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.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

Regulation of interstitial cell function by seminiferous tubules in intact and cryptorchid rats

The effects of experimental cryptorchidism on seminiferous tubule secretions and interstitial cell testosterone production were studied in vitro. Spent media obtained from incubations of seminiferous tubules (SMST) from cryptorchid rats caused a significant increase in testosterone production when added to interstitial cells isolated from intact rats. The previously noticed inhibitory activity of the SMST from stages VIII–XI of the sperma-togenic epithelial cycle gradually disappeared after the induction of experimental cryptorchidism. SMST obtained from both sham-operated or cryptorchid rats stimulated basal testosterone production when added to interstitial cells from cryptrochid rats. SMST from rats had been cryptorchid for 7, 14 and 28 days stimulated testosterone production when added to interstitial cells prepared from intact animals. Seminiferous tubules from cryptorchid rats therefore appear to be the source of a heat stable, trypsin-resistant factor with an apparent molecular weight of between 5000 and 10 000 daltons which stimulates testosterone production when added to interstitial cells in vitro. Its activity could not be blocked by an LRH antagonist. This factor enhances both basal and LH-stimulated secretion of testosterone in contrast to the inhibitory activity which involves only a partial blockade of LH-dependent steroidogenesis.     

睾丸间质细胞的发生与调控

睾丸间质细胞分布于生精小管的疏松间质组织中,是产生雄性激素的主要场所。睾丸间质细胞的功能障碍是导致男性原发性性腺功能低下、隐睾、尿道下裂等疾病的重要原因,因此睾丸间质细胞对男性生殖具有重要意义。本文对近年来关于睾丸间质细胞的发生、发育及调节的研究进展予以综述。     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.     

Enhancement of Leydig cell testosterone secretion by isolated seminiferous tubules during co-perifusion in vitro: comparison with static co-culture systems

The aim of this study was to identify an in-vitro test system for the reproducible demonstration of a modulatory effect of isolated seminiferous tubules (s-tubules) on testosterone production by purified rat Leydig cells. Co-incubation of s-tubules with various numbers of Leydig cells had no significant effect on basal and hCG-stimulated testosterone production over 4-24 h incubation. In contrast, addition of s-tubule conditioned medium (STCM) to Leydig cells enhanced both basal and hCG-stimulated testosterone production over 5 h, but this effect was variable in magnitude and was not completely reproducible. Co-perifusion of isolated s-tubules with Percoll-purified Leydig cells for 6 h produced significant and consistent increases in Leydig cell testosterone secretion compared with Leydig cells perifused on their own. In six experiments, s-tubules enhanced Leydig cell testosterone secretion by 26 +/- 5% (P less than 0.001) in the absence of LH stimulation and by 48 +/- 11% following pulsatile stimulation with 1 ng/ml ovine LH (oLH). The presence of s-tubules enhanced (P less than 0.01-0.001) testosterone secretion by Leydig cells in response to pulses of oLH at doses ranging from 0.1 to 10 ng/ml, but the magnitude of enhancement was greatest with 0.1 and 1 ng/ml doses. These stimulatory effects were not explained by Leydig cell contamination or by testosterone leakage from the isolated s-tubules. Co-perifusion of Leydig cells with isolated epididymal tubules as a control tissue had no significant effect on LH-stimulated Leydig cell testosterone production. Stimulatory effects of s-tubules on Leydig cell testosterone secretion were observed at a 'physiological' ratio of s-tubules to Leydig cells (200 cm tubules/3 million cells) and was mediated by a humoural agent(s), since perifusion of s-tubules and Leydig cells in series gave similar results to co-perifusion of these tissues. This system proved to be robust and, in contrast to static culture systems, gave highly reproducible results, which should allow detailed investigation of the dynamic interactions between s-tubules and Leydig cells and the hormonal control of these events.     

Ontogeny and cellular origin of a rat seminiferous tubule factor(s) that inhibits LH-dependent testosterone production by interstitial cells in vitro

Previous studies have shown the presence of a peptide in spent media from incubated seminiferous tubules (SMST), which inhibits LH stimulation of testosterone production by rat Leydig cells in vitro. The present study has investigated whether the secretion of this inhibitor changes during development in the rat. Seminiferous tubules obtained from rats aged 10, 20, 25, 30, 35, 40, 42, 50 or 60 days were incubated at 32 degrees C for 24 h. Spent media from these incubations were then added to interstitial cells isolated from the testes of rats aged 60 days. Spent media from rats aged 10-30 days had no effect on basal or oLH-stimulated testosterone production by interstitial cells during 3-h incubation. Significant inhibition of LH-stimulated testosterone production was, however, observed with SMST from rats aged 35-60 days. Spent media prepared using tubules from normal, prenatally irradiated (Sertoli cell-enriched) or seminiferous tubules, depleted of peritubular cells, had no effect on basal, but inhibited LH-stimulated, testosterone production. Spent media from peritubular cell cultures had no effect on basal or LH-stimulated testosterone production by interstitial cells. The inhibitory effect of SMST was also dependent on the age of the rats providing the target cells. Interstitial cells from rats aged 10, 20, 50 or 60 days were responsive to the inhibitor while cells from rats aged 30 and 40 days were not. The results of the present study demonstrate that the seminiferous tubule factor(s), which inhibits LH action on interstitial cells, is first secreted at 35 days, a time when the most mature germ cells present are in the early maturation phase. Moreover, interstitial cells are responsive to this factor in both immature (10-20 day-old) and mature (50-60 day-old) rats, but not at ages in between these times. It is suggested that the adult Sertoli cell is the major source of the interstitial cell inhibitor.     

