Ultrastructural observations on spermatozoa retained within the seminiferous epithelium after treatment with dibutyryl cyclic AMP

Spermiation was inhibited in the Syrian hamster by administering large doses of dibutyryl cyclic AMP. After treatment with dibutyryl cyclic AMP most stage VIII, IX, and X seminiferous tubules contained some mature spermatozoa within the seminiferous epithelium. The acrosomal membranes and plasma membranes of the unreleased spermatozoa remained intact, indicating that the spermatozoa had not been phagocytized by the Sertoli cells. Sertoli-spermatid junctional specializations were usually applied to the heads of the mature spermatozoa. The unreleased spermatozoa often appeared swollen with accumulated fluid located in the subacrosomal space. The accumulation of subacrosomal fluid in the unreleased spermatozoa seems to result from the absence of tubulobulbar complexes. That is, when tubulobular complexes fail to form the normal flow of cytoplasm into the tubulobular complexes is blocked resulting in an accumulation of fluid around the nucleus. Inhibition of spermiation may result from the absence of tubulobulbar complex formation. It is postulated that the tubulobulbar complex functions to transfer a chemical trigger from the maturing spermatid into the Sertoli cell. This chemical trigger may initiate the disappearance of the Sertoli-spermatid junctional specialization and induce spermiation.     

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.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Effect of the microtubule disrupting agents,colchicine and vinblastine,on seminiferous tubule structure in the rat

Injections of colchicine or vinblastine were given intratesticularly and rats sacrificed 6 and 12 hr later. Colchicine and vinblastine produced identical morphological patterns of response in the seminiferous tubules resulting in arrest of germcell mitoses and meioses and a rapid depletion of the microtubules normally found within the Sertoli cell. Sloughing of cells into the lumen of seminiferous tubules was the most prominent feature noted. Germ cells and portions of the apical Sertoli cells were frequently sloughed together where they remained in close association. Usually germ cells and associated Sertoli cell fragments were cleaved from the wall of the seminiferous tubule at a level between dissimilar generations of germ cells, e.g. between spermatocytes and spermatids. This selective sloughing probably occurred as the result of the support normally provided by intercellular bridges which link clones of like germ cell types. Sequential steps in the process leading to sloughing of Sertoli-germ cell associations could be inferred from observations made in plastic 1 μm sections. Cell sloughing at 12 hr post-injection was generally more extensive. It was frequently noted that germ cells and the apical portions of Sertoli cells had been extruded to the level of the most adluminal tight junctions forming the blood-testis barrier. It was concluded that disruption of Sertoli microtubules was responsible for sloughing of Sertoli fragments and associated germ cells, and that the cytoskeletal support of the Sertoli cell was, at least in part, dependent upon the integrity of Sertoli microtubules. The Sertoli cell could not round-up after loss of its cytoskeletal support, due to the numerous attachment devices known to link it with various apically positioned germ cells. Thus, the cell was severed at some point along its delicate apical processes, as the consequence of forces produced by the ‘rounding-up’ process. Long-term sacrifice after vinblastine or colchicine treatment allowed the Sertoli cells to regain microtubules and long processes but not their typical configuration. Spermatogenesis remained severely impaired.     

Stage-specific effects of the fungicide carbendazim on Sertoli cell microtubules in rat testis

The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

Transforming growth factor beta gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions

The potential role of transforming growth factor beta (TGF beta) as a mediator of cell-cell interactions within the seminiferous tubule was investigated through an examination of the local production and action of TGF beta. Sertoli cells and peritubular (myoid) cells were isolated and cultured under serum-free conditions. Secreted proteins from Sertoli cells and peritubular cells were found to contain a component that bound to TGF beta receptors in RRA. Reverse-phase chromatography of Sertoli cell and peritubular cell secreted proteins fractionated a protein with similar biochemical properties as TGF beta 1. This fractionated protein also contained TGF beta bioactivity in its ability to inhibit growth of an epidermal growth factor-dependent cell line. Both peritubular cells and Sertoli cells contained a 2.4 kilobase mRNA species that hybridized in a Northern blot analysis with a TGF beta 1 cDNA probe. TGF beta 1 gene expression was not detected in freshly isolated germ cells. TGF beta 1 alone was not found to influence Sertoli cell nor peritubular cell proliferation with cells isolated from a midpubertal stage of development. The effects of hormones and TGF beta on Sertoli cell differentiation and function were assessed through an examination of transferrin production by Sertoli cells. TGF beta 1 had no effect on transferrin production nor the ability of hormones to influence transferrin production. The presence of peritubular cells in a coculture with Sertoli cells also did not affect the inability of TGF beta 1 to act on Sertoli cells. Although Sertoli cell function did not appear to be influenced by TGF beta 1, peritubular cells responded to TGF beta 1 through an increase in the production of a number of radiolabeled secreted proteins. TGF beta 1 also had relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of Sertoli cells and peritubular cells responded to TGF beta 1 by the formation of large cell clusters with ball-like structures. Data indicate that TGF beta may have an important role in influencing the differentiation and migration of peritubular cells. Observations demonstrate the local production of TGF beta within the seminiferous tubule by Sertoli cells and peritubular cells and suggest that TGF beta may have a role as a paracrine-autocrine factor involved in the maintenance of testicular function.     

