犊牛睾丸支持细胞的分离与冷冻保存

以新生犊牛睾丸为实验对象,应用组合酶法进行支持细胞分离培养,并研究了冷冻保存后支持细胞的生长特性。结果表明:在细胞分离时,消化睾丸组织,分离曲细精管法所获得的细胞悬液中的有效细胞数高于组织剪碎法。支持细胞体外培养,4 h后开始贴壁,3~4 d铺满培养皿底壁,传代后细胞生长较快,2 d即可增殖一代。HE染色,胞质染色较淡,而细胞核染色较深,呈圆形或椭圆形位于细胞质中央或偏位,核仁明显。采用10%FBS+10%DMSO的DMEM液做冷冻液,对细胞进行冷冻保存时,支持细胞的复苏率在65%以上。解冻后的支持细胞体外培养,4h开始有细胞贴壁,24h后大部分细胞贴壁,3~4d铺满培养皿底壁。     

新生牛睾丸支持细胞的体外成熟与分化

为建立支持细胞体外分化成熟的细胞模型,本试验使用犊牛睾丸支持细胞进行传代培养,观察细胞形态的变化,通过免疫荧光染色分析波形蛋白和ZO-1的表达,通过荧光定量RT-PCR和Western blot检测细胞增殖相关基因(PI3K、Akt)、细胞紧密连接相关基因(ZO-1、Connexin-34)的mRNA和蛋白表达的变化。结果表明,随着传代次数增加,支持细胞的细胞质充分扩展,逐渐趋于成熟状态;波形蛋白和ZO-1在未成熟与成熟的支持细胞都呈阳性;支持细胞成熟分化后,PI3K的mRNA和蛋白表达水平显著下降(P<0.05),Akt的mRNA和蛋白表达水平未见明显改变(P>0.05);ZO-1和Connexin-34的mRNA及蛋白表达水平升高,但差异不显著(P>0.05)。体外传代培养能够促进支持细胞的分化成熟。     

新生牛睾丸未成熟支持细胞的分离培养与鉴定

为了探究中国西门塔尔牛新生牛睾丸未成熟支持细胞的体外培养特性,为细胞水平研究与繁殖性状相关候选基因奠定基础.以新生牛曲细精管为试验材料,利用组合酶消化法将其解离成单细胞悬液,采用差速贴壁法纯化未成熟支持细胞后进行体外培养与鉴定.结果 表明:福尔根染色结果可见所分离的细胞具有明显的双极小体;RT-PCR检测结果显示,所分...     

牛乳腺组织的冷冻保存及乳腺细胞的分离

为研究牛乳腺组织冷冻保存的可行性,将牛乳腺组织剪成直径小于0.5 mm的组织块,以含20% NBS和10% DMSO的DMEM/F12作为冷冻保护液,按如下程序进行冷冻保存:4 ℃平衡1 h,－20 ℃平衡2 h,－40 ℃平衡2 h,－80 ℃保存。1周后,复苏牛乳腺组织,检测组织的存活率,分离培养乳腺细胞,检测细胞的存活率,观察细胞生长状态。结果显示,复苏后的牛乳腺组织可重新贴壁生长,贴壁存活率为62.5%,经消化离散,可分离培养出乳腺细胞,细胞存活率为58.5%,培养24 h后细胞存活率为46.3%。随着培养时间延长,细胞活力恢复,第1代细胞存活率为93.2%,生长状态良好。该乳腺组织的冷冻方法操作简单、方便、快捷,且很好地保持了乳腺细胞的活力,为稳定和增加乳腺细胞的来源提供了很好的途径,为进一步研究乳腺的结构和发育提供了素材。     

用液氮冷冻保存犊牛睾丸原质细胞的试验

犊牛睾丸细胞作为多种病增殖的基质,己经应用在疫苗生产中,并有广泛的开发前景,但是由于产犊季节的不均衡,给生产带来了困难,为了使生产不受犊牛睾丸来源不足的影响,我厂自1994年起,做了用液氮冷冻保存犊牛宰丸原质细胞的试验,取得了令人满意的结果。     

新生牛睾丸生殖细胞体外培养

实验探讨牛雄性生殖干细胞的培养体系,以期为牛精原干细胞多能性的维持和诱导分化成精子的深入研究奠定基础。为此,从新生犊牛睾丸组织中分离纯化牛雄性生殖干细胞,并在LIF和EGF等不同条件下培养,观察新生牛睾丸生殖细胞在体外的生长行为。结果表明:体外培养新生牛睾丸生殖细胞能得到包括EG细胞集落在内的多种细胞集落,LIF,EGF促进EG细胞集落形成。EG细胞集落接种于无分化抑制剂的环境下培养,3 d后细胞开始分化,1周后分化为多种细胞。     

