Immature rat seminiferous tubules reconstructed in vitro express markers of Sertoli cell maturation after xenografting into nude mouse hosts.

Sertoli cells undergo a maturation process during post-natal testicular development that leads to the adult-type Sertoli cell, which is required for spermatogenesis. Understanding Sertoli cell maturation is therefore necessary to gain insight into the underlying causes of impaired spermatogenesis and male infertility. The present study characterized the cellular and molecular differentiation of Sertoli cells in a xenograft model of mammalian testicular development. Immature rat Sertoli cells were cultured in a three-dimensional culture system to allow the formation of cord-like structures. The in vitro Sertoli cell cultures were then grafted into nude mice. Sertoli cell proliferation, morphological differentiation and mRNA expression of Sertoli cell maturation markers were evaluated in xenografts. Sertoli cell proliferation significantly decreased between 1 and 4 weeks (6.7 +/- 0.9 versus 1.2+/- 0.1%, P < 0.001), and was maintained at low levels thereafter. Sertoli cell cord-like structures significantly decreased between 1 and 4 weeks (59.6 versus 21%, P < 0.05), whereas Sertoli cell tubules were more frequently observed after 4 weeks (13.3 versus 73.1%, P < 0.05). Furthermore, expression of androgen binding protein, transferrin and follicle stimulating hormone receptor, markers for mature Sertoli cells, was detected after 1 week of grafting and increased significantly thereafter. We conclude from these results that rat Sertoli cells continue maturation after xenografting to the physiological environment of a host. This model of in vitro tubule formation will be helpful in future investigations addressing testicular maturation in the mammalian testis.

NF-kappaB induces cAMP-response element-binding protein gene transcription in sertoli cells.

Spermatogenesis is dependent upon Sertoli cells, which relay hormonal signals and provide factors required for the differentiation and proliferation of germ cells. NF-kappaB transcription factors are constitutively expressed in the nuclei of Sertoli cells in rodent testis. Electrophoretic mobility shift assays demonstrated that Sertoli NF-kappaB proteins specifically bind to kappaB enhancer motifs within the promoter of the cAMP-response element-binding protein (CREB) gene, an important mediator of hormonal signals that control spermatogenesis. Overexpression of NF-kappaB proteins in primary Sertoli and NIH 3T3 fibroblast cells induced the CREB promoter in transient transfection assays. Stimulation of Sertoli cells with tumor necrosis factor-alpha, an NF-kappaB-activating cytokine produced by round spermatids located adjacent to Sertoli cells, stimulated the elimination of IkappaB, the translocation of additional NF-kappaB to the nucleus, and increased NF-kappaB binding to CREB promoter kappaB enhancer elements. Tumor necrosis factor-alpha also stimulated transcription from the CREB promoter. These data demonstrate that NF-kappaB contributes to the up-regulation of CREB expression in Sertoli cells and raises the possibility that NF-kappaB may induce other Sertoli genes required for spermatogenesis. Furthermore, the CREB promoter is also inducible by NF-kappaB in NIH 3T3 cells suggesting that NF-kappaB may be a general regulator of CREB in non-testis tissues.

Hormonal regulation of the NF-kappaB signaling pathway.

The NF-kappaB transcription factor modulates a number of gene responses to hormonal stimuli. NF-kappaB can be induced by growth promoting hormones and cytokines, has been shown to counteract the effectiveness of steroid hormones and has recently been found to be regulated during mammalian spermatogenesis. Recent advances in the characterization of the NF-kappaB signaling pathway offer new opportunities to examine how hormonal stimuli regulate NF-kappaB mediated gene expression. In this mini-review we outline the signal pathways responsible for activating NF-kappaB, discuss the hormonal regulation of NF-kappaB and the regulation of hormonal responses by NF-kappaB, as well as summarize new studies characterizing NF-kappaB expression and activity in the mammalian testis.

RNA processing and the control of spermatogenesis.

The testis is a rich source for expression of mechanisms for gene regulation. Germ cell expansion and differentiation require many cellular changes and regulatory steps. In the developing germ cells, the length of mRNA transcripts often vary as the cells mature, reflecting the ongoing regulatory changes. Due to the numerous maturation stages that germ cells must undergo, novel gene regulation strategies have been developed that provide for flexible gene expression and protein function. Some of the methods employed in the testis to alter gene expression and function include the initiation of transcription at alternative start sites, the splicing in or out exons to alter the properties of the resulting protein, changes in the site of polyadenylation to control mRNA stability, and delays in the translation of transcripts to ensure a source of protein late in germ cell development after transcription ceases. Using these varied expression strategies, individual genes are able to perform different functions that can be directed to specific development timepoints.