The consequences of actin disruption at Sertoli ectoplasmic specialization sites facing spermatids after in vivo exposure of rat testis to cytochalasin D

Cytochalasin D (CD) was used to perturb actin filaments of the Sertoli ectoplasmic specialization (ES)--a cytoskeletal complex of the Sertoli cell related to spermatids. CD (500 microM for 6 h) produced a loss of 88% of the ES facing the head region of early (Step 8) elongating spermatids as compared to vehicle (dimethylsulfoxide:saline) controls. Nitrobenzoxadiazole-phallacidin staining of F-actin revealed a CD-related loss of uniform fluorescence over the head of elongated spermatids. To examine for a possible relationship between the presence of actin and cell attachment at ES sites, hypertonic fixatives were introduced to provoke cell shrinkage and stress ES-associated junctions. After osmotic stress, cell-to-cell adhesion at ES sites remained intact in vehicle-treated animals. CD treatment caused Sertoli cells to separate from elongating spermatids at sites where ES had been lost from the Sertoli cell surface. It is suggested that actin of the ES plays a role in cell-to-cell interaction analogous to its possible role at the Sertoli cell barrier. In CD-treated animals, structures resembling tubulobulbar complexes frequently developed at sites where ES was lost, suggesting that the loss of ES has a facilitatory role in tubulobulbar complex formation. It is hypothesized that tubulobulbar complexes are devices that rid the cells of ES-associated junctional links to effect dissociation of the spermatid from the Sertoli cell during spermiation. Spermatids at Step 8 of development are known to become oriented with their acrosomes facing the base of the Sertoli cell. After CD treatment, a 5.8-fold increase in malorientation of Step 8 spermatids was noted. A role for the ES cytoskeletal complex in orienting the spermatid acrosome toward the basal aspect of the Sertoli cell is also suggested.     

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

Relationship of intratesticular testosterone content of stallions to age, spermatogenesis, Sertoli cell distribution and germ cell-Sertoli cell ratios

Testes were obtained from 47 1-20-year-old stallions during the natural breeding season. Total testicular testosterone and testosterone/g testis increased with age (P less than 0.005), and total testicular testosterone was associated with larger testis size (P less than 0.05). Neither testosterone per gram nor per paired testes were related to total Sertoli cell number (P greater than 0.05), but greater testosterone per paired testes was associated with fewer Sertoli cells per unit of seminiferous tubule length (P less than 0.005) or basement membrane area (P less than 0.02) and with a higher number of germ cells supported per Sertoli cell (P less than 0.05). Although values for testosterone per gram and per paired testes were unrelated (P greater than 0.10) to sperm production/g testis or to the yield of spermatids/spermatogonium, testosterone per paired testes was positively related to sperm production per paired testes (P less than 0.05). It is concluded that intratesticular testosterone increases with age, is related in a positive manner to quantitative rates of sperm production, and can account for some of the differences in sperm production among individual stallions within a single breeding season.     

Postnatal development of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow

The postnatal development of the Sertoli cell barrier, tubular lumen, fluid flow, and cytoskeletal elements in Sertoli and myoid cells was investigated in the Sprague-Dawley rat. With the aid of hypertonic fixatives, a barrier to the rapid entry of fluid was noted in the majority of tubules on the 15th and 16th postnatal (p.n.) days and was completely formed in all tubules prior to p.n. day 18. The actin forming the ectoplasmic specialization (ES), a cytoskeletal complex related to the occluding junctions composing the barrier, began its development during the period of initial barrier formation (16 p.n. day) and progressively attained its adult prominence. The ES developed its characteristic adult pattern and adult fluorescent intensity at about p.n. day 22. Some seminiferous tubules showed very small lumina as early as p.n. day 10. All tubules were not open until p.n. day 30. The size (diameter) of the lumen increased slowly from p.n. day 10 until p.n. day 30 when it started to increase rapidly until about p.n. day 50. Fluid flow in seminiferous tubules was detected as early as p.n. day 20 and increased in amount thereafter. Myoid cell actin filament bundles, running in parallel, were present at p.n. day 10. Actin formed a meshwork pattern characteristic of the adult on, or slightly prior to, p.n. day 22. These data indicate that there is a temporal relationship between the development of the actin cytoskeleton within the Sertoli cell and initial formation of the Sertoli cell barrier. Similarly, there is a temporal relationship between the development of the actin cytoskeleton of myoid cells and tubular fluid flow. The rapid increase in tubular lumen diameter, however, does not correlate with the initial development of Sertoli and myoid cytoskeletal elements.     

