Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes

In adult male mice exposed to 300 R X-irradiation, the sper-matogonial population was selectively killed except for the radioresistant type A_s stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stages 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of ³HTdR were given between 2 and 3.5 days post-irradiation, 90–95% of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and at times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of ³HTdR prior to irradiation, there was appreciable retention of label by those type A_s sper-matogonia that had originally incorporated ³HTdR in stages 2-4. This labeling pattern was identical to that of the long-cycling A_s stem cells in nonirradiated testes. Since the long-cycling A_s stem cells are thought to be characterized by a prolonged G₁ or “A-phase” which is known to be a highly radioresistant portion of the cell cycle, it was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed that following germ cell depletion, as after irradiation injury, the long-cycling A_s survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population.     

Spermatogonial intercellular bridges in whole-mounted seminiferous tubules from normal and irradiated rodent testes

Whole-mounted seminiferous tubules from normal and irradiated rodent testes were examined by light microscopy. These studies reveal the presence of intercellular bridges in all classes of spermatogonia except for the single A_s stem cells. It was demonstrated that A_s stem cells divide to produce new A_s spermatogonia or paired daughter cells that are united by a cytoplasmic bridge. Evidence was given that all subsequent progeny of these paired A's up to and including the production of type B spermatogonia remain linked by cytoplasmic bridges in increasingly larger and more complex syncytial networks. It is proposed that the intercellular bridges mediate both differentiation and degeneration of spermatogonia. The maintenance of synchronous development within cohorts of spermatogonia is attributed to the bridges. Moreover, the fact that spermatogonia in both normal and irradiated testes degenerate in clusters is determined by the presence of intercellular bridges. Lastly, the integrity of the bridges appears essential for normal germ cell development.     

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     

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.     

Regulation of the density of spermatogonia in the seminiferous epithelium of the Chinese hamster: I. Undifferentiated spermatogonia

The topographical arrangement of the clones of A single, A paired, and A aligned (As, Apr, and Aal) spermatogonia on the basement membrane of seminiferous tubules of the Chinese hamster was studied. It was found that at least some of these clones are not distributed at random as clones of similar cell number were often seen in clusters. Areas were found with few or many As spermatogonia. Also, clusters of Apr spermatogonia were found, indicating that in such an area many As spermatogonia more or less synchronously formed Apr spermatogonia. Since clusters of clones of 16 Aal spermatogonia were observed, it can be concluded that these clusters of Apr spermatogonia may proliferate in at least a roughly synchronous way. It was found that over large areas the densities of undifferentiated spermatogonia may be very low or high in comparison to the mean density in the animal. Whether the ratio of self-renewal and differentiation of the stem cells changed locally in response to the high or low density of undifferentiated spermatogonia in particular areas was investigated. No indications for a regulatory mechanism to keep the density of stem cells and/or the density of undifferentiated spermatogonial clones at a certain level could be detected in the normal Chinese hamster. This lack of regulation was at least partly responsible for the widely different numbers of A1 spermatogonia that were formed in the various areas studied in stage IX.     

Staging equine seminiferous tubules by Nomarski optics in unstained histologic sections and in tubules mounted in toto to reveal the spermatogenic wave

Nomarski optics were used to identify stages of the spermatogenic cycle of seminiferous tubules in sectioned tissue or in whole dispersed tubules and to characterize the equine spermatogenic wave. Embedded tissues were sectioned at 20 microns. Whole dispersed tubules were obtained by enzymatic digestion of thin slices of fresh testis. Dispersed tubules were fixed, dehydrated in graded levels of alcohol, infiltrated with Epon, and mounted in toto on glass slides. Stages of the spermatogenic cycle could be identified under Nomarski optics in both histologic sections and tubules mounted in toto. Stage dependent nuclear chromatic and cytoplasmic changes in spermatogonia, spermatocytes, and spermatids were evident. Spermatid development included chromatin condensation, nuclear elongation, acrosomal development from the Golgi and proacrosomic granules, migration of the annulus and mitochondrial alignment, and the transient appearance of the chromatoid body and manchette. Both nuclear and cytoplasmic details of Sertoli cells were revealed. In tubules mounted in toto, the spermatogenic wave along the length of the tubules occurred as a consecutive set of stages occupying small regions along the tubular length. The spermatogenic wave in the horse is more similar to that of humans than that of rats. The combination of enzymatic isolation of seminiferous tubules and identification of spermatogenic stages by Nomarski optics facilitates examination of the spermatogenic wave in species whose tubules are tightly bound and not easily teased apart.     

