改良法分离睾丸支持细胞体外诱导异种淋巴细胞凋亡

目的改进睾丸支持细胞（Sertoli细胞）的分离方法及培养条件,探讨Sertoli细胞体外诱导异种淋巴细胞凋亡的作用机制。方法取2～4周龄的SD大鼠睾丸,采用Ⅴ型胶原酶、胰蛋白酶及脱氧核糖核酸酶二步消化法制备Sertoli细胞。用免疫组织化学法（SABC法）检测Sertoli细胞中Fas配体（FasL）、转化生长因子p,（TGF-β1）、clusterin的表达;流式细胞仪检测Sertoli细胞诱导异种淋巴细胞凋亡情况。结果在分离培养纯化过程中,Sertoli细胞占培养细胞总数的90％以上,生长状况良好,能稳定表达FasL、TGF-β1和clusterin,并且能够诱导异种淋巴细胞发生凋亡。结论应用本实验方法能够稳定获得大量高纯度的Sertoli细胞,并且能够发挥其免疫豁免的功能,从而用于细胞联合移植。     

Proliferation of Sertoli cells during development of the human testis assessed by stereological methods

Sertoli cells were studied using stereological methods in testes obtained from five children who were stillborn, and 31 individuals between 3 months and 40 years of age, who had suffered from sudden, unexpected death. The mean nuclear volume of the Sertoli cells, the numerical density of Sertoli cells, and the total number of Sertoli cells per individual were determined by point- and profile-counting of 0.5 micron sections. The nuclear volume of Sertoli cells increased from a median of 120 microns3 (range 53-130) during the period of 3 months to 10 years to 210 microns3 (170-260) in adults (greater than 25 years). The numerical density of Sertoli cells decreased from a median of 1200 X 10(6)/cm3 (870-1400) during childhood (3 months to 10 years) to 140 X 10(6)/cm3 (110-260) in adults (greater than 25 years). The total number of Sertoli cells per individual increased significantly from a median of 260 X 10(6) (130-520) during the late foetal period to 1500 X 10(6) (850-2900) in individuals from 3 months to 10 years of age. A further increase was found during puberty as the number of Sertoli cells in adults (greater than 25 years) was 3700 X 10(6) (2500-5600). These results indicate that significant qualitative and quantitative changes in the population of Sertoli cells take place after birth.     

大鼠睾丸支持细胞对骨髓问充质干细胞免疫抑制作用的影响

目的探索睾丸支持细胞（Sertoli细胞）对骨髓间充质干细胞（BMSC）免疫抑制作用的影响,为二者在移植免疫中的联合应用提供思路。方法二步酶解法处理大鼠睾丸,分离Sertoli细胞;Percoll法分离大鼠BMSC;Ficoll法分离淋巴细胞;刀豆蛋白A（ConA）刺激进行T细胞转化试验;将Sertoli细胞、BMSC和Sertoli细胞＋BMSC分别加入未经ConA处理的静止淋巴细胞培养体系和经ConA处理后的T细胞转化体系,MTT法测定淋巴细胞增殖情况,观察BMSC、Sertoli细胞或二者共培养对T细胞活化、增殖的影响。结果 BMSC、Sertoli细胞以及二者共培养对静止的淋巴细胞无明显作用。BMSC、Sertoli细胞及二者共培养对T细胞的活化、增殖均有明显的抑制作用,且Sertoli细胞与BMSC共培养时抑制作用呈现一定的协同性。结论 BMSC和Sertoli细胞均具有负性免疫调节作用,二者共培养可以进一步增强BMSC的免疫抑制效应。     

