Immunomagnetic separation of normal rat testicular cells from Roser's T-cell leukaemia cells is ineffective

Elimination of contaminating malignant cells is crucial to avoiding cancer relapse in association with transplantation of autologous spermatogonial stem cells. In the clinical setting, there is presently no effective procedure for separating testicular cells from cancer cells. Here, CD4, a selective surface marker for Roser's rat leukaemic T-cells, was utilized to eliminate cancer cells from testicular cell samples from leukaemic piebald variegated (PVG) rats by magnetic-activated cell sorting (MACS). All animals receiving MACS-selected testicular cells died within 14–15 days. Only one-third of the contaminating leukaemic cells could be removed from testicular samples. An increase in antibody concentration enhanced the proportion of leukaemic cells removed from 27 to 49%. Variations in the cell size and expression of surface antigens on testicular leukaemic cells were the major obstacles to purification based on this marker. It is concluded that MACS does not prevent transmission of leukaemia to syngenic PVG rats when cells from leukaemic testes are used for testicular cell transplantation.     

Acute airway obstruction in a child with acute lymphoblastic leukaemia during central venous catheterization

We report the case of a seven-year-old girl with recently diagnosed acute lymphoblastic leukaemia (ALL) who suffered acute airway obstruction during insertion of a central venous catheter under general anaesthesia. The central airway obstruction was due to a mixture of leukaemic cells, blood clot and fibrin. There is discussion about airway obstruction both as a complication of central line insertion and secondary to ALL. The pulmonary complications of ALL, with particular reference to pulmonary haemorrhage, are detailed. The management of blood clot obstructing the central airway is discussed.     

干细胞向生殖细胞分化的研究进展

干细胞(SCs)具有在体外分化为生殖细胞的潜能,为研究生殖细胞(GCs)早期发育提供了良好的模型,并将为干细胞移植修复生殖功能提供细胞资源。本文综述了胚胎干细胞/诱导多能干细胞(ESCs/iPSCs)、新生儿附属物来源干细胞(NDSCs)以及成体干细胞(ASCs)向生殖细胞分化所取得的研究进展,同时总结了各类干细胞向生殖细胞分化时所遇到的障碍及所面临的挑战,为干细胞在生殖医学领域的应用提供理论依据。     

咖啡因对胎鼠及乳鼠睾丸生殖细胞数量及合成DNA的影响

为了深入了解咖啡因对胎儿、新生儿生殖细胞的数量及其合成ＤＮＡ的影响，本实验采用低（３×１０－４ｍｏｌ／Ｌ）、中（６×１０－４ｍｏｌ／Ｌ）、高（１．２×１０－３ｍｏｌ／Ｌ）浓度咖啡因体外培养ＳＤ孕１８天胎鼠、０天及４天乳鼠睾丸组织块，培养时间分别为１周、２周、３周，观察咖啡因作用后睾丸内生殖细胞数量的变化，并且利用计算机图象分析系统测定生殖细胞ＤＮＡ的含量，以探讨两者的关系。结果如下：（１）１８天胎鼠睾丸培养组织的生殖细胞受咖啡因影响最少，０天乳鼠次之，４天乳鼠受影响较大。（２）低浓度的咖啡因对生殖细胞数量影响较少；中等浓度的咖啡因在培养３周后才有生殖细胞数量的降低；而高浓度的咖啡因在培养２周后，已有下降，３周更明显。（３）生殖细胞数量的减少往往伴随ＤＮＡ含量的降低，由此推测高浓度咖啡因长时间培养后使生殖细胞数量减少，可能通过抑制ＤＮＡ复制发挥作用。     

激活态雪旺细胞源性神经营养因子对胚芽干细胞向神经细胞分化的影响

目的 探索小鼠胚芽干细胞(embryonic germ cells,EGCs)分离、培养的有效方法;观察激活态雪旺细胞(activated Schwann cells,ASCs)源性神经营养因子对小鼠EGCs向神经细胞分化的影响.方法 分离受精11d胚胎小鼠生殖腺嵴及同时取少量的腹壁组织共同消化培养,经胰蛋白酶消化制备成EGCs悬液,将细胞种植在饲养层细胞上.观察小鼠EGCs集落形成情况,并采用阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色对其进行鉴定.采用双酶消化组织块法结合机械分离法分离培养ASCs.神经诱导实验采用基础培养基+小鼠EGCs(空白对照组)和ASCs培养基与小鼠EGCs共培养(实验组),培养3周后对神经诱导结果进行NeuN、MBP、GFAP免疫荧光染色鉴定,计算细胞染色阳性率,对实验结果进行统计学分析.结果 小鼠EGCs呈集落性生长,集落隆起、多数呈鸟巢状、与周围饲养层细胞存在明显界限;集落内的细胞密集,呈现一个或几个核仁、核质比高的圆形或卯圆形;阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色均阳性.小鼠EGCs神经诱导1周后,倒置相差显微镜下可见EGCs克隆的边缘出现向外迁移的细胞,圆形或椭圆形,部分细胞有突起伸出.3周后,迁移的、有细长突起的细胞越来越多,NeuN、MBP、GFAP免疫荧光染色均阳性.细胞染色阳性率比较差异有统计学意义.结论 通过一种简单、经济的方法体外成功分离并获得小鼠EGCs;ASCs源性神经营养因子能够促进小鼠EGCs在体外向神经细胞分化.     