Morphological events occuring in the seminiferous tubules of the Brazilian Nelore zebu associated with puberty.

The morphological events occurring in the Brazilian Nelore zebu testis from 3 to 18 months of age are described. Spermatozoa were seen only at 16--18 months of age indicating a considerable delay in the sexual development of the Nelore zebu when compared with taurine breeds.     

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.     

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.     

Morphological aspects of cultured rat seminiferous tubules in the presence of fetal calf serum and follicle-stimulating hormone

Culturing seminiferous tubules allows the analysis of spermatogenesis under controlled conditions. Reproducing the specific microenvironment for germ cells is a challenge taken up by various studies. The difficulty in supplementing all nutrients and the physical disturbances during isolation procedures cause degeneration of many cells in the seminiferous epithelium. We tested some culture conditions in order to preserve an acceptable morphology of cells inside the tubules. Seminiferous tubules were cultured during fifteen days in HAM F10 medium supplemented with fetal calf serum (FCS) and/or follicle-stimulating hormone (FSH). The cellular morphology was analyzed using scanning and transmission electron microscopy. During the culture period cellular degeneration occurred progressively. Morphological modifications of the Sertoli cells and germ cells such as accumulation of lipid droplets, nuclear and cytoplasmatic vacuolization and the presence of cell debris were observed. The addition of FCS activated the myoid cells causing nuclear rounding and thickening of the tubular wall. The best results were obtained with a serum-free culture medium supplemented with FSH.     

Organization of the membrane skeleton in spreading mouse blastomeres. I. Morphological analysis.

Differentiation in the mouse embryo begins at the 8-cell stage when the blastomeres spread against each other in a process called compaction. The spreading behavior of blastomeres on lectin-coated coverslips mimics that of blastomeres in the embryo, and we have utilized this model system to obtain an en face view of the membrane skeleton in the spreading blastomeres. Embryos were cultured on the coverslips for periods ranging from 20 sec to 6 hr, and the cells were disrupted to expose the cytoplasmic face of the adherent membranes and their associated filaments. The "membrane lawn" preparations were fixed, critical point dried, rotary shadowed, and the replicas examined by transmission electron microscopy. Using this technique we found that the plasmalemma of rounded blastomeres is associated with a lacy 3-dimensional filamentous meshwork that is transformed into a thin mat of densely woven filaments when the cells flatten. The overall organization of the membrane skeleton is similar in flattening 2- and 8-cell embryos, but there are significant differences in the time required for spreading to take place, in the means whereby the membrane skeletons are reorganized, and in the extent of maximal flattening. The significance of these observations for the compaction process is discussed.     

Regulation of the density of spermatogonia in the seminiferous epithelium of the Chinese hamster: II. Differentiating spermatogonia

In this study the yield of the proliferation of the differentiating spermatogonia into spermatocytes was determined in five Chinese hamsters. Large differences of up to a factor 2 were found between the numbers of A1 spermatogonia in the various animals. However, the numbers of leptotene spermatocytes varied only by up to a factor 1.2 between animals. It is concluded that more spermatogonial degeneration takes place in animals with a relatively large number of A1 spermatogonia than in those with a small number of these cells. In such a way in all animals ultimately about the same number of spermatocytes is formed. An experiment was done in which the number of A1 spermatogonia was lowered with the S-phase killer cytosine arabinoside (Ara-C). It was found that this greatly increased the yield of the spermatogonial proliferation, showing a direct relationship between the number of A1 spermatogonia in an animal and the extent of the spermatogonial degeneration. In addition to the variation in the number of A1 spermatogonia found between various animals, an even larger variation of up to a factor 3.7 was found between the numbers of A1 spermatogonia in different areas of seminiferous tubules within each animal. Nevertheless the variation in the number of leptotene spermatocytes in different areas within each animal was not larger than a factor 1.3. It is concluded that in the normal animal the phenomenon of spermatogonial degeneration depends on the local density of these spermatogonia. Apparently, when too many spermatogonia are present the surplus of cells degenerates.     

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.     

