Carcinoma-in-situ cells in cultured human seminiferous tubules

For the first time, early germ-cell derived tumour cells were studied in an in-vitro system of cultured seminiferous tubules. The intratubular tumour cells not only survived in culture for 7 days but were also able to multiply. Dividing tumour cells were identified in semi-thin sections and electron micrographs by morphological criteria. Additionally, mitotic activity was demonstrated by [3H]thymidine histo-autoradiography. There are numerous reports on cell lines established from solid non-seminomas, but up to now no references to seminoma cell lines or cultures of intratubular tumour cells are available. The culture of seminiferous tubules offers a tool in making carcinoma-in-situ cells accessible for experimental work.     

Serum reproductive hormone levels and sperm production in male adult rats after treatment with arresting, a fraction obtained from seminiferous tubules conditioned medium

This study used seminiferous tubule (ST) segments from adult rats to condition culture medium that had been concentrated, size fractioned and administered 10-84 days to adult rats by subcutaneous or intratesticular injection and the effects on testes weight, testosterone, luteinizing hormone (LH) and FSH levels and (homogenization-resistant) epididymal sperm count were determined. The conditioned medium obtained 2 days after culture of ST was fractionated in a 30-100 kDa component. The fraction was injected subcutaneously or intratesticularly. This factor(s), named arresting, decreases sperm count in the epididymis from 13 days to 84 days of treatment without changes in serum LH or testosterone levels. The results of the present study suggest that arresting acts on spermiogenesis/spermiation and/or the entry of sperm into the epididymis from the efferent ductules.     

生精小管异位重构研究进展

哺乳动物睾丸的形成和精子发生是一个复杂的过程,包括细胞的迁移、增殖、分化和细胞间的相互作用。尽管近年来对此进行了大量的研究,但对许多方面仍然是未知。缺少一个合适的研究睾丸形成与精子发生的模型是阻碍研究取得进展的一个重要原因。在过去的几年中,相继发现2种对研究哺乳动物精子发生很有价值的模型:一是未成熟的睾丸组织异位移植于免疫缺陷鼠的皮下可以成活并产生有功能的精子;另一是未成熟睾丸组织消化成单细胞后异位移植于免疫缺陷鼠的皮下可以重新形成生精小管样结构并完成完整的生精过程。本文对这两种方法的研究进展、应用及前景进行了综述。     

7-Alpha-hydroxytestosterone in seminiferous tubules of rat testis

Production of 4-androstene-7 alpha,17 beta-diol-3-one (7 alpha-hydroxytestosterone) from testosterone was measured in seminiferous tubule and interstitial fractions of rat testis. In adults, specific activity of the 7 alpha-hydroxylase was about 10 times higher in interstitial cells than in seminiferous tubules, but tubule production was a significant portion of the total. Seminiferous tubule 7 alpha-hydroxylase was not detectable in weanlings. Also, we confirmed that 7 alpha-hydroxytestosterone inhibits the activity of the 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase active on testosterone and suggest a role in maturational changes.     

Microvasculature of the human testis in correlation to Leydig cells and seminiferous tubules

Summary. The microvasculature of the human testis is closely related to the Leydig cells and the seminiferous tubules. Semi-thin sections of testicular tissue serve as a basis for the computer-aided 3-D reconstruction of the microvasculature, the seminiferous tubules and the Leydig cells. After vascular perfusion with glutaraldehyde (5.5%) and paraformaldehyde (4%), it is possible by means of light and electron microscopy, to analyse the organization of the capillaries between the Leydig cells (inter-Leydig cell capillaries) as well as of those within the lamina propria (intramural capillaries). These arise from arterioles, deriving from branches of the segmental arteries. The capillaries ramify between the Leydig cells and run either semi-circumferentially around the seminiferous tubules (peritubular capillaries) or penetrate the lamina propria of the neighbouring tubules. This is the beginning of the intramural capillary which after leaving the tubular wall continues to a further capillary path. Consequently, the microvasculature of the human testis with regard to the seminiferous tubules is subdivided into afferent, intramural and efferent capillaries. Leydig cell clusters are present on both the arterial and the venous sides of the microvasculature.     

