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.     

Repeated interruptions of the testicular blood flow do not have long-term effects on spermatogenesis in the ram

Summary. The effect of repeated interruptions of the testicular blood flow on spermatogenesis was studied in mature Texel rams. Reversible interruption of the blood flow was achieved by an inflatable occluder, placed around the testicular artery at the level of the spermatic cord. In eight testes the blood flow was successfully interrupted six times for 1 h within 3 weeks and in 14 testes nine times for 1 h within 3 weeks. Nine weeks after the last blood flow interruption spermatogenesis was evaluated in histological sections of the testes. Both after six and nine blood flow interruptions a qualitatively complete epithelium was found in at least 90% of the seminiferous tubules. Cell counts in stages VII and VIII of the spermatogenic cycle revealed a slight decrease of spermatocytes and spermatids in the tubules with a complete epithelium after nine occlusions, which was only statistically significant for Preleptotene Spermatocytes. After six occlusions the numbers of all cell types were at or even slightly above control levels. These results show that repeated periods of ischaemia for 1 h do not result in conspicious long-term damage to spermatogenesis.     

Influence of histological degree of seminiferous tubular degeneration and stage of seminiferous cycle on the proliferation of spermatogonia in aged Syrian hamster (Mesocricetus auratus)

The ageing testis is associated with germ loss in the seminiferous epithelium and a decrease in spermatogonia proliferation. In this work, we study whether the stages of the seminiferous epithelium cycle and/or the degree of histological tubular degeneration resulting from ageing is related with this decrease in spermatogonia proliferation. Eleven hamsters were used, five aged 6 months and six aged 24 months. In both groups, the proliferative activity was studied by BrdU immunostaining. The number of BrdU‐positive and BrdU‐negative cells was measured, providing the overall proliferation index in adult and aged testes. The mean number of BrdU‐positive cells was also determined for each degree of histological degeneration of seminiferous epithelium, and a spermatogonia proliferation index was obtained for each stage of the seminiferous cycle. Ageing caused an overall decrease in the BrdU‐positive cell percentage and a decrease in the number of BrdU‐positive cells in the tubular sections with hypospermatogenesis, the sloughing of germ cells and maturation arrest, these changes being similar in both young and old animals. The spermatogonia proliferation index was only seen to be significantly lower in ageing hamster in stages VII–VIII of the seminiferous epithelium cycle. In conclusion, the overall decrease in proliferation observed in aged seminiferous epithelium is correlated with an increase in the number of degenerated sections of the seminiferous tubules, and this decrease is a phenomenon which occurs in specific stages of the seminiferous cycle.     

Marmoset spermatogenesis: organizational similarities to the human

The common marmoset (Callithrix jacchus), a small New World primate of high fecundity, is widely used in reproductive research. The aim of the present study was to determine the organization of the germ cells within the seminiferous epithelium, the duration of the spermatogenic cycle and the number of spermatogonial mitoses. Antibodies to cAMP response element modulator (CREM) and proliferating nuclear cell antigen (PCNA) and a cRNA directed against protamine P2 and morphological criteria were used to discriminate between stages of the spermatogenic cycle. Plastic sections were used to document the cell associations present in each of the nine stages of spermatogenesis. Up to five such stages could be observed within individual cross-sections of seminiferous tubules. Based on the pattern of incorporation of bromodeoxyuridine the length of the spermatogenic cycle was estimated to be 10 days and the duration of spermatogenesis to be 37 days. Four mitotic divisions were noted in spermatogonia. It is concluded that the organization of spermatogenesis in the marmoset has similarities to the human ('helical') and this makes the marmoset a suitable model for studies relevant to human testicular function.     