睾丸支持细胞的基本生物学特性

目的通过了解睾丸支持细胞(Sertoli cells,SC)的基本生物学特性,探讨SC鉴定的良好方法。方法联合应用复合胶原酶及差速贴壁法从睾丸组织中分离、培养SC,光镜和电镜下观察细胞的形态、MTT法测定细胞的生长曲线,观察其在体外培养条件下的增殖特性;利用免疫细胞染色及免疫荧光染色的方法,检测Fas配体(FasL)的表达,观察其免疫功能;应用吖啶橙荧光染色进行细胞鉴定。结果联合应用复合胶原酶及差速贴壁法分离、培养的细胞在光镜下呈长柱状及三角形,增殖能力强,电镜下胞核中可见特异性卫星小体,胞质中细胞器丰富,免疫细胞染色及免疫荧光染色证实其高表达FasL,吖啶橙荧光染色可见胞核中含有大量异染色质,核仁明显,证实其为SC。结论复合胶原酶及差速贴壁法分离的SC具有良好的增殖能力及免疫功能,电镜、吖啶橙荧光染色是鉴定SC方便、有效的方法。     

Selected Enzyme Histochemistry of Sertoli Cells 1. Immature Rat Sertoli Cells in Vitro

Sertoli cells were collected from the testes of 21 day old, sexually immature Wistar rats. The cells were then incubated for 10 or 14 days in culture medium with or without the addition of FSH. This time period corresponds to the time period in vivo when rat Sertoli cells undergo active differentiation with concomitant histochemical and morphological changes. Cells cultured 10 and 14 days after initial plating were processed for the histochemical detection of three esterases and four dehydrogenases by observing relative staining intensities of azo dye precipitation and formazan reaction product, respectively. Appropriate controls were established. The presence of FSH mildly increased the staining activity of LDH, SDH and G-6-PDH in both 10 and 14 day cultured cells. However, SDH staining intensity/cell did not increase to surpass LDH staining intensity/cell as it does in vivo during this period of time. Addition of FSH also slightly increased staining of non-specific esterase. Type B esterase and 3 beta-ol DH activity was not evident in 10 and 14 day cultured cells, even in the presence of FSH. Our results indicate that, based on histochemical parameters, immature Sertoli cells do not mature in culture commensurate with the cell in vivo.     

大鼠发育中睾丸支持细胞中间丝蛋白的研究

对胎龄１７天到生后４０天的大鼠睾丸支持细胞的细胞角蛋白和波蛋白进行了免疫组化观察。结果表明：胎龄１７天至出生，睾丸支持细胞的核下区波蛋白和细胞角蛋白皆呈阳性反应。纵隔处的睾丸索（将发育成直细精管和睾丸网）的细胞角蛋白反应明显增强，而波蛋白反应弱。随着发育，在将成为曲细精管的睾丸索，细胞角蛋白反应减弱，生后大多数支持细胞呈角蛋白阴性，而波蛋白的表达逐渐增强。但是确有少数支持细胞仍呈细胞角蛋白阳性。     

猪睾丸Sertoli细胞的分离、培养及其免疫豁免相关细胞因子的检测

目的 探讨猪睾丸Sertoli细胞分离、培养的方法 ,并对其体外培养的状态及免疫豁免相关细胞因子的表达进行检测,为Sertoli细胞用于移植提供依据.方法 无菌条件下取10～15日龄的湖北白猪睾丸,剥离睾丸白膜及表面血管,剪碎后用2.0 g/L V型胶原酶以及2.5 g/L胰蛋白酶和0.5 g/L DNaseI消化(两步法)分离Sertoli细胞,于37℃、含体积分数为5%CO<,2>的培养箱中培养.采用倒置相差显微镜观察细胞形态,透射电镜进行超微结构鉴定,流式细胞仪检测细胞体外活率.逆转录聚合酶链反应检测Sertoli细胞sox9、FasL、转化生长因子β(TGF-β)及Clusterin的表达,四甲基偶氮唑盐(MTT)检测细胞的活性.结果 猪睾丸经分离、培养,所获得的Sertoli细胞纯度达90%以上,培养后的细胞凋亡率为(2.61±0.96)%,死亡率为(2.12±0.74)%;培养的Sertoli细胞稳定表达sox9、TGF-β、Clusterin,而FasL表达较弱.体外培养的Sertoli细胞可以保持良好活性达21 d.结论 采用V型胶原酶以及胰蛋白酶和DNaseI两步法消化分离可获得大量高纯度的Sertoli细胞,该细胞在培养过程中能够稳定表达FasL、TGF-β和Clusterin分子.     