Cell type-specific regulation of CREB gene expression: mutational analysis of CREB promoter activity.

Previous studies have shown that activation of the cyclic AMP (cAMP) pathway down-regulates CREB expression in CATH.a cells, an effect that appears to be mediated via inhibition of CREB gene transcription. In the current study, we compared this effect in CATH.a cells with regulation of CREB expression in another cell line, C6 glioma cells. In contrast to the findings in CATH.a cells, activation of the cAMP pathway up-regulates CREB expression in C6 glioma cells. To determine whether these opposite effects can be explained by regulation of CREB promoter activity, chloramphenicol acetyltransferase (CAT) assays were performed in CATH.a and C6 glioma cells that were transiently transfected with a CREB promoter-CAT fusion plasmid. Activation of the cAMP pathway decreased levels of CAT activity in transfected CATH.a cells but increased CAT activity in transfected C6 glioma cells. We next investigated the effect of mutations in the CREB promoter on such regulation in these two cell lines. Mutations of single CRE or Sp1 binding sites in the CREB promoter reduced basal levels of CAT activity but did not significantly attenuate regulation of the promoter in CATH.a or C6 glioma cells. However, mutation or deletion of two CRE sites in the CREB promoter completely abolished up-regulation of CAT activity in the C6 glioma cells and abolished basal levels of CAT activity in CATH.a cells. CREB promoter activity was also studied in cultured SHSY5Y cells and in primary cultures of striatal neurons as further comparisons. Activation of the cAMP pathway was found to increase CAT activity in both cell types. In the striatal cultures, this effect was obliterated by mutation or deletion of either of the two CREs in the promoter. These findings demonstrate cell type-specific effects of the cAMP pathway on CREB expression, which appear to be mediated via differential regulation of the CREB promoter.

Inducible cAMP early repressor ICER down-regulation of CREB gene expression in Sertoli cells.

The cAMP response element binding protein (CREB) and the cAMP-responsive element modulator (CREM) are cyclically expressed in the seminiferous tubules during spermatogenesis. In the somatic Sertoli cells, which are the major supporters of germ cell development in the seminiferous tubules, the expression of CREB is cyclical and appears to be regulated by the levels of cAMP produced in response to the pituitary derived follicle-stimulating hormone FSH. Cyclic AMP response elements (CREs) located in the promoter of the CREB gene were shown earlier to be implicated in an autopositive feedback loop that up-regulates the expression of CREB. Here we show that in Sertoli cells FSH-mediated induction of the CREM repressor isoform, ICER (inducible cAMP early repressor) is correlated with the inhibition and delay of CREB gene expression in the seminiferous tubules. ICER binds to the two CREs located in the promoter of the CREB gene and in transient transfection assays of Sertoli cells, ICER expression vectors down-regulate transcription of a reporter gene driven by the CREB gene promoter. In addition, analyses of ICER and CREB gene expression in isolated segments of rat seminiferous tubules reveals stage-specific and cycle-dependent expression of ICER. The periods of enhanced expression of ICER correspond to the stages of spermatogenesis with the lowest levels of CREB expression. We suggest that the expression of ICER in Sertoli cells may contribute to the periodic repression of CREB gene expression during the repeated 12-day cycles of spermatogenesis, and may be required to reset the levels of activator CREB prior to the initiation of each new cycle of spermatogenesis.

Stage-specific nuclear expression of NF-kappaB in mammalian testis.