The effect of gossypol acetic acid on the different stages of the spermatogenic cycle in the rat

The reversibility of the effect of gossypol on testicular histology and fertility was studied in rats. Adult males of proven fertility were treated orally with gossypol acetic acid (15 mg/kg) for 9 or 16 weeks (groups 1 and 2, respectively). Another groups of animals (group 3) was given gossypol (15 mg/kg) for 16 weeks and killed 6 weeks after the end of treatment. Control animals (group 4) were given the vehicle only by oral intubation. In the mating studies, although only 33% of the animals in group 1 were infertile, 100% infertility was observed following 16 weeks of gossypol treatment (group 2). All animals in group 3 regained their fertility 6 weeks after cessation of drug treatment. Damage was observed to 15.7% of the seminiferous tubules after 9 weeks of drug treatment, and to 78% after 16 weeks of treatment. Extensive vacuolization, increased numbers of lipid droplets, degeneration of germ cells, loosening of the epithelium, and a significant decrease in the number of pachytene spermatocytes (stages VII-X) and spermatids (steps 7-10 at stages VII-X) were observed after gossypol treatment. There was a decrease in the diameter of only stage VIII seminiferous tubules after 9 weeks of treatment, whereas a reduction was observed in the tubules of all stages after 16 weeks of gossypol treatment. In the recovery phase, the diameter of seminiferous tubules was similar to that of controls, except for tubules at stage VIII. No change in the area of the lumen of the seminiferous tubules and lipid bodies was observed after 9 weeks of drug treatment, but a marked reduction in the area of the lumen (stages II-X) and an increase in lipid bodies (all stages) was observed after 16 weeks of gossypol treatment. Six weeks after cessation of treatment, the area of the lumen and the number of lipid bodies were comparable to values in controls. A reduction in the area of the epithelium was restricted to just a few stages (VIII-XIV) in treated animals at 9 weeks, whereas after 16 weeks the area of the epithelium was decreased in all tubules. In the recovery phase, except for tubules at stage VIII, the area of the seminiferous epithelium was comparable to that in controls.     

Transillumination to evaluate spermatogenesis: effect of testosterone enanthate in adult male rats

The present study has been designed to determine which stages of seminiferous cycle were modified by in vivo intramuscular administration of one or two doses of 25 mg of testosterone enanthate. Testicular weight was significantly reduced after treatment with two doses of testosterone enanthate (p < .05). Assessment of total lengths of stages per 100 mm of seminiferous tubules scored by transillumination showed that testosterone enanthate increased significantly the length of stages VI-VII, but it reduced total lengths of stages I, XII, and XIII-XIV. A dose-dependent reduction in the absolute and relative frequency of stage I was observed with testosterone enanthate treatment, whereas stages VI-VII were significantly increased. Analysis of relative frequencies of stages showed that stages XIII-XIV and stages I-V were significantly below the control value (1), whereas stages VI-VIII were over 1. In summary, results of the present study showed that transillumination is an adequate technique to assess the effect of drugs on spermatogenesis, and that testosterone enanthate modifies the pattern of the stages of the seminiferous epithelium cycle, before testicular weight is affected.     

Transformation,migration and outcome of residual bodies in the seminiferous tubules of the rat testis

Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague–Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.     

睾丸细胞生物学研究进展

睾丸是男性生殖腺,由生精小管和间质构成。生精小管主要由生精细胞和支持细胞组成,是精子发生场所;间质中主要是间质细胞,间质细胞合成与分泌雄激素。本文介绍睾丸3种细胞的发育分化,以及成年期睾丸细胞的结构和生物学研究进展。     

The selective removal of pachytene spermatocytes using methoxy acetic acid as an approach to the study in vivo of paracrine interactions in the testis

