A morphological study of the testes in patients with idiopathic male infertility--immunohistochemical analysis of collagens and laminin in human testes

In the human testis, the distribution of extracellular components, such as types I, III, IV and V collagens and laminin, was investigated immunohistochemically by light microscopy. Specimens were obtained by testicular biopsy from 40 patients with idiopathic male infertility and 14 normal adult males. In the normal testes, the basement membrane was positive for types I, III, IV and V collagens and laminin. However, the distribution patterns of these components were different. Furthermore, a reactivity for types I and III collagens was found in the interstitial connective tissue matrix. Immunoreactivity for types I and III collagens was markedly positive in the limiting membranes around the Leydig cells. In the pathological testes, all the layers of the basement membrane of both thickened and obstructed tubules were positive for types I and III collagens. On the other hand, reaction products of type IV collagen were localized in the inner layer of the basement membrane and the peritubular cell (myoid cell) layer, and those of laminin were only found in the inner layer. Type V collagen-reactivity was observed in the basement membrane of thickened tubules. Positive reactions for types IV and V collagens and laminin were seldom recognized in the obstructed tubules. In the interstitial space, the connective tissues were significantly increased as compared with normal testes, which included extracellular components that reacted for types I and III collagens. Histological findings in normal adult testes and pathological testes were compared. Quantitative analysis of mean thickness of the basement membrane (W), mean seminiferous tubular diameter (T), T/W ratio and Leydig cell index demonstrated significant differences between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Leydig cell hyperplasia in cryptorchid patients: Quantitative evaluation of Leydig cells in undescended and contralateral scrotal testes

Summary Leydig cell number was evaluated quantitatively in testicular biopsies from post-pubertal cryptorchid patients and normal controls. For this quantitative evaluation we used the following method. This is based on the determination of the total number of Leydig cells, Leydig cell clusters and seminiferous tubules in the entire histologic sections of each biopsy and the determination of the following indices; mean Leydig cells per tubule, mean Leydig cell clusters per tubule and mean Leydig cells per cluster. In addition, the numbers of Sertoli cells were counted, and Leydig-Sertoli cell ratio was also determined. These indices were correlated with each other. All indices were significantly elevated not only in undescended but in contralateral scrotal testes of the cryptorchid patients in comparison to those in normal controls. Between undescended and descended scrotal testes of the same individual patients, those indices were significantly higher in the descended scrotal testes than in the undescended ones. Thus, Leydig cell hyperplasia was noted in the testes of post-pubertal cryptorchid patients, and was more prominent in the contralateral scrotal testes than in the undescended ones.     

Ischemia,whether from ligation or torsion,causes ultrastructural changes on the contralateral testis

OBJECTIVE: Unilateral testicular torsion results with a decrease in contralateral testicular blood flow caused by a reflexive sympathetic response. The aim of this study was to investigate whether twisting of the spermatic cord, or testicular ischemia without twisting, activates this reflex mechanism and causes ultrastructural changes in the contralateral side. MATERIALS AND METHODS: Thirty adult male albino Wistar rats were randomly allocated into three groups of sham, torsion, and ligation. Right testes were twisted 720 degrees counterclockwise in the torsion group. Right spermatic cords were ligated permanently with a silk suture including the vas deferens in the ligation group. After 24 h of testicular ischemia, contralateral left testes were removed for electron microscopic evaluation. RESULTS: Contralateral testes showed similar ultrastructural changes in the torsion and ligation groups. The fibrous tunica propria enveloping the seminiferous tubule was thickened due to increased collagen fibers. The basal lamina was continuous but thickened and showed several foldings. The gap between basal lamina and the germ cells was increased because of collagen fibers. Leydig cells showed mitochondrial degeneration with the loss of its cristae. Leydig cells lost their contact with its neighborhood cells in some areas, and these gaps were filled with collagen fibers. Germ cells showed dilated cisternae of smooth endoplasmic reticulum, cytoplasmic electron-dense bodies and clear regions. CONCLUSIONS: Similar electron microscopic findings observed in the torsion and ligation groups indicate that testicular ischemia rather than twisting of the spermatic cord is responsible for the ultrastructural changes in the contralateral side.     

