Germ cell-Sertoli cell interactions: analysis of the biosynthesis and secretion of cyclic protein-2

Cyclic protein-2 (CP-2) is secreted in vitro in substantial amounts by mature rat Sertoli cells in intact Stage VI and Stage VII seminiferous tubules. This stage-dependent secretion has led us to postulate that the biosynthesis of this molecule is stimulated by germ cells at a specific state of development. In order to explore this hypothesis and to examine the steps in CP-2's biosynthesis, we generated a polyclonal antisera against this protein and used it to analyze the biosynthesis and secretion of CP-2. Analysis of the steps in the biosynthesis of CP-2 indicated that its polypeptide core represented most if not all of the translation product of the CP-2 mRNA and that a single aspargine-linked oligosaccharide became attached to this core. Analysis of the rate of biosynthesis of CP-2 at specific stages of the cycle of the seminiferous epithelium was also conducted. Two-millimeter segments of tubules at Stage II, VI, VIIa, b, VIII, and XII were cultured for 1 hr in the presence of [35S]methionine and radiolabeled CP-2 immunoprecipitated from the tubules. Data (35S-CP-2 synthesized per hour) demonstrated that the rate of CP-2's biosynthesis increased 9-fold from Stage II to Stages VI and VIIa, b and then decreased 13-fold by Stage XII. To determine whether these rates of biosynthesis were identical to the rates of secretion, tubules were cultured for 17 hr with [35S]methionine, CP-2 was immunoprecipitated from the culture medium and data were expressed as 35S-CP-2 secreted per hour. This analysis demonstrated that the rate of secretion of CP-2 varied in the same stage-specific manner as its rate of synthesis. However, at each stage, the apparent rate of biosynthesis of the molecule exceeded its apparent rate of secretion. In order to explain this observation, we analyzed the rate of export of newly synthesized CP-2 out of the tubules. This demonstrated that quantitative export of the protein into culture medium required at least 17 hr. This period of time was most likely due to the retention of the protein within the tubular lumen, since primary cultures of Sertoli cells were shown to rapidly secrete newly synthesized CP-2. We, therefore, concluded that CP-2 was biosynthesized in a stage-dependent manner and that all CP-2 was secreted.     

Isolation of cyclic protein-2 from rat seminiferous tubule fluid and Sertoli cell culture medium

Cyclic Protein-2 (CP-2), a stage-specific secretory product of the rat seminiferous epithelium, has been isolated from seminiferous tubule fluid (STF) and Sertoli cell culture medium. Isolation from STF was accomplished by mixing STF with radiolabeled proteins secreted by Stage VI-VII seminiferous tubules and sequential fractionation of these proteins by hydroxylapatite, DEAE-agarose, and quaternary amine ion-exchange chromatography. Radiolabeled proteins were used to identify the chromatographic fractions that contained CP-2. Through use of these procedures, a highly purified preparation of radioinert CP-2 was obtained from seminiferous tubule fluid. Cyclic Protein-2 was also isolated from Sertoli cell culture medium, indicating that the Sertoli cell is its most likely source. Preliminary characterization of CP-2 was conducted. First, CP-2 appeared to be highly enriched in methionine. Second, the molecular weight of CP-2 was found to be 20,000. Third, analysis by reverse-phase hydrophobic chromatography indicated that CP-2 was relatively hydrophobic. We conclude that CP-2 is a small hydrophobic glycoprotein secreted in vivo and in vitro in a stage-specific manner by Sertoli cells.     

Germ cell-Sertoli cell interactions: the effect of testicular maturation on the synthesis of cyclic protein-2 by rat Sertoli cells

Cyclic Protein-2 (CP-2) is synthesized in a stage-specific manner by mature rat Sertoli cells within stage VI and VII seminiferous tubules. To determine how testicular maturation affects CP-2 synthesis, we cultured 20 cm of tubules encompassing all stages of the cycle from rats 17, 35, 45, and 75 days old. The greatest increase in CP-2 synthesis was found to occur between 35 and 45 days and exceeded that observed for transferrin and sulfated glycoprotein (SGP)-2. Additionally, two-dimensional gel analysis indicated that secretion of CP-2 increased from 35 to 45 days to a greater extent than the secretion of SGP-1 and SGP-2 and transferrin. Biochemical analysis also demonstrated that CP-2 synthesis was stage-specific by 45 days. Immunocytochemistry expanded these observations; CP-2 was not detected in 7-35-day-old Sertoli cells. However, at 36 days, CP-2 was detected in Sertoli cells in stage VI and VII tubules but not at any other stage. CP-2 concentration in stage VI-VII tubules was increased by 38 days, but was unchanged thereafter. Finally, we immunocytochemically examined age-related changes in CP-2 concentration of the proximal convoluted kidney tubule. This analysis revealed that, at 1 wk, CP-2 was present in all proximal tubules except those in the subcapsular area; however, by 14 days, CP-2 was detected in all proximal tubules. This comparison of Sertoli cells and proximal tubule cells indicates that CP-2 content is determined by the maturity of a cell and not by the age of the animal.     

Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.     

Proceedings: The role of the Sertoli cell in phagocytosis of the residual bodies of spermatids

The fate of residual bodies which form as spermatids are released from the seminiferous epithelium has been studied as part of a cytological investigation of the Sertoli cells during the stages of rat spermatogenesis. Testes from normal adult rats were fixed by whole body perfusion. All 14 stages of rat spermatogenesis were identified and studied by light and electron microscopy. Residual bodies are released at Stage 8 and are found in the luminal spaces of the seminiferous epithelium. During Stage 9 they appear to migrate peripherally in channels of the Seroli cell cytoplasm. During this migration, lysosomal-like bodies surround the residual bodies and appear to be involved in the degradative process. A considerable proportion of the lipid material persists and forms basal collections in the Sertoli cells. The lipid inclusions reach a peak at Stages 13 and 14 of the cycle and persist until Stage 2 and 3. Some lipid inclusions persist until Stage 4 to 7 when noticeable decrease occurs corresponding to the peripheral migration of maturing spermatids.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

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.     

Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule

Electrophoretic and morphologic methods have been used to study the time course of [35S] methionine-labeled proteins accumulated in the incubation medium of rat fetal testes and seminiferous cords/tubules during their development. We have found that Sertoli cell-specific secretory proteins S70, S45 and S35 became progressively prominent as premeiotic, meiotic and postmeiotic spermatogenic events were established in the seminiferous tubules. In the sexually mature rat, S70, S45 and S35 were expressed in a spermatogenic stage-dependent manner. While S70, S45 and S35 were present in Stage VII-VIII, S45 and S35 were observed in Stages X and XIV. Neither S70, S45 nor S35 were detected in Stage IV. A relevant group of high molecular weight proteins, previously reported as characteristic products of cultured peritubular cells, accumulated in the incubation medium of seminiferous cords from postnatal Day 0-15 rats. This group of high molecular weight proteins appears when peritubular cells are proliferative and are engaged in the organization of the seminiferous tubular wall. A low molecular weight protein, designated T35, was also detected. T35 was prominent in the medium of incubated fetal testes and seminiferous cords of postnatal rats 0- to 5-days-old and disappeared gradually thereafter. A set of proteins (designated SP1 and SP2) previously ascribed to both cultured Sertoli and peritubular cells, were recognized during the early postnatal stages of seminiferous tubular development. SP1 and SP2 displayed age-dependent fluctuations in their [35S] methionine labeling. The timing of appearance of S70, S45, and S35 indicates both age- and spermatogenic stage-related activity that, in the future, may prove to be functionally significant in the spermatogenic process.     

The cycle of the seminiferous epithelium in Bali cattle (Bos sondaicus)

The duration of the cycle of the seminiferous epithelium in 5 mature Bali bulls was 11.75 days (standard error of estimate 0.52 days). The relative frequencies of the stages of the cycle of the seminiferous epithelium (morphological classification) of Bali cattle differed from other cattle and buffalo in that there were lower frequencies of Stage 1 and 2 tubules, and higher frequencies of Stage 3, 6 and 7 tubules.     

Cytoskeletal involvement in spermiation and sperm transport

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2–3 months (Stage 1, 10 g), 4–5 months (Stage 2, 35 g), and 6–7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.     

Testins are localized to the junctional complexes of rat Sertoli and epididymal cells.

Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. Conclusions: This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

Stability of spermatogenic synchronization achieved by depletion and restoration of vitamin A in rats

Studies of synchronization of spermatogenesis following vitamin A deficiency have suggested that this may provide an in vivo model for the study of stage-dependent changes in hormonal action and protein secretion within the seminiferous epithelium. However, until now, no information on the stability or durability of this condition has been available. In this study, 200 seminiferous tubules from each of 40 rats (including controls) were classified according to their spermatogenic stage after withdrawal and replenishment of vitamin A. Following 15 wk withdrawal and subsequent replenishment of vitamin A, spermatogenesis was initiated in a synchronous fashion. This synchrony remained stable for more than 10 cycles of the seminiferous epithelium (2.5 spermatogenic cycles). In association with the extended period of vitamin A deficiency, a proportion of tubules (30%) showed morphological characteristics of either Sertoli cells only or Sertoli cells plus spermatogonia with occasional pachytene spermatocytes. During the 11-wk period of observation in this study, no significant change in proportions of damaged tubules were observed. Testicular testosterone concentrations, although elevated with respect to controls, showed no correlation with the stage of the cycle of the seminiferous epithelium observed, whereas pituitary and serum follicle-stimulating hormone levels were elevated, probably due to the number of damaged tubules observed. The persistence of synchrony in spermatogenesis following vitamin A treatment suggests that this model is applicable for studies of paracrine actions within the testis. However, the decreased ratio of synchrony observed with time may provide evidence that duration of the individual stages of the cycle of the seminiferous epithelium might be subject to temporal variation, leading to a progressive desynchronization of spermatogenesis in this model system.