Histopathological diagnosis of hypercurved seminiferous tubules

A simple method for the recognition of hypercurvature of the seminiferous tubules in otherwise normal biopsies from infertile males consists of the recognition of characteristic 'figure 8' profiles of sectioned seminiferous tubules. Nineteen cases of morphometrically verified hypercurvature and 24 controls were tested in order to determine the validity of the method, which showed virtually no overlap in the two groups when at least four x 100 fields were assessed for 'figure 8' and grazing profiles. A recommended diagnostic approach, with cautions concerning pitfalls, is presented, and incidence, pathogenesis, and potential treatment are briefly discussed.     

Three‐dimensional structure of seminiferous tubules in the adult mouse

Seminiferous tubules develop from sex cords, which are embryonic structures with simple C‐shaped arches. Histologically, the epithelium of adult mouse seminiferous tubules has been divided into 12 stages based on the associations of spermatogenic cells in four cycles of spermatogenesis. However, the gross characteristics of the seminiferous tubules themselves, including their number, length, run, and mutual relationships remain largely unknown. In the present study, we analyzed all seminiferous tubules in a single adult mouse testis with high resolution using serial paraffin sections and high‐perfomance three‐dimensional reconstruction software. There were 11 seminiferous tubules with an average length of 140 mm. Each tubule ran along circular paths within the testis while making convolutions with cranial and caudal hairpin turns. The cranial turns of all tubules were in contact with the tunica albuginea, whereas the caudal turns were not, resulting in funnel‐shaped networks of these tubules with tapered caudal portions. The caudally located networks surrounded the preceding cranially located networks from the bottom and outside, similar to stacked paper cups. Five out of the 11 seminiferous tubules were continuous from one end to the other both connected with the rete testis (10 connection points). Nine branching points, one blind end, and 18 more connection points with the rete testis were detected in the remaining six seminiferous tubules, making the paths of these tubules complicated to various degrees. The present study revealed that the 3D structures of seminiferous tubules were highly regular as a whole in the adult mouse testis.     

Flagellum abnormalities of spermatozoa in seminiferous tubules after a short term vasectomy.

Electron microscopic observation was performed to examine whether spermiogenesis in the hamster might be affected by a short term vasectomy. When viewed by light microscopy, spermiogenesis was temporarily inhibited at 2 weeks, though to a limited extent among individuals, and had apparently recovered to the control level at 4 and 8 weeks after vasectomy in all hamsters. It was revealed at the electron microscopic level that mature spermatozoa with an abnormal flagellum were intermingled among numerous normal spermatozoa. The flagellum of the mature spermatozoa was composed of four different components: a mitochondrial sheath, outer dense fiber, fibrous sheath and axoneme. Abnormalities of the mitochondrial sheath were of three types: its discontinuity, displasia and deformation. The appearance of these abnormalities increased with the time after vasectomy and finally reached 52% as the highest value at 8 weeks. Additional noteworthy findings were the bisectioning of the outer dense fibers 2, 4 and 7, the fusion of two or three fibers, and the partial deletion of the fibrous sheath. These defects were characteristic in the vasectomized hamsters. Possible correlating microenvironmental factors are discussed as to how these abnormalities of the four flagellar components occur in the vasectomized hamsters.     

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.     

Effect of testicular macrophage conditioned media from rats with autoimmune orchitis on Leydig cell function

PROBLEM: The aim of this study was to investigate the influence of immune-activated testicular macrophages obtained from rats with autoimmune orchitis (EAO) on Leydig cell steroidogenesis. METHOD OF STUDY: EAO was induced in rats by active immunization with testis homogenate and adjuvants. Testicular and peritoneal macrophages from rats with EAO were isolated and cultured for 24 hr. Testosterone (T) production by purified Leydig cells incubated in vitro with macrophage-conditioned media (CM) from rats with EAO or control rats was measured. RESULTS: An increase in T production by Leydig cells incubated with CM from testicular, but not peritoneal, macrophages of rats with EAO was observed. This increase was dose-dependent up to a concentration of 30% CM; proportions higher than 35% exhibited an inhibitory effect. CONCLUSIONS: Immune-activated testicular macrophages obtained from rats with EAO induced both stimulatory and inhibiting steroidogenic effects on Leydig cells in vitro and not the exclusively inhibitory action that has widely been attributed to activated macrophages. This dual effect probably depends on the ability of these cells to synthesize different molecules that may exert opposite effects.     