Effect of unilateral cryptorchidism on the intertubular tissue of the adult rat testis: evidence for intracellular changes within the Leydig cells

Adult male rats were made unilaterally cryptorchid for 1, 2 or 4 weeks, and the morphological response of the Leydig cells was then studied using morphometric assessment of total Leydig cell volume and number per testis in abdominal and scrotal testes. Serum hormone levels were measured and the steroidogenic properties of isolated Leydig cells were evaluated by in-vitro stimulation with hCG and interstitial fluid (IF) obtained from normal rat testes. Total Leydig cell volume and number per testis were not altered in abdominal vs scrotal testes, although the volume of the abdominal testis was 46, 29 and 21%, respectively, of the volume of the contralateral scrotal testis after 1, 2 and 4 weeks. This reduction was accompanied by significant (P less than 0.05) elevation of the serum levels of FSH and LH, although serum testosterone levels were unchanged from the normal range. Despite the lack of quantitative alterations in Leydig cell morphology, hCG- and IF-stimulated testosterone production was significantly (P less than 0.01) greater by abdominal Leydig cells when compared with scrotal Leydig cells derived from the same animals. Ultrastructural examination of Leydig cells in situ suggested an increase in volumetric density of mitochondria in abdominal Leydig cells. Together with the enhanced steroidogenic responses of these cells, these findings suggest that disruption of spermatogenesis in the cryptorchid testis is accompanied by intracellular activation of Leydig cells. Since these effects were not exhibited by Leydig cells from the scrotal testis it is concluded that local factors within the cryptorchid testis are responsible, at least in part, for regulation of Leydig cell activity.     

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.     

Hormonal regulation of a rat seminiferous tubule factor which inhibits LH action on interstitial cells

The cytosol from rat testes or seminiferous tubules contains a factor that markedly reduces the responsiveness of interstitial cells to stimulation by LH. It was noted previously that the inhibitor cannot be found until 35 days of age, suggesting that gonadotrophic stimulation of the testes is of importance for its formation. In the present studies, treatment of intact 20-day-old rats with FSH or with a combination of FSH and LH caused a premature appearance of the inhibitory activity. LH alone had a weak effect. However, hypophysectomy at 20 or 35 days of age did not influence the inhibitor content of the testes. Moreover, when the Leydig cells of adult rats were destroyed selectively by treatment with ethylene dimethane sulphonate, inhibitor levels were unchanged. It is suggested that induction of the Leydig cell inhibitor is under the control of FSH. However, once induced, its regulation seems to be independent of the pituitary gland. In separate experiments, ligation of the efferent ducts of the testes in adult animals did not cause any accumulation of inhibitory activity in the ligated testes, nor could the inhibitor be traced in the caput epididymis. Thus, it does not seem to be secreted into the epididymis, but rather may act as a paracrine factor in the testis.     

Non-tumoural parenchyma in Leydig cell tumours: pathogenetic considerations

Little is known about the pathogenesis of Leydig cell tumours (LCTs) of the testis. The observation of several associated dysgenetic features in the non-tumoural parenchyma and in the contralateral testes of men with testicular germ cell neoplasms has served as the basis to propose that there may be a common mechanism for different male reproductive disorders. However, the possible relationship between LCTs and other testicular lesions has not been explored. Here we describe the presence of primary lesions in the non-tumoural parenchyma of testes with LCT, from which we try to establish possible pathogenetic associations. We studied the non-tumoural parenchyma adjacent to 16 LCT specimens. Parameters as Leydig cell hyperplasia (LCHY), qualitative evaluation of the germinal epithelium and spermatogenesis, the presence of Sertoli cell-only tubules (SCOT), and the Sertoli cell nuclear morphology were consistently assessed in all cases. SCOT associated with Sertoli cell dysgenetic morphology was the most frequent finding, present in 50% of the cases. Another interesting finding was the presence of LCHY in four cases (25%). Abnormal spermatogenesis was found in 81.25% of the cases, and it consisted of lesions of the adluminal or basal compartments of seminiferous tubules. The occurrence of either dysgenetic Sertoli cells or LCHY adjacent to LCTs could represent primary anomalies, resulting from a common insult also involved in tumourigenesis. The abnormalities in spermatogenesis observed here are likely to represent consequences of either tumour compression or abnormal hormonal production. The significance of these associations merits further investigation regarding a common pathogenesis.     