Multinucleated spermatocytes and spermatids in human seminiferous tubules

The cytology of multinucleated spermatocytes and spermatid giant cells in the seminiferous tubules of men with oligozoospermia and of men older than 65 years has been investigated electron microscopically. Two different processes which are responsible for the origin of multinucleated germ cells have been analysed: Defects of the intercellular bridges (IB): they move apart and thus allow the confluence of one clone's cells to symplasms. The confluence of membranes: within one clone the membranes of neighbouring germ cells are dissolved and thus intercellular bridges are found in the cytoplasm of the resulting giant cells. The spermatid giant cells reveal a new organization of the cell nuclei and the cell organelles. Yet they disintegrate in the lumen of the seminiferous tubules. The appearance of giant cells therefore is an expression of the germ cell degeneration.     

Paracrine regulation of Leydig cells by the seminiferous tubules

Testes of adult control and unilateral cryptorchid rats were fixed by vascular perfusion. The cell profile area of peritubular Leydig cells surrounding tubules in different stages of spermatogenesis, and the cell profile area of perivascular Leydig cells were determined. The size of peritubular Leydig cells was dependent on which type of tubulus the cells were surrounding. Some peritubular Leydig cells, especially those surrounding stages VII–VIII (88.1 ± 7.1 μm², mean ± SD, n = 6 rats), were larger than perivascular Leydig cells (69.3 ± 5.9 μm²). The size of Leydig cells surrounding stages IX–XIV was similar to that of perivascular cells. In the abdominal testes no spermatogenic cycle was present and the sizes of peritubular and perivascular Leydig cells were equal (63.0 ± 5.1, vs 66.7 ± 7.3 μm², mean ± SD, n = 5 rats). It is suggested that the tubules and the spermatogenic cycle locally modulate Leydig cell activity and that Leydig cell malfunction in abdominal testes may be due to a decreased stimulatory influence from the damaged tubules.     

Ultrastructure of the seminiferous tubules in oligoasthenoteratozoospermic men associated with varicocele

Varicocele is associated with venous reflux that may cause increased heat and interstitial pressure within the testes, with variable pathological effects on spermatogenesis. This study aimed to study the ultrastructural testicular changes in the seminiferous tubules of 20 infertile severe oligoasthenoteratozoospermia (OAT) men associated with varicocele and five patients with obstructive azoospermia without varicocele as controls. They were subjected to testicular biopsy which was evaluated by transmission electron microscopy. Ultrastructurally, the seminiferous epithelium in the testicular biopsies of infertile severe OAT men associated with varicocele was variably affected in the form of thickening of the peritubular connective tissue, vacuolation of Sertoli cell and germ cell cytoplasm, presence of degenerated and apoptotic cells among the germinal epithelium, altered spermatids and abnormal spermatozoa. It is concluded that varicocele in severe OAT men is associated with ultrastructural changes in the seminiferous tubule.     

Cytoprotective and anti‐apoptotic effects of Satureja khuzestanica essential oil against busulfan‐mediated sperm damage and seminiferous tubules destruction in adult male mice

We studied the protective effect of Satureja khuzestanica essential oil (SKEO) against damage caused by busulfan on testis in male mice. The NMRI mice (n = 40) were assigned to four groups including: G1: control, G2: treated with busulfan for 4 days (3.2 mg kg⁻¹), G3: receive busulfan (4 days, 3.2 mg kg⁻¹) and SKEO (28 days, 225 mg kg⁻¹) at the same time, G4: pre‐treated with SKEO (7 days, 225 mg kg⁻¹) and subsequently cotreated with busulfan (4 days, 3.2 mg kg⁻¹) and SKEO (28 days, 225 mg kg⁻¹). The histological changes of testis were analysed using H&E staining. Sperm parameters, cytotoxic and apoptotic factors were also studied by computer‐aided sperm analyzer, MTT and TUNEL assays respectively. Our results showed that SKEO pre‐administration significantly improved all parameters of epididymal spermatozoa and decreased germinal epithelium destruction following busulfan chemotherapy. We also found lower MTT levels and TUNEL‐positive cells in SKEO pre‐treated groups. In conclusion, SKEO possesses beneficial effects on sperm parameters when taken before chemotherapy and continued during and after chemotherapy for a long time, than when used short‐term coinciding with the chemotherapy. Our results support valuable data about the application of SKEO for protection against adverse effects of busulfan on male genital system in patients under chemotherapy.     