A retrospective analysis of pathological changes of testicular tissue in normal adult rats

Rat testicular model is widely used in experiments on andrology, pharmacology and reproductive toxicology. Generally, normal adult rat is considered to have normal testes. However, whether normal adult rats appeared abnormal testes have not been evaluated. The objective of this study was to evaluate the incidence of abnormal testes in normal adult Sprague Dawley (SD) rats and pathological changes in testicular tissues. Six hundred and sixteen adult male SD rats used in previous studies as controls were retrospectively analysed. Testicular tissues were stained with haematoxylin–eosin for observation of pathology. Among 616 rats, 14 rats had pathological testes, and the incidence of abnormal testis was 2.3%. In the 14 rats with abnormal testes, 10 rats were microrchidia (71.4%) and four rats showed normal testicular size. Testicular abnormality included complete interruption of spermatogenesis, partial germ cell arrest, progressive hypospermatogenesis, seminiferous epithelia vacuolation and inflammatory status. Bilateral testicular tissues had similar pathological changes in abnormal testes. The findings in this study demonstrate that the normal adult rats have abnormal testes. We should pay attention to the possibility of abnormal testes when using normal adult male rats for establishing a testicular model.     

Histological and morphometric studies on the kinetics of germ cells and immature Sertoli cells during human prespermatogenesis.

Human prespermatogenesis between the 8th week of pregnancy and six months after birth was studied in testis material of 28 male foetuses from spontaneous abortions and 81 infants who died from sudden infant death. The foetuses and infants were grouped in 10 age groups. A first steep raise in the numbers of germ cells per 20 tubular cross sections from 22.3 in the first group up to 69.5 in group 3 was observed, i.e. up to the end of the 22nd week of pregnancy. Thereafter, a continuous decrease could be observed modulated by a second slighter increase during the first 4 months after birth. The ratio of germ cells and immature Sertoli cells improves from about 1:20 at the beginning to 1:8 in group 3; afterwards it changes in favour of the immature Sertoli cells down to 1:140 at the end of the study. The initial augmentation of germ cells is interpreted as the effect of a first proliferation wave comparable to that of M-prospermatogonia in other species. The decrease of germ cells is due to the stop of germ cell proliferation and simultaneous high proliferative activity of the immature Sertoli cells.     

Serum testosterone response to single injection of hCG ovine-LH and LHRH in male rats

A biphasic pattern of testosterone secretion in response to a single injection of 100 IU hCG has been observed in the rat. Serum testosterone increased from basal levels of 8.7 pL 3.1 ng/ml (mean pL SEM) to 23.0 pL 1.4 ng/ml within 2 h of hCG-stimulation and returned to control levels by 2 days. A second, delayed, but significant increase in serum testosterone occurred, reaching a peak of 24.6 pL 4.0 ng/ml at 3 days and declining to basal values at 5 days. To study this response further, lower doses of hCG were tried. Administration of 10 IU hCG produced a single peak of testosterone, which did not occur until 24 h. Differences in the serum testosterone response were related to the concentration of hCG measured in the serum after injection, as injection of 1 IU, which failed to increase serum hCG levels above detection, was also inadequate to increase serum testosterone. The response after stimulatin with 500 μg ovine-LH or 0.1–10.0 μg LHRH was also evaluated. Injection of 500 μg ovine-LH produced a significant rise in serum testosterone reaching a peak at 2 h of 25.2 pL 2.6 ng/ml and subsequently declining over the next 48 h to control levels where it remained for 5 days. Stimulation with doses of 0.1–10.0 μg LHRH produced rapid and short increases in serum LH concentration which induced peaks of testosterone up to 48.8 pL 14.1 ng/ml 1 h post injection. No secondary peak of testosterone followed. Failure of ovine-LH and LHRH to produce a second testosterone peak suggests that this response may be due to a re-stimulation of the Leydig cell by elevated levels of hCG which persist until the fourth day after injection.     

