Relative roles of testosterone and the germ cell complement in determining stage-dependent changes in protein secretion by isolated rat seminiferous tubules

This study has compared the effect of withdrawal of testosterone (+/- replacement) with that of selective depletion of pachytene spermatocytes (PS) or round/elongating spermatids (RS), or both PS and RS, on the level of overall protein secretion by seminiferous tubules (ST) isolated at particular stage-groups of the spermatogenic cycle. Testosterone withdrawal was induced by destroying the Leydig cells with a single injection of ethane-dimethane sulphonate (EDS), with or without concomitant replacement of testosterone by injection; ST at stages II-V, VI-VIII or IX-XII were then isolated from control and treated rats at 4 days after treatment. Methoxyacetic acid (MAA) was administered, in either one or two doses, to selectively destroy 80-100% of pachytene and later spermatocytes; ST at stages I-V, VI-VIII or IX-XIV were then isolated at specific times after treatment such that ST were depleted selectively of either PS, RS or PS+RS. Isolated ST (5 cm) were then cultured for 22 h at 34 degrees C with 35S-methionine and its incorporation into secreted proteins then quantified. Based on the incorporation of 35S-methionine, ST at stages VI-VIII showed a significantly higher level of protein secretion than did ST at earlier or later stages. This difference was abolished following testosterone withdrawal but was maintained by testosterone replacement. The normal increase in protein secretion by ST at stages VI-VIII was also prevented if either PS or RS were depleted, whereas depletion of either PS or RS alone had no significant effect on protein secretion by ST at stages I-V, and only the depletion of RS significantly reduced protein secretion by ST at stages IX-XIV. Depletion of both PS+RS reduced protein secretion significantly by ST at all stages. In contrast to the data for total protein secretion, the levels of sulphated glycoprotein (SGP)-1 and SGP-2 secreted by ST at stages VI-VIII showed that these two Sertoli cell proteins were unaffected by germ cell depletion except after co-depletion of PS+RS when secretion of SGP-1 was halved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification, ontogeny, and regulation of an interleukin-6-like factor in the rat seminiferous tubule

The present study's aims are to search for the presence of interleukin-6 bioactivity (IL-6) in medium conditioned by various testicular cell types and to investigate the cellular and hormonal regulation of testicular IL-6 production. Sertoli cells prepared from rats of increasing ages (20, 35, and 45 days) secreted IL-6 in vitro, whereas medium conditioned by pachytene spermatocytes, early spermatids, and peritubular cells showed no activity. Lipopolysaccharide (LPS) and latex beads, two known stimulators of monocyte/macrophage IL-6 production, markedly stimulated IL-6 secretion by Sertoli cells at all the ages investigated. Maximum levels of IL-6 were reached after 6 h of culture of Sertoli cells with LPS and after 24 h with latex beads. When Sertoli cells were cocultured with pachytene spermatocytes, early spermatids, or fractions containing residual bodies and cytoplasts from elongated spermatids, only the latter significantly stimulated IL-6 levels. Maximum levels of IL-6 were attained by adding 2 x 10(6) residual bodies to Sertoli cells; a significant increase in IL-6 secretion was seen after 6 h, and maximum levels were observed after 24 h. The levels of IL-6 varied throughout different stages of the seminiferous epithelium cycle; highest levels were observed in stages II-VI and lowest in stages VII-VIII. IL-6 bioactivities induced by LPS and residual bodies and cytoplasts from elongated spermatids could be totally neutralized with a specific monoclonal antibody at all of the ages studied. FSH, phorbol myristate acetate, and IL-1 alpha augmented Sertoli cell IL-6 secretion in a dose-dependent manner. Furthermore, FSH and (Bu)2cAMP differentially stimulated IL-6 secretion during the seminiferous epithelial cycle. It is concluded that the release of IL-6 from Sertoli cells is regulated by a complex interplay between residual bodies and humoral factors.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.     

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.     