Ectopic Leydig Cells in Seminiferous Tubules of an Infertile Human Male with a Chromosomal Aberration

A case of a human male infertility with chromosomal aberration is reported. The patient showed neither mental retardation nor physical abnormalities except that the testes were somewhat small and soft. Plasma follicle stimulating hormone and luteinizing hormone were 49.0 and 19.0 mIU/ml. Plasma testosterone was 2.6 ng/ml. Karyotype was considered to be 46 XY q-, long arms of the Y chromosome being deleted. Histological features of the testis were peculiar. Seminiferous tubules were small and devoid of spermatogenic cells, consisting only of Sertoli cells. Peritubular boundary layer of the tubules showed a marked increase in width due to the increase of collagen fibers. The base of some Sertoli cells was seen to protrude into the thickened peritubular boundary layer or, though rare, into the interstitial space. Unusual cells which had a round vesicular nucleus and abundant, dense cytoplasms also occurred in the boundary layer of most tubules. These cells were identified as Leydig cells because of an extensively developed smooth endoplasmic reticulum in their cytoplasm, although they lacked Reinke's crystals. These ectopic Leydig cells sometimes lay in direct contact with Sertoli cells in the seminiferous tubule.     

Histochemical study on glutathione S-transferase in patients with testicular tumor

Tumor tissue and nontumorous tissue of 31 patients with testicular tumor were examined by the peroxidase antiperoxidase (PAP) procedure using the primary antibody against glutathione S-transferase (GST). Histology of primary tumor was classified as seminoma in 10 cases, non-seminoma in 18 (including 2 cases of yolk sac tumor), and malignant lymphoma in 3. Tumorous tissues except one with yolk sac tumor failed to be stained with GST. The seminiferous tubules of the nontumorous testicular tissue had a positive reaction in the infant cases, but not in the adult cases. The degenerated seminiferous tubules involved in the testicular tumors also had a positive reaction in all the cases. Leydig cells had a positive reaction in all the cases. In particular, diffuse Leydig cell's hyperplasia was observed in a case with high serum beta hCG and urinary hCG levels. These data may be relevant in explaining the inherent hypofertility of these patients.     

Melatonin prevents testicular damage in hyperlipidaemic mice

The damaging effect of hyperlipidaemia on testicular structure was determined, and the influence of melatonin was evaluated in testicular damage related to hyperlipidaemia. Hyperlipidaemia was induced in ApoE-knockout C57BL/6J male mice fed with high-fat diet alone (group A), or with high-fat diet and melatonin (group B). Six ApoE wild-type C57BL/6J male mice were fed with normal diet, served as controls. At the end of the experimental period, ultrastructural observations showed dramatically histopathological alterations in testicular tissues of group A. The basement membranes of seminiferous tubules were partially thickened and wavy-like in testes of mice with hyperlipidaemia, and vacuolar degeneration of mitochondria and dilation of endoplasmic reticulum were identified as well as the number of mitochondria and lipid droplets decreased significantly in Leydig cells and Sertoli cells. Electrondense deposits were observed in cytoplasms of germ cells. The testicular histostructure in group B treated with melatonin was similar to that of control. Apoptosis was determined by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labelling. Apoptotic germ cells were significantly more numerous in group A than in group B and controls. The results suggest that melatonin may be potential to attenuate testicular damage by improving histopathological changes and reducing germ cell apoptosis in hyperlipidaemic mice.     

The leprous testis

A clinical investigative study of 148 male leprous patients demonstrated the presence of testicular lesions in 35 cases. Semen analysis revealed marked oligo-athenozoospermia in 10 cases and azoospermia in 25 cases. Testicular biopsies from leprous testes showed different histologic patterns ranging from spermatogenic arrest to complete hyalinization of both seminiferous tubules and interstitial tissue. Histochemical staining for neurovascular supply revealed degenerative nerve change in addition to altered permeability of the testicular capillaries. There was good correlation between the results of semen analysis and histological and histochemical examination of testicular biopsies.     

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.     

The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors

PURPOSE: The role of androgen receptor coactivators in testicular development and cancer formation is unclear. p44/Mep50 was identified as an androgen receptor coactivator that functions in a complex with protein arginine methyltransferase 5. We studied the expression of p44 and protein arginine methyltransferase 5 in developing fetal testis and adult testicular tumors, including seminomas and Leydig cell tumors. MATERIALS AND METHODS: A total of 30 human fetal testes from abortuses at a gestational age of 10 to 40 weeks, 33 human seminomas and 11 human Leydig cell tumors were retrieved from the archives of the departments of pathology. Immunohistochemistry was performed with affinity purified p44 and IgG purified protein arginine methyltransferase 5 polyclonal antibodies. RESULTS: Protein arginine methyltransferase 5 and p44 were expressed predominantly as nuclear proteins in fetal Leydig cells and human adult nonneoplastic testes, including germ cells and Leydig cells, while they were expressed in the cytoplasm of germ cells of the fetal testis. Expression was strongest in the fetal testis during the second trimester. Compared to adult nonneoplastic testes, human seminoma and Leydig tumor cells showed a marked decrease in nuclear expression of p44 and protein arginine methyltransferase 5 with a concomitant marked increase in cytoplasmic expression of these proteins. Furthermore, average testicular size was increased by 29% in p44(+/-) heterzygotic mice. CONCLUSIONS: These results suggest distinct functions of the nuclear and the p44/protein arginine methyltransferase 5 complexes in the developing fetal testis and in the oncogenesis of testicular tumors. Further studies are needed to confirm the functional relevance of these findings.     

