Morphometrical analysis of Sertoli cell ultrastructure during the seminiferous epithelial cycle in rats

Stage-dependent variations of Sertoli cell organelles during the seminiferous epithelial cycle were analyzed morphometrically in rats by use of a point-counting method. Cyclic changes in volume and surface area of various organelles were observed. Mitochondria and rough endoplasmic reticulum increased at stages VII-VIII and stage VII, respectively. Lipid droplets accumulated markedly after spermiation, decreased rapidly after meiotic division, and remained at low levels for stages IV-VIII. The most prominent change was a topographic alteration of the Golgi apparatus. It was usually located exclusively in the basal cytoplasm, but shifted upwards to the mid and apical cytoplasm at stages VII-VIII. This shift may be implicated in an increase of plasma membrane and lysosomes in these regions. Consecutive increases of primary and secondary lysosomes were observed twice in the basal and mid cytoplasm. The first peak of the primary lysosomes at stage IV was followed by the first peak of the secondary lysosomes at stage VI; and the second peak of the primary lysosomes at stages VII-IX was followed by the second peak of the secondary lysosomes at stage IX. These consecutive increases may indicate that Sertoli cells anticipate the increase of structures to be removed and accordingly produce primary lysosomes before their appearance.     

Implication of amphiphysin 1 and dynamin 2 in tubulobulbar complex formation and spermatid release

Tubulobulbar complexes (TBCs) are composed of several tubular invaginations formed at the plasma membrane of testicular Sertoli cells. TBCs are transiently formed at the contact region with spermatids at spermatogenic stage VII in rat and mouse, and such TBC formation is prerequisite for spermatid release. Since the characteristic structure of TBCs suggests that the molecules implicated in endocytosis could be involved in TBC formation, we here investigated the localization and physiological roles of endocytic proteins, amphiphysin 1 and dynamin 2, at TBCs. We demonstrated by immunofluorescence that the endocytic proteins were concentrated at TBCs, where they colocalized with cytoskeletal proteins, such as actin and vinculin. Immunoelectron microscopy disclosed that both amphiphysin 1 and dynamin 2 were localized on TBC membrane. Next, we histologically examined the testis from amphiphysin 1 deficient {Amph(-/-)} mice. Morphometric analysis revealed that the number of TBCs was significantly reduced in Amph(-/-). The ratio of stage VIII seminiferous tubules was increased, and the ratio of stage IX was conversely decreased in Amph(-/-). Moreover, unreleased spermatids in stage VIII seminiferous tubules were increased in Amph(-/-), indicating that spermatid release and the following transition from stage VIII to IX was prolonged in Amph(-/-) mice. These results suggest that amphiphysin 1 and dynamin 2 are involved in TBC formation and spermatid release at Sertoli cells.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Morphometric studies on lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat

Summary The volume and surface area of lipid inclusions often present in the cytoplasm of rat Sertoli cells was measured directly from semi-thin sections of perfusion-fixed testicular tissues using an image analyser linked to a light microscope. Sertoli cell nuclei were used as a reference for comparing any variations in the measured parameters of lipid inclusions during the rat spermatogenic cycle. Volume density of Sertoli cell lipid inclusions was assessed by morphometric analysis of Sertoli cells photographically reconstructed from electron micrographs. Maximum lipid content in Sertoli cells occurred during stages IX–XIV of the spermatogenic cycle, then declined at stages I–III and remained low from stages IV–VIII. The persistence and increase in number of many large Sertoli cell lipid inclusions beyond the stage where spermatid residual bodies are phagocytosed within the Sertoli cells (stage IX) suggests that the synthesis and lipolysis of Sertoli cell lipid inclusions represents an intrinsic functional cycle of the Sertoli cells. Stage-dependent variations in the lipid content of rat Sertoli cells offers morphological evidence that the metabolic duties of the Sertoli cells are synchronised with the spermatogenic cycle to provide local coordination of the proliferation and maturation of the germ cells.     

Molecular dynamics of the blood–testis barrier components during murine spermatogenesis

The blood–testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII–IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin‐3, claudin‐11, occludin, and zonula occludens‐1 (ZO‐1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII–IX). Although we detected claudin‐11, occludin, and ZO‐1 throughout spermatogenesis, claudin‐3 was only detected during stages VI–IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II–VI, Middle: VII–VIII, Late: IX–I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage‐specific localization of claudin‐3 protein plays an important role in regulating BTB permeability. Mol. Reprod. Dev. 77: 630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.     

Stage specific effect during one seminiferous epithelial cycle following ethylene glycol monomethyl ether exposure in rats.

Stage specific effect of single oral dose (500 mg/kg body wt) of ethylene glycol monomethyl ether (EGME) was characterised during one cycle of seminiferous epithelium in rats. Maximum peritubular membrane damage and germinal epithelial distortion were observed at stages IX-XII. Cell death occurred during conversion of zygotene to pachytene spermatocytes (stage XIII) and between dividing spermatocytes and step I spermatids (stage late XIII-XIV). Profound effect was noted during first meiotic division than during second meiotic division. Presence of multinucleated secondary spermatocytes indicated cytokinesis arrest. The spermatogenesis was delayed and consequently frequency of tubules at stages I-VIII was reduced by day 10. Many of the tubules were devoid of round spermatids on day 12. Possibly, EGME (or it's metabolite) distorted the barrier system at stages IX-XIV and damaged the cells mostly at stages XII-early XIV.     

Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle.

The occurrence of degenerating germ cells in the cycle of the seminiferous epithelium was measured in testicular tissues from eight normal adult rats. Testes were perfusion fixed, embedded in epoxy resin and, after sectioning a total of 180 randomly selected blocks at 1 microns, stained sections were examined by light microscopy; all cross-sectioned seminiferous tubules were categorized into one of 14 stages of the spermatogenic cycle. The number of degenerating cells per tubule was recorded in 2103 tubules. Degenerating germ cells were not detected at stages II-VI, and only rarely at stage VII (n = 366 tubules) in which one primary spermatocyte and one step 19 spermatid degenerated. All other stages exhibited a greater incidence of degenerative germ cells, particularly at stage XIV where, on average, the frequency of degenerating cells per round seminiferous tubule was about 40 times greater than at stage VII. The results indicated that, in the normal adult rat testis, the germ cells are least at risk of degeneration as they pass through stage VII.     

The effect of cryptorchidism on the quantitative histology, histochemistry and hydrolytic enzyme activity of the rat testis.

Cryptorchidism of the mature rat testis led to degeneration of the seminiferous tubules and changes in enzyme patterns and activities. Spermatogenic stages 1-4, containing pachytene primary spermatocytes in late meiotic prophase, and stage 5, containing recently formed round spermatids, were damaged by 48 h. Within 96 h stages showed a loss of germinal cells into the lumen and this was almost complete by 192 h. Acid phosphatase showed increased histochemical activity in the basal area of the seminiferous tubule up to 96 h of cryptorchidism, and at 192 h much of the activity was located in large lipidcontaining bodies within the remaining seminiferous epithelium. Total and free biochemical acid phosphatase decreased during cryptorchidism in parallel with cell loss; there were no significant changes in total cathepsin D activity but free enzyme activity was increased throughout the experimental period indicating increased lability of lysosomes in the Sertoli cell. Lactate dehydrogenase activity was mainly tubular but succinate dehydrogenase also showed interstitial activity. Lipoamide dehydrogenase (NADH) was found mainly in the interstitium. During cryptorchidism both lactate and succinate dehydrogenase activity decreased in the tubules parallel to the loss of germinal cells, whereas lipoamide dehydrogenase (NADH) activity increased in both interstitial and tubular areas. It is suggested that the initial lesion in the seminiferous epithelium, produced by cryptorchidism is in the Sertoli cell and that germ cell damage may result from reduced function of the Sertoli cell.     

Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Fine structure of seminiferous tubules in antarctic seals

Summary The fine structure of seminiferous tubules from 5 crabeater, 2 leopard and 2 Ross seals showed that during the nonbreeding season the tubules were essentially similar in possessing spermatogenic and Sertoli cells. However, the tubules of leopard and Ross seals had more primary and secondary spermatocytes and spermatids than the crabeater seals. In general, the tubules were devoid of spermatozoa. The spermatids showed stages of maturation such as Golgi phase of acrosome formation, acrosomal cap formation and condensation of nuclei. Some spermatids degenerated in tubules. Both maturing and degenerating spermatids were closely associated with Sertoli cells. Junctional complexes with plaques of filaments were observed between Sertoli cells and the spermatogenic cells. Sertoli cells, irregular and polygonal, contained highly convoluted nuclei, strands of rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi complexes, small mitochondria, variable amounts of lipid droplets, lysosomes, lipofuscin granules and highly plicated plasma membranes. In brief, the spermatogenic activity had practically ceased in the testes and the animals probably secreted low levels of testosterone during the nonbreeding season.This research was supported in part by National Science Foundation Grants G.U. 30270 and G.U. 29829X from the Office of Polar Program and by NIH Grant 5 R01 AM11-376     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.     

Altered phosphorylation and distribution status of vimentin in rat seminiferous epithelium following 17β-estradiol treatment

Vimentin, type III intermediate filament, has stage-specific localization in the Sertoli cell. In the rat, during stages I–V and XI–XIV of the seminiferous epithelium, vimentin is localized in the perinuclear area with filaments projecting into the apical region toward the developing germ cells. These filaments decrease in length at stages VI–VII with perinuclear staining in stages VIII–IX, when spermiation occurs. Our earlier studies following 17β-estradiol treatment to adult male rats demonstrated an increase in germ cell apoptosis, spermiation failure and disruption of Sertoli cell microfilaments and microtubules. The present study was undertaken to determine the stage-specific distribution of vimentin and its involvement in spermiation failure and germ cell apoptosis. Immunofluorescence studies revealed that in contrast to the perinuclear localization with small extensions in control stages VII–IX, long extensions radiating apically to the spermatids in deep recess were observed in the treated group. Immunoprecipitation studies showed marked absence of phosphorylated vimentin in stages VII–VIII in the treated group. Further, localization of plectin, cytoskeletal linker protein, showed decrease in all the stages of spermatogenesis following estradiol treatment. Interestingly, for the first time the localization of plectin in the tubulobulbar complex was observed. In conclusion, the study suggests that estradiol treatment leads to an effect on vimentin phosphorylation, which could have inhibited the disassembly of vimentin leading to retention of apical projection in stages VII–VIII. These effects could be presumably due to a decrease in plectin, affecting the reorganization of vimentin and therefore the apical movement of spermatids, leading to spermiation failure.