Retinyl palmitate, a major retinyl ester in rat testis seminiferous tubules. II. The Sertoli cell, site of retinol esterification

The study was designed to 1) isolate and quantitate retinyl esters normally present in the testis seminiferous tubules (ST) and 2) determine whether the Sertoli (SE) cells in the ST have the enzyme system necessary to esterify retinol. In the first study testes from normal adult male rats were removed, the ST were isolated mechanically and retinol was extracted and separated into free and retinyl esters in a prestandardized alumina column. The fraction containing retinyl esters was hydrolyzed, and the fatty acid composition was determined gas chromatographically. The results showed that retinyl palmitate was the major retinyl ester in the testis ST constituting almost 55% of the total esters. In the second study adult male rats were given Busulfan (1,4, dimethylsulfonoxybutane) intraperitoneally twice six weeks apart. At the end of six weeks following injection, the testes from a few animals were examined histologically, and the results showed that Busulfan treatment destroyed the germinal epithelium completely but had no effect on the structural integrity of the SE cells. Labeled retinol (free) mixed in rat albumin solution was injected intratesticularly (in vivo study) or incubated (in vitro study) with ST which had been isolated from the testes of rats treated with Busulfan. The results of the studies indicate that labeled retinol (free) was converted to retinyl esters in the ST. Since the ratio of conversion of free retinol (labeled) to retinyl esters remained constant in the treated ST (which contained only Sertoli cells, the germinal epithelium having been destroyed) and in the control ST (which contained both Sertoli cells and germinal epithelium), we conclude that the Sertoli cells are the site of retinol esterification in the testis ST.     

Localization of the glucocorticoid receptor in testis and accessory sexual organs of male rat

Localization of glucocorticoid receptor-like immunoreactivity (GR-LI) was studied in adult rat testis, epididymis, ejaculatory duct, seminal vesicle and prostate by light and electron microscopic immunocytochemistry. In the interstitium of the testis GR-LI was seen in the nuclei of Leydig cells, macrophages, fibroblasts, smooth muscle cells and endothelial cells of blood vessels. Furthermore, GR-LI was observed in zygotene and early pachytene primary spermatocytes of some seminiferous tubules during stages XIII-XIV and I-III of the spermatogenic cycle. Other spermatogenic cells and Sertoli cells were devoid of staining. GR-LI was also found in peritubular myoid cells, fibroblasts and basal cells of the epididymis, vas deferens and prostate. Localization of GR-LI in primary spermatocytes and Leydig cells suggests that glucocorticoids directly affect spermato- and steroidogenesis in the testis. The absence of GR-LI from functional, stromal cells of the male accessory sexual organs suggests that they are not targets for glucocorticoid hormones.     

Germ cell genotype controls cell cycle during spermatogenesis in the rat

Spermatogenesis is one of the most productive self-renewing systems in the body: on the order of 10(7) spermatozoa are produced daily per gram of testis tissue. In each mammalian species, the time required for completion of the process is unique and unalterable. Because the process is supported by somatic Sertoli cells, it has generally been thought that cell-cell interaction between germ and Sertoli cells controls the duration of cell cycles and cellular organization. We have used the newly developed technique of spermatogonial transplantation to examine which cell type(s) determines the rate at which germ cells proceed through spermatogenesis. Rat germ cells were transplanted into a mouse testis, and the mouse was killed 12.9-13 days after administration of a single dose of [3H]thymidine. The most advanced rat cell type labeled was the pachytene spermatocyte at stages VI-VIII of the spermatogenic cycle. In animals given only rat cells, some endogenous spermatogenesis of the mouse recovered. The most advanced labeled mouse cell types in recipients killed 12.9-13 days after administration of a single dose of [3H]thymidine were meiotic cells or young spermatids, which is consistent with a spermatogenic cycle length comparable to the 8.6 days reported for the mouse. The same results were obtained if a mixture of rat and mouse cells were transplanted. There existed two separate timing regimens for germ cell development in the recipient mouse testis; one of rat and one of mouse duration. Rat germ cells that were supported by mouse Sertoli cells always differentiated with cell cycle timing characteristic of the rat and generated the spermatogenic structural pattern of the rat, demonstrating that the cell differentiation process of spermatogenesis is regulated by germ cells alone.     

Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels

To elucidate the role of germ cells in the regulation of inhibin B secretion, serum inhibin B levels in prepubertal boys and adult men whom had a concurrent testicular biopsy showing either normal or impaired testicular function were compared. In addition, by immunohistochemistry the cellular localization of the two subunits of inhibin B (alpha and betaB) were examined in adult testicular tissue with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only tubules (SCO) as well as in normal testicular tissue from an infant and a prepubertal boy. Adult men with testicular biopsy showing normal spermatogenesis (n=8) or spermatogenic arrest (n=5) had median inhibin B levels of 148 pg/mL (range, 37-463 pg/mL) and 68 pg/mL (range, 29-186 pg/mL), respectively, corresponding to normal or near-normal levels of our reference population (165 and 31-443 pg/mL; n=358). Men with SCO (n=9) had undetectable or barely detectable (n=1) serum levels of inhibin B. In contrast to adults, prepubertal boys with SCO (n=12) all had measurable serum inhibin B levels that corresponded to our previously determined normal range in healthy prepubertal boys (n=114). However, in postpubertal samples from the same SCO boys, inhibin B levels were undetectable as in the adult SCO men. Intense inhibin alpha-subunit immunostaining was evident in Sertoli cells in both prepubertal and adult testes. In the prepubertal testis, positive immunostaining for the betaB-subunit was observed in Sertoli cells. In the adult testis, intense immunostaining for the betaB-subunit was evident in germ cells from the pachytene spermatocyte to early spermatid stages and to a lesser degree in Leydig cells, but not in Sertoli cells or other stages of germ cells. Thus, surprisingly, in adult men the two subunits constituting inhibin B were expressed by different cell types. We speculate that during puberty Sertoli cell maturation induces a change in inhibin subunit expression. Thus, immature Sertoli cells express both alpha and betaB inhibin subunits, whereas fully differentiated Sertoli cells only express the alpha-subunit. The correlation in adult men between serum inhibin B levels and spermatogenesis may be due to the fact that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells, including the stages from pachytene spermatocytes to early spermatids.     

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.     

Histopathology of the seminiferous tubules in mice injected with syngeneic testicular germ cells alone

Previously, a model of murine experimental autoimmune orchitis was produced by active immunization with viable syngeneic testicular germ cells without resorting to any adjuvants. The histological mode of the spermatogenic disturbance of this autoimmunity was investigated in A/J mice. A significant spermatogenic disturbance was consistently induced after the appearance of inflammatory cell responses around the tubuli recti. It first appeared seminiferous epithelium adjacent to the tubuli recti, then spread to the peripheral epithelium. The histopathology of the seminiferous tubules in the early phase ranged from partial degeneration and depletion of all kinds of germ cells to complete loss of germ cells other than some remaining spermatogonia, while both Sertoli cells and the basal lamina of the tubules appeared intact. In the late phase, depletion of Sertoli cells, disorganization of the seminiferous tubular wall or filling with many round-shaped degenerating germ cells, appearance of malformed spermatids with signet ring nuclei, depletion of immature germ cells with remaining elongated spermatids, or complete loss of the seminiferous epithelium were observed in addition to the early histopathological features.     

Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p < 0.001) in DES-treated animals. Plasma FSH levels were suppressed markedly at 18 and 25 days, but not at 35 days, in GnRHa-treated rats, whereas in DES-treated rats the FSH levels were suppressed significantly only at 35 days. Both treatments suppressed plasma levels of inhibin B, though this was more pronounced (p < 0.05) in DES- than in GnRHa-treated rats. In controls, Sertoli cell immunoexpression of inhibin alpha, sulfated glycoprotein-1 (SGP-1), and androgen receptor (AR) increased in intensity and changed to an adult, stage-dependent pattern by 25 days. In GnRHa-treated rats these changes were reduced in intensity but were similar to those in controls at 35 days. In DES-treated rats, the increase in intensity and stage-dependent pattern of immunoexpression of inhibin alpha, SGP-1, and AR were virtually absent at 25 days but were present by 35 days. Germ cell volume per Sertoli cell was reduced in GnRHa- and DES-treated rats compared with controls at 18 and 25 days but was significantly greater (p < 0. 001) in DES- than in GnRHa-treated rats at 35 days. The proportion of apoptotic to viable germ cells was increased (p < 0.01) in GnRHa- and DES-treated rats compared with controls at 18 and 25 days; but at 35 days, values in GnRHa-treated rats had declined to control values whereas those for DES-treated rats remained 10-fold elevated (p < 0.001). In adulthood, testis weight and daily sperm production were reduced by 43% and 44%, respectively, in GnRHa-treated rats, but spermatogenesis was grossly normal. Comparable changes were observed in approximately 25% of DES-treated rats, but the majority exhibited > 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.     