Male infertility with chromosomal abnormalities. II. XX-male syndrome

We report two cases of the XX-male syndrome, and review the literature. The first case was a 31-year-old married man, a welder, complaining of infertility. His height was 158 cm, weight 82 kg and distance of extended hand 155 cm. The external genitalia showed a normal male type, but bilateral small testes and gynecomastia were noticed. The second case was a 32-year-old married man, a shopkeeper, complaining of infertility. His height was 165 cm, weight 60 kg and distance of extended hand 167 cm. No gynecomastia was noted. The external genitalia showed a normal male type, but bilateral small testes were noticed. In each case, azoospermia was identified in semen analysis. Urethrography revealed the prostatic utricle in the second case. The testicular biopsy specimens revealed hyalinization of seminiferous tubules and proliferation of Leydig cells. X chromatin was positive in buccal smears, and Y chromatin negative in cultured lymphocytes. Chromosomal analysis showed 46, XX karyotype in the first case and 46, XXp+ in the second case. H-Y antigen was positive in each case. Basal serum levels of LH and FSH were moderately elevated and the serum testosterone level was low. Serum levels of PRL, TSH, estradiol, GH, T3 and T4 were normal. An impaired response by testicular Leydig cells to hCG was observed. The LH and FSH responses to LH-RH were almost normal. Clomiphene citrate administration resulted in a decrease in the serum testosterone and gonadotropins levels. These results indicate hypergonadotropic hypogonadism secondary to testicular failure in both XX-males. Twenty-six cases of the XX-male syndrome have been cited in the Japanese literature. The clinical features and etiology of this syndrome are discussed.     

Morphological and Endocrinological Differences between the Abdominal Testes in Bilateral and Unilateral Cryptorchid Rats

Using a new experimental model of cryptorchism in rats, where testicular descent was prevented, testicular development and function were studied in bilateral and unilateral cryptorchid animals Morphometric and radioimmunological techniques were used. Up to 30 days after birth testicular development was identical in the two types of abdominal testes but in adult rats differences were observed. In these rats spermatogenesis was damaged to a similar extent, but total tubular length and testicular weight were increased in the bilateral abdominal testes. Moreover, in these testes, the volume density of Leydig cells, the total Leydig cell mass, the average Leydig cell size and the testis testosterone concentration were larger than in unilateral abdominal testes. It is suggested that the impaired spermatogenesis seen in both kinds of abdominal testes may be unrelated to Leydig cell function.     

Immunohistochemical and ultrastructural study of Sertoli cells in androgen insensitivity

The ultrastructure of a perfusion-fixed gonad in a case of androgen insensitivity was studied using thin sections and freeze-fracture replicas. The distribution and arrangement of intermediate filaments in Sertoli cells was visualized immunohistochemically using an antibody against vimentin. Leydig cells lacked Reinke crystals, but contained all of the cytoplasmic organelles involved in steroid synthesis and additionally several lysosomes. The basement membrane and the basal lamina of the testicular tubules were considerably thickened. The testicular tubules consisted of gonocytes and Sertoli cells which had an immature nuclear structure, incomplete development of intercellular junctions and a primitive distribution pattern of intermediate cytoplasmic filaments. The previously reported differences in electron density of Sertoli cell cytoplasm are a non-specific feature without significance to Sertoli cell maturation.     

Testicular needle biopsy: Is it a safe and adequate method?

We aimed to investigate if testicular needle biopsy is adequate and safe for the examination of testis. Needle biopsies were performed on 21 testes of 5 patients with advanced prostate cancer and 11 patients with cryptorchidism before orchiectomy. Biopsies were done with the prostatic tru-cut needle. After needle biopsy, the tract and puncture sites were explored and an incisional biopsy was performed on each testis. Both needle specimens and open biopsy specimens were fixed in Bouin's solution and sent for histologic examination. There were small haematomas in two testes and moderate haemorrhage between tunica vaginalis layers in another. The tissues obtained by needle biopsy were sufficient except for two specimens and diagnostic accuracy was perfect. Nevertheless, measurement of the seminiferous tubules of the lamina propria could not be achieved in many cases.     