Age-dependent Sertoli cell responsiveness to germ cells in vitro

This study investigated the effect of germ cells (greater than 80% mid- and late-pachytene spermatocytes) on the secretion of androgen binding protein (ABP) and transferrin by monolayer cultures of Sertoli cells isolated from rats aged 10, 18 or 26 days. There was an age-dependent increase in secretion of ABP and transferrin. Treatment of the Sertoli cell monolayers with hypotonic buffer to remove residual germ cells reduced this increase significantly. On the other hand, addition of germ cells to hypotonic-treated Sertoli cell monolayers increased both basal and FSH + testosterone-stimulated ABP and transferrin secretion at all three ages, although Sertoli cells from 10-day-old animals showed the greatest response. Moreover, addition of germ cells reduced responsiveness to FSH + testosterone in Sertoli cell monolayers obtained from rats aged 18 or 26 days. In monolayers obtained from 10-day-old rats, the opposite effect was noted in the case of ABP secretion. The stimulatory effect of germ cells on ABP and transferrin secretion was proportional to their number, and was reversed 48 h after the germ cells added previously were removed by hypotonic treatment. Whereas the reversal was complete with cultures of Sertoli cells isolated from 18- and 26-day-old rats, approximately 40% of the stimulatory effect remained after removal of germ cells from cultures from the 10-day-old age group. Adhesion of germ cells to Sertoli cell monolayers was also found to be age-dependent, with the largest proportion of added germ cells adhering to Sertoli cells isolated at 18 and 26 days of age. It is concluded that germ cells can significantly and differentially modulate the basal and hormone-stimulated secretory activity of Sertoli cells in vitro and that Sertoli cell responsiveness to germ cells (pachytene spermatocytes) is age-dependent and seems to appear early during the maturation process, before these germ cells appear in the testis.     

Clusterin produced by Sertoli cells inhibits heat stress‐induced apoptosis in the rat testis

The objectives of this study were to determine whether the inhibition of clusterin expression in rat Sertoli cells enhances heat stress‐induced apoptosis. The scrotums of rats were immersed in a water bath of 43 °C for 15 min. Testicular weight and germ cell number markedly decreased after the heat treatment in a time‐dependent manner. In contrast, clusterin mRNA and protein expression levels were significantly up‐regulated and peaked on day 21. The apoptotic index was markedly increased 1 day after the heat treatment. We then purified Sertoli cells from the rat testes, and an expression vector containing siRNA targeting the clusterin gene was transiently transfected into Sertoli cells. Following exposure to heat stress at 41 °C for 12 h, clusterin mRNA was markedly up‐regulated after transfection with the control vector; however, the transfection of siRNA targeting the clusterin resulted in >70% reduction in the expression of clusterin mRNA. Furthermore, the apoptotic index in these Sertoli cells was significantly higher after the treatment with siRNA targeting the clusterin than control, and the most prominent difference was observed within 24 h after the heat treatment. These results suggest that an increase in the secretion of clusterin by Sertoli cells protects the testes from heat stress‐induced injury.     

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.     

Influence of age, hormones and germ cells on glutathione S-transferase activity in cultured Sertoli cells

Glutathione S-transferase (GSH-S-T) activity was measured, using 1-Cl-2,4-dinitrobenzene as substrate, in Sertoli cell cultures obtained from rats aged 10, 18, and 26 days. The GSH-S-T activity showed a significant increase with age of the Sertoli cell donor. When cultures were treated with hypotonic solution, in order to eliminate residual contaminating germ cells, the age dependent increase in enzyme activity was less pronounced. FSH, but not testosterone, increased enzyme activity in all cultures. Addition of freshly isolated germ cells (mainly pachytene spermatocytes) to hypotonic-treated Sertoli cell monolayers enhanced GSH-S-T activity at all ages. It is concluded that GSH-S-T activity can be measured in cultured Sertoli cells during the period of onset of spermatogenesis (10-26 days). This enzyme activity is dependent on age of the Sertoli cell donor and is influenced by FSH and germ cells. Since GSH-S-Ts are actively engaged in cell detoxificative functions through conjugation of xenobiotics with glutathione, the present findings suggest that this enzyme may have a relevant protective role during the critical period when spermatogenesis is being established.     