On the synthesis and secretion of rat seminiferous tubule proteins in vivo after ischemia and germ cell loss

This study was undertaken to determine whether alterations in Sertoli cell protein synthesis and secretion were important precursors to germ cell loss after ischemic insult to the testis. Ischemia was induced by a 1-h, 720 degrees spermatic cord torsion, and this was shown to cause a loss of germ cells over a 15-day period. Seminiferous tubules were perifused in vivo with [35S]methionine. Lumen fluid (LF) was collected by in vivo micropuncture, and seminiferous tubule extract (TE) was collected after tubule homogenization and centrifugation. Electrophoresis of proteins in these fluids followed by autoradiography of radiolabeled proteins allowed examination of synthesized, i.e., TE, and secreted, i.e., LF proteins. No consistent changes were detected in synthesized or secreted proteins prior to the major loss of germ cells; thus, major changes in the capacity of Sertoli cells for protein assembly and transport are not a preliminary feature of post-ischemia germ cell loss. Changes in specific protein synthesis and secretion were also modest in this in vivo environment after germ cell loss. Overall protein synthesis appeared reduced as loss of germ cells progressed, but one protein whose amino acid sequence confirmed identity with a testis-specific stress protein (hst70) was up-regulated after ischemia and germ cell loss.     

Motor function of the proximal stomach and visceral perception in gastro-oesophageal reflux disease

Oxytocin (OT) is present in the mammalian testis and has been shown to play a role in the modulation of seminiferous tubule contractility and steroidogenesis. However, stage-specific effects of the peptide have not been previously investigated. In this study, computer-assisted analysis and time-lapse videomicrography were used to investigate basal contractility and the response to OT of seminiferous tubules at specific stages of the spermatogenic cycle. Adult rat testes were placed in fresh oxygenated DMEM F12 medium, decapsulated, and the tubules gently teased apart. Stages were identified by transillumination and a 10 mm section of tubule at each of stages IV-V, VII-VIII and XIII-I was placed in a microslide chamber and perifused with medium. After a control period of 3 h, OT (2 nM) was given for 1 h, followed by another control period of 1 h. The experiment was repeated using tubules from different rats and data were analysed to give arbitrary units of tubule contractility. Contractility was observed in all the tubules studied and the contractile activity was shown to vary depending on the stage of the spermatogenic cycle. Mean basal contractility at stages VII-VIII, the time when sperm are shed from the epithelium, was significantly lower than that at stages IV-V and XIII-I. The response of the tubules to OT was also stage-dependent, with the peptide producing the largest increases in contractile activity at stages VII-VIII and having no effect at stages IV-V. We postulate that these stage-specific differences in basal and OT-stimulated contractility may be important in co-ordinating the movement of developing germ cells towards the lumen of the seminiferous epithelium and in the process of spermiation.     

Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men

Testicular biopsies of infertile patients are often characterized by a mixed atrophy, in which different types of spermatogenic lesions are found in adjacent tubules. In order to evaluate a possible involvement of the state of differentiation of the Sertoli cells, the distribution pattern of cytokeratin and vimentin intermediate filaments within the seminiferous epithelium of 228 biopsy specimens with normal spermatogenesis (n = 10), mixed atrophy (n = 206) or Sertoli Cell Only Syndrome (n = 12) were investigated by means of immunohistochemical techniques. Sertoli cells were regularly found to show vimentin expression in tubules with normal spermatogenesis as well as in tubules with any kind of spermatogenic impairment including SCO. Cytokeratin expression as a marker showing lack of differentiation was common in Sertoli cells of tubules with arrest of spermatogenesis at the level of spermatogonia, and was occasionally associated with arrest at the level of primary spermatocytes or with SCO. Ultrastructural examination of tubules with spermatogonial arrest revealed Sertoli cells with features of typical fetal or prepubertal Sertoli cells, such as round to ovoid nuclei without indentations, stacks of rough ER and spot desmosomes. These data suggest that spermatogenic arrest at the level of spermatogonia might be due to functional impairment of the associated Sertoli cells, which have maintained or regained an undifferentiated state and are not able to initiate or trigger the process of spermatogonial differentiation.