Abnormal ultrasonic pattern in contralateral testes in patients with unilateral testicular cancer

Summary Ultrasound examination and biopsy of the nonaffected testis was performed in 78 men with a unilateral testicular cancer. Each testis was measured in three planes and the volume was calculated using the formula of an ellipsoid. The ultrasonic texture of each testis was given a score ranging from 1 to 5 as follows: 1, very regular; 2, slightly irregular; 3, irregular with small echogenic points; 4, very irregular or with coarse echogenic points; and 5, irregular with demarcated areas raising suspicion of tumor. Biopsies were examined for the presence of tubules with carcinoma in situ (CIS), germinative epithelium, Sertoli cell only, and obliterations; the thickness of tubular membranes and the amount of Leydig cells were registered. The mean ultrasonic testicular volume was 12.88 ml (range 3–24 ml), which was smaller than that previously reported for normal men and larger than that previously reported for infertile men. The ultrasonic testicular volume was inversely correlated to the score. Score 4 was given to 46% of the testes (median score, 4), and the score distribution was different from that reported in normal men (median, 2) and in infertile men (median, 3). In all, 9 testes contained CIS tubules, and 8 of these were given score 4; 1 testis with CIS in only 5% of the tubules was given score 3. The predictive value of score 4 for the testis to contain CIS was 22.2%, and the predictive value of a score different from 4 that the testis would not contain CIS was 97.6%. We conclude that a large percentage of contralateral testes in men with unilateral testicular cancer have an abnormal echotexture and that CIS is most likely found in testes given score 4 by ultrasound.     

Immunohistochemical, histological and ultrastructural evaluation of the effects of leptin on testes in mice

This study was undertaken to examine the effects of leptin on testes in mice. For this purpose, 12 male mice were divided into two groups. Animals in Group I were designated as control. Mice in Group II were injected daily with leptin for 5 days. All animals were decapitated at the end of the experiment. The testes were removed and weighed out. Testicular tissue specimens were processed for light and electron microscopic examination and semi-quantitative evaluation of immunohistochemical testosterone staining. Intensity of immunostaining was determined on a scale between 0 (no staining) and 5 (heavy staining). For morphometric comparison, diameters of seminiferous tubules from each group were measured. In the leptin injected group, testicular weights and diameters of seminiferous tubules were significantly increased in comparison to control values. In light microscopic examination, an increase in secretory granules in the cytoplasm of Leydig cells was observed after leptin treatment. In the same group, distinct changes indicative of increased cell activation were seen in the ultrastructure of Leydig cells. Amount of mitochondria, lysosomes and cytoplasmic secretory granules were increased. Furthermore, an increase in extensiveness of rough endoplasmic reticulum was noted in this group. Immunohistochemical testosterone staining of the cytoplasm of Leydig cells was heavy (5+) in the leptin treated mice compared to mild score (2+) in the control mice. Additionally, heavy immunostaining of testosterone was also observed in the interstitial space after injection of leptin. The present findings indicate that testicular functions and synthesis of testosterone increase after administration of leptin.     

The testicular regression syndrome--do remnants require routine excision?

Aim

Excision of testicular remnants is debatable in the scenario where hypoplastic vas and vessels can be seen entering a closed internal ring during laparoscopy for impalpable testes. We aimed to establish how frequently excised remnants have identifiable testicular tissue and, hence, malignant potential.

Methods

This study is a retrospective review of all excised testicular remnants in children with impalpable testis. Specimens that were excised for indications other than testicular regression syndrome were excluded. Pathology reports of excised specimens were reviewed, and the presence of multiple histologic features was noted. Histologic confirmation of testicular/paratesticular tissue required the presence of 1 or more of the following: seminiferous tubules, germ cells, Sertoli cells, Leydig cells, vas deferens, or epididymal structures. Malignancy potential was defined by the presence of germ cells or seminiferous tubules. All patients with seminiferous tubules were further examined by a single histopathologist.

Results

A total of 208 testicular remnants from 206 children were excised over the 11-year period (1999-2009). Histologic evidence confirmed excision of testicular/paratesticular tissue in 180 cases (87%). Seminiferous tubules were noted in 27 (15%), and germ cells were present in 19 (11%) cases.

Conclusion

Viable germ cells were found in 11% of examined remnants, which, in our opinion, justifies their removal.     

