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.     

Mutation frequency and type during ageing in mouse seminiferous tubules.

Mutations arise in the germline by errors of replication, recombination and repair, and the movement of transposable elements. Transgenic mice bearing reporter genes such as lacZ have proven useful for measurements of spontaneous and induced mutation frequencies, as well as studies of the effects of ageing. In this study, testicular DNA from lacZ transgenic mice was examined for age-related effects on mutation frequency and type. The recovered transgene was tested for simple substitutions and rearrangements including transposition of endogenous mobile elements. There was no evidence for either an age-related accumulation of mutations, or for the insertion of retrotransposons into the lacZ reporter gene in the testis. We conclude that the frequency of retrotransposition of several mouse mobile elements into the lacZ reporter gene is less than 3.73x10(-8). This is significantly less than the known frequency of approximately 7% of all spontaneous mutations in the mouse being due to retrotransposition of these elements.     

Ultrastructure of the seminiferous tubules in human testes before and after varicocelectomy

Ultrastructure of the membrana propria and the seminiferous epithelium was studied in infertile human testis both before and 3–6 months after varicocelectomy. The frequent alterations, observed before and after the operation, were extremely thickened membrana propria, deep invaginations, multilamination and knob-like formation of basal laminae and formation of multinucleated spermatids, which were all considered as the common response of the testis to different noxious agents. Although the cells of the seminiferous epithelium were clearly affected by varicocele before varicocelectomy, many areas exhibited normal features after the operation. Furthermore, multinucleated cells, sharing common features of Sertoli cell and spermatogonium, were observed, as well as presence of well-developed annulate lamellae in the Sertoli cells, exhibiting centrioles in the vicinity of their nuclei after varicocelectomy. These multiple ultrastructural observations indicate that Sertoli cell division takes place. This study suggests that if the observation period of the tissue samples after varicocelectomy is long enough, the reversible changes of the tubular cells would be seen much more frequently.Part of this study was submitted as a PhD thesis by Asst. Prof. Hülya Özgür and presented at the 12th National Congress on Electron Microscopy held in Antalya, Turkey, 1995     

Loss of laminin and type IV collagen in uterine luminal epithelial basement membranes during blastocyst lmplantation in the mouse

Background: Removal of the uterine luminal epithelium and its basement membrane is necessary for successful implantation of invasive blastocysts. Few reports, however, have specifically addressed the penetration and loss of the uterine luminal epithelial basement membrane (UEBM). We investigated the loss of UEBM by examining the distribution of laminin and type IV collagen. Methods: Blastocyst implantation sites were collected from mice on days 5,6, and 7 of pregnancy. Paraffin sections were prepared from these tissues and processed with standard immunoperoxidase techniques to reveal the distribution of laminin and type IV collagen. Results: On day 5 of pregnancy blastocysts were adherent to the uterine epithelium. The epithelium and UEBM were complete and uninterrupted. On day 6 the juxtaembryonic uterine epithelium was lost and focal discontinuities were seen along the UEBM. By 1200 hr the UEBM had receded to the region near the ectoplacental cone, but staining was reduced for both antigens over the entire region surrounded by decidual cells. This decreased staining of the UEBM occurred in areas not yet occupied by trophoblast cells. On day 7 the UEBM was lost over the entire embryonic half of the uterine lining, corresponding to the distribution of decidual cells. Conclusions: Progressive loss of the UEBM occurred in a consistent spatiotemporal pattern following the onset of blastocyst implantation. Diminished immunoreactivity of laminin and type IV collagen in the UEBM was closely correlated with the area occupied by decidualized endometrial stroma and occurred in areas not yet in contact with trophoblast cells. We conclude that decidual cells are instrumental in the removal of UEBM during early pregnancy. © 1995 Wiley-Liss, Inc.     

Localization of dynamin 2 in rat seminiferous tubules during the spermatogenic cycle

Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.     

Histochemical demonstration of disulfide-groups in the lamina propria of human seminiferous tubules

Summary The distribution of disulfide-groups was investigated in the tunica propria of human seminiferous tubules by means of a thiosulfation/Alcian Blue+0.8 Mol MgCl₂-staining reaction. Controls had shown the absence of significant amounts of sulfhydryl- or sulfate-groups in the lamina propria, which groups would also be demonstrated by the method employed.The lamina propria of human seminiferous tubules is rich in disulfide-groups. The staining reaction decreases in the region of the tubulus rectus, is only faint in the connective tissue which underlies the epithelium of the rete testis, and is absent in the lamina propria of efferent ducts.It is suggested that microfibrils and type IV collagen (both rich in cystine) are the materials responsible for the histochemical reaction described. The occurrence of multiple layers of basal lamina material (type IV collagen) and bundles of microfibrils is shown in comparative electron microscopic studies.     