The Rel/nuclear factor (NF)-kappaB family of transcription factors are important intracellular conveyors of extracellular signals in a number of systems. However, little is known of their roles in the specialized, hormonally regulated environment of the mammalian testis. In this study NF-kappaB p50 and p65 proteins were found to be constitutively present and active in the nucleus of Sertoli cells cultured from rat testis. In vivo, NF-kappaB proteins are present in the nucleus of Sertoli cells during all 14 (I-XIV) cyclical stages of spermatogenesis; however, nuclear NF-kappaB expression was elevated in stage XIV and remained high in stages I-VII. In contrast, NF-kappaB p50 and p65 subunits are transiently expressed in the nuclei of germ cells with peak levels found in pachytene spermatocytes during stages VII-XI and lower levels in stage I-VII spermatids. Tumor necrosis factor-alpha, which is produced by round spermatids in the testis, increased nuclear NF-kappaB binding activity when added to Sertoli cells. Stimulation of Sertoli cells with activators of the cAMP-protein kinase A (PKA) signaling pathway such as forskolin or FSH also increased NF-kappaB DNA binding activity. Consistent with the cellular localization studies, NF-kappaB was found to be activated as high basal levels of NF-kappaB-stimulated reporter gene expression were detected in transient transfection studies of Sertoli cells. Addition of tumor necrosis factor-alpha to Sertoli cells further stimulated kappaB enhancer-mediated transcription. These findings suggest that NF-kappaB proteins are stage specifically localized to Sertoli cell and spermatocyte nuclei and may play a role in the regulation of stage-specific gene expression during the process of spermatogenesis.

Cyclic AMP signaling and gene regulation.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger produced in cells in response to hormones and nutrients. The production of cAMP is dependent upon the actions of many different proteins that affect its synthesis and degradation. An important function of cAMP is to activate the phosphorylating enzyme, protein kinase A. The key roles of cAMP and protein kinase A in the phosphorylation and regulation of enzyme substrates involved in intermediary metabolism are well known. A newly discovered role for protein kinase A is in the phosphorylation and activation of transcription factors that are critical for the control of the transcription of genes in response to elevated levels of cAMP.

Alternative exon splicing controls a translational switch from activator to repressor isoforms of transcription factor CREB during spermatogenesis.

The cAMP/protein kinase A signaling pathway activates the cAMP-responsive transcription factor CREB. Here we describe a unique alternative RNA splicing event that occurs during the development of germ cells in the testis, resulting in a translational switch from an mRNA encoding activator CREB to an mRNA encoding novel inhibitor CREB isoforms (I-CREBs). Alternative splicing of an additional exon into the CREB mRNA in mid to late pachytene spermatocytes results in the premature termination of translation and consequent downstream reinitiation of translation producing I-CREBs. The I-CREBs down-regulate cAMP-activated gene expression by inhibiting activator CREB from binding to cAMP response elements. Further, the developmental stage-specific expression of I-CREBs in germ cells of the seminiferous tubules correlates with the cyclical down-regulation of activator CREB, suggesting that I-CREBs repress expression of the cAMP-inducible CREB gene as well as other genes transiently induced by cAMP during the 12-day cycle of spermatogenesis.

An alternatively spliced polycistronic mRNA encoding cyclic adenosine 3',5'-monophosphate (cAMP)-responsive transcription factor CREB (cAMP response element-binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform.

Cyclic AMP response element-binding protein (CREB) regulates the expression of cAMP-responsive genes. In the rat testis, several isoforms of CREB arise from alternative exon splicing that occurs cyclically during the 12-day cell association cycles of spermatogenesis. Insertion of alternatively spliced exon W into CREB mRNA during spermatogenesis results in a polycistronic RNA that encodes two novel internally translated CREB repressor isoforms called I-CREBs, consisting of the carboxy-terminal DNA-binding domain devoid of the transactivation domains. Here we report the alternative splicing of an additional novel exon Z in CREB mRNA expressed in human but not in mouse or rat testis. Insertion of exon Z abolishes the synthesis of one of the two inhibitor CREBs due to the introduction of an inframe stop codon within exon Z. We show that exon Z is not spliced into mRNAs in mouse and rat testes due to the evolution of mutations in the splice signals flanking exon Z. These findings suggest that the splicing in of exon Z may be part of a human-specific mechanism to regulate cAMP-dependent regulatory pathways in spermatogenesis by extinguishing the expression of a CREB repressor.

Role of transcription factors CREB and CREM in cAMP-regulated transcription during spermatogenesis.

The cAMP response element binding protein (CREB) and the cAMP-responsive element modulator (CREM) are cyclically expressed at high levels during spermatogenesis. Cyclical expression of CREB and CREM in germ and somatic Sertoli cells correlates with the fluctuations in cAMP signaling induced by the pituitary gonadotropic hormones FSH and LH both during sexual maturation of the testis and during the 12-day cycles of spermatogenesis that occur in the adult testis. CREB and CREM are expressed at different times during the spermatogenic cycle, undergo programmed sequential switches from activator to repressor isoforms by mechanisms of alternative exon splicing and promoter usage, and are autoregulated by cAMP signaling in opposing directions. cAMP response elements located in the promoter of the CREB gene upregulate the expression of activator CREBs, whereas cAMP autoregulatory response elements in the internal promoter of the CREM gene induce expression of repressor CREM isoforms. The complex mechanisms for the regulation of the expression of CREB and CREM in the testis appear to reflect critical adaptations for regulating key target genes essential for the development of germ cells.

Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in CATH.a cells.

We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element-binding protein (CREB) is down-regulated in CATH.a cells (a neural-derived cell line) by activation of the cAMP pathway. We now demonstrate that this down-regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin-induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run-on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed.

Expression of the gene encoding transcription factor cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB): regulation by follicle-stimulating hormone-induced cAMP signaling in primary rat Sertoli cells.

The somatic Sertoli cells of the testis are major targets for FSH and are important for the regulation of spermatogenesis. The binding of FSH to Sertoli cells activates the cAMP-dependent protein kinase A signaling pathway, resulting in phosphorylation of the cAMP response element-binding protein (CREB), which is required to transactivate genes containing cAMP response elements (CREs). Here we show that the addition of forskolin to cultured primary Sertoli cells results in the phosphorylation of CREB within 2-5 min. Phospho-CREB levels remain elevated with continued forskolin stimulation, but fall by 60% within 5 min after the removal of forskolin. In addition, we found that 8-bromo-cAMP induces CREB RNA accumulation in the Sertoli cells. Transient transfections of primary Sertoli cells with CREB promoter-chloramphenicol acetyltransferase reporter plasmids define a conserved 300-base pair region of the CREB promoter surrounding the transcription start site that is required for both basal and cAMP-inducible expression of the CREB gene. This region of the promoter contains three Sp1-binding sites flanking the transcription initiation site and two CREs located 65 and 85 base pairs downstream of the transcription initiation site. We show that the Sp1 motifs bind Sp1 in Sertoli extracts and contribute to basal promoter activity, and that the CREs bind CREB and are essential for cAMP induction of CREB gene transcription. These findings support the model of FSH- and cAMP-mediated CREB autoregulation of its own promoter and may explain the dramatic stage-specific oscillations in Sertoli cells of CREB messenger RNA levels during the 12-day cycles of spermatogenesis in rat seminiferous tubules.

An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription.

cAMP response element-binding protein (CREB) and modulator protein (CREM) regulate the transcription of cAMP-responsive genes via phosphorylation by cAMP-dependent protein kinase A. Reverse transcription and polymerase chain amplification of RNA from male germ cells identify an alternatively spliced CREM isoform, CREM delta C-G, lacking four exons including those encoding the protein kinase A-regulated phosphorylation domain and the flanking glutamine-rich transcriptional activation domains. CREM delta C-G retains exons that encode the basic-leucine zipper (bZIP) DNA-binding domain, binds to cAMP response elements (CREs), and competitively inhibits binding of CREB and CREM to CREs. Expression of CREM delta C-G inhibits transcription of a CRE-containing chloramphenicol acetyltransferase reporter plasmid induced by endogenous CREB. Antiserum to CREM detects CREM delta C-G in elongated spermatids from rat testis. These observations indicate that CREM delta C-G is a unique form of a competitive negative regulator of CREB-mediated gene transcription expressed in a maturation-dependent manner in haploid germ cells. The developmental specificity of CREM delta C-G suggests that it may play a role in transcriptional regulation during spermatogenesis.

Chemiluminescence receptor assay for measuring vitamin B12 in serum evaluated.

We evaluated a chemiluminescence receptor assay for vitamin B12 in serum (Magic Lite; Ciba Corning Diagnostics), in which an acridinium ester label is used with magnetic particle separation. Within- and between-batch precisions were generally acceptable, except at low analyte concentrations. The reference range determined from 104 elective preoperative patients was 120-610 pmol/L, compared with 150-590 pmol/L for our in-house radioligand-binding assay. Magic Lite discriminated between normal and abnormal results as effectively as the in-house method when local reference ranges were applied. Magic Lite demonstrated a negative bias at low analyte concentrations and was unable to detect any vitamin B12 in two B12-deficient patients. Assay accuracy--judged from analytical recovery and comparisons with the in-house method and two other radioassay kits (Quantaphase, Bio-Rad Labs., and Immophase, Ciba Corning Diagnostics)--was poor at low B12 concentrations when the manufacturer's recommended two-point calibration was used. This problem was partially corrected by using a full set of calibrators.

