Evaluation of blood–testis barrier integrity in terms of adhesion molecules in nonobstructive azoospermia

Blood–testis barrier (BTB) is critical for maintaining fertility. The integrity of tight junctions (TJs) provides restricted permeability of BTB. The aim of this study was to evaluate the relationship between BTB and Sertoli cells. Testicular sperm extraction (TESE) obtained from nonobstructive azoospermia (NOA) patients was examined: Group I (spermatozoa + ) and Group II (spermatozoa − ). The tissues were stained with haematoxylin eosin, periodic acid–Schiff and Masson's trichrome for Johnsen's score evaluation. Apoptosis and adhesion molecules such as claudin-11, occludin and ZO-1 were assessed. In Group I, the integrity of the seminiferous tubules was intact. In Group II, some seminiferous tubule walls were lined only with Sertoli cells, had a thickening of the basement membrane, and oedema in interstitial spaces. In Group I, the seminiferous tubule consisted of a stratified columnar epithelium, claudin-11 expressions were observed as linear staining in the basal zone of the tubule, while seminiferous tubules, with low epithelium, displayed a punctate type of staining. Immunohistochemical observations were consistent with the ultrastructural findings. In Group II, high apoptosis and unstained/irregular TJ formation in claudin-11, occludin and ZO-1 were observed. In conclusion, disruption of relation between BTB and TJs may reveal inadequate spermatogenesis, which is one of the mechanisms behind azoospermia.     

Changes in lanthanum permeability of rat Sertoli cell tight junction after CDDP administration

Using electron microscope, distribution of lanthanum tracer in the seminiferous epithelium was evaluated in adult male rats 2, 7, 25 and 53 days after single intraperitoneal injection of 3 mg/kg of CDDP. Lanthanum penetrated beyond the tight junction between Sertoli cells and the maximum penetration around the spermatid was found on the 7th day, when any degenerative changes of seminiferous epithelium were not recognized light microscopically. And 4 weeks pretreatment with Cephalantin and Kallikulein had no effects on this lanthanum tracer penetration around the spermatid. On decreasing this lanthanum tracer penetration after 7 days, some lanthanum was also found around the spermatocytes on the 53rd day but not around the spermatid. It is proposed that lanthanum tracer penetration beyond the Sertoli cell tight junction may be a morphological marker of early Sertoli cell dysfunction after CDDP administration.     

高功率微波辐射对大鼠血睾屏障的影响

目的:探讨高功率微波(HPM)辐射对血睾屏障(BTB)结构与功能的影响。方法:166只二级雄性Wistar大鼠经0、10、30、100mW/cm2HPM辐射5min,于辐射后6h,1、3、7、14d,采用镧醛(含20g/L硝酸镧,50mg/L戊二醛)心脏灌注和2%伊文思蓝(EB)尾静脉注射,经光镜、电镜和激光共聚焦显微镜(LSCM)观察BTB结构变化及硝酸镧和EB分布。结果:10～100mW/cm2HPM辐射后大鼠睾丸间质水肿伴血管充血、出血;生精小管近腔室内血浆蛋白和红细胞积聚,病变在辐射后6h出现,1～7d明显加重,范围扩大,14d有所减轻;30、100mW/cm2辐射组明显重于10mW/cm2辐射组。电镜下见各辐射组硝酸镧颗粒于近腔室内精母细胞、精子细胞和精子之间沉淀。LSCM观察见生精小管近腔室内弥散分布的EB颗粒。结论:HPM辐射可引起BTB结构破坏,通透性增加。     

On the Postnatal Development of the Terminal Segment of the Seminiferous Tubules in Normal and Germ Cell Depleted Rat Testes

The development of the terminal segment of the seminiferous tubules was studied in 5 to 50 days old normal rats. At the age of 5, 10, and 15 days the terminal segment contained fewer gonocytes or spermatogonia than did the corresponding seminiferous tubule. The differentiation of the terminal segment was obvious at 20 days of age due to the high number of germ cells in the seminiferous tubules, where the epithelium became stratified at this stage. The blood-testis barrier in the terminal segment was chiefly established between 15 and 20 days of age as revealed by the lanthanum tracer technique.
To study the effect of the germ cells on the differentiation, the germ cell depleted testes of prenatally irradiated rats were also studied. The modified Sertoli cells of the terminal segment were more vacoulated and had fewer lipid droplets and inter-Sertoli cell junctions than did the Sertoli cells of the seminiferous tubules. The ultrastructure of the modified Sertoli cells of the terminal segment was similar in adult normal and adult SCO (Sertoli cell only) rats. The amount of lipid droplets in the Sertoli cells of SCO rats showed considerable variation among different tubular cross-sections within one testis.     

