Insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor mediates paracrine interactions during spermatogonial development.

The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor transduces signals after binding IGF-II or M6P-bearing growth factors. We hypothesized that this receptor relays paracrine signals between Sertoli cells and spermatogonia in the basal compartment of the seminiferous epithelium. For these studies spermatogonia were isolated from 8-day-old mice with purity >95% and viability >85% after overnight culture. The IGF-II/M6P receptors were present on the surface of spermatogonia, as detected by indirect immunofluorescence. We determined that both IGF-II and M6P-glycoproteins in Sertoli cell conditioned medium (SCM) modulate gene expression in isolated spermatogonia. The IGF-II produced dose-dependent increases in both rRNA and c-fos mRNA. These effects were mediated specifically by IGF-II/M6P receptors, as shown by studies using IGF-II analogues that are specific agonists for either IGF-I or IGF-II receptors. The SCM treatment also induced dose-dependent increases in rRNA levels, and M6P competition showed that this response required interaction with IGF-II/M6P receptors. The M6P-glycoproteins isolated from SCM by IGF-II/M6P receptor affinity chromatography increased spermatogonial rRNA levels at much lower concentrations than required by SCM treatment, providing further evidence for the paracrine activity of Sertoli M6P-glycoproteins. These results demonstrate that Sertoli cells secrete paracrine factors that modulate spermatogonial gene expression after interacting with cell-surface IGF-II/M6P receptors.

Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis.

Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand-activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3-kb transcript generally expressed at higher levels than the larger 7.4- or 8.0-kb transcript. In both the mouse and rat, the 2.3- and 7.4-kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4-kb mRNA appearing earlier during testicular development. The major 2.3-kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady-state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3' termination site of the 2.3-kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation.

Overexpression of androgen-binding protein/sex hormone-binding globulin in male transgenic mice: tissue distribution and phenotypic disorders.

The rat androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) gene in transgenic mice was previously shown to be specifically expressed in the testes. This study verifies a Sertoli cell location of ABP and translation of testicular ABP mRNA in the transgenic mice by dihydrotestosterone (DHT)-binding assays and immunohistochemistry. DHT-binding activities in the testis and epididymis of the hemizygous transgenic mice were elevated 20-fold as compared to activity in the wild-type tissues. DHT-binding activities were also elevated in blood plasma at least 25- to 50-fold in the transgenic mice; binding was undetectable in the plasma from control mice. Immunohistochemical analysis revealed that the transgenic testicular ABP was primarily in the cytoplasm of Sertoli cells and lumen of the seminiferous tubules. In some tubules, intense staining also was associated with spermatids. After transport to the epididymis, there were large amounts of immunoreactive ABP internalized in the epithelium of the initial segment and proximal caput. The increased levels of plasma and testicular ABP had no effect on levels of testosterone; there was a 30-fold range of plasma and testicular testosterone levels in the wild-type and transgenic mice. Increased ABP levels in the transgenic mice were associated with structural and functional abnormalities in the testis. Abnormal spermatogenesis resulted in extensive structural changes in the transgenic testis; the degree of the defect varied from near normality to the loss of most germ cells. In the affected mice, seminiferous tubules had smaller diameters and decreased numbers of germ cells, particularly in the spermatid stages of differentiation. Pyknotic nuclei and multinucleated cells were associated with the spermatids in the defective tubules, but not in the wild-type tubules. Consequently, mice with the spermatogenic disorder had reduced epididymal sperm numbers. The variable spermatogenic disorder was associated with variable male fertility. The homozygous transgenic male and female mice also had a serious motor dysfunction affecting their hind limbs. This study demonstrates how the transgenic mouse model can be used to study ABP's function, and the data support several hypotheses on its function in the testis and epididymis.

Stage-specific expression of B cell translocation gene 1 in rat testis.

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Sertoli cell-spermatogenic cell interaction: the insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor mediates changes in spermatogenic cell gene expression in mice.

The insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional receptor with distinct binding sites for IGF-II and mannose 6-phosphate (M6P)-bearing glycoproteins. We used the immediate-early response gene c-fos to assay early changes in gene expression in spermatogenic cells in response to ligands for this receptor that are present in the seminiferous epithelium. We confirmed that c-fos behaves as an immediate-early response gene in spermatogenic cells after stimulation of protein kinase C with phorbol ester or after intercellular calcium levels are raised with calcium ionophore. After determining that IGF-II mRNA is present in Sertoli cells, we treated spermatogenic cells with this growth factor and found that it increased c-fos mRNA levels in a dose-dependent manner. Similarly, Sertoli cell-conditioned medium (SCM) caused a dose-dependent increase in c-fos levels in spermatogenic cells isolated from adult mice. This effect was inhibited in the presence of 5 mM M6P, demonstrating that this change in c-fos gene expression was mediated by the IGF-II/Cl-MPR. In addition, SCM treatment of purified pachytene spermatocytes and round spermatids caused a dose-dependent increase in 18S rRNA levels that was completely abolished in the presence of M6P. Our results provide direct evidence that IGF-II/Cl-MPR ligands secreted by Sertoli cells can modulate gene expression in spermatogenic cells and strongly suggest that they are important in the regulation of spermatogenesis.

Sertoli cell and germ cell cystatin C: stage-dependent expression of two distinct messenger ribonucleic acid transcripts in rat testes.

Sertoli cells were shown to synthesize and secrete cystatin C, a potent inhibitor of cysteine proteases. The evidence for this observation was obtained from protein sequencing. Western analysis using antiserum specific to cystatin C, and immunoprecipitation of 35S protein secreted by cultured cells. The Western analysis with an antiserum to human cystatin C showed that cultured Sertoli cells secrete three previously reported immunoreactive forms of cystatin C: a predominant pair of proteins at 13-14 kDa and a less abundant 20-kDa protein. Immunohistochemical localization of cystatin C in sections of rat testes showed intense staining in Sertoli cells; no immunoreactivity was observed in spermatogonia or spermatocytes. A cDNA fragment for rat cystatin C was obtained by use of the polymerase chain reaction and was used as a probe in Northern analyses to examine the steady-state levels of cystatin C mRNA in intact testes and in Sertoli and spermatogenic cells. Sertoli cells contained a 700-nucleotide cystatin C transcript, and a mixed population of spermatids and spermatocytes contained a 550-nucleotide transcript. Analysis of RNA from purified spermatogenic cells revealed that round and condensing spermatids contained the 550-nucleotide transcript, while pachytene spermatocytes contained a smaller 500-nucleotide transcript. The 700-nucleotide transcript was present in testes isolated from rats of 5-79 days of age, the 500-nucleotide transcript was detected initially in testes from 24-day-old rats, and the 550-nucleotide transcript was detected initially at 35 days of age. Both the 500- and 550-nucleotide transcripts increased in abundance until 50 days of age. RNA from stage-synchronized testes showed that steady-state levels of both the 550- and 700-nucleotide transcripts were lowest in stages VI-VII of the cycle. These data suggest that the role of cystatin C in the testis may be to inhibit the proteolytic activity of the cysteine protease cathepsin L in all stages except stages VI-VII.

Structural analysis of sulphated glycoprotein 2 from amino acid sequence. Relationship to clusterin and serum protein 40,40.

Sulphated glycoprotein 2 (SGP-2) is the major secreted protein product of rat Sertoli cells; likewise, clusterin is a major constituent of ram rete testis fluid. Isolation and sequencing of the intact subunits and peptides derived from clusterin show that it is the ram homologue of rat SGP-2. Human serum protein 40,40 (SP-40,40), a component of the SC5b-9 complex of complement, has recently been reported to be the human homologue of rat SGP-2. Analysis of the amino acid sequences of rat SGP-2 and human SP-40,40 show that both of these proteins have a significant relationship to the heavy chain of myosin. The regions of highest sequence similarity correspond to the major amphipathic domains in SGP-2/SP-40,40 and the long alpha-helical-tail domain of myosin, which forms a rod-like structure. SGP-2 has anomalous sedimentation behaviour which indicates that it probably exists in an extended conformation. A putative dinucleotide-binding structure has been identified in the longest stretch of identity between SGP-2 and SP-40,40. Elucidation of these features of SGP-2 and SP-40,40 may help to direct future studies into the role of these proteins in the reproductive and complement systems.

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator.

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.