Molecular andrology as related to sperm DNA fragmentation/sperm chromatin biotechnology

Genetic male infertility occurs throughout the life cycle from genetic traits carried by the sperm, to fertilization and post-fertilization genome alterations, and subsequent developmental changes in the blastocyst and fetus as well as errors in meiosis and abnormalities in spermatogenesis/spermatogenesis. Genes encoding proteins for normal development include SRY, SOX9, INSL3 and LGR8. Genetic abnormalities affect spermatogenesis whereas polymorphisms affect receptor affinity and hormone bioactivity. Transgenic animal models, the human genome project, and other techniques have identified numerous genes related to male fertility. Several techniques have been developed to measure the amount of sperm DNA damage in an effort to identify more objective parameters for evaluation of infertile men. The integrity of sperm DNA influences a couple's fertility and helps predict the chances of pregnancy and its successful outcome. The available tests of sperm DNA damage require additional large-scale clinical trials before their integration into routine clinical practice. The physiological/molecular integrity of sperm DNA is a novel parameter of semen quality and a potential fertility predictor. Although DNA integrity assessment appears to be a logical biomarker of sperm quality, it is not being assessed as a routine part of semen analysis by clinical andrologists. Extensive investigation has been conducted for the comparative evaluation of these techniques. However, some of these techniques require expensive instrumentation for optimal and unbiased analysis, are labor intensive, or require the use of enzymes whose activity and accessibility to DNA breaks may be irregular. Thus, these techniques are recommended for basic research rather than for routine andrology laboratories.     

小鼠睾丸消化细胞异体移植重构生精小管的研究

目的:采用免疫缺陷小鼠作为受体,通过对小鼠睾丸消化细胞异位移植后不同时期移植物的研究,观察生精小管重构、生精细胞归巢及精子发生情况。方法:取新生ICR小鼠的睾丸消化成单细胞悬液,将其与Matrigel基质胶混匀后移植于雄性裸鼠背部皮下,术后裸鼠行去势。移植后分别于4、6、8、10周处死5只裸鼠,计算移植成功率,取移植物测量直径,并进行HE染色和免疫组化检测,观察生精小管的重构、生精细胞归巢及精子发生情况。结果:20只受体鼠接受睾丸消化细胞移植后全部存活。睾丸消化细胞移植后10周内可见明显隆起的包块,包块直径由第4周的(3.91±0.71)mm增加到(6.69±0.50)mm,移植物表面有血管生成。对移植物石蜡切片进行HE染色可见生精小管样结构,部分生精小管管腔内可见由精原细胞发育至精子细胞的各级生殖细胞,未见明显精子产生。对8周移植物进行免疫组化观察,可见生殖细胞标志物Mvh、支持细胞标志物Gata4和间质细胞标志物P450Scc表达。结论:新生小鼠睾丸消化细胞移植于裸鼠背部皮下后可重构生精小管,为研究睾丸组织工程及睾丸发育和精子发生过程中睾丸各组成细胞之间的相互作用提供了理想的研究模型。     

A study on budding protrusions of human seminiferous tubules

In order to elucidate the morphogenesis of seminiferous tubular protrusions, histometric, microscopic and electron microscopic studies were performed on the testes of 202 Japanese men, including 117 sudden deaths, 75 hospital deaths and 10 prostatic cancer cases. Protrusions usually occurred at outer convexes of multi-bending tubular portions and were divided into dome, sessile, pedunculated and multi-branched types. Aggregated Sertoli cells were present in dome-type protrusions as a major component, and spermatogenesis associated with active mitoses of spermatogonia was induced with development of protrusions. Protruding walls consisted of inner compact and outer loose layers. Distribution of lipid droplets in Sertoli cell cytoplasm in protrusions was different from those in the original tubules. The incidence of protrusions peaked in the forties and sixties, respectively, in the case of hospital and sudden death cases with underlying tubular atrophy. The findings suggest that tubular protrusions take place as a compensatory reaction for declining spermatogenesis, and therefore, probably represent a regenerative phenomenon in hypospermatogenic testes.     

