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     

氟致小鼠睾丸组织形态结构损伤的观察

选取20只20日龄的昆明小鼠,随机分为4组,分别给予含0,50,100和150 mg/L氟化钠的饮水120 d,通过石蜡组织切片观察氟对睾丸的损伤作用。结果发现,小鼠饮用含有氟化钠的水后,曲细精管管壁断裂、结构排列紊乱;生精细胞、次级精母细胞从生精上皮脱落;精母细胞胞质和胞核中出现大量的空泡样病变,且细胞的微核率增加;精子出现畸形。这说明氟对睾丸组织结构具有明显的损伤作用。     

F1代犏牛睾丸的解剖组织结构研究

牦牛和普通牛杂交繁殖的第一代(F1)犏牛雄性不育问题已引起了牦牛学界的关注。本研究将性成熟后的F1代犏牛和牦牛睾丸制成石蜡切片,HE染色后在显微镜下观察睾丸的解剖组织结构特点。结果发现犏牛睾丸的曲细精管管壁薄,其管壁上的支持细胞和精原细胞、初级精母细胞、次级精母细胞数量较少,且一部分精原细胞死亡脱落于曲细精管中,未见精子细胞和精子。说明F1代犏牛睾丸组织结构异常,精子发生受阻。     

太湖猪不同日龄睾丸组织形态变化观察

实验旨在观察太湖猪不同日龄睾丸组织的生长发育,探讨猪睾丸曲精细管的增长及生精细胞发育规律。选取60、90、310日龄正常个体、310日龄晚熟个体(每组30头)的太湖猪睾丸组织,制样片进行组织学观察并测量分析。结果显示:随着日龄增加,太湖猪睾丸曲细精管、精原细胞、初级精母细胞以及精子细胞直径均逐渐增加;其中90日龄的睾丸曲细精管直径较60日龄极显著增加,310日龄正常个体及310日龄晚熟个体的曲精细管直径、精原细胞直径、初级精母细胞直径以及精子细胞直径较前期阶段均极显著增加,且310日龄晚熟个体的各指标均低于310日龄正常个体。结果证明90～310日龄太湖猪公猪的睾丸生长发育较快,310日龄(晚熟个体)的睾丸增长速度较正常同龄个体慢,但仍呈增加趋势。     

氟暴露小鼠睾丸形态计量学研究

为了观察小鼠氟中毒睾丸形态变化特点,研究氟对睾丸的毒性影响,选用45只健康性成熟雄性昆明系小鼠,随机分为对照组、低氟组(25 mg/L Na F)、高氟组(100 mg/L Na F),连续饲喂60 d,制作睾丸石蜡组织切片,H.E.染色后,显微镜下观察曲细精管形态变化,通过定量分析方法探讨氟对睾丸的毒性影响。结果表明,与对照组相比,低氟组和高氟组曲细精管半径、面积以及曲细精管管腔半径、面积均无显著性差异(P0.05);高氟组小鼠曲细精管细胞层数和厚度极明显低于对照组(P0.01);形态学观察显示,25 mg/L氟中毒小鼠睾丸生精细胞减少,100 mg/L Na F可导致生精细胞排列紊乱、各级生精细胞减少等。本试验可为氟的雄性生殖毒性研究提供直观、科学的参考。     

精原干细胞研究进展

精原干细胞位于雄性哺乳动物的曲细精管生精上皮基膜内侧,是一群具有高度自我更新能力和分化潜能的细胞,它能向子代传递遗传信息,在哺乳动物中一般是指未分化的A型精原细胞。在精子发生这一特殊的细胞分化过程中,精原干细胞的地位极其重要,它既具有自我更新生成新的干细胞能力,又能够不断的增殖分化形成各阶段的生殖细胞,并最终分化产生精子,承载起向下一代传递遗传信息的使命。精原干细胞凭借其特有的生物学特性,在医学、畜牧业生产等领域都有着广阔应用前景。本文就精原干细胞的生物学特征、分离纯化、培养和移植技术等研究概况作简要概述。     

不同日龄猪睾丸的组织学观察

以不同日龄二元杂交长大猪睾丸为实验材料,探讨猪睾丸生精上皮细胞的增殖和发育规律。取出7、30、60、90、150日龄猪的右侧睾丸,制样进行组织学观察。研究结果表明：随着日龄的增加,睾丸曲细精管的直径逐渐增加,30日龄后增加的比较显著,至90日龄发育的显著,然后又快速的增加;二元杂交长大公猪30日龄内,支持细胞增殖达到最大数量,随后保持恒定;随着日龄的增加,睾丸曲细精管的直径逐渐增加,在30日龄内增加的比较显著,30～90日龄内增加的不显著,然后又快速的增加,达到最大。支持细胞的个数在30日龄内增加的显著,之后增加的不显著。7日龄猪睾丸中,精原细胞处于曲细精管管腔中间。60日龄睾丸中,精原细胞基本都贴附在曲细精管基膜上。150日龄猪睾丸中有精子生成。     

