表达的影响。方法选用出生后3周雄性SD大鼠23只,随机分为对照组（普通饲料喂养）和营养组（自制的营养性饲料喂养）,分别于喂养后第1~5周末测量大鼠体质量、体长,计算Lee’s指数,喂养5周后以营养组致肥成功大鼠8只为肥胖组,继续喂养4周后,打击头部处死,睾丸做石蜡切片,免疫组化染色,并与对照组比较。结果喂养9周后,肥胖组大鼠睾丸间质细胞eNOS阳性表达增强,PCNA阳性精原细胞数明显减少（P〈0.05）。结论营养性肥胖对雄性SD大鼠的生殖发育产生了影响,可能与睾丸间质细胞NOS和精原细胞PCNA的表达改变有关。     

食蟹猴自发性膝骨关节炎模型的中药干预实验

目的分析独活寄生汤干预对食蟹猴自发性膝骨关节炎(KOA)模型炎症调节的作用,为进一步药物治疗的疗效检验及安全性评估提供可靠保证。方法选取清洁级青年型和老年型食蟹猴各6只,对食蟹猴自发性KOA模型进行评价;将老年型食蟹猴随机分为干预组和空白对照组,采用独活寄生汤进行干预,对比两组全血白细胞、C反应蛋白、红细胞沉降率及关节液性状和肿瘤坏死因子(TNF)-α含量的表达变化。结果老年型食蟹猴自发性KOA模型会出现典型与人类相似的骨关节炎相关指标改变。独活寄生汤干预可下调TNF-α表达,降低全血白细胞、C反应蛋白水平,干预前后差异有统计学意义(P0. 05)。结论老年型食蟹猴自发性KOA模型能有效模拟人类自发性KOA发生发展;独活寄生汤干预可能通过抑制TNF-α的表达,降低关节软骨的炎症因子水平,从而缓解KOA病程,产生治疗作用;全血白细胞、C反应蛋白可作为疗效判断的重要参考指标。     

氯胺酮联合丙泊酚在食蟹猴动物实验中的麻醉效果观察

目的观察氯胺酮联合丙泊酚在食蟹猴动物实验中的麻醉效果。方法选取24只4～7岁雄性食蟹猴,按随机数字表法分为小牛血清组、四氯化碳组、酒精组及对照组,每组6只,造模18周后采用氯胺酮基础麻醉+丙泊酚静脉麻醉对食蟹猴行枪式肝穿活检进行麻醉,术中监测生命体征和观察麻醉效果,并对其进行统计学分析。结果造模18周后,3个造模组食蟹猴均出现肝纤维化,但以四氯化碳组肝纤维化明显;24例食蟹猴氯胺酮肌注起效时间为(4.20±1.55)min,各组氯胺酮肌注起效时间比较差异无统计学意义(P0.05);24例食蟹猴刺激睁眼时间为(6.50±2.84)min,各组刺激睁眼时间比较差异无统计学意义(P0.05);在氯胺酮联合丙泊酚麻醉实验中,四组心率、呼吸均平稳,肝穿前后呼吸末CO_2分压、心率在组内比较及3个造模组与对照组比较差异均无统计学意义(P0.05);四组肝穿前后血糖均变化明显,组内肝穿前后比较差异有统计学意义(P0.05),但3个造模组与对照组比较差异均无统计学意义(P0.05)。四组食蟹猴麻醉过程中均无无躁动、嚎叫表现,且苏醒迅速,术后无呕吐、抽搐等不良反应。结论四氯化碳可有效地进行食蟹猴肝纤维化模型造模,氯胺酮基础麻醉+丙泊酚静脉麻醉在食蟹猴动物实验中应用麻醉效果良好,具有推广意义。     

脑源性神经营养因子、TNF-α及巨噬细胞迁移抑制因子在抑郁症食蟹猴中枢边缘系统奖赏环路中的作用

目的探讨脑源性神经营养因子(BDNF)、肿瘤坏死因子(TNF)-α及巨噬细胞迁移抑制因子(MIF)在抑郁症食蟹猴中脑边缘奖赏环路中的作用。方法选取成年雌性食蟹猴12只,按照随机数字表法分为对照组(n=6)和实验组(n=6)。实验组食蟹猴于vertical-chamber拘禁笼中拘禁60 d,对照组食蟹猴仍群居于大笼中。定时观察两组动物行为时间,重点包括社会交流、进食、休息、焦虑、情感依附等时间,并注意有无外伤、疾病发生。分别在拘禁前、拘禁期间第10、30、60天、拘禁后10 d采集静脉血,检测两组各时间点BDNF、TNF-α及MIF含量。结果与对照组相比,实验组食蟹猴运动、探索和进食行为时间显著减少,抑郁行为持续时间较长(P<0.05)。与对照组相比,实验组食蟹猴BDNF含量降低,MIF及TNF-α含量增高(P<0.05);且实验组食蟹猴随着拘禁时间延长,BDNF含量逐渐降低,MIF及TNF-α含量逐渐升高(P<0.05)。结论增加BDNF含量、降低TNF-α及MIF含量,可能是中枢边缘系统奖赏环路减缓抑郁症的机制之一。     