诊断超声照射后大鼠睾丸生殖细胞的凋亡变化

目的　探讨诊断超声照射对大鼠睾丸组织细胞凋亡的影响。　方法　应用HP 85 0 0彩色多普勒超声诊断仪 ,对 72只大鼠固定持续照射睾丸。随机分为 10min组、2 0min组、30min组和空照组 ,根据取材时间不同随机分为 12h组、2 4h组和 48h组。应用原位末端标记技术 (TUNEL)检测各组凋亡细胞。　结果　各实验组凋亡细胞数均大于空照组 ,以持续照射 30min、2 4h组凋亡细胞数最多 ,达 (12 .86± 3.18)个 /HP 10 0 0×油镜 ,48h各组凋亡细胞开始下降 ,但仍高于空照组。　结论　诊断超声在同一切面持续照射 10min以上 ,可引起大鼠睾丸生精细胞凋亡 ,照射后取材时间不同 ,凋亡细胞量也不同。     

Apoptosis precedes detachment of germ cells from the seminiferous epithelium after hormone suppression by short-term oestradiol treatment of rats

Spermatogenesis is a highly synchronized process in which FSH and testosterone are considered the major regulators. Nevertheless, the mechanism by which these hormones act on germ cells is unclear. Cell adhesion has been proved to play an essential role in the regulation of programmed cell death in epithelial cells and it is now known that FSH and testosterone withdrawal results in the triggering of apoptosis as well as germ cell detachment from the seminiferous epithelium. Therefore, it seemed important to investigate whether the triggering of apoptosis in germ cells by experimental hormone suppression occurred as a result of their previous detachment from the epithelium. To achieve this goal, adult male rats were injected with 50 μg oestradiol benzoate for 1, 2, 3, 4, 5 or 10 days to suppress gonadotrophin secretion and thus intratesticular levels of testosterone. Germ cell apoptosis was assessed in testes from these animals by in situ 3' end-labelling of DNA fragments and quantified in seminiferous tubule sections at stages VII–VIII. Serial sections throughout the epididymides from these animals were analysed to search for immature germ cells detached from the epithelium. These cells were scored and quantified in non-consecutive randomly selected epididymal sections. Our data indicate that the triggering of apoptosis in germ cells precedes germ cell detachment, suggesting that detachment of germ cells from the epithelium, occurring after hormone suppression, is not necessary for germ cell apoptosis.     

Male fertility in long-term survivors of childhood acute lymphoblastic leukaemia

To study long-term testicular function following the treatment of acute lymphoblastic leukaemia (ALL) in childhood, 37 young adult males were assessed at two separate time points. The initial assessment was made by a wedge testicular biopsy after completion of treatment (median 9.7 years; range 4.1-16.3 years) and the subsequent assessment (median 18.6 years; range 15.4-26.8 years) consisted of the clinical examination of pubertal stage, measurement of serum gonadotrophins and testosterone and, in 19 patients, semen analysis. All 37 men completed pubertal development normally and had a testosterone concentration within the normal adult range. Six men showed evidence of severe damage to the seminiferous epithelium, five were azoospermic and one, who did not provide semen for analysis, had a reduced mean testicular volume (11 mls; normal greater than or equal to 15 mls) and a raised basal FSH level (13 UI 1-1; normal less than or equal to 6 IU 1-1). All six men with germ-cell damage had received either cyclophosphamide or both cyclophosphamide and cytosine arabinoside as part of their chemotherapy regimen. Approximately 10.7 years earlier all 37 men had undergone a testicular biopsy after completion of their chemotherapy. Morphological damage to the seminiferous epithelium had been calculated by estimating the tubular fertility index (TFI), which is the percentage of seminiferous tubules containing identifiable spermatogonia (age-matched normal = 100%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of the blood-testis barrier and changes in vascular permeability at puberty in rats

The effectiveness of the blood-testis barrier to water-soluble substances was assessed in rats of various ages by measuring the volumes of distribution of Cr-EDTA and albumin, and estimating the proportion of the testis made up by interstitial tissue and tubular lumen by morphometric techniques on cryostat sections of frozen tissue. The interstitial tissue volume fell from 15 days to reach adult values at about 30 days of age. A lumen was present in some animals at 15 days, and it enlarged progressively to reach adult levels at about 45 days of age. The 1-h Cr-EDTA space began to fall after 25 days, and reached adult values by 33 days; in rats aged 25 and 30 days, the Cr-EDTA space was almost twice the measured interstitial tissue volume, but even in the older rats, the Cr-EDTA space remained appreciably greater than the interstitial tissue volume. The 20-h albumin space did not begin to fall until after 33 days, and had still not reached adult values in rats aged 44 days. Thus, the functional barrier to water-soluble markers develops later and more gradually than the barrier to electron-opaque markers as used by previous authors, and its appearance correlates more closely with enlargement of the tubular lumen than with formation of the inter-Sertoli cell junctions. The rate at which the albumin space approached its final value was used to calculate the vascular permeability to albumin. This rose to a maximum between 25 days and 33 days of age, and then fell again, although adult values had still not been reached by 44 days of age.     