野外采集牦牛睾丸组织的冷冻保存及其生精细胞的复苏培养研究

采用玻璃化冷冻方法对野外采集的牦牛睾丸组织进行冷冻,通过HE(hematoxylin-eosin)染色分析发现玻璃化冷冻后的牦牛睾丸组织曲细精管结构保存较完好,曲细精管可见大量形态完好的各类生精细胞。采用台盼蓝染色检测细胞活率,发现冷冻复苏后细胞活率可达80.20%。分别通过生精细胞和精原干细胞标志蛋白DDX4和GFRA1免疫荧光染色发现复苏后培养14天后的生精细胞和精原干细胞的数量明显减少。通过RT-qPCR对复苏后不同实验处理组牦牛睾丸细胞标志基因的表达分析,发现培养30天的生精细胞中的精原干细胞标志基因Thy1和UCHL1的表达量显著增高。因此,玻璃化冷冻保存的牦牛睾丸组织中曲细精管及其生精细胞得到了较好的保护,该方法对于其他哺乳动物生精细胞的长久有效保存具有重要的参考价值。     

牛精原干细胞的分离和纯化及体外培养的一般特性

采用两步酶消化法制备5月龄的牛生殖细胞悬液,用Percoll不连续密度梯度法分离精原细胞,接种于含10%胎牛血清的DMEM/F12培养基中,37℃,5%CO2饱和湿度培养,观察培养细胞的生长和形态变化。结果5月龄牛的曲细精管主要包含细胞为精原细胞、Sertoli细胞,每克睾丸实质收获生精上皮细胞总数平均为3.18×106个细胞,精原细胞纯化后纯度达69.27%,精原细胞主要分布于27%~35%的Percoll梯度中。牛精原干细胞体外培养6~7 d后开始分裂,20 d后精原干细胞形成小集落。结果表明用两步酶消化、Percoll不连续密度梯度法分离的精原细胞能满足体外培养的需要,可以存活并发生增殖。     

精原干细胞移植技术研究进展

精原干细胞位于雄性哺乳动物体内曲细精管生精上皮基膜内，在复杂且高度有序的精子发生过程中占有极其重要的地位，它一方面自我更新生成新和干细胞，另一方面源源不断的增殖分化形成各阶段的生殖细胞直至精子，更重要的是它能向下一代传递遗传信息，1994年，美国宾夕法尼亚大学《兽医学院的Brinster等人首次将可育供体雄性小鼠的精原干细胞注射到不育小鼠的曲细精管中，结果受体鼠产生了具有受精能力的精子并产出了正常后代^[2,3]，1996年，他们又将大鼠的精原干细胞移入10只免疫缺陷性小鼠曲细精管中，在小鼠的睾丸中产生了大鼠的精子，更另人震惊的是，在1999年同类实验中Brinster等人又使得兔和狗的精原干细胞在小鼠睾丸中增殖并分化，这些成果被视为“跨物种产生精子中的重大突破”，在干细胞领域稳定向前发展的今天，精原干细胞移植技术的发展和应用为深入探讨精子发生，干细胞状态的维持，自我更新的方式，自增殖及分化过程的启动和调控等的生物学机制的研究提供了优良的途径，可能成为一种极为有用的生物方法，本文简要概述了这项技术的主要方法以及最新进展和成果。     

Classification of the spermatogenic cycle,seasonal changes of seminiferous tubule morphology and estimation of the breeding season of the large Japanese field mouse (Apodemus speciosus) in Toyama and Aomori prefectures, Japan

The large Japanese field mouse, Apodemus speciosus, is a potential indicator of environmental stress, but this function has not been confirmed by histological studies. Since environmental stress affects the reproductive function of mice, we determined the reproductive characteristics of this species at two locations: Toyama (36°35ʹN, 137°24ʹE) and Aomori (40°35ʹN, 140°57ʹE). Mice were captured during May–November (n=119) and July–November (n=146) at these locations, respectively. We classified the breeding season from the numbers of pregnant females and young, in addition to the spermatogenic cycle and seasonal changes in seminiferous tubule morphology of males. Testicular weight was measured, and seminiferous tubule morphology was examined histologically. Fourteen stages were found in the seminiferous epithelium cycle based on acrosome formation and spermatid head morphology. At both locations, the breeding season peaked from late summer to early autumn and possibly in spring. Spermatogenic activity was classified into 4 periods from June to November: resting around June and October–November; resumptive around July; active around August; and degenerative around September. During the resting period, the seminiferous tubules consisted of Sertoli cells, spermatogonia and spermatocytes. Spermatogenesis began during the resumptive period, and spermatids were observed. During the active period, active spermatogenesis and a broad lumen were observed. During the degenerative period, spermatogenesis ended, and Sertoli cells, spermatogonia, spermatocytes and degenerating exfoliated round spermatids were observed. This study provides scientific information about the testicular histopathological evaluations of the large Japanese field mouse for its use as an index species of environmental pollution.     