利用血清冷冻保存犊牛睾丸原代细胞试验

在不利用冷冻保护剂的条件下,用犊牛血清对经胰酶消化分散的犊牛睾丸原代细胞进行超低温冷冻保存试验。结果表明,解冻后细胞成活率达75.73%以上,培养时可有效形成单层细胞,满足常规试验要求。复苏后的犊牛睾丸原代细胞于体外培养时存在贴壁迟缓,前期生长速度较慢等现象,且要求pH值低于7.0。     

不同浓度海藻糖对奶山羊睾丸组织冷冻保存效果的影响

为了研究含有不同浓度海藻糖的冷冻保护液对关中奶山羊睾丸组织冷冻保存的效果,将关中奶山羊的睾丸组织切碎后,冷冻保存在含有不同浓度海藻糖的冷冻保护液中,保存1个月后解冻,分别检测冷冻保存前后睾丸组织总细胞活力、总抗氧化酶(T-AOC)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性以及谷胱甘肽(GSH)、丙二醛(MDA)含量。结果:各试验组总细胞活力、T-AOC、SOD、CAT活性以及GSH含量均高于对照组,各试验组MDA含量低于对照组。另外,经含有15%海藻糖的冷冻保护液保存后,总细胞活力显著高于其他试验组(P0.05),T-AOC、SOD、CAT活性和GSH含量均显著高于其他试验组(P0.05),MDA含量显著低于其他试验组(P0.05)。研究结果表明,在冷冻保护液中添加海藻糖能够通过保护组织中抗氧化酶活性,提高奶山羊睾丸组织冷冻保存的效果;添加海藻糖的最佳浓度为15%。     

成年兔睾丸支持细胞的分离纯化研究

本试验以5月龄公兔为试验动物,去除睾丸被膜及血管,分离出完整的曲精细管后用0．5％胰蛋白酶和0．1％胶原酶消化并进行低渗处理,将制成的细胞悬液植入25cm^2的培养瓶,于38．7℃、5％CO2条件下培养,结果表明：经1：3Hanks超纯水低渗处理后,离体支持细胞纯度和活率分别达到72．04％和84．5％,显著提高了离体支持细胞的获取量和活率。     

成年兔睾丸支持细胞的分离纯化研究

本试验以5月龄公兔为试验动物,去除睾丸被膜及血管,分离出完整的曲精细管后 用0．5％胰蛋白酶和0．1％胶原酶消化并进行低渗处理,将制成的细胞悬液植入25 cm2的培 养瓶,于38．7℃、5％CO2条件下培养,结果表明:经1:3Hanks超纯水低渗处理后,离体支持 细胞纯度和活率分别达到72．04％和84．5％,显著提高了离体支持细胞的获取量和活率。     

Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls

Testes were obtained from 34 Hereford or Angus bulls at about 1.5 yr of age and were used to investigate the relationship between the absolute number of Sertoli cells vs testicular size and daily spermatozoal production (DSP). Quantitative determination of DSP was based upon enumeration of elongated spermatids in testicular homogenates. The ratio of step 8 spermatids to Sertoli cells (S:SC) was established by direct counts of these cells in each of 20 round stage VIII seminiferous tubular cross sections for each bull. The number of Sertoli cells per paired testes was calculated as (total spermatids divided by S:SC)/.394, where total spermatids equalled the number of homogenization-resistant spermatids. The factor of .394 adjusted for the fact that the latter cells are present for only 39.4% of the spermatogenic cycle. All data were subjected to simple linear and second-order regression analyses. A positive linear relationship (P less than .005) was found between testicular weight (Y, in grams) and the absolute number of Sertoli cells per paired testes (X, in billions), which was characterized by the equation Y = 315.2 + 10.74X and a coefficient of correlation (r) of .56 (P less than .01). A similar relationship was observed between DSP (Y, in billions) and Sertoli cell numbers (X, in billions). This was characterized by the equation Y = 1.36 + .222X (P less than .005) and a coefficient of correlation of .70 (P less than .01). Daily sperm production was unrelated to the S:SC ratio (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro‐matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.     