Degenerated tubules in the guinea pig testis after long-term vasectomy or sham operation

The testes of eight unilaterally vasectomized and six sham-operated Dunkin Hartley guinea pigs were examined 3 years after operation by wax and resin histology and transmission electron microscopy. Degenerated tubules are reported that were common on the side of vasectomy but also found in the contralateral testes and in the controls. A central accumulation of macrophages, rich in phagocytosed debris including spermatozoal fragments, was surrounded by attenuated Sertoli cells, a markedly thickened basement membrane and myoid cells. At some sites macrophages impinged directly on the basement membrane. They probably represented highly degenerated seminiferous tubules. The study suggests that the response to injury of seminiferous tubules may show species variations. Macrophages did not feature in the degenerated seminiferous tubules we reported following vasectomy in the rat. However, the rat showed striking changes in the morphology of the basal laminae and myoid cells which did not occur in the guinea pig. Pathological changes have been reported in the human testis following vasectomy but their etiology is unclear. Studies in the guinea pig are enhancing understanding of the mechanisms and features of testicular damage.     

Three-dimensional structure of seminiferous tubules in the Syrian hamster

The hamster is useful for the study of male reproductive biology. However, unlike in the mouse and rat, the gross structure of seminiferous tubules in the hamster is largely unknown. The aim of the present study was to clarify the precise 3-dimensional (3D) structure of seminiferous tubules in hamsters. We reconstructed all seminiferous tubules in 3 and 1 testes from 0-day (P0) and 10-week (adult) Syrian hamsters, respectively, using serial paraffin sections and high-performance 3D reconstruction software. In P0 hamsters, the average numbers of seminiferous tubules, terminating points, branching points, and blind ends per testis were 9.0, 89.7, 93.0, and 0.7, respectively. There were two types of tubules: shorter and dominant ones. The dominant tubules, 2–4 in number per testis and accounting for 86% of the total tubule length, had many terminating and branching points and appeared to be derived from the anastomosis of many shorter tubules. In an adult hamster, there were 11 seminiferous tubules with a total length of 22 m, 98 terminating points, 88 branching points, and 2 blind ends per testis. Three of the 11 tubules were dominant ones, accounting for 83% of the total length, and occupied the testis from the surface over the circumference to the center, while the others were short and occupied only one side of the testis. The amplitude and direction of the curves of tubules were random, and there were no funnel-shaped networks of tubules present, in contrast to the mouse testis. The present study revealed the 3D structure of seminiferous tubules in developing and adult Syrian hamsters, which is different from that in mice and rats.     

Development of the seminiferous tubules and rete testis during prenatal human development

The source of origin and particular features of the formation of the seminiferous tubules and tubules of the rete testis during prenatal human development were studied. The seminiferous tubules and tubules of the rete testis were shown to be formed from bands of cells of the celomic epithelium and primordial germ cells, which appear simultaneously in the anlage of the mediastinum testis and the central part of its parenchyma in embryos 13.0–17.0 mm long. It was shown by methods of plastic and graphic reconstruction and fine dissection under the control of the MBS-1 binocular microscope that the seminiferous tubules anastomose with each other both in the same and in adjacent lobules. Tubules of the rete testis do not form anastomoses but are superposed one above the other to give the impression of a network. They merge as the approach the tunica albuginea and continue into the efferent ductules.Department of Human Anatomy, Chernovtsy Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 227–229, February, 1978.     

Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rabbits

Using stereological methods, especially the optical disector for unbiased estimation of nuclear number, our recent study demonstrated that long-term (6 or 12 months) vasectomy in the rhesus monkey had no significant effects on spermatogenesis (Peng et al. Reproduction 2002, 124, 847-856). This study aimed to determine the scenario in the rabbit using the same morphometric methodology. Three groups of normal male Japanese white rabbits (aged 4-5 months) were subjected to unilateral vasectomy; 10 days, 6 months and 12 months later both testes and epididymides were removed. Testicular and epididymal methacrylate-embedded sections were obtained for stereology. Vasectomy-induced damage to spermatogenesis was observed, primarily sloughing of spermatogenic cells with a greater reduction in the number of advanced (adluminal) cells. The damage was most severe at 10 days, occurring in all the testes on the vasectomized side and involving sloughing of even type A spermatogonia, the number of which returned to normal at 6 and 12 months. Damage was less severe at 6 and 12 months, being found in half of the testes of the vasectomy side, in which the total numbers of later germ cell types were 24.0-59.1% (spermatocytes) and 0.3-11.6% (spermatids) of control at 6 months, and 20.1-22.1% (spermatocytes) and 0.4-12.0% (spermatids) of control at 12 months. By contrast, Sertoli cell number per testis was unchanged following vasectomy in any group. Epididymis on the vasectomy side, especially at 10 days and 6 months, appeared larger than on the contralateral side, but this difference was not statistically significant, and no sperm granuloma was seen in the epididymis.     

Morphogenesis of the bovine rete testis: the intratesticular rete and its connection to the seminiferous tubules

The development of the intragonadal rete testis and the establishment of the connection between seminiferous and straight testicular tubules was studied using ultrastructural and histochemical methods in 60 bovine embryos and fetuses ranging from day 39 through day 225 post conceptionem. The methodology included a modified acetylcholinesterase (AChE) reaction as a selective marker for pre-Sertoli cells and a modified microsomal aminopeptidase (MAP) reaction as a selective marker for the epithelia of rete testis and straight testicular tubules. Between 40 and 45 days, the rete testis is predominantly an extratesticular rete situated in the cranial peduncle of the gonadal fold and in broad contact with the pro/mesonephric giant corpuscle. During this period, the intragonadal rete enters the gonad proper from its craniodorsal pole and extends into the cranial fourth of the testis. Between 60 and 110 days the rete testis attains its definitive position, extending into the central longitudinal axis as far as to the caudal fourth of the testis. For the caudal expansion of the rete testis the preceding proliferation of the mediastinal stroma is an important prerequisite. In the 40 to 45-day-old embryo the area of the testicular cords may be divided into two zones. A narrow outer zone contains plate-like cords with a thick diameter, and a larger central zone is filled with a network of thinner cords. Only the thick outer cords transform into the permanent seminiferous tubules, whereas the thinner cords in the central zone are transitory structures that disappear between 45 and 110 days. One important function of these transitory cords is to establish a continuous system of basal laminae that allows a direct connection between the central ends of the growing seminiferous tubules and the peripheral extensions of the rete testis (future straight testicular tubules). The first true straight testicular tubules become visible between 85 and 110 days. Due to a strong proliferation of the tubulus rectus-cells the straight testicular tubules elongate continuously, and the border between the rete system and the seminiferous tubules is slowly shifted towards the testicular periphery. This shift is not restricted to the prenatal period, but proceeds until after birth. At the cytological level, the formation and elongation of the straight testicular tubules is effected by proliferating cells that advance along the continuous basal lamina into the area of the seminiferous tubules. The pre-Sertoli and germ cells in this zone of invasion are separated from each other and overgrown by the tubulus rectus-cells. Exposed to the special milieu of the straight testicular tubules, pre-Sertoli and germ cells apparently cannot survive and finally disappear. Accepted: 31 July 2000     

High doses of nandrolone decanoate reduce volume of testis and length of seminiferous tubules in rats

Anabolic-androgenic steroid (AAS) compounds rank among the drugs most widely abused with the goal of improving athletic ability, appearance, or muscle mass. It has been shown that these compounds have adverse effects on human and animal physiology and sperm quality, but quantitative structural changes of the testis have received less attention. The present study was conducted to evaluate the effects of nandrolone decanoate, which is one of the AAS compounds, on testis weight and volume, diameter and length of seminiferous tubules in rats by unbiased stereological methods. Adult rats were divided into three groups. The first comprised control rats; the second and third groups received low and high doses of nandrolone decanoate for 14 weeks. The rats were then left untreated for 14 weeks. After removal of the testis, stereological study of these tissues showed that the mean volume of testis and length of the seminiferous tubules in the animals that received high doses of nandrolone decanoate were reduced approximately 32% (p<0.01) and approximately 31% (p<0.04), respectively, in comparison with the control group. It can be concluded that the high doses of nandrolone decanoate produce structural changes in the rat testis that remain 14 weeks after stopping injection of the drug.     