Testicular weight and morphology, serum gonadotropins, intratesticular levels of testosterone and ABP levels in testicular interstitial fluid were studied in adult rats at intervals of 1 to 70 days after a single oral dose of 650 mg/kg methoxy acetic acid. At 3 days, this treatment resulted in the selective loss or depletion of pachytene and later spermatocytes from seminiferous tubules at all stages other than VIII to XI of the spermatogenic cycle. At later times this lesion was expressed as an absence mainly of round (14 days) or elongated (21 days) spermatids from the majority of seminiferous tubules. Other than these changes, spermatogenesis did not appear to be affected by treatment and was qualitatively normal in all tubules at 70 days after treatment. As deduced from cell counts at 3 days posttreatment, the initial action of methoxy acetic acid was restricted to late zygotene spermatocytes (stage XII) and pachytene spermatocytes at all stages other than early- to mid-stage VII. Levels of FSH in serum and those of ABP in testicular interstitial fluid indicated that Sertoli cell function was altered in rats treated with methoxy acetic acid. Both were increased at 1 to 3 days posttreatment, returned to normal at 7 to 14 days but were increased again at 21 days before finally returning to control levels at 28 days. In contrast, the levels of testosterone in serum, isolated seminiferous tubules and testicular interstitial fluid were unaffected by treatment, as also were the serum levels of LH. The two periods of increase in FSH and ABP levels coincided with the times of greatest decrease (approximately 20%) in testicular weight, and may be related either to the type of germ cell missing from the affected tubules and/or to the stage of the cycle of the affected (or unaffected) tubules. These data suggest that chemicals such as methoxy acetic acid may prove useful in the study of paracrine interactions in vivo.     

The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium

This study was designed to explore the relationship between the intratesticular distribution of testosterone and spermatogenesis by completely destroying the Leydig cells of mature male rats with injection of a single i.p. dose of ethane dimethanesulphonate. After such treatment, testosterone levels in serum, testicular interstitial fluid, seminiferous tubules, and whole testis declined significantly 6 to 24 hours after injection and fell below assay detection limits between 3 and 7 days. At 3 and 7 days, serum LH and FSH levels rose significantly and remained elevated up to 4 and 6 weeks, respectively, in comparison with vehicle-treated controls. Leydig cells disappeared from the interstitium by day 3, but between 2 and 4 weeks postinjection a new generation of fetal-like Leydig cells repopulated the testicular interstitium and, during weeks 6 to 10, were transformed into, or replaced by, Leydig cells with an adult type of morphology. Histologic examination of the seminiferous tubules showed progressive disruption of spermatogenesis between 3 and 14 days post-ethane dimethanesulphonate. The first histologic sign of spermatogenic damage was noted at day 3, with the occurrence of stage-specific degenerating pachytene primary spermatocytes at stages VII to VIII of the spermatogenic cycle. On day 7, these cells and degenerating round, or step 19, spermatids often were observed during stages VII to XI, although qualitatively normal spermatogenesis also was seen in these and all other stages of the cycle. Maximum impairment of spermatogenesis occurred 2 weeks post-ethane dimethane sulphonate, at which time the tubules commonly lacked one or more germ cell generations or, alternatively, showed accumulation of lipid inclusions, extracellular spaces, and variable numbers of degenerating germ cells. Following repopulation of the testis by Leydig cells during weeks 3 and 4, spermatogenesis recovered. By 10 weeks after treatment, qualitatively normal spermatogenesis was seen in the great majority of seminiferous tubules, although a few tubules still remained in which the germ cell complement was severely reduced, and contained only Sertoli cells and spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of stage-specific paracrine regulation of Leydig cells by the seminiferous tubules

The size of peritubular Leydig cells surrounding tubules in different stages of the spermatogenic cycle was determined in 43- and 47-day-old male rats. A stage-dependent variation in the size of peritubular Leydig cells was not present in 43-day-old rats, but by 47 days those Leydig cells closely adjacent to tubules at stages VII-VIII were larger than others. At 43 days of age spermatogenesis had developed up to step 18 spermatids in late stage VI tubules. At 47 days of age the first mature sperm had just been released from the seminiferous epithelium, and consequently the first wave of the spermatogenic cycle was completed. Tubules at stages VII-VIII therefore acquire the ability to influence surrounding Leydig cells when they contain step 19 spermatids. It remains to be shown whether this maturation step is due to inherent maturation of the Sertoli cells or if step 19 spermatids specifically modulate Sertoli cell function.     

