Laminin, type IV collagen, and fibronectin in normal and cryptorchid human testes. An immunohistochemical study

An immunohistochemical study of laminin, type IV collagen, and fibronectin was carried out in the testes of normal men and in the cryptorchid and contralateral scrotal testes of cryptorchid men from 2 to 40 years of age. The integrated optical density (IOD) per unit area of the lamina propria was measured in the immunostained sections. Fibronectin was found throughout the thickness of the lamina propria of the seminiferous tubules and in the interstitial connective tissue. No differences between normal and cryptorchid testes were found. Laminin was observed in the innermost part of the lamina propria of the seminiferous tubules and surrounding the endothelium of blood capillaries from infancy. No differences were found between normal and cryptorchid testes in the prepubertal period. In adult cryptorchid testes, laminin formed more numerous and deeper invaginations towards the seminiferous epithelium than in normal adult testes. Type IV collagen appeared throughout the thickness of the lamina propria of normal testes as well as in the wall of interstitial blood vessels. From infancy, the lamina propria of seminiferous tubules, but not blood vessel walls, showed lesser immunostaining for type IV collagen and a lower IOD of this component than did control tests from men of the same age. No differences between unilateral and bilateral cryptorchidism were found. The contralateral scrotal testes of cryptorchid males showed intermediate immunostaining for type IV collagen between that of normal control testes and that of cryptorchid testes. These findings suggest that the lamina propria of seminiferous tubules is lesioned at an early age in both cryptorchid and contralateral scrotal testes of cryptorchid men.     

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.     

Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18-day-old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6-, 8-, 17-, and 18-20-day-old mice, and peritubular cells from 18-20 day old mice, all recovered from outbred white Swiss (CD-1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 Bl6/J, C3 HEB, BDF-1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20-day-old Sprague-Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85-98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni-Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 x 10(5) to 2.9 x 10(8) plaque-forming units, and insert sizes averaged 1.0-1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided.     

Pattern of Sertoli cell degeneration in cryptorchid prepubertal tests

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.     

Bilateral prepubertal testicular biopsies predict significance of cryptorchidism-associated mixed testicular atrophy, and allow assessment of fertility

INTRODUCTION: Mixed atrophy of the testis (MAT), a frequent finding in biopsies of formerly cryptorchid and/or infertile patients, is defined as the synchronous occurrence of both seminiferous tubules containing germ cells and Sertoli cell only-tubules in variable proportions. In tubules containing germ cells, different types of abnormalities in spermatogenesis may be seen. The presence of adult spermatids in the biopsy, even in small numbers, correlates with successful spermatozoa retrieval for "in vitro" fertilization techniques. Currently, it is unknown whether precursor lesions of MAT can be identified in cryptorchid patients during childhood. MATERIAL AND METHODS: Eighteen formerly cryptorchid adults who had undergone testicular biopsies in childhood had a repeat testicular biopsy to evaluate infertility. In prepubertal biopsies, abnormalities of the testicular parenchyma were classified into types I (slight alterations), II (marked germinal hypoplasia), and III (severe germinal hypoplasia). In postpubertal biopsies, the percentage of tubules containing germ cells and Sertoli cell only-tubules were estimated, as well as the presence of complete spermatogenesis. Abnormalities in spermatogenesis were classified into lesions of the adluminal or basal compartments of seminiferous tubules. RESULTS: Comparison between prepubertal and postpubertal biopsies revealed that most specimens developing from type III lesions presented with incomplete spermatogenesis (P<0.0001) and more severe lesions of the germinal epithelium (P=0.049). DISCUSSION: Type III lesions correlated with MAT characteristics that confer a worse prognosis for in vitro fertilization. Thus, MAT characteristics may be predicted in prepubertal cryptorchid patients, allowing a fertility prognosis. The pathogenesis of these lesions, and their possible inclusion into the spectrum of the testicular dysgenesis syndrome, are discussed.     