Influence of the male reproductive tract on the reproductive potential of round spermatids abnormally released from the seminiferous epithelium.

Round spermatids can be collected from testicular biopsy material or occasionally from semen samples. We evaluated the influence of the passage of round spermatids through the male reproductive tract on their reproductive potential. A model of abnormal release of round spermatids from the seminiferous epithelium was created in mature male rats (group A). Additional sham-treated rats of the same age served as a control group (group B). Round spermatids were collected from the testicles of rats of both groups, the epididymides of rats of group A, and the vaginae of mature female rats mated with rats of group A. Isolated round spermatids were processed for ooplasmic injections. Injected oocytes were cultured. At 96 h post-injection, the blastocyst development rate was significantly higher in the groups of oocytes injected with testicular spermatids than the groups of oocytes injected with spermatids recovered from the vaginae, or the head, body, or tail of the epididymides. It appears that round spermatids recovered from testicular biopsy material have larger reproductive capacity than ejaculated round spermatids, due to mechanical or chemical detrimental influences of storage/passage through the male reproductive tract (outside the testicle) on the capacity of round spermatids to induce optimal early embryonic development.     

Role of estrogen in spermatogenesis in initial phase males of the three‐spot wrasse (Halichoeres trimaculatus): Effect of aromatase inhibitor on the testis

The three‐spot wrasse, Halichoeres trimaculatus, is a protogynous hermaphrodite. Under appropriate social conditions, female fish can become male. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the role of estrogen in the male is not known. To clarify the involvement of estrogen in spermatogenesis in hermaphrodite fish, we treated initial phase (IP) males for 10 weeks with exemestane, an aromatase inhibitor (AI), to block estrogen synthesis. Fish treated with AI exhibited decreases in gonadal weight, plasma estrogen levels, and spermatogonial proliferation in the testis, together with increases in androgen levels. Additionally, we confirmed that exogenous estrogen treatments stimulated the renewal and proliferation of spermatogonia in the testis of IP males. These results indicate that estrogens play an important role in regulating spermatogenesis in this fish. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Germ cell apoptosis in autoimmune orchitis: involvement of the Fas-FasL system

PROBLEM: The aim of this study was to determine the mechanism of germ cell death in experimental autoimmune orchitis (EAO) and the involvement of the Fas-FasL system in this process. METHOD OF STUDY: The EAO was induced in rats by immunization with testis homogenate and adjuvants. Apoptosis was studied by light microscopy, in situ end labeling of apoptotic DNA and DNA fragmentation techniques. Fas, FasL and caspase 3 expression was detected by immunohistochemistry. RESULTS: In rats with orchitis the number of Fas+ and FasL+ apoptotic germ cells increased from day 50, when the lesion develops, to 150 days, and correlates with the degree of testicular damage. Most spermatocytes expressing Fas were apoptotic. Many Fas+ germ cells were also immunoreactive for FasL. Moreover, these cells also expressed caspase 3. CONCLUSIONS: In rats with EAO germ cell death occurs through an apoptotic mechanism preceding germ cell sloughing. Immunohistochemical data suggest that the Fas-FasL system mediates germ cell apoptosis in an autocrine and/or paracrine way.     