动脉粥样硬化相关基因在高血脂食蟹猴中的差异表达

目的研究高血脂与动脉粥样硬化基因的相关性,从中筛选动脉粥样硬化早期诊断基因。方法采用温和的动脉粥样硬化膳食(0.22 mg胆固醇/卡路里,40%的能量来源于脂肪)单笼喂养中老年雄性食蟹猴,膳食诱导12个月后,从膳食诱导实验猴中筛选出10只高血脂猴。然后采用荧光定量PCR方法检测对照组和高血脂组食蟹猴外周血白细胞内113个动脉粥样硬化相关基因的表达差异。结果在食蟹猴外周血白细胞内共检测到66个动脉粥样硬化相关基因,其中18个基因随血脂升高表达显著上调(P<0.05),21个基因表达显著下调(P<0.05),另外还有27个基因无表达差异(P>0.05)。结论以上结果为后续大样本相关研究缩小了基因遴选范围,通过进一步研究可能从中筛选出基因表达水平上的动脉粥样硬化早期诊断指标。     

建立疟疾近期复发动物模型的研究 Ⅰ.食蟹猴疟原虫红外期裂殖体发育成熟的非同步性

用引自越南的食蟹猴疟原虫子孢子感染恒河猴,第8d出现原虫血症,原虫密度为17/100白细胞,隔22h后,从肝活体组织中可检查到大量红外期裂殖体,其平均密度为3.74±0.66个/mm~3肝组织,它们绝大多数尚未发育至完全成熟,其分化程度参差不齐,平均直径为34.23±7.28μm,有的甚至近似于发育至第6d的红外期裂殖体,平均直径仅为15.75±2.47μm。结果说明红外期裂殖体发育成熟并非同步。     

诱导食蟹猴疟原虫对氯喹产生适应性的初步研究

本研究用食蟹猴疟原虫—猴模型进行。初步结果表明,引自越南的食蟹猴疟原虫对氯喹可产生适应性,它较易于子孢子感染后经诱导而产生。适应性表现为受感染猴原虫再现时,再给服氯喹,随后原虫转阴,但在短间隔期内又见原虫再现。已对氯喹产生了适应性的食蟹猴疟原虫,血传转种给另外的健康猴,排除了红外期引起的复发,原虫再现的现象继续发生。适应氯喹而引起的原虫再现易误认为都是红外期引起的复发。     

Heat treatment induces liver receptor homolog-1 expression in monkey and rat sertoli cells

We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Sertoli cells. After local warming (43 C) the monkey testis, however, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Sertoli cell dedifferentiated marker. Furthermore, we isolated rat primary Sertoli cells from testes at various stages of development and treated with 43 C water in vitro. The changes in LRH-1 as well as CK-18 expression were analyzed by confocal immunohistochemistry and Western blot. The results showed that LRH-1 was stage-dependently expressed in the Sertoli cells; no LRH-1-positive signal was detected in the cells obtained from the testes of adult rat on d 60 after birth when mature spermatozoa in the testis was completed. However, the mature Sertoli cells were warmed at the 43 C water bath for 15 min, and the LRH-1 signal was remarkably induced in a time-dependent manner, just like the changes of CK-18 expression in the Sertoli cells, suggesting that the heat-induced dedifferentiation of the mature Sertoli cells might be related to LRH-1 regulation. LRH-1 expression induced by the heat treatment was completely inhibited by the addition of ERK inhibitor U0126 in the culture, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated via ERK1/2 activation pathway. Testosterone was found to have no such effect on LRH-1 expression in the monkey and rat Sertoli cells.     