Flow cytometry evaluation of testicular and sperm cells obtained from bulls implanted with zeranol

Flow cytometry of testicular and sperm cells was used to evaluate effects of pre-weaning zeranol implants on spermatogenesis. Forty five Angus-Simmental bulls were randomly assigned to three treatment groups of 15 bulls each: no implant, one implant at 30 d of age and two implants, one at 30 and the second at 120 d of age. Prior to slaughter at approximately 15 mo, semen was collected from 30 bulls, 10 of each group. Following slaughter, testes were weighed, and testicular biopsies and vas deferens sperm obtained from the same 30 bulls. Testicular and sperm cells were stained with acridine orange and measured by flow cytometry. Proportions of testicular haploid, diploid and tetraploid cells were determined by relative amounts of green (DNA) and red (RNA) fluorescence. Treatment of sperm at low pH prior to acridine orange staining potentially induces partial denaturation of DNA, detectable by the metachromatic shift from green (native DNA) to red (single-stranded DNA) fluorescence. The effect of this shift was quantified by alpha-t [alpha t = red/red + green) fluorescence]. Nonimplanted bulls had heavier (P less than .01) testicular weights than treated bulls. The proportion of haploid cells was greater (P less than .02) and diploid cells less (P less than .03) in testes of nonimplanted bulls. Sperm from implanted bulls had altered chromatin structure, indicated by higher (P less than .05) alpha t values. Flow cytometry is an effective means for detecting changes in testicular cell subpopulations and chromatin structure of sperm.     

奶山羊睾丸组织冷冻保存及复苏后精原干细胞的分离培养

以关中奶山羊为材料,比较了2种玻璃化冷冻法和1种慢速冷冻法对睾丸组织保存的效率,复苏后发现3种冻存方法均有较高的成活率(超过50%),其中玻璃化Ⅱ组达65%以上,培养及检测后均有精原干细胞存活,在体外培养可以形成胚胎干细胞样克隆,其表达精原干细胞和多能性干细胞的相关特异性标记:碱性磷酸酶(AP)、CD49f、CD133、C-Myc、Oct4和Klf4阳性,证明睾丸组织冷冻可以提供一种有效的奶山羊精原干细胞的保存方法。试验中所使用的低温保存方法简便易行,效果良好,表明这些方法可为癌症治疗等造成的无精症及少精症等不育症患者的医治以及一些珍稀濒危物种和优良畜禽的生殖细胞保存提供一种简捷有效的途径。     

Testosterone production after in vitro stimulation of testicular cells with gonadotropins in bulls

The response of bulls' testicular cells to the stimulation by serum gonadotropin ad us. vet. (Bioveta, Ivanovice in Haná) was investigated in in vitro tests. The testicular tissue was sliced, collagenase-split and the loosened cells were stimulated by gonadotropin concentrations of 7.8 to 250 I U per l; control samples without gonadotropin treatment were used for comparisons. The testicular cells responded to the increasing stimulation by higher production of testosterone.     

Restoration of spermatogenesis in infertile male chickens after transplantation of cryopreserved testicular cells

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze).

2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks.

3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype.

4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue.

5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.     

Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue

Lung dendritic cells (DCs) are potent antigen presenting cells (APCs) that initiate and modulate the adaptive immune response upon microbial infection within the pulmonary environment. For the first time, neonatal and adult lung DCs in a large animal model were compared in these studies. Here, we isolated and identified lung DCs in both neonatal and adult sheep, a valuable experimental animal utilized in pulmonary studies of naturally occurring respiratory diseases. Neonatal lung DCs exhibited characteristic dendrites and morphology when observed by transmission electron microscopy and expressed low to moderate DEC-205, CD80/86, MHC class II and CD 14. Regardless of age, lung DCs were functionally able to endocytose FITC conjugated ovalbumin but to a lesser degree than monocyte-derived DCs. In addition, neonatal lung DCs were demonstrated to be potent stimulators of allogeneic T cell proliferation. Together, these results demonstrate that neonatal and adult lung DCs are functionally similar. It is apparent from the data presented that neonatal pulmonary DCs do not exhibit an intrinsic functional defect that would impair their ability to take up antigen and stimulate na?ve T cells. These data support growing evidence that neonatal immune responses may differ from adults due to different microenvironmental influences rather than differences in dendritic cell maturation states.     

Effects of testicular biopsy in clinically normal bulls

OBJECTIVE: To determine whether testicular needle biopsy is detrimental to testicular function in clinically normal bulls. DESIGN: Prospective study. ANIMALS: 6 mixed-breed mature bulls. PROCEDURE: A randomly selected testicle from each bull was biopsied with a 14-gauge needle biopsy instrument. Bulls were then evaluated over a 90-day period for changes in scrotal temperature and thermal patterns, ultrasonographic appearance, and quality of spermatozoa. At the end of the 90-day study, bulls were castrated, and testicles were examined grossly and histologically. RESULTS: Changes were detected in scrotal temperatures and thermal patterns and in the breeding soundness examination results during the first 2 weeks of the study. However, there were no long-term changes in semen quality over the course of the experiment. Hyperechoic areas were detected on ultrasonographic examination and corresponded to the areas of penetration by the biopsy instrument. Microscopic lesions that were indicative of testicular dysfunction were not found. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that testicular biopsy is a safe procedure in bulls. Testicular biopsy could possibly be used to further examine bulls that have less than satisfactory results for breeding soundness examinations.