移植大鼠生精干细胞到裸鼠生精小管实验系统的建立

目的 建立移植大鼠生精干细胞到裸鼠生精小管的实验系统.方法 采用一次腹腔注射busulfan 30mg/kg以消除裸鼠生精小管内源性生精细胞,制备生精干细胞移植受体小鼠;采用laminin粘附的方法分离大鼠生精干细胞;应用生精小管直接注射和输出管注射的方法移植分离到的大鼠生精干细胞进入受体裸鼠的生精小管内.结果 busulfan腹腔注射4周后受体裸鼠生精小管中的精子发生明显得到抑制.大鼠生精干细胞移植2～3个月后在受体裸鼠的生精小管中发现了大鼠的长型精子细胞.结论 成功建立了移植大鼠生精干细胞到受体裸鼠生精小管的试验系统.     

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.     

Cyproterone acetate induced changes in the behaviour of hydroxysteroid dehydrogenases in rat Leydig cells during perinatal development

The influence of cyproterone acetate (CA) upon the behaviour of hydroxysteroid dehydrogenases (HSDH) in the Wistar rat Leydig cells was investigated during the perinatal phase with the help of enzymhistochemical cum morphometrical techniques. The pregnant rats as well as their offsprings were injected with CA (dosage: 35 mg/kg body wt) sc, daily from 14 fetal day upto 31 postnatal day (p.n.d.). The animals were killed on 5, 20, and 32 p.n.d.; the enzymhistochemical reactions for 3-beta-HSDH, 11-beta-HSDH, 17-HSDH and 3-alpha-HSDH were performed in the cryostat sections of the testis, and the morphometric evaluation of HSDH positive Leydig cells was carried out. On 5 p.n.d. the activity of 17-beta-HSDH was slightly impaired in the intertubular Leydig cells of the CA treated animals. On 20 p.n.d., CA prevented nearly completely the HSDH activity in the newsly built peritubular Leydig cells; the activities of 3-beta-HSDH, 17-beta-HSDH, and 3-alpha-HSDH resided mainly in the intertubular Leydig cells. On 32 p.n.d. the HSDH activities in the Leydig cells were observed in the control as well as in treated animals. It seems that the differentiation of peritubular Leydig cells, and thereby the steroid production, is delayed by CA, but not entirely blocked.     

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.     

Tamoxifen induced multinucleated cells （symplasts） and distortion of seminiferous tubules in rat testis

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg·kg-1·d-1, 400 mg·kg-1·d-1 or 800 mg·kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.     

Studies on LH modulated 8kDa peptide involved regulation of testosterone production in rat Leydig cells

Aim: To demonstrate the role of the 8 kDa peptide in regulation of testosterone production by mt Leydig cells. Meth-ods: A peptide similar to 8 kDa peptide purified from immature rat Leydig cells was isolated and purified from rat lungcytosol. Immunological and structural similarity between the peptides purified from lung and Leydig cells was estab-lished by Western blot and tryptic map comparison respectively. Results: Addition of the 8 kDa peptide 10, 50, 100;and 150 ug decreased the production of testosterone in Leydig cells dose-dependently. But the addition of the peptide150 ug along with hCG had no effect on hCG-stimulated increase in testosterone production. Conclusion: In vitro ad-dition of the peptide purified from lung cytosol to adult rat Leydig cells resulted in a concentration-dependent decrease inbasal testosterone production although it had no effect on hCG-stimulated testosterone production. (Asian J Androl1999 Dec; 1: 191-194)     

小鼠Leydig肿瘤细胞膜受体LHR减量调节中受体基因转录变化及其机制

膜受体的减量调节是受体在转导跨膜信号的同时，其数目呈进行性下降的一种细胞生物学行为。本文运用ＲＴＣＲ、放免分析及“ｒｕｎｏｎ”等技术研究小鼠Ｌｅｙｄｉｇ 细胞株ＬＨ 受体（ＬＨＲ） 减量调节过程中ＬＨＲ ｍＲＮＡ 水平变化及其调控机制。结果揭示，在ｈＣＧ作用后ＬＨＲ 减量调节的过程中，ＬＨＲｍＲＮＡ 水平发生了“升高降低”的变化，这种变化是ＬＨＲ基因转录活性的变化决定的，与ＬＨＲｍＲＮＡ 的降解无关，ｃＡＭＰ 以“正相关”的方式调控着ＬＨＲ基因的转录速率。