Germ cell-specific cyclic adenosine 3',5'-monophosphate response element modulator expression in rodent and primate testis is maintained despite gonadotropin deficiency.

cAMP response element modulator (CREM) is an important component of the cAMP-mediated signaling pathway and is essential for differentiation of haploid male germ cells. In the rodent, testicular expression of CREM is believed to be controlled by FSH. We studied the expression pattern of CREM and gonadotropic control in the nonhuman primate and rodent testis. Adult cynomolgus monkeys (Macaca fascicularis) received daily either vehicle or the potent GnRH antagonist (ANT) cetrorelix for periods of 25 and 56 days. Rats were also exposed to vehicle or ANT for periods of 14 and 42 days. ANT treatment suppressed pituitary gonadotropin secretion, reduced testis size, and altered spermatogenesis. A rabbit polyclonal antibody raised against recombinant CREM tau and reacting with CREM alpha, -beta, -gamma, -tau1, and -tau2 at similar affinities was used for immunocytochemistry and Western blotting. CREM expression was seen in round spermatids, with highest levels during spermatogenic stages V-VII, but declined with progression of spermatid development in the primate. Similar observations were made for the rat testis. Thus, CREM expression was maximal at the onset of acrosome formation and was low or undetectable upon initiation of spermatid elongation in both species. A weak, but specific, CREM signal was seen in mid- to late pachytene spermatocytes and during meiotic division in both species. After ANT exposure, the germ cell- and stage-specific pattern of CREM expression was quantitatively retained at all time points and in both species. Northern and Western blot analysis confirmed the maintenance of testicular CREM expression despite 25 days of ANT treatment. A retrospective immunocytochemical analysis of rat testes 14 days posthypophysectomy revealed CREM signals in round spermatids. These findings demonstrate that the testicular expression of CREM is not entirely dependent on gonadotropic hormones but, rather, on the maturational stage of haploid round germ cells.     

EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro

Epidermal growth factor (EGF) superfamily of peptide growth factors (EGF-GFs) plays a role in male germ cell development, but the precise function is yet to be defined. The present study shows that EGF-GFs stimulate spermatogonial proliferation in vitro. The EGF-GF ligands, EGF, transforming growth factor-alpha and betacellulin all stimulated DNA synthesis in microdissected stage I segments of rat testis seminiferous tubules in vitro, as revealed by 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU) labeling. A fourfold increase over control of BrdU labeled cells, identified as spermatogonia, was seen after treatment with EGF. RT-PCR analysis revealed that the EGF receptors erbB1, erbB2, erbB3 and erbB4 were expressed at all stages of the spermatogenic wave, whereas differential expression was found in isolated Leydig, Sertoli and peritubular cells. The results show that EGF-GFs is spermatogonial growth factor(s) in vitro, although we have not discriminated between a direct action and an indirect effect via somatic cells. We suggest that EGF-GFs is involved in the paracrine control of spermatogenesis in vivo.     

Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment

We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII-XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd-VIII in premeiotic cells is under control of the hypothalamic-pituitary-testicular axis.     

The expression and regulation of Bcl-2-related ovarian killer (Bok) mRNA in the developing and adult rat testis.

OBJECTIVE: To study the role of Bcl-2-related ovarian killer (Bok) in the regulation of apoptosis in the testis of developing and adult rat. METHODS: Bok mRNA expression was analyzed by Northern hybridization before and after culturing rat seminiferous tubules in vitro. Seminiferous tubules were cultured with different hormones and growth factors. Changes in the expression level of Bok mRNA during testicular development was analyzed by Northern hybridization. Localization of Bok mRNA was verified by in situ hybridization. RESULTS: Bok mRNA was highly expressed in the rat testis, varying during development. Highest expression levels were found in immature rats. Highest hybridization intensity appeared to be in spermatogonia, pachytene spermatocytes and Sertoli cells. Treatment with FSH was able to inhibit spontaneous increase of Bok mRNA expression that occurred in the defined stages of the rat seminiferous epithelium. CONCLUSIONS: FSH protects germ cells from apoptosis and this protective effect may at least partly be due to the inhibition of Bok gene expression. The amount of apoptosis varies during testicular development and highest expression of Bok mRNA occurs at the time of apoptosis, suggesting a possible role for Bok in its regulation.     

Development of l-CDB-4022 as a nonsteroidal male oral contraceptive: induction and recovery from severe oligospermia in the adult male cynomolgus monkey (Macaca fascicularis)