Experimental cryptorchidism in the adult mouse: I. Qualitative and quantitative light microscopic morphology

Morphologic changes in the testes of adult mice after experimentally induced cryptorchidism were studied by light microscopy and stereology. Increasing duration of cryptorchidism resulted in a gradual decrease in the volume of seminiferous tubules per testis, and this was associated with germ cell degeneration. The volumes of Sertoli cell lipid droplets increased, and dilations of the intercellular space between the Sertoli cell junctions was observed in the cryptorchid testis. The luminal volume of the seminiferous tubule was reduced by 50% after 28 days of cryptorchidism. However, the volumes of intertubular tissue and Leydig cells in control and cryptorchid testes were not significantly different. Leydig cell number per testis increased, and the average volume of a Leydig cell decreased gradually with the progression of the cryptorchid state. The volume of the connective tissue cells in the intertubular area increased, but no significant volume change was observed in the volume of intertubular macrophages. After 28 days, the cryptorchid testis contained a significantly increased volume of blood vessels and a reduced volume of lymphatic space per testis. These observations clearly demonstrate that, although the mouse is a species closely related to the rat, the morphologic changes that occur in the Leydig cell population after induction of experimental cryptorchidism in this species is different.     

Elastic fibers in tunica propria of undescended and contralateral scrotal testes from cryptorchid patients

Elastic fibers in the tunica propria of the testes from cryptorchid patients and normal fertile adults were examined by light (Weigert resorcin-fuchsin stain) and electron microscopic techniques. In the testes from normal fertile adults, elastic fibers were proved to exist in the tunica propria by light and electron microscopy, and were located in the fibrous and cellular layers of the tunica propria. In the undescended testes from the cryptorchid patients, during prepubertal and pubertal periods, elastic fibers could not be visualized in the tunica propria, but were found after puberty. The positive Weigert reaction of the tunica propria in the undescended testes from postpubertal cryptorchid patients, however, was markedly weaker than that in normal control patients, suggesting diminution of elastic fibers. This diminution of elastic fibers in the undescended testes from postpubertal cryptorchid patients was also substantiated by electron microscopy. However, in the contralateral scrotal testes, elastic fibers appeared during puberty and were observed after puberty in the same manner as in normal testes. Thus, the present study suggested retarded appearance of elastic fibers in puberty and impaired development of those fibers after puberty, in the undescended testes of cryptorchid patients.     

Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism

Cytoskeletons in Sertoli cell play an important role in process of spermatogenesis. The expression and distribution of the intermediate filaments, vimentin, keratin and desmin, were studied in the Sertoli cells of the cryptorchid testis of rhesus monkey. Vimentin was localized in the perinuclear region of Sertoli cells of the normal testis. An intense increase in vimentin immunoreactivity was observed with appearance of disorganized staining in the Sertoli cells of the cryptorchid testes. Cytokeratin 18, a marker of immature Sertoli cells, re-expressed in the cells of the adult cryptorchid testes. Desmin was also observed in the Sertoli cells in addition to the peritubular myoid cells on 30 days after the cryptorchid operation. These data suggest that Sertoli cells in primate can be affected by the heat stress. The altered changes in intermediate filaments could be possible to induce the Sertoli cell functional changes that would partially contribute to the germ cell apoptosis leading to azoospermia or oligozoospermia.     

Number and DNA content of hypertrophic spermatogonia in normal and cryptorchid human testes.

The number and the DNA content of hypertrophic spermatogonia were studied in normal and cryptorchid testes. The number of hypertrophic spermatogonia in cryptorchid testes is higher than in control testes at the same age. This finding suggests alterations in the spermatogonia of cryptorchid males. The results show a polyploid DNA content in more than 80% of hypertrophic spermatogonia in both normal and cryptorchid testes. There were hypertrophic spermatogonia with a DNA content between 2c and 4c (0.5%) and between 4c and 8c (9%). These spermatogonia might be cells in the S phase of the cell cycle.     

Development of bovine fetal testis tissue after ectopic xenografting in mice

Testis tissue xenografting represents a versatile model to study testis biology, and to preserve fertility in immature animals. To evaluate whether bovine fetal testes can mature when grafted into mouse hosts, small fragments of testes from midgestation (125 to 145 days of gestation) bovine fetuses were grafted ectopically into immunodeficient castrated male mice. At grafting, donor tissue displayed the typical seminiferous cords composed of gonocytes and primitive Sertoli cells. At 5 or 10 months after grafting, weight of the seminal vesicles in recipient mice was indicative of production of bioactive testosterone by xenografts. Xenografts showed similar development regardless of donor age. At 5 months, tubule formation occurred but germ cell differentiation had not proceeded beyond the spermatogonia stage. At 10 months, an increase in tubule size was evident and pachytene spermatocytes were observed as the most advanced type of germ cells in the xenografts of 2 donors. The number of tubules with germ cells was reduced in xenografts compared to donor tissue, but at 10 months the number of germ cells per tubule was higher than in donors. Germ cell proliferation was similar in donor tissue and xenografts. However, Sertoli cells showed a higher proliferation rate in xenografts collected at 5 months than in donor fetal testes and xenografts collected at 10 months. Sertoli cells in xenografts showed a progressive but incomplete loss of expression of Müllerian inhibiting substance and weak androgen receptor expression, indicating an incomplete Sertoli cell maturation. In conclusion, fetal testis tissue developed partially, qualitatively similar to pubertal testes in situ.     