Effect of vasectomy on the seminiferous tubule boundary zone in the Albino Swiss rat

The boundary zone of a seminiferous tubule consists of the basement membrane of the seminiferous epithelium, its myoid cells, and their basal laminae. This study examines the boundary zones of seminiferous tubules in healthy and degenerated testes following long-term, left-sided vasectomy in the rat and compares them to those of sham-operated controls and adult rats exposed in utero to the antiandrogen, flutamide. Degenerated tubular profiles showed similar changes, irrespective of whether the degeneration was ipsilateral or bilateral. In transverse tubular profiles, the basal laminae of the seminiferous epithelium and the myoid cells became more undulating, that of seminiferous epithelium showing complex folding. The collagen layer of the boundary zone, which lies between the basal laminae of the seminiferous epithelium and the myoid cells, thickened and its fibers became irregularly orientated. Rather than being flattened as in controls, the region of the myoid cell near the nucleus and the nucleus itself developed triangular profiles in the transversely sectioned tubules. Similar features were also seen in the degenerated tubules of rats exposed to flutamide. The changes in the boundary zone are not specific for vasectomy and probably reflect reduction in the cross-sectional area of tubular profiles and possibly in their length. We also noted occasional leukocytes infiltrating the boundary zone; they may have increased in number in those tubules that showed degeneration following vasectomy.     

The seminiferous epithelium cycle and its duration in different breeds of dog (Canis familiaris)

Testis structure and function in dogs are relatively poorly investigated. The aim of the present study was to carry out a comparative investigation of the stages of the seminiferous epithelium cycle and its duration in different breeds of dog. Fifty-six sexually mature dogs (mongrel, n  = 12; pinscher, n  = 12; beagle, n  = 5; American pit bull, n  = 9; poodle, n  = 12; and Labrador retriever, n  = 6) were analysed. Intratesticular injections of tritiated thymidine were given to determine the duration of spermatogenesis. Orchiectomy was performed at different time periods following injection (1 h, 2 and 4 weeks). Testis fragments were embedded in plastic and routinely prepared for histological and autoradiographic evaluations. Eight stages were characterized based on the acrosome system. Significant ( P  < 0.05) differences were found for the frequencies of the different stages characterized (except Stages V, VI and VIII), particularly for the mongrel. Stage IV (when spermiation occurs) was the most frequent in all six breeds (~25%), whereas Stages II and VIII were the least frequent (< 8%). Each spermatogenic cycle and the total duration of spermatogenesis lasted 13.73 ± 0.03 and 61.9 ± 0.14 days, respectively, for the mongrel, poodle, pinscher, beagle, and Labrador retriever. These values were ~10% lower ( P  < 0.03) for the American pit bull (12.55 ± 0.26 and 56.5 ± 1.17 days, respectively). To our knowledge, this is the first comprehensive study to perform a careful investigation of stage frequencies and seminiferous epithelium cycle duration in this very important domestic species.     

ORIGINAL ARTICLE: LPS Increases the Expression Levels of IL‐18, ICE and IL‐18 R in Mouse Testes

Problem This study examined the effect of intraperitoneal administration of lipopolysaccharide (LPS) to adult male mouse on the expression levels of interleukin‐18 (IL‐18), IL‐18 receptor (IL‐18R) and the IL‐1β converting enzyme (ICE) (IL‐18 family) in their testes and spleen (control). Method of study Adult mice were injected (intraperitoneally; i.p.) with saline (control) or LPS (2, 20, 100 μg/mL; 100 μL/mouse). After 3 and 24 hr, testes and spleen were collected. Testicular tissue was examined for IL‐18 (by ELISA, real time PCR, and western blot analysis), IL‐18R and ICE (western blot and real time PCR analysis) and spleen tissue was examined for the IL‐18 family by real time PCR analysis. Student’s t‐test was used for statistical analysis. Results Homogenates of mouse testes contain and express basal levels of IL‐18, ICE and IL‐18 R. The expression levels of IL‐18, ICE and IL‐18R were significantly increased 3 and 24 hr after intraperitoneal injection of LPS to mature mouse, as examined by ELISA, western blot and real time PCR analysis. However, the expression levels of IL‐18, ICE and IL‐18Rα in the spleen increased significantly only after 24 hr of LPS stimulation, as examined by real time PCR. Conclusion Our results demonstrate that LPS increases the expression levels of the IL‐18 family in mouse testis and spleen, but the time of expression differs between the two organs. The presence of IL‐18 in the testes might be involved in the regulation of physiological and infection/inflammatory processes, and may be part of the autocrine/paracrine factors that control spermatogenesis. Further studies should be performed to confirm this possibility.