Intracellular potentials in cells of the seminiferous tubules of rats.

1. Membrane potentials have been recorded from cells of seminiferous tubules of rats in vitro using micro-electrodes. The value in 808 impalements was -28-2 +/- 0-3 mV (mean +/- S.E.) at 33 degrees C. 2. Increasing the potassium concentration depolarized the cells, a tenfold increase in concentration causing a depolarization of 16 mV. Removal of sodium from the bathing solution caused a hyperpolarization of 3 mV at a potassium concentration of 5-9 m-equiv/l. Removal of chloride and replacement with impermeant anions had no effect on potential. Removal of calcium from the bathing solution caused a minor but significant depolarization. 3. Ouabain (10-3 M), dinitrophenol (2-5 times 10-4 M) or removal of glucose from the bathing fluid all caused depolarization. The membrane potentials of the cells were sensitive to temperature over the range 10-33 degrees C, the apparent activation energy for the reactions maintaining the potential being approximately 6 kcal/mole. 4. Membrane potentials in seminiferous tubules were independent of age of the animal, were insensitive to previous hypophysectomy and were insensitive to a number of hormones (FSH, LH, HCG, oxytocin). In high concentration prostaglandin E1 caused depolarization. 5. Acetazoleamide (4 times 10-5 M) caused a rapid, but reversible, depolarization of the tubular cells. This was also true in conditions when the HCO'3/CO2 buffer system was replaced with Tris-buffer. Another carbonic anhydrase inhibitor (p-sulphonamido-benzoic acid) had similar effects on cell potentials as acetazoleamide. These results are discussed in relation to the nature of the ionic secretion produced in the tubules. 6. Occasional cells showed phasic variations in membrane potential. A possible connexion between these variations and the contractile activity of the tubules is discussed.     

IgG and IgA antigens in human renal basement membranes.

Cryostat sections of fresh human kidney studied by immunofluorescence were negative for IgG, IgA, IgM, Fc and Fab fragments of IgG, and kappa and lambda light chains. All of these conjugates except anti-IgM stained glomerular basement membranes and several other connective tissues if the cryostat sections were washed in phosphate-buffered saline (PBS) before being studied. Staining was inhibited by blocking reactions as well as by fixation before washing in PBS or by washing in non-ionized or hypotonic solutions. Results of several other aspects of this reaction are also reported.     

Synthesis and expression of laminin during human foetal lung development

Backgrounds: The lung develops by epithelial tubes budding and branching into a flexible mesenchyme. This growth is associated with the remodelling of the epithelial basement membrane, of which laminin is a major component. Methods: Both the synthesis and expression of laminin were studied in the human lung between 10 and 31 weeks of gestation, using in sity hybridization and immunohistochemistry. Results: The synthesis of the β chain was active in the epithelial and surrounding mesenchymal cells. The mRNAs coding for the γ chain were less abundant and mainly found in the epithelium. The synthesis of these two chains continued throughout gestation, and no significant difference in the density of hybridization grains could be detected between the tips of the expanding buds and the proximal portions. Immunohistochemical localization of laminin showed important modifications of the basement membrane during gestation. In the first part of the pseudoglandular stage the epithelial basement membrane stained continuously for laminin. Later, the basement membrane was labelled in a graded fashion: at the apex of the growing buds the staining became weak with focal disruptions. Both epithelial and mesenchymal synthesis of laminin remained active, while the polypeptide was undetectable using immunohistochemistry. Conclusions: These findings suggest that the remodelling of the basement membrane during human lung morphogenesis is probably not related to a decreasing synthesis of laminin, but to either a proteolytic degradation or the assembly of an inadequate complex undetectable with the polyclonal antibody antilaminin. © 1995 Wiley-Liss, Inc.     

Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.

To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.     

Loss and rearrangement of glomerular basement membrane laminin during acute nephrotoxic nephritis in the rat.

Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.     

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.     