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).     

Localization of integrin β1, α1, α5 and α9 subunits in the rat testis

Very late activation ( VLA, β₁; α₁; α₅, α₉) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti β₁ antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, β₁ integrin was localized to the acrosome. The α₁ subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the α₁ subunit. Immunoreactivity for α₁ integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The α₅ subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III–VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII–VIII. The α9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.     

Laminin and type IV collagen in the human testis

Specimens of normal human testis and biopsies from testes with Sertoli-cell-only syndrome in which the seminiferous tubules had a remarkably thickened lamina propria, were investigated immunohistochemically using specific antibodies against human laminin and human type IV collagen. In the normal testis, both laminin and type IV collagen were localized to the epithelial basement membranes and the peritubular cell layers. In addition, laminin was found in the Sertoli cells. In the pathological testis, structures representing invaginations of the tubular basement membrane were positive for both laminin and type IV collagen. The presence of laminin and type IV collagen in the myoid cell layers, and laminin in the Sertoli cells from both normal and pathological testis and its indication for the secretion of these substances by the myoid and Sertoli cells is discussed.     

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).     

Postpubertal cryptorchism. Morphofunctional study with special reference to Leydig's cells

In a group of 17 patients of postpubertal age with unilateral (n = 15) or bilateral (n = 2) cryptorchism, a significant decrease in the tubular diameter was observed, in addition to Leydig cell hyperplasia (many with cytoplasm vacuolization and/or atrophy) in both the cryptorchid testes and in the contralateral scrotal testes. The number of testosterone-positive Leydig cells in testicular tissue sections, studied with peroxidase-antiperoxidase, was diminished in the cryptorchid testes, whereas in the contralateral scrotal testes it was similar to the control group. Together with normal testosterone levels and elevated luteinizing hormone and follicle-stimulating hormone levels in peripheral blood, this leads us to think of a compensated dysfunction of the Leydig cells. This possible lower testosterone production by the Leydig cells in the cryptorchid testis is not borne out morphologically, where the volume of the organelles is similar to the contralateral scrotal testes.     

Postnatal germ cell development during first 18 months of life in testes from boys with non-syndromic cryptorchidism and complete or partial androgen insensitivity syndrome

IntroductionNeonatal testicular germ cells/gonocytes, transform into stem cells for spermatogenesis during ‘minipuberty’, driving change in timing of surgery. This study examined gonocyte transformation in cryptorchid testes in children ≤ 18 months of age with unilateral, bilateral undescended testes (UDT), complete or partial androgen insensitivity syndrome (CAIS, PAIS) [3,4].Material and methodsTesticular biopsies were taken from patients with unilateral or bilateral UDT, PAIS or CAIS, aged 10 days–18 months. These testicular sections underwent immunohistochemistry with antibodies (Oct4, Ki67, C-Kit, Sox9) followed by confocal imaging, cell counting and statistical analysis.ResultsBoth Sertoli cells/tubule and germ cells (GC)/tubule decreased with age, and % empty tubules (no GC) increased with age but with no significant differences between patient groups. Oct4⁺ germ cells/tubule decreased with age. There are some GCs and Sertoli cells proliferating during the first year and most proliferating Oct4⁺ germ cells (Oct4⁺/Ki67⁺) were located off tubular basement membrane.ConclusionOur study showed that Oct4 expression gradually decreased after minipuberty and transformation into spermatogonia. Germ cells and Sertoli cells undergo mitosis during the first 12 months although not abundantly. We propose that Oct4⁺ gonocyte transformation into spermatogonia via proliferation and migration to the basement membrane may be delayed in UDT.     