Classification of the germinal epithelium in Java monkeys (Macaca cynomolgus) using digital image processing

Testicular tissue of two adult crab-eating macaques was used to investigate the distribution of the spermatids in the seminiferous tubules. For this purpose, morphological criteria for distinction among the various degrees of development of these cells were elaborated. Subsequently, based upon analyses of serial sections, three-dimensional reconstructions of the cellular distribution pattern were generated by means of digital image processing. In these reconstructions it became evident that the spatial extension of a single differentiation stage can vary considerably. Large tubular portions that contained a single differentiation stage alternated with those exhibiting several differentiation stages which occupied only small patch-like areas of the seminiferous epithelium. Particularly in the latter portions of the tubule a helical continuity of subsequent stages in the sense of a wave of spermatogenesis became immediately obvious. Transitory changes in the direction of the wave (modulations) occurred frequently. The present findings support the suggestion that an uniform kinetic principle of mammalian spermatogenesis exists, which is subjected to slight modifications during the evolution of primates.     

Morphological alterations and intratubular lipid inclusions as indicative of spermatogenic damage in cimetidine-treated rats

Doses of cimetidine (50 mg/kg b/w) were administered to adult male Wistar rats over 52 consecutive days. Besides plasma testosterone levels, morphological and morphometric aspects of the seminiferous tubules as well as histochemical analysis of the lipid content by oil red O were emphasized. Abnormal tubules exhibiting disorganization of their cellular association, loss of germ cells, and multinucleated giant spermatids were usually found. Significant reductions of testis weight and tubular diameter at specific stages (VII-IX), as well as lack of contact between Sertoli cells and spermatids in tubules at stage IX, suggest a possible interference of cimetidine on the histoarchitecture of the seminiferous epithelium. The dense concentration of lipid inclusions in tubules at postspermiation stages indicates phagocytosis and degradation of germ cells. Since no change in serum testosterone levels was verified in cimetidine-treated rats, the authors could not exclude the possibility that besides an antiandrogenic effect, other biochemical factors necessary for normal spermatogenesis could be involved in the testicular alterations.     

Carcinoma in situ in testicular biopsies

Carcinoma in situ of the testis is an abnormality of the seminiferous epithelium characterized by aneuploid cells with clear cytoplasm located along the tubular basement membrane or in the lumina of tubules. Morphological, cytogenetic, and histochemical features of this anomaly are presented. An adequate fixation of testicular tissue samples is necessary for correct diagnosis. The fact that approximately 50% of carcinoma in situ may develop into malignant germ cell tumours of the testis in 5 years and the investigations of the testicular tissue adjacent to these tumours support the malignant nature and invasive potential of testicular carcinoma in situ. The incidence of carcinoma in situ in persons at risk is reviewed.     

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.     

Carcinoma-in situ of the human testis: Tumour cells are distributed focally in the seminiferous tubules

Summary. The distribution of carcinoma- in-situ was investigated in the longitudinal course of human seminiferous tubules. Serial sections of tubular segments, measuring 9330 μm and 1600 μm in length respectively, were analysed.
The tubules exhibited a local accumulation of tumour cells. Areas with abundant tumour cells followed areas that were free of tumour cells. The original coiled configuration of the seminiferous tubules was reconstructed with the help of a three-dimensional crepe rubber model. The model showed that tumour-bearing tubular segments lay close together, even if they were actually situated far distant from each other in the longitudinal course of the tubule.
Consequently, distant tubular segments can be attached to a common area of the interstitial tissue. This corresponds to the observation that histological sections frequently show clustered CIS-tubules surrounded by tubules exhibiting spermatogenesis. It is a subject for debate whether the interstitial tissue connects tubular segments to functional testicular units.     

Spermatogenese

Spermatogenesis takes place within the testicular seminiferous tubules which consist of the peritubular lamina propria and the seminiferous epithelium. The latter is composed of germ cells and somatic Sertoli cells. Sertoli cells trigger germ cell development by mediating follicle-stimulating hormone and androgen hormonal stimuli. Spermatogenesis comprises proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of spermatids into spermatozoa (spermiogenesis). There are six distinct and specific germ cell associations (I–VI). These “stages of spermatogenesis” occur sequentially along the length of a tubule. Different defects in spermatogenesis occur in adjacent seminiferous tubules (mixed atrophy) and are associated with deficits in differentiation of Sertoli cells. Biopsy specimens should be fixed in Bouin’s solution. Diagnosis of preinvasive carcinoma in situ is based on the immunohistochemical demonstration of placental-like alkaline phosphatase (PLAP), which is expressed exclusively in carcinoma in situ cells. Histological evaluation should be performed using a score count system, and the use of histological techniques for protein and mRNA expression. Testicular biopsy should only be performed in accordance with strict indication criteria, and histological evaluation should be carried out in specialist centres, i.e. as recommended by the European Academy of Andrology (EAA).     