The v-rel oncogene: insights into the mechanism of transcriptional activation, repression, and transformation.

The v-rel oncogene product from the avian reticuloendotheliosis virus strain T corresponds to a member of the Rel-related family of enhancer-binding proteins that includes both the mammalian 50- and 65-kDa subunits of the NF-kappa B transcription factor complex. However, in contrast to NF-kappa B, v-Rel has been shown to function as a dominant-negative repressor of kappa B-dependent transcription in many mature cell types. We now demonstrate that a highly conserved motif within the Rel homology domain of v-Rel containing a consensus protein kinase A phosphorylation site is required for DNA binding, transcriptional repression, and cellular transformation mediated by this oncoprotein. However, replacement of the serine phosphate acceptor within the protein kinase A site with an alanine did not alter any of these functions of v-Rel, suggesting that phosphorylation at this site is not central to the regulation of this oncogene product. Rather, the inactive mutations appear to identify a functional domain within v-Rel required for these various biological activities. It is notable that these same mutations do not impair the ability of v-Rel to heterodimerize with the 50-kDa subunit of NF-kappa B, suggesting that v-Rel-mediated transcriptional repression likely involves direct nuclear blockade of the kappa B enhancer rather than indirect alterations in the composition of preformed cytoplasmic NF-kappa B complexes. Paradoxically, when introduced into undifferentiated F9 cells, v-Rel functions as a kappa B-specific transcriptional activator rather than as a dominant-negative repressor. These stimulatory effects of v-Rel require both the conserved protein kinase A phosphorylation site and additional unique C-terminal sequences not needed for v-Rel-mediated repression in mature cells. Retinoic acid-induced differentiation of these F9 cells restores the repressor function of v-Rel. These opposing biological actions of v-Rel occurring in cells at distinct stages of differentiation may have important implications for the mechanism of v-Rel-mediated transformation occurring in avian splenocytes.

The human placental lactogen genes: structure, function, evolution and transcriptional regulation.

hPL is a member of an evolutionarily related gene family including hGH and hPRL. Expression of hPL is limited to the placenta but its physiological actions are far reaching. hPL has a direct somatotropic effect on fetal tissues, it alters maternal carbohydrate and lipid metabolism to provide for fetal nutrient requirements, and aids in stimulation of mammary cell proliferation. Two hPL genes (hPL3 and hPL4) encoding identical proteins are responsible for the production of up to 1-3 g PL hormone/day. Recent studies have characterized the regulatory controls of hPL expression. At the post transcriptional level, RNA stability may contribute to variable levels of hPL3 vs. hPL4 production. In addition, non-tissue-specific protein-promoter interactions involving the Sp1 transcription factor are necessary for hPL transcription initiation. A transcriptional enhancer located 3' to the hPL3 gene is responsible for the placenta-specific expression of this gene, while an additional enhancer may be located 3' to the hPl4 gene. The hPL enhancer is bound by multiple proteins including at least one placental specific protein that interacts with a TEF-1 motif. Therefore, enhancer-protein interactions most likely play a large part in the high levels of placenta-specific hPL expression.

DNA sequences involved in the transcriptional activation of a human placental lactogen gene.

To identify regulatory elements in the promoter of a human placental lactogen gene (hPL3) that are important for its transcriptional activation, sequences 5' to the start of transcription were linked to the reporter gene chloramphenicol acetyltransferase (CAT) and transiently transfected into JEG-3 cells, a human placental choriocarcinoma cell line. In the presence of the hPL3 enhancer, deletion of the promoter sequence between -142 and -129 basepairs resulted in an 8-fold decrease in CAT activity. Similar results were seen with the SV40 enhancer and the hPL3 promoter in HepG2 liver cells. Nuclear proteins from HepG2, HeLa, and JEG-3 cells formed specific binding complexes with this region of the hPL3 promoter by a gel mobility shift assay, indicating that the DNA-binding protein was not tissue specific. The -142 to -129 basepair region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. An oligonucleotide containing Sp1-binding sites specifically competes for proteins binding the hPL3 promoter, and the methylation interference pattern is similar to that for an Sp1-binding site. This suggests that the hPL3 promoter binds Sp1- or an Sp1-like trans-acting factor, and this binding site is important for transcriptional regulation by the hPL3 enhancer in PL-producing cells.

NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to kappa B enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-kappa B-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.