Patterns and composition of basement membranes in colon adenomas and adenocarcinomas

We studied the distribution of type IV collagen and type VII collagen in the basement membranes of normal mucosa of the colon, adenomas, and adenocarcinomas using immunoperoxidase and immunofluorescence techniques. In normal mucosa, we found regular type IV collagen-positive basement membranes, lining vascular structures and mucosal epithelia. These basement membranes, however, lacked type VII collagen. In adenomas of the colon, intact basement membranes were observed through type IV collagen staining. Type VII collagen staining was also detected, but only in connection with dysplastic epithelium. Adjacent to the dysplastic epithelium in adenomas, histologically normal epithelium also showed type VII collagen staining along the basement membrane, but this was restricted to the epithelium of the luminal surface. These areas were also investigated for expression of keratins 8, 18, and 19, and keratins 5 and 8 (monoclonal antibodies NCL-5D3 and RCK 102, respectively), but altered differentiation was not detected using this technique. In adenocarcinomas of the colon, type IV collagen was irregularly deposited in the basement membrane of neoplastic tubules. Type VII collagen staining was detected only in well or moderately differentiated carcinomas and in higher amounts. Our findings therefore reveal a transient expression of type VII collagen in the transition of dysplastic epithelium into carcinoma, suggesting the involvement of type VII collagen in the process of early invasion.     

The fine structure and development of the peritubular contractile cell component in the seminiferous tubules of the mouse

Testes of sexually mature, as well as newborn and young mice of varying ages were studied by electron microscopy. The seminiferous tubules in the mature mouse possess a single cell layer of extremely flattened cells which form a sheath-like structure around the epithelium of the tubule. These peritubular cells are characterized by cytoplasmic filaments and other features which are typical of smooth muscle cells. A basement lamina is associated with the interstitial or peripheral surface of the cell. Peripherally, there is an additional cellular layer consisting of connective tissue fibrocytes. In newborn animals, the cells surrounding the tubule epithelium consist of a homogeneous population of fibroblasts, 3–4 layers in thickness. With growth and development of the testes the number of cell layers is reduced and the cells become more attenuated. At 13 days, those cells which are closest to the epithelium show localized aggregates of fine filaments, as well as what appears to be the elaboration of a basement lamina. By 17 days, the cytoplasmic filaments are more numerous and the basement lamina is well defined: by 19 days, the cells closely resemble the peritubular muscle cells of the adult. The probable functional role of these cells is discussed with respect to both sperm transport and the production and maintenance of the surrounding connective tissue stroma.     

Ultrastructural study of Sertoli cells in rat seminiferous tubules during intrauterine life and the postnatal period

Summary Sertoli cells have various functions: mechanical (creation of two compartments in the seminiferous tubules, migration of germinal cells), secretory (secretion of anti-Müllerian hormone, inhibin, androgen-binding-protein and estrogen) and phagocytic.We report an ultrastructural study of the rat Sertoli cell during maturation and consider possible correlations between the acquisition of certain morphological characteristics and certain functions.During fetal life, the Sertoli cell possesses differentiated zones of junction with the gonocytes and seems to have a role in the migration of the gonocytes towards the periphery of the seminiferous tubule. The Sertoli cell performs the phagocytosis of the gonocytes which degenerate during their migration, and seems to be the site of production of protein granules, whose presence can be related to the synthesis of anti-Müllerian hormone.After birth and before puberty, when the inclusions resembling secretory granules disappear, the Sertoli cell membranes in contact with spermatocytes II and spermatids differentiate, forming, through the differentiated junctional complexes, two compartments (adluminal and luminal) in the seminiferous tubules. Finally, they acquire the characteristics of active secretory cells, capable, in particular, of steroid synthesis.     

Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action

Introduction

Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation.

Material and methods

Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment – tT3) or until pnd 15 (continuous treatment – cT3) or solvent – control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats.

Result

tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased.

Conclusions

Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone.     

Quantitative assessment of basement membranes in soft tissue tumours. Computerized image analysis of laminin and type IV collagen

Basement membranes (BMs) in 201 soft tissue tumours were quantified using computerized image analysis of tissues immunostained for laminin and type IV collagen. The purpose of the study was to compare and quantify the extent of BM deposition in a large and varied group of benign and malignant tumours. Laminin and type IV collagen gave similar results. The difference between benign and malignant was statistically highly significant (P=0·0001), with greater deposition in benign tumours. BM deposition was homogeneous in benign tumours and heterogeneous in sarcomas and appeared to correlate with the degree of differentiation. Some poorly differentiated sarcomas showed cytoplasmic laminin staining but little or no extracellular BM. Immunohistochemical evaluation of BM has some advantages over electron microscopy; specialized equipment is not needed and since large samples can be studied with little sampling error, heterogeneity can be studied more readily. Subjective visual assessment gives a good overall indication of the extent of BM deposition and in many situations is likely to be a suitable alternative to image analysis. Because of staining heterogeneity, BM immunohistochemistry is unlikely to be of significant value in the diagnosis of specific types of sarcoma. © 1998 John Wiley & Sons, Ltd.