Immunohistochemical localization of inhibin-α in the testes of normal men and in men with testicular disorders

Testicular biopsies from normal men and from men with testicular disorders were examined by immunohistochemistry for the presence of the inhibin-alpha subunit using two different antisera. Immunoreactive inhibin-alpha (irI-alpha) was found in Leydig cells in normal, oligospermic, and azoospermic men and in men with Klinefelter's syndrome, and it was also found in a Leydig cell tumour. hCG-treatment apparently increased the amount of immunoreactive inhibin-alpha, particularly in Leydig cells. Sertoli cells also contained irI-alpha but the staining intensity was considerably stronger in testes with impaired spermatogenesis or Sertoli-cell-only syndrome than in normal testes. It is suggested that the serum concentration of irI-alpha and inhibin in humans may, in a complex way, be related to both Leydig and Sertoli cell function, and that the relative contribution from these cells may change in cases of testicular malfunction.     

Reproductive aging in the Brown Norway rat is characterized by accelerated germ cell apoptosis and is not altered by luteinizing hormone replacement.

Reproductive aging in the male Brown Norway (BN) rat is characterized by decreased Leydig cell steroidogenesis associated with seminiferous tubule dysfunction. This could be a result of a combination of a primary testicular defect and a secondary hypothalamic pituitary dysfunction. In the present study, we determined in the BN rat whether germ cell loss occurred via apoptosis. We then defined the age of onset of Leydig cell dysfunction and germ cell loss and examined whether chronic luteinizing hormone (LH) replacement would delay or prevent reproductive aging. Plasma hormone levels, testicular sperm concentrations, and germ cell apoptosis were studied in 6, 9, 12, 15, 18, and 21-month-old BN rats. Beginning at 15 months, testicular weight, sperm concentration, total sperm counts, plasma testosterone, LH, and inhibin decreased, whereas the proportion of regressed testes and plasma follicle-stimulating hormone (FSH) levels increased with aging. Accelerated germ cell apoptosis involving spermatogonia, preleptotene and pachytene spermatocytes, and spermatids was evident in some tubules of the relatively normal testes from 21-month-old rats. In the regressed testes, complete cessation of spermatogenesis occurred. The apoptotic index was higher in the testes of old (21-month-old) rats in particular at stages XII-XIV when compared with younger animals. Chronic LH replacement (0.5 microg i.p. twice per day) administered to 15-month-old BN rats for 6 months did not alter plasma hormone levels, testes weight, sperm concentration or content, or the germ cell apoptotic index. In the control group, 3 out of 10 testes were regressed, whereas in the LH-replaced group, only 1 out of 12 testes was regressed. We show in this study that early reproductive aging in the BN rat began at around 15 months. Germ cell loss associated with aging occurs via apoptosis. Replacement therapy with LH for 6 months does not decrease or delay the testicular dysfunction associated with aging. It is unlikely that hypothalamic-pituitary dysregulation is the major cause of testicular aging.     

Effects of photoperiod on the ultrastructure of Leydig cells in rat

The aim of this study was to examine effects of photoperiod on the ultrastructure of Leydig cells in rat. For this purpose, 21 male Wistar rats were used. Animals were divided into three groups: Control rats in group I were kept under 12 hrs light: 12 hrs dark conditions (12L: 12D) for 10 weeks. Animals in group II were exposed to long photoperiods (18L: 6D), while rats in group III were exposed to short photoperiods (6L:18D) for 10 weeks. At the end of the experiment, all animals were killed by decapitation and blood samples were obtained. Serum testosterone levels were determined with the use of a chemiluminescent enzyme immunoassay. The testes of all rats were removed and weighed, then processed for light and electron microscopy. For morphometric comparison, diameters of seminiferous tubules in each group were measured. In rats exposed to long photoperiods, testicular weights, diameters of seminiferous tubules and serum testosterone levels were significantly increased as compared to those in control rats, whereas exposure of rats to short photoperiods resulted in a significant decrease of testicular weights, diameters of seminiferous tubules and serum testosterone levels as compared to those in control rats and rats maintained in long photoperiods. The amount of mitochondria and cytoplasmic secretory granules were increased in the cytoplasm of Leydig cells of rats exposed to long photoperiods. Furthermore, an increase in extensiveness of rough endoplasmic reticulum in the cell cytoplasm was noticed in this group, whereas a decrease in mitochondria and cytoplasmic secretory granules of the Leydig cell cytoplasm was seen in rats exposed to short photoperiods. The results of our study indicate that testicular functions increase after exposure to long photoperiods and decrease after exposure to short photoperiods.