Preservation of common carp germ cells under hypothermic conditions: Whole tissue vs isolated cells

The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L‐15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The 2‐week trial demonstrated that storage media have a profound influence, while regular medium exchange does not have a positive effect on cell viability. Viability of SSCs and OSCs after two weeks was approximately 40% and 25%, respectively; however, survival of ~70% was obtained after 10 days of storage for SSCs and 7 days for OSCs. Hypothermic storage developed in this study has many practical applications during the development of surrogate broodstock technologies for common carp, but also in carp hatcheries and for the conservation of genetic resources of closely related cyprinid species.     

Fine Structure of Spermatogonia and Spermatocytes in the Rlue Fox (Alopex Lagopus)

The ultrastructural features, characterizing the different types of spermatogonia and spermatocytes in the blue fox, have been studied within and near the reproductive season, and also in the summer and autumn.Two distinct types of spermatogonia — A and Β — are described. The A-spermatogonia often have a prominent nucleolus and numerous cytoplasmic organelles including characteristic whorls of AER. Large vacuoles containing electron dense particles are sometimes observed. In the B-spermatogonia the chromatin forms condensed areas of varying size, and the nucleolus is usually absent. The number of cytoplasmic organelles is generally small.Ultrastructural characteristics are further used to distinguish between the different stages in the prophase of the primary spermatocytes. In leptotene the nucleus contains a thread-like chromatin with electron dense peripheral areas. Towards the end of the stage the mitochondria display dilated cristae, and aggregations of a granular material can be observed in the intermitochondrial matrix. Zytogene is characterized by the appearance of syniaptinemal complexes in the nucleus, and of the chromatoid body and piles of annulate lamellae in the juxtanuclear cytoplasm. In pachytene the chromosomes become apparent as aggregations of condensed chromatin associated with the synaptinemal complexes. The Golgi complex is more prominent than in the previous stages, and the number of the other cytoplasmic organelles is increasing. In the last stages of the prophase (diplotene and diakenesis) the chromosomes become still more electron dense, the nucleolus appears as a very prominent structure, and there is a marked vesiculation of the cytoplasm.The secondary spermatocytes have a characteristic nucleus with a somewhat irregular outline and larger peripheral areas of condensed chromatin. In the cytoplasm a double Golgi complex is frequently observed.In the summer and autumn spermatocytes in zygotene seem to represent the most advanced form of spermatogenic cells.     

鸡精原细胞分离纯化与体外培养初探

本研究以组合酶分步消化法并利用Percoll梯度分离、贴壁纯化的方法，从不同时期的鸡睾丸组织中分离精原细胞。结果发现：①Percoll分离后，出壳6d雏鸡获得的细胞总数、精原细胞总数、精原细胞纯度分别为282．1、174．6、61．9％，其中精原细胞纯度比孵化15d、19d鸡胚和出壳后13d雏鸡分别高10．2％、5．7％和2．5％；②贴壁纯化后精原细胞纯度达到82％，比未经贴壁纯化的精原细胞纯度高出15．6％；③在无饲养层培养条件下，比较Percoll梯度分离前、后鸡精原细胞体外培养的情况，Percoll梯度分离前精原细胞贴壁在时间上比分离后较快，且分离前精原细胞体外存活时间亦比分离后长。     

精原细胞分化与凋亡的影响因素

人们对哺乳动物精子发生过程已经有了相当多的了解，但对精子发生过程的调控机制仍然了解甚少。生殖激素及原癌基因和肿瘤抑制基因的表达产物对维持正常精子发生中各类细胞的存活、增殖和凋亡，以及各类细胞受损伤后的清除均具有十分重要的作用。本文论述了促卵泡激素、雌激素、雄激素、干细胞因子、Bcl-2家族蛋白及p53蛋白在原细胞的分化和凋亡中的作用，以便人们进一步了解精子发生的调控机制。     

鸡精原细胞分离与纯化初探

采用二酶三步消化法处理鸡睾丸组织,获得的细胞悬液以Percoll作为介质并结合细胞选择性贴壁培养纯化精原细胞。结果发现：1mg/mL胶原酶 0．25％胰蛋白酶 0．25％胰蛋白酶处理鸡睾丸组织,获得的细胞总数多达5．8×10~6个；Percoll离心后精原细胞主要位于19％～35％梯度层中,纯度平均达到66．4％；贴壁纯化后精原细胞纯度则达到82％,比未经贴壁纯化的精原细胞纯度高15．6％。     

Differentiation and development of Leydig cells,and changes of testosterone levels during testicular differentiation in tilapiaOreochromis niloticus

Initial appearance and development of Leydig cells (LCs) during testicular differentiation in tilapia,Oreochromis niloticus, were investigated histologically. In addition, changes of testosterone levels in gonadal tissue and serum were examined by radioimmunoassay. In the gonads of fry at 23–26 days after hatching, initial testicular differentiation was confirmed by the observation of the differentiation of connective tissues into tissues which are characteristic of the adult testis. LCs, which were identified by the ultrastructural features (a moderate number of mitochondria with tubular cristae, well developed smooth endoplasmic reticulum and many free ribosomes) appeared initially at the time of testicular differentiation. LCs increased in number rapidly in the testes of fish at 70 days after hatching. Concomitant with this increase, spermatogonia increased in number. Testosterone was detectable in the fish at 40–50 days after hatching, but levels in tissue and serum were low. Testosterone levels increased gradually in the fish beginning at 70 days after hatching and increased still more at 100–150 days accompanying active spermatogenesis.     

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.     

小鼠精原细胞体外增殖与冷冻保存

利用一步酶法将7日龄昆白系小鼠睾丸分离获得生精上皮单细胞悬液(主要含精原细胞和支持细胞),分别接种于D-MEM、D-MEM/F12和KSOM后,系统研究其精原细胞的存活和增殖情况,并对生精上皮单细胞、曲细精管及完整睾丸进行了-70℃冷冻保存,同时对生精上皮单细胞进行了液氮冷冻(-196℃)保存。结果表明,小鼠精原细胞在D-MEM和D-MEM/F12中均能存活并增殖,表现为在培养的第1～9天/1～8天呈逐渐减少趋势,第9～13天/8～12天呈增殖趋势,第17天/16天以后增殖减缓,而在KSOM中只短暂存活不能增殖。生精上皮单细胞、曲细精管及完整睾丸-70℃短期(1周和2周)冷冻保存及单细胞悬液液氮短期(2周)和中长期(1和3个月)冷冻保存后,均可获得较理想的细胞存活率(均大于85%)。     

小鼠实验性隐睾诱发生殖细胞类型变化

利用 3 0～ 3 5日龄昆白系小鼠制作实验性隐睾 ,定期分批朴杀取样 ,检查隐睾组织学及生殖细胞群体变化 ,为生殖细胞富集及提高体内精原干细胞转基因效率提供条件和依据。结果表明 ,盆腔隐睾精子发生被阻断于精子形成阶段 ;经历 1 5d以上 ,曲细精管内精子数量较少 ;腹腔隐睾精子发生被阻断于精原细胞向精母细胞过渡阶段 ;经历 3 0 d以上 ,曲细精管仅由精原细胞、少量精母细胞及支持细胞组成。由此可知 ,制作盆腔隐睾 ,可得到含少量精子的生殖细胞群体以及主要含精原细胞的生殖细胞群体