In-vitro contractility of human seminiferous tubules in response to testosterone,dihydrotestosterone and estradiol

Summary The effects of steroids (testosterone, dihydrotestosterone and estradiol) on human seminiferous tubules in vitro were ascertained by recording the intratubular pressure with a servonull micropressure measuring device. We describe here the first response of the human seminiferous tubule to steroids. Testosterone and dihydrotestosterone had a biphasic effect on tubular contractility. Higher doses of both testosterone and dihydrotestosterone induced contractions of the seminiferous tubules whereas lower doses of these compounds induced relaxation. Estradiol (10^-9 M to 10^-6 M) induced relaxation of the seminiferous tubules in a dose-dependent manner. The results from these experiments suggested that steroids may be involved in the control of contraction of the human seminiferous tubule and may regulate the movement of spermatozoa from the tests.     

Pathological observation on lamina propria of seminiferous tubules in infertile men

In this paper the authors observed the pathological alterationsin lamina propria of seminiferous tubules from 41 patients witholigospermia and azoospermia by using morphometry and immuno-histochemistry. Morphological alterations in seminiferous tubules ofthese patients were classified into three types; hypospermato-genesis, spermatogenic arrest and Sertoli-cell-only syndrome. Thelimiting membrane of the seminiferous tubules was found thicker invarious degrees. Morphometrically, there were significant differ-ences between the three types of alterations in terms of thickness ofthe lamina propria and the diameter of the seminiferous tubules.Immunohistochemically, the inner and outer layers of the laminapropria were positive for type Ⅳ collagen and laminin, while theintermediate layer, the hyperplastic zone, was negative, but thiszone was positive for type Ⅰ and Ⅲ collagen. The results indicatedthat the basic component of the altered lamina propria was of colla-gen fibers but not of type Ⅳ collagen, and     

Effect of testicular capsulotomy on lipid droplets in the seminiferous tubules of rats

Aim: In order to reveal the histochemical alteration that might occur during the processes of the spermatogenic disruption induced by testicular capsulotomy, the location and alteration of lipid droplets in the seminiferous tubules were observed in the present study. Methods: Osmium tetroxide was used to demonstrate the lipid droplets in the seminiferous tubules of capsulotomized and sham-operated control testes. Results: In the seminiferous tubules of the sham-operated rat testes, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in size and number, with many lipid droplets migrated towards the lumen of the tubules. Conclusion: The results indicated that a progressive fatty degeneration occurred in the seminiferous tubules after testicular capsulotomy.     

不育症患者睾丸曲细精管界膜的病理学观察

应用形态计量学和免疫组织化学方法观察４１例少精子症和无精子症患者的睾丸活检曲细精管界膜的病理学变化。曲细精管生精上皮的病理变化主要有三种类型：生精功能低下、生精阻滞和唯支持细胞综合征。三种病变的曲细精管均表现有不同程度的界膜增厚。形态计量学结果显示三种病变的曲细精管界膜厚度、管内径与正常对照相比均有显著性差异，管内径与界膜厚度呈负相关。免疫组化层粘连蛋白Ⅳ型胶原染色显示界膜的内外层呈阳性反应，而中间增厚带为阴性，Ⅰ型、Ⅲ型胶原免疫组化染色界膜增厚的中间带为阳性。提示：病变界膜的成分为胶原纤维，但与正常成分－Ⅳ型胶原不同，表现为Ⅰ型及Ⅲ型胶原增生。     

Stage-specific degeneration of germ cells in the seminiferous tubules of non-obese diabetic mice

The cause of fertility problems in insulin-dependent diabetes is largely unknown. To evaluate the role of autoimmunity-associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage-specific apoptosis of germ cells in the insulitis phase of pre-diabetes was quantified in the testes of non-obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in-situ end-labelling (ISEL) of DNA. ISEL-positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL-positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17–20 weeks, the number of ISEL-positive cells/mm tubule was significantly lower in segments at stages I–II of the seminiferous epithelial wave but higher at stages III–IV in comparison to BALB/c mice. The numbers of ISEL-positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of ³²P-3' -end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.