A quantitative study of seminiferous tubular cells in the developing Murrah buffalo testis

We report here a systematic quantitative study of the seminiferous tubular cells of Murrah buffaloes. The most advanced germ cell types in the different age groups (months) were A(0) spermatogonia (SG) (1 and 3), early pachytene (6 and 9), late pachytene (12), secondary spermatocytes (15 and 18), elongating spermatids (21 and 24), elongated spermatids attached to Sertoli cells (30), elongated spermatids detached from Sertoli cells (36) and spermatozoa (42 and 48). Central primitive Sertoli cells (CPSC) and basal primitive Sertoli cells (BPSC) were present in the sex cord of one-month-old calves, while Sertoli cells (SC) were first seen in nine-month-old calves. The number of gonocytes were maximal at six months but they were not seen after this time. Prespermatogonia (PSG) and SG were at a maximum at nine months of age but PSG were not seen after 36 months. The number of SG decreased significantly after nine months up to 36 months of age.Although spermatocytes and spermatids appeared in earlier developmental stages, a rapid increase in their number was recorded after 36 months. The number of SC was maximal in 18-month-old animals. BPSC predominated in the sex cord of animals aged one to six months, SG at 9-12 months of age, primary spermatocytes from 15-30 months and spermatids from 36 to 72 months and in older animals. We concluded that a decrease in the number of SG in buffalo calves after nine months of age might be responsible for a delay in sexual maturity. Moreover, the small number of spermatocytes and spermatids present before 36 months of age may be associated with the low yield of different germ cell divisions and with the cellular degeneration. A rapid increase in the number of spermatocytes and spermatids after 36 months resulted in sexual maturity between 42 and 48 months.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

体外培养小鼠生殖细胞的碱性磷酸酶活性研究

研究小鼠生殖细胞体外培养时碱性磷酸酶 (AP)活性表达 ,为鉴别精原细胞提供依据。分别培养 2日龄 ,6日龄 ,12日龄仔鼠及成年鼠生精上皮细胞 2 4～ 4 8h,进行 Gomori- Takamat-su组化染色 ,观察各类细胞形态及染色特性。结果表明 ,体外培养 2 4～ 4 8h,管周细胞为铺展面积较大的不规则形状 ,细胞表达 AP活性。支持细胞 (Sertoli cell)为不规则形状 ,不表达 AP活性。生殖细胞均为圆球形。性原细胞和精原细胞均表达 AP活性 ,部分精母细胞也表达 AP活性。 AP活性表达可作为检测培养体系中小鼠精原细胞的一个辅助指标     

Spermatid giant cells, tubular hypospermatogenesis, spermatogonial swelling, cytoplasmic vacuoles, and tubular dilatation in the testes of normal rabbits

Testes of 36 normal New Zealand white rabbits (8, 15, 18, 26, and greater than 52 weeks of age) were examined by light and electron microscopy. The incidence and number of affected tubules were determined for spermatid giant cells, focal tubular hypospermatogenesis, cytoplasmic swelling of spermatogonia, intracytoplasmic vacuoles in seminiferous epithelium, and tubular dilatation. Spermatogenesis commenced at 15-18 weeks of age and was present in all rabbits by 18 weeks. Spermatid giant cells occurred in 96% of rabbits 15 weeks of age and older. Focal hypospermatogenesis was present in 14-57% of testes once active spermatogenesis began. Ninety-seven percent of testes in all age groups combined had spermatogonial swelling. Infrequent dilated seminiferous tubules were present in five rabbits. Ultrastructurally, spermatid giant cells were round cells with multiple nuclei that appeared to arise by widening of narrow intercellular bridges that normally connect spermatogenic epithelial cells. Pale-staining spermatogonia consisted of cytoplasmic and nuclear swelling with disruption of plasma and nuclear membranes. Tubules with spermatogonial swelling were more numerous in 15- and 18-week-old rabbits. There were no significant differences in incidence or extent of spermatid giant cells, focal hypospermatogenesis, cytoplasmic vacuoles, or tubular dilatation between age groups after spermatogenesis commenced. Although the cause of these changes is not known, they were frequent findings in normal rabbits 15 weeks of age and older.     

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。     

Enhanced Proliferation of Undifferentiated Spermatogonia after Treatment of Short Photoperiod Exposure in the Syrian Hamster, Mesocricetus auratus

Five Syrian hamsters were exposed to a short photoperiod (8L:16D) during 159 days. Atrophied testes were removed, fixed in Allen's solution; paraffin sections of the testicular tissues and whole-mounted seminiferous tubules were prepared. The numbers of various types of spermatogonia were investigated and compared with those in animals maintained in natural photoperiod (12L:12D). All the types of differentiated spermatogonia (A1, A2, In, B1, B2) were significantly decreased in number after the treatment of short photoperiod exposure, while undifferentiated spermatogonia (isolated, paired and aligned type) were significantly increased at stages V-VI and VII-VIII of the seminiferous epithelial cycle. This strictly local reaction of the undifferentiated spermatogonia to the loss of the differentiated spermatogonia suggests the presence of a feedback effect of a certain type(s) of differentiated cells to the undifferentiated spermatogonial proliferation. This feedback mechanism may also play an important role for regulating annual changes in spermatogenesis of seasonal breeders, not only in laboratory but also in natural habitat.