The number of spermatogonia in various congenital testicular disorders

Various congenital testicular disorders, including monorchism, retractile testis, cryptorchidism and male intersex, were investigated by counting the number of spermatogonia per seminiferous tubule. The results showed that all 7 cases of monorchism had normal numbers of spermatogonia per seminiferous tubule. However, in 29 cases of a retractile testis a normal testis was observed in 13 (44.8 per cent). Therefore testicular dysgenesis is suggested to exist in more than half of cases of the retractile testis. Of 150 cases of cryptorchidism 82 were bilateral and 68 were unilateral. There was no significant difference in the number of spermatogonia per seminiferous tubule between these 2 groups. The higher the testes were located the worse the ratio of spermatogonia per seminiferous tubule. Fewer or absent spermatogonia were observed in 2 patients less than 2 years old. Of 28 contralateral scrotal testes in patients with unilateral cryptorchidism 4 (14.3 per cent) had no spermatogonia per seminiferous tubule and 8 (28.0 per cent) had a decreased number of spermatogonia per seminiferous tubule. The male intersex patients had much damage even in the scrotal testes. From these results it is suggested that these congenital testicular disorders, except monorchism, have similar histological features. Moreover, these conditions are possibly related in etiology to the phenomenon of deficient androgen stimulation.     

A study of cryptorchidism. I. Light and electron microscopic study of Leydig's cells in the testes of cryptorchid patients

A morphological study of the Leydig's cells in the testes of cryptorchid patients was made by light and electron microscopy. Seventy four unilateral and bilateral cryptorchids (aged 2 to 37 years) were selected for light microscopic observation, and 28 of these specimens were also examined by electron microscopy. In 5 cases of pre-pubertal and pubertal cryptorchids, tissue specimens were biopsied after 20,000 units of HCG had been given and examined similarly. In addition, Leydig's cell density was evaluated quantitatively in the undescended and contralateral scrotal testes of 12 post-pubertal patients. This was based on the determination of the total number of Leydig's cells, Leydig's cell clusters and seminiferous tubules in the entire histologic section of each biopsy and the calculation of the following indices; mean number of Leydig's cells per tubule, mean number of Leydig's cell clusters per tubule and mean number of Leydig's cells per cluster. In addition, the number of Sertoli's cells was counted, and the ratio of Leydig's cells to Sertoli's cells was also calculated. In the undescended testes, almost no mature Leydig's cells were found by light or electron microscopy during pre-pubertal periods; and, even in puberty they were few, while immature precursor Leydig's cells were abundant. In the 5 cases treated preoperatively with HCG, even at 5 years, mature Leydig's cells were observed by light and electron microscopy. On the contrary, after puberty, not only the undescended but also the contralateral scrotal testes of the cryptorchids had more mature Leydig's cells than the normal controls. This Leydig's cell hyperplasia was also confirmed by the quantitative analysis of Leydig's cell density. In the mature Leydig's cells of the undescended testes, however, the electron microscopic observation showed marked regressive changes, in cytology especially prominent depletion of the smooth endoplasmic reticulum and frequent occurrence of specific cytoplasmic inclusion bodies. Such regressive changes as found in the cells of the undescended testes were not observed in the cells of the contralateral scrotal testes. Thus, the morphological alteration of Leydig's cells observed here suggest that the cells are in a dysfunctional condition and that the androgen production is consequently decreased in the undescended testes of cryptorchid patients.     

Effects of steel mutation on spermatogenesis stimulated by fetuin

The effect of the steel mutation on spermatogenesis was investigated using organ culture. Cultured cryptorchid testes from WB-+/+ mice showed an effective differentiation of type A spermatogonia in response to Pedersen type III fetuin. In contrast, the cryptorchid testes from WB-S1/+ showed neither differentiation nor division of type A spermatogonia. The findings reported here are the first demonstration of retarded response of steel mutation on a well-known agent, fetuin.     

Nicotine toxicity to the ultrastructure of the testis in rats.

OBJECTIVE: To investigate the influence of nicotine exposure on the ultrastructure of the rat testis. MATERIALS AND METHODS: Twenty rats were injected with nicotine at a dose of 0.4 mg/100 g body weight daily for 3 months; a group of 20 control rats matched for weight and age were injected with saline only for the same duration. The testes were then harvested and examined by transmission electron microscopy. RESULTS: Rats given nicotine showed: thickening of the tunica propria, caused by an increase in the collagen fibres under the irregular basal lamina; degeneration of junctional specializations between the Sertoli cells, with malformed nuclei showing condensed chromatin; Sertoli cells with numerous polymorphic mitochondria with irregular cristae and an electron-dense matrix. The germ cells were degenerated, spermatids retained excess cytoplasm and accumulated electron-dense lipid droplets in the cytoplasm. The acrosomes were irregular and abnormally configured. CONCLUSION: There were ultrastructural alterations in rats exposed to nicotine that could be attributed to the detrimental effects of nicotine on germ cells, peritubular structures and Sertoli cells.