内皮素对大鼠睾丸间质细胞睾酮生成的影响

本研究采用大鼠睾丸间质细胞体外培养的技术 ,观察了内皮素 1对离体间质细胞睾酮分泌的影响。研究发现 ,10 -9mol/L的ET 1可显著抑制间质细胞睾酮的基础分泌 (P <0 .0 5 ) ,并且ET 1对人绒毛膜促性腺激素 (hCG)刺激睾丸间质细胞睾酮分泌也有抑制作用 ,其有效抑制浓度为 10 -10 mol/L(P <0 .0 5 )。本实验结果提示 ,ET 1呈剂量依赖性抑制睾丸间质细胞睾酮的基础分泌和hCG诱导的分泌 ,ET 1可能为睾丸内的一种局部调节多肽     

The function of the primordial germ cell in extragonadal tissues

The identity of the germ cell tumours of the pineal and the thymus with those of the testis and ovary suggests that the widely disseminated primordial germ cells might subserve some special function in these sanctuaries. It is proposed that thymic localization might be required for the conveyance of genetic haematological and immunological information and that the pineal-diencephalic localization could programme neuroophthalmic tissues prior to the development of the blood-brain barrier. The latter speculation was tested by producing allergic encephalomyelitis in thymectomized, bursectomized and thymobursectomized chickens. It was found that although thymectomy and, to a lesser extent, bursectomy decreased the severity of the experimental encephalomyelitis the combined procedure resulted in more severe inflammatory lesions. This may be due to release of suppressed intraneural immunological mechanisms in the somatically impaired bird.     

Lamellar germ cell processes: structures for possible interaction between germs cells and Sertoli cells during spermatogonial differentiation and early meiotic prophase

The recent finding of a stage-dependent topographical relationship of basally located germ cells to Sertoli-Sertoli interspaces in the rat testis (Ulvik 1983a) has suggested a focal interaction between Sertoli cells and early germ cells at the sites where 2 Sertoli cells meet over a basal germ cell. Focal, stage-dependent interaction between Sertoli cells and germ cells may require specializations of the germ cell membrane. Therefore, in the present work the surface of spermatogonia and spermatocytes has been studied under the electron microscope throughout the cycle of the seminiferous epithelium. Lamellar processes, 1–3 μm long, were found to emerge from germ cells and protrude into the spaces between the overlying Sertoli cells. The processes appeared on differentiating type A spermatogonia after stage VI, on intermediate and type B spermatogonia, and on leptotene, zygotene, and early pachytene spermatocytes; however, they were not found on type A spermatogonia prior to stage VII or on preleptotene spermatocytes at stage VII. The lamellar processes are thus stage-specific and may be sites of focal interaction with the Sertoli cells. Since the cell processes always protrude into the Sertoli-Sertoli interspaces this finding supports the hypothesis of focal, stage-dependent interaction between early germ cells and Sertoli cells via the narrow Sertoli-Sertoli interspaces.     

Differentiation potential of adipose tissue-derived mesenchymal stem cells into germ cells with and without growth factors

This study aimed to culture the adipose tissue-derived mesenchymal stem cells (AT-MSCs) with and without leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and retinoic acid (RA), and investigate their impact on the differentiation of these cells into germ cells. MSCs were separated from adipose tissue of mice, and the nature of these cells is confirmed by flow cytometry. The cells were cultured in different conditions, including MSCs grown in the presence of the growth factors, MSCs without the growth factors, MSCs cultured with combined growth factors and RA, and MSCs cultured with RA. After 2 weeks, the gene expression of c-Kit, Gcnf, Mvh and Scp3 and the protein expression of c-Kit and Gcnf were assessed by real-time PCR and Western blot, respectively. Scp3 was overexpressed in the groups supplemented with RA (p < .01). The expression of c-Kit and Mvh in the growth factor-supplemented groups was increased (p < .01). Western blot analysis confirmed the real-time PCR results. The use of the growth factors for the long-term culture of stem cells can be beneficial. However, to promote germ cell differentiation, the growth factors might be used by other meiosis inducer factors, such as RA.     

Quantitative （stereological） study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate(TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B sperrnatogonia and late elongated spermatids per testis were reduced to 51%, 66% and 14% of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51%-65% of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0% of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular test-osterone levels. (Asian J Androl 2004 Dec; 6: 291-297)