The present study was undertaken to examine the antispermatogenic effect of l-CDB-4022 in the adult male cynomolgus monkey. Monkeys (four per group) were dosed via nasogastric tube for 7 d with l-CDB-4022 at 12.5 mg/kg.d or vehicle (d 0=first day of dosing). Plasma levels of l-CDB-4022 and its deesterified metabolite were nondetectable prior to treatment and in all vehicle-treated monkeys. Peak levels of l-CDB-4022 and its metabolite were observed at 4 h after dosing with steady-state levels apparent around d 4. Sperm concentration and total sperm per ejaculate were decreased to levels below 1x10(6) sperm/ml or sperm/ejaculate in l-CDB-4022-treated monkeys by d 17 and remained suppressed through wk 6. Sperm motility also declined to 0% for 6 wk. Testicular volume was reduced in l-CDB-4022-treated monkeys through d 21. The left testis and epididymis were removed from all monkeys on d 24. At this time, the most mature germ cells in the seminiferous tubules of testes from l-CDB-4022-treated monkeys were either spermatocytes or round spermatids. Immature germ cells, but not mature sperm, were found in the efferent ducts and collapsed epididymal lumen of l-CDB-4022-treated monkeys. A steady recovery in sperm motility, concentration, and total sperm per ejaculate was observed in l-CDB-4022-treated monkeys such that these parameters were not different from those of vehicle-treated monkeys by wk 16. Volume of the remaining testis increased in vehicle- and l-CDB-4022-treated monkeys after hemicastration; however, the increase in l-CDB-4022-treated monkeys was delayed compared with that observed in the vehicle-treated monkeys. The morphology of the remaining testis and epididymis, which were removed on wk 17, was normal. Serum inhibin B levels were increased in l-CDB-4022-treated monkeys during the dosing interval; thereafter serum inhibin B levels declined such that there was no difference between the groups by wk 3. l-CDB-4022 treatment did not affect circulating levels of testosterone, LH, FSH, or estradiol. In conclusion, these data indicate that in the cynomolgus monkey, a representative higher primate, l-CDB-4022 exerts a selective antispermatogenic action, which was reversible under the conditions of this study and thus has potential as a nonhormonal oral male contraceptive.     

Expression of Hsp70-2 in unilateral cryptorchid testis of rhesus monkey during germ cell apoptosis

We investigated the possible role of Hsp70-2 in germ cell apoptosis induced by heat stress in monkey unilateral cryptorchid testis. The study focused on in situ analysis of the testicular cell DNA fragmentation and on the possible relationship between Hsp70-2 expression and germ cell apoptosis. The TUNEL result showed that most of the germ cells were labeled in the cryptorchid testis on d 5 after induction of cryptorchidism; that with most of the apoptotic germ cells depleted, only a few germ cells were labeled on d 10; and that almost no apoptotic signal was observed in the cryptorchid testis on d 15 and thereafter. This indicates that the increasing germ cell degeneration in cryptorchid testis may take the form of apoptosis. Using in situ hybridization, immunohistochemistry, and Northern blot, we examined the changes of Hsp70-2 expression in the monkey cryptorchid testis. The level of Hsp70-2 mRNA decreased slightly, while the expression of HSP70-2 protein was almost unchanged at the early stage of germ cell apoptosis in the cryptorchid testis on d 5 and dropped dramatically along with the loss of apoptotic germ cells in the cryptorchid testis on d 10 after operation. It is therefore suggested that Hsp70-2 might not take part in inhibiting the apoptosis of germ cells at the early stage during operation-induced cryptorchid testis, and that Hsp70-2 gene does not belong to the immediate early related gene responsible for germ cell apoptosis induced by heat stress.     

Sertoli cell proliferation during prepubertal development in the rhesus monkey (Macaca mulatta) is maximal during infancy when gonadotropin secretion is robust

Although a marked pubertal increase in Sertoli cell number is a hallmark of testicular development in the rhesus monkey, the ontogeny of this somatic cell type before puberty is less clear. To clarify this issue, groups (n = 4) of neonate (1-2 d old), infant (4-5 months old), juvenile (14-17 months old), and adult male rhesus monkeys were injected with 5-bromo-2'-deoxyuridine (BrdU) 2 h before castration. Tissue was fixed in Bouin's fluid, and the percentage of BrdU-labeled Sertoli cells at each developmental stage was calculated. In addition to the labeling index, Sertoli cell number per testis for the neonate and infant groups was enumerated using standard histomorphometry and compared with that previously reported by this laboratory for juvenile and adult rhesus monkeys. The number of Sertoli cells per testis in infants (156 +/- 49 x 10(6), mean +/- SD) was 4-fold greater than that in neonates (42 +/- 12 x 10(6)). The previously established value for this parameter in juvenile monkeys was 286 +/- 121 x 10(6). Incorporation of BrdU into nuclei of Sertoli cells indicated that these cells were mitotically active at all three stages of prepubertal development. The labeling index in the neonate and infant groups (1.33% in both cases), however, was significantly greater than that in juveniles (0.25%). From the foregoing results, we conclude that Sertoli cell proliferation during prepubertal development in the rhesus monkey occurs predominantly during infancy, when gonadotropin secretion is elevated, and to a lesser extent during the juvenile phase of development, when circulating gonadotropin concentrations are undetectable.