Transport of transferrin-bound iron into rat Sertoli cells and spermatids

Transferrin (Tf), a major secretory protein of Sertoli cells, may transport iron to spermatogenic cells. This was assessed by measuring the uptake of Fe from 59Fe-125I-labelled rat Tf by Sertoli cells and round spermatids in vitro. Uptake of Fe from labelled Tf by Sertoli cells after a 72-h pre-incubation period was linear for 20 h (approximately 18 pmol/10(6) cells/20 h), whereas the uptake of Fe from labelled Tf by round spermatids after a 16-h pre-incubation period reached a plateau by 2 h (approximately 5 pmol/10(6) cells/2 h). The corresponding net uptake of Tf by both cell types was less than 0.1 pmol. High speed supernatants prepared from Sertoli cells and spermatids labelled with 59Fe-125I-Tf were fractionated by gel permeation chromatography. Separate peaks of protein-bound 59Fe and 125I-Tf were observed. Protein bound 59Fe could be precipitated with an antiserum to rat ferritin. It is concluded that iron from exogenous Tf is transported into Sertoli cells and round spermatids in vitro, and is complexed to intracellular ferritin. However, the present results do not exclude the possibility that Sertoli cell Tf may serve purposes other than iron transport.     

大鼠SERTOLI细胞对骨髓间充质干细胞体外诱导分化的影响

目的探索Sertoli细胞(SCs)对骨髓间充质干细胞(BMSCs)成脂、成骨和成神经诱导分化的影响。方法密度梯度离心法分离BMSCs,二步酶消化法分离SCs;对第3、4代BMSCs进行成脂、成骨及成神经诱导,并与SCs共培养,观察其形态学变化。结果油红O染色显示BMSCs成脂诱导后,胞浆出现折光性强的脂滴,加入SCs后,脂滴明显减少,染色不明显;茜素红S染色显示BMSCs成骨诱导后可显示钙结节,加入SCs后,钙结节更多,结节内红染更明显;成神经诱导在加入SCs前后没有明显差异。结论 SCs可促进BMSCs成骨分化,而抑制其成脂分化,对成神经分化则没有明显影响。     

An assessment of inhibin-like activity secreted by Sertoli cells in culture using castrated adult male rats

Sertoli cells isolated from testes of 16–18 days old male rats were maintained in culture. Incubation media from these culture were pooled on day 7 and tested for its inhibin-like activity either with (SCCM) or after charcoal treatment (CSCCM) in castrated adult male rats. The assay was based on the tacit assumption that SCCM or CSCCM would specifically lower circulating blood serum levels of FSH. Subcutaneous (sc)injections of CSCCM at a dose level of 1 mg protein per rat, per day, x 3 days caused a specific suppression of FSH levels, while lower dosages of CSCCM (Protein content of 300 μg or 600 μg/rat/day, x 3 days) were without any affect on basal levels of FSH and LH. SCCM was ineffective at all dose levels tested. Intracardiac injections of varying doses of LHRH (25 to 400 ng/rat) to CSCCM pre-treated rats (200 μg/rat/day, x 3 days) failed to increase the levels of LH and FSH. These results support the presence of inhibin like activity in SCCM by a bioassay procedure alternate to in vitro pituitary cell culture system used by other investigators.     

Identification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells

Androgen and androgen receptor (AR) play important roles in male spermatogenesis and fertility, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR(-/y)) and their littermate wild-type (WT) mice. Digital gene expression analysis identified 2276 genes downregulated and 2865 genes upregulated in the S-AR(-/y) mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing 10 times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/AR-regulated genes, including 164 upregulated and 439 downregulated, were found in both S-AR(-/y) mice testis and TM4/AR cells. Using informatics analysis, the gene ontology was applied to analyze these androgen-/AR-regulated genes to predict the potential roles of androgen/AR in the process of spermatogenesis. Together, using gene analysis in both S-AR(-/y) mice testis and TM4/AR cells may help us to better understand the androgen/AR signals in Sertoli cells and their influences in spermatogenesis.     