Sertoli cell junctional complexes in gossypol-treated neonatal and adult guinea pigs

The effect of gossypol, an experimental male contraceptive agent, on the development and maintenance of the blood-testis barrier was determined by feeding gossypol daily to prepubertal and adult guinea pigs, and then examining their testes by electron microscopy of thin sections and freeze-fracture replicas. In guinea pigs of 10 to 30 and 10 to 40 days of age that were fed gossypol, impermeable continuous junctional zones did not develop between adjacent Sertoli cells. Compartmentalization of germ cells in the seminiferous epithelium, therefore, was nonexistent. These findings were obtained by use of the sterol-binding polyene, filipin, used as a low molecular weight tracer in combination with freeze-fracture. In general, the seminiferous tubules lacked lumina and spermatogenesis did not progress beyond the pachytene spermatocyte stage. In adult guinea pigs fed gossypol daily for five weeks, continuous zonules at the base of the seminiferous epithelium appeared intact and were impermeable to filipin. Discontinuous zonules found higher in the epithelium showed distensions between interrupted junctional strands and were permeated by filipin. In addition, vacuolated spaces between Sertoli cells and clumps of heterochromatin were conspicuous in some of the Sertoli cell nuclei. Spermatogenesis was disturbed in about 10% of the seminiferous tubules examined. These perturbations included exfoliation of round and elongated spermatids with concomitant formation of multi-nucleated giant cells. Spermatozoa from these adult male guinea pigs were immotile. These findings suggest that, in neonatal animals, gossypol appears to prevent the maturation of Sertoli cells and this effect is expressed as the failure of focal Sertoli cell tight junctional strands to assemble into continuous zonules. In adult animals, gossypol appears to have no effect on the maintenance of the blood-testis barrier.     

鹿藿根4种提取物抗雄性小鼠生育作用比较

目的:比较4种鹿藿根提取物(乙醇提取物、乙酸乙酯提取物、正丁醇提取物及水溶物)的抗生育作用。方法:60只雄性昆明种小鼠分为生理盐水对照组、雷公藤多苷对照组、乙醇提取物组、乙酸乙酯提取物组、正丁醇提取物组及水溶物组,每组10只。各组药物浓度均为1%,雷公藤多苷为0.1%水溶液。每次0.1ml/10g,灌胃,1次/d,连续给药,共11周。在用药10周后,一对一雌雄合笼交配1周。分笼10d后处死雌鼠,观察妊娠率、活胎数、死胎数。11周后处死雄性小鼠观察附睾精子、附睾及睾丸病理变化情况及血清睾酮含量测定。结果:连续用药11周,与生理盐水对照组相比,实验组小鼠妊娠率下降,其中水溶物组最为明显(P<0.05)。雷公藤多苷对照组和水溶组精子明显减少(P<0.05)。组织学观察鹿藿根提取物对精原细胞、初级精母细胞影响不显著。各提取物组血清睾酮差异无显著性(P>0.05)。生理盐水对照组、雷公藤多苷对照组、乙酸乙酯提取物组、正丁醇提取物组、乙醇提取物组及水溶物组的抗精子发生效应积分分别0、(5.3±1.5)、(2.6±1.7)、(2.9±1.2)、(2.2±0.9)、(2.1±1.0)分。结论:4种鹿藿根提取物均有抗生育作用,但水溶物作用更强且对睾丸组织及睾丸生精能力影响较小。     

促性腺激素释放激素拮抗剂对大鼠睾丸支持细胞雄激素受体和Fas配体基因表达的影响

本实验利用注射促性腺激素释放激素拮抗剂（ＧｎＲＨ Ａ）引起睾丸生精细胞程序化死亡的大鼠模型，用地高辛标记的大鼠雄激素受体和Ｆａｓ配体的ＲＮＡ探针对大鼠睾丸石蜡切片进行原位杂交，观察雄激素受体和Ｆａｓ配体基因在生精细胞凋亡中的变化。结果显示，ＧｎＲＨ Ａ处理后曲细精管有退行性变，原位杂交表明此时Ｓｅｒｔｏｌｉ细胞的雄激素受体表达明显减低，而Ｆａｓ配体表达显著升高。提示Ｆａｓ配体的表达可能与生精上皮细胞程序化死亡关系密切。     

Development of the blood-testis barrier in the domestic fowl (Gallus domesticus)

The formation of the blood-testis barrier (BTB) in the domestic fowl was studied at the electronmicroscopic level employing lanthanum as a tracer. No effective barrier could be demonstrated in testes before puberty, although several components of the Sertoli junctional complex such as focal tight junctions and desmosomes were already existent. The time of onset of meiosis after hatching showed great individual variation and meiosis did not occur synchronously in the tubules of a given testis. An effective barrier could first be detected in tubules containing early spermatids, and in which spermatogonia and primary spermatocytes at the leptotene stage were still within the open compartment. Thus, barrier formation was correlated with the occurrence of haploid germ cells. Complete compartmentation of seminiferous tubules, leaving only spermatogonia within the open compartment, was attained in tubules containing elongated spermatids of the maturation phase. In these tubules, primary spermatocytes at the leptotene stage were situated in an intermediate compartment.     

Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

AIM: To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone (FSH) or both agents given together. METHODS: From postnatal day (PND) 5 to 15 male rats were daily injected with 12.5 microg of 17beta-estradiol benzoate (EB) or 7.5 IU of human purified FSH (hFSH) or EB + hFSH or solvents (control). On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUNEL method. RESULTS: Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB + hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB + hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB + hFSH. CONCLUSION: At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.