The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen

The quantitative distribution pattern of Ki-67 protein and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied in human testis biopsies. In normal seminiferous epithelium Ki-67 is expressed in nuclei of spermatogonia, while PCNA additionally occurs in nuclei of primary spermatocytes. The staining pattern of spermatogonia is as follows (Ki-67-positive/PCNA-positive): 26.6 +/- 12.4%/46.3 +/- 9.5%. No stage-dependent differences were found. Biopsies with mixed atrophy (score < or =7) showed a significant (P < 0.05) decrease of immunopositive spermatogonia to 19.9 +/- 3.0%/31.4 +/- 5.7% (score 1) with minimal variation between different samples (score 7 to 1). Associated with defined histological defects such as hypospermatogenesis (hyp), spermatogenic arrest at the level of spermatids (sda), spermatocytes (sca) or spermatogonia (sga), however, there was a significant (P < 0.05) decrease of Ki-67 staining in tubules showing hyp (28.6 +/- 8.8%), sda (25.6 +/- 9.3%), sca (23.7 +/- 9.3%) and sga (16.2 +/- 6.0%) and of PCNA staining in sca (32.2 +/- 11.8%) and sga (20.0 +/- 9.5%), respectively. The decrease of immunoreactive spermatogonia did not correspond to elevation of follicle stimulating hormone (FSH). These data demonstrate that the low spermatogenic efficiency in infertile men is not only due to postmeiotic events, but also to a decrease in the meiotic activity of spermatogonia, and is not related to serum FSH.      

Re-examination of spermatogonial renewal in the rat by means of seminiferous tubules mounted “in toto”

Observations on dissected tubules, fixed in Carnoy, stained with hematoxylin and mounted “in toto” revealed that there were five distinct classes of type A spermatogonia. The type A₁ found in stages II–VIII of the cycle of the seminiferous epithelium had round, pale-stained nuclei, typically arranged in linear clusters of four or eight along the tubular wall. They all divided at stage IX to produce type A₂ cells. These in turn divided at stage XII to produce type A₃ spermatogonia. The type A₂ and A₃ cells had large ovoid nuclei containing globular masses of deeply stained chromatin and were randomly distributed in the space between Sertoli nuclei. The type A₃ spermatogonia divided at stage XIV to produce type A₄ cells. These had smaller nuclei, sometimes lobulated, containing more deeply stained chromatin granulation, free in the nucleus or adhering to the nuclear membrane. They divided in stage I of the cycle to yield two classes of spermatogonia: intermediate type and new type A₁. Hence, type A₁–type A₄ spermatogonia were considered as “renewing stem cells.” The fifth class of type A spermatogonia (A₀) was found at all stages of the cycle. Rare, isolated or in pairs, they had oval nuclei with deeply stained chromatin granulations. Seldom seen to divide, they did not appear to be actively involved in cell renewal and were tentatively considered as “reserve stem cells”.     

Effects of alpha-amanitin on RNA synthesis in cultured rat seminiferous tubules.

The effect of the specific RNA polymerase II inhibitor α-amanitin on the cell morphology and RNA synthesis during spermatogenesis was investigated. Light microscopic autoradiography showed that α-amanitin largely decreased the rate of RNA synthesis in the pachytene spermatocytes and in the spermatids. Degenerative changes occurred most rapidly in the mid pachytene spermatocytes and in the intermediate and type B spermatogonia. Already after 14 hr of culture with α-amanitin (10 μg/ml) a part of these cells showed progressive degenerative changes beginning with the clumping together of the chromosomes. This indicates that at least a part of the HnRNA which is synthesized in the lampbrush loops of the pachytene chromosomes is needed to maintain the normal structure and function of the pachytene spermatocytes. Biochemical evidence suggested also that the formation of HnRNA was specifically inhibited in the pachytene spermatocytes by α-amanitin.     

Morphological studies on the synthesis of secretory granules in convoluted tubules of mouse submandibular gland.

The synthesis of zymogen-like secretory granules in convoluted tubules of mouse submandibular gland (SMG) was investigated by histometry, light microscopy and electron microscopy. In normal males secretory granules in the SMG increased greatly from 25 days after birth and reached a maximum level 50 days after birth. Castration of adult male mice markedly decreased the level, but it was completely restored by testosterone administration. A parallel was found between change in the granule level and the amount of rough endoplasmic reticulum (RER) in the convoluted tubular cells during development or after various treatments. Development of the Golgi apparatus was also observed in the cells when the granules increase. Both the increase in the granules and in the RER induced by testosterone were prevented by actinomycin D or puromycin. These results indicate that the granule contents are synthesized on the RER under the control of testosterone, and then condensed in the Golgi apparatus.