Pattern of Sertoli cell degeneration in cryptorchid prepubertal tests

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.     

Quantitative and ultrastructural alterations in the lamina propria and Sertoli cells in human cryptorchid testes

A quantitative and ultrastructural study was performed on biopsies of human cryptorchid testes to investigate lesions in the lamina propria and Sertoli cells. Prepubertal cryptorchid testes (1-9 years of age) were classified into four groups: Type 1, testes with minimal lesions; Type II, testes with a moderate decrease in tubule diameter and spermatogonal number; Type III, testes with Sertoli cell hypoplasia and a marked reduction in tubule diameter and spermatogonal number; and Type IV, testes with Sertoli cell hyperplasia and a variable reduction in spermatogonal number. An increase in thickness of the lamina propria was found in Type II and III testes from 5 years of age onwards. These testes also showed a decrease in both the average number of peritubular cells per cross-sectioned tubule and in the average nuclear volume of these cells. Most of the postpubertal cryptorchid testes from 13- to 18-year-old youths presented a prepubertal pattern suggestive of delayed testicular maturation. Postpubertal testes from 19- to 27-year-old men were classified into three types: Type A testes showed complete spermatogenesis, mature Sertoli cells and no lesions in the lamina propria; Type B testes showed isolated spermatogonia, mature Sertoli cells, and a marked thickening of the lamina propria; and Type C testes showed isolated spermatogonia, hyperplasia of immature Sertoli cells, and a slightly thickened lamina propria. Maturation of the lamina propria was always associated with maturation of the Sertoli cells. Thickening of the lamina propria was associated with peritubular cell alterations consisting of decreases in the nuclear volume (average and total per testis) of peritubular cells and increases in the number of these cells per cross-sectioned tubule. The three types of adult cryptorchid testes appear to be the postpubertal transformation of Type 1 testes (Type A), Type II and Type III testes (Type B), and Type IV testes (Type C).     

Changes in the expression of claudins and transepithelial electrical resistance of mouse Sertoli cells by Leydig cell coculture

In the testis, tight junctions (TJs) between adjacent Sertoli cells are important for the formation of blood-testis barrier (BTB). To verify the role of paracrine interactions between the Sertoli and Leydig cells in the structure and function of BTB in testis, the expression of claudin-1 and -11, and transepithelial electrical resistance (TER) of the mouse Sertoli cells were examined under the Leydig cell coculture. TER of Sertoli cell monolayer was significantly larger under the Leydig cell coculture in comparison with the control culture. Meanwhile, the expression of claudin-1 slightly decreased and claudin-11 significantly increased in the Sertoli cells in the Leydig cell coculture compared with control. Testosterone significantly increased claudin-11 expression in cultured Sertoli cells. Taken together, it suggested that Leydig cell coculture changed the structure and functions of inter-Sertoli TJs in vitro. Interactions between Leydig and Sertoli cells might be involved in the development of functional blood testis barrier in mouse testis.     

Epidermal growth factor stimulates lactate production and inhibits aromatization in cultured Sertoli cells from immature rats

The addition of epidermal growth factor (EGF) at nanomolar concentrations to cultures of Sertoli cells from 16-day-old rats induced more than a 2-fold stimulation of lactate production after 12 h of incubation. The effects of EGF, insulin and FSH on lactate production were very similar and the concentrations that produced half maximal stimulation were 11, 22 and 25 ng/ml or, in molar terms, 1.8, 3.5 and 0.6 nM for EGF, insulin and FSH, respectively. No synergistic or additive effects of these hormones on lactate production could be demonstrated. In contrast, EGF inhibited FSH-stimulated oestradiol synthesis by more than 50%. Insulin had no effect on FSH-stimulated aromatization.