The Sertoli cell in vivo and in vitro

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo “spermiation” at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood–testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood–tissue barriers.     

Development of the seminiferous tubules after neonatal hemicastration in the boar

Development of the prepubertal seminiferous tubules of the right testis was characterized morphometrically every 14 days from 10 to 122 days of age in intact boars (I) and boars hemicastrated (HC) on Day 10 of life from two herds (Trial 1 and Trial 2). Comparisons were made between the remaining testis of Group HC boars and one testis in Group I boars. By 38 days of age seminiferous tubule length in Group HC boars was double (P less than 0.0001) that in Group I boars. Seminiferous tubule length did not differ between trials within treatments. The diameter of the seminiferous tubule was similar in Group HC and I boars but was greater (P less than 0.05) in Trial-1 than Trial-2 boars from Day 80 to 122 of life. Relative mass (mass of tissue/body mass) of Sertoli cells became 2-fold greater (P less than 0.0001), in Group HC than in one testis of Group I boars by 38 days of age and this difference was maintained throughout the experimental period. The relative mass of Sertoli cells was greater (P less than 0.05) in Trial-1 than Trial-2 boars within each treatment between 80 and 122 days of age. The relative mass of gonocytes was similar for all groups and treatments of boars. By 122 days of age the relative mass of spermatogenic cells was greater (P less than 0.05) in Group HC than in one testis of Group I boars and greater (P less than 0.01) in Trial-1 than Trial-2 boars within each treatment. Onset of spermatogenesis was first observed at 80 and 94 days of age in boars in Groups HC and I, respectively. Development of seminiferous tubule lumen was first observed at 94 and 108 days of age in boars in Groups HC and I respectively. Seminiferous tubule lumen, taken as a measure of fluid secretion of the Sertoli cells, occupied a greater (P less than 0.01) portion of seminiferous tubule in Trial-1 than Trial-2 boars within each treatment at the end of the experimental period. It is concluded that neonatal hemicastration of boars rapidly caused a compensatory seminiferous tubule elongation apparently due to Sertoli cell proliferation and an earlier onset of spermatogenesis. However, the gonocytes do not proliferate until they transform into spermatogonia.     

Microtubule polarity in Sertoli cells: a model for microtubule-based spermatid transport

Bundles of microtubules occur adjacent to ectoplasmic specializations (ESs) that line Sertoli cell crypts and support developing spermatids. These microtubules are oriented parallel to the direction of spermatid movement during spermatogenesis. We propose a model in which ESs function as vehicles, and microtubules as tracks, for microtubule-based transport of spermatids through the seminiferous epithelium. Microtubule polarity provides the basis for the direction of force generation by available mechanoenzymes. As part of a more general study designed to investigate the potential role of microtubule-based transport during spermatogenesis, we have studied the polarity of cytoplasmic microtubules of Sertoli cells. Rat testis blocks were incubated in a lysis/decoration buffer, with and without exogenous purified bovine brain tubulin. This treatment results in the decoration of endogenous microtubules with curved tubulin protofilament sheets (seen as hooks in cross section). The direction of curvature of the hooks indicates microtubule polarity; that is, clockwise hooks are seen when viewing microtubules from the plus to the minus end. We found that, in Sertoli cells, most of the hooks were orientated in the same direction. Significantly, when viewed from the base of the epithelium, hooks pointed in a clockwise direction. The clockwise direction of dynein arms on axonemes of sperm tails, in the same section, provided an internal check of the section orientation. Electron micrographs of fields of seminiferous epithelium were assembled into montages for quantitative analysis of microtubule polarity. Our data indicate that Sertoli cell cytoplasmic microtubules are of uniform polarity and are orientated with their minus ends toward the cell periphery. These observations have significant implications for our proposed model of microtubule-based transport of spermatids through the seminiferous epithelium.     

Non-muscle cofilin is a component of tubulobulbar complexes in the testis

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.