Spermatogenesis--physiology and pathophysiology

Spermatogenesis takes place within the testicular seminiferous tubules which consist of the peritubular lamina propria and the seminiferous epithelium. The latter is composed of germ cells and somatic Sertoli cells. Sertoli cells trigger germ cell development by mediating follicle-stimulating hormone and androgen hormonal stimuli.Spermatogenesis comprises proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of spermatids into spermatozoa (spermiogenesis). There are six distinct and specific germ cell associations (I-VI). These "stages of spermatogenesis" occur sequentially along the length of a tubule. Different defects in spermatogenesis occur in adjacent seminiferous tubules (mixed atrophy) and are associated with deficits in differentiation of Sertoli cells. Biopsy specimens should be fixed in Bouin's solution. Diagnosis of preinvasive carcinoma in situ is based on the immunohistochemical demonstration of placental-like alkaline phosphatase (PLAP), which is expressed exclusively in carcinoma in situ cells. Histological evaluation should be performed using a score count system, and the use of histological techniques for protein and mRNA expression. Testicular biopsy should only be performed in accordance with strict indication criteria, and histological evaluation should be carried out in specialist centres, i.e. as recommended by the European Academy of Andrology (EAA).     

Distribution of carcinoma-in-situ in testes from infertile men

Quantitative histologial studies on 4 testes removed because of carcinoma-in-situ (CIS) were performed in order to determine the distribution of CIS within the testis and to estimate the likelihood of diagnosing testicular CIS by biopsy. The CIS changes were distributed in all parts of the testes but were less frequent in the parts adjacent to the epididymis. In the 4 testes examined 1.4 to 599 of the entire testicular volume contained seminiferous tubules with CIS. In parts of the testes where more than approximately 10% of the testicular volume consisted of tubules with CIS all simulated biopsies measuring 3 mm contained the lesion. The same was true for simulated biopsies measuring 1.5 mm when more then approximately 30% of the testicular volume consisted of tubules with CIS.
If the distribution of CIS generally is similar to that found in the 4 analysed testes there seems to be a high probability of detecting the disease by one or two testicular biopsies.     

Possible role of elongated spermatids in control of stage-dependent changes in the diameter of the lumen of the rat seminiferous tubule

Adult male rats were treated with a single dose of 650 mg/kg methoxy acetic acid to deplete the seminiferous tubules specifically of pachytene and later spermatocytes. The effect of this treatment and the subsequent maturation-depletion of later germ cell types on the diameter of the seminiferous tubule and its lumen and the area of the seminiferous epithelium were studied in relation to the stages of the spermatogenic cycle. At 21 days after methoxy acetic acid treatment, the diameter of the tubule and the area of the epithelium were reduced below control values at all stages, consistent with the reduced number of early (stage VIII) or late (all other stages) spermatids. Unexpectedly, diameter of the lumen was also reduced at all stages other than VIII, and especially at stage VII. In controls, lumen diameter at stages VII and VIII was increased by approximately 50% compared with earlier and later stages. In rats treated 21 days previously with methoxy acetic acid no change occurred at stage VII (lacking elongated spermatids) while a normal increase did occur at stage VIII (lacking round but not elongated spermatids). At earlier times after methoxy acetic acid treatment when stage VII tubules were depleted of pachytene spermatocytes alone (3 days) or together with early spermatids (7 days), the diameter of the lumen was not significantly different from the control value. These data suggest that lumen diameter may be regulated by elongated spermatids, especially at stages VII and VIII.