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.

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.

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.

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.

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.

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.

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.

Nucleotide sequence of a transcriptional enhancer located 2.2 kb 3' of a human placental lactogen-encoding gene.

The nucleotide sequence of the human placental lactogen-encoding gene enhancer was determined. This tissue-specific enhancer is contained in a region flanked by a 284-bp Alu repeat.

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.

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.

Adult Fanconi syndrome and multiple myelomatosis.

A case is described of a 59-year-old woman presenting with multiple renal tubular defects. The aminoaciduria was of a generalized type. When investigated initially the only feature of myelomatosis was urinary Bence-Jones protein. Two years later radiologically classical multiple myelomatosis developed and rapidly progressed to the patient's death nine months later.

A rapid radioimmunoassay method for plasma luteinizing hormone.

1. A rapid double antibody radioummunoassay method for the determination of plasma luteinizing hormone (hLH) is described. 2. The method involves preincubation of the anti-LH serum with the precipitating second antibody and incubation of this mixture with unknows and standards at room temperature overnight. The incubation tubes are then centrifuged, supernatants decanted and the precipitates counted. 3. A total of two working days is required to complete the assay and report the results. 4. This method is more rapid than the conventional ones currently used for the estimation of LH in blood.

Studies of thyroxine binding to plasma proteins in health and disease.

Thyroxine binding protein characteristics were defined in 6 normal subjects, 4 with thyroxine binding globulin (TBG) deficiency, 3 with TBG increase, one with hyperthyroxinemic dysalbuminemia, 7 with severe non-thyroidal illness, and 3 with chronic renal failure. Free thyroxine was measured by Sephadex partition in plasma to which increasing thyroxine concentrations were added. Deconvolution of the resultant titration data was performed by computer modelling. Abnormalities of thyroxine binding capacities or of binding affinities occur in non-thyroidal illness, chronic renal failure, and sporadically. The patient with hyperthyroxinemic dysalbuminemia had increased thyroxine binding affinity to thyroxine binding prealbumin as well as to albumin. "Free-T4 assays" or estimates of Free-T4 by calculation from total thyroxine and measures of protein binding such as T3-uptake must be expected to be perturbed by these binding protein abnormalities unless such interferences are explicitly demonstrated to be absent.

Role of serum androgens and sex hormone binding globulin capacity in the evaluation of hirsutism in women.

Serum samples from 23 hirsute women and 23 non-hirsute women matched for age and deviation from ideal weight were analyzed for total testosterone (T), percent free testosterone (% FT) and sex hormone binding globulin capacity (SHBG). The % FT was assayed by 2 methods, using diluted serum in equilibrium dialysis (EQD) and using undiluted serum in centrifugal ultra-filtration (UF). SHBG was measured by a DEAE cellulose filter assay and T by radioimmunoassay. The two methods for determining % FT correlated well. There was considerable overlap between the hirsute and control groups for all of the measured parameters. The discrimination between the 2 groups provided by the indirect estimate of free testosterone obtained from the ratio of T to SHBG was at least as good as that provided by the free testosterone derived from ultrafiltration or dialysis.

Studies of intestinal permeability in inflammatory diseases using polyethylene glycol 400.

It has been proposed that increased bowel permeability might play a role in the pathogenesis of inflammatory disease. Intestinal permeation was investigated by measuring the 6-hour urinary excretion of polyethylene glycol (PEG) 400 in 40 adult volunteer controls and in patients with inflammatory disease. Of the patients, 15 had Crohn's disease; 7, ulcerative colitis; 2, celiac disease; and 7, rheumatoid arthritis. No significant difference in total urinary excretion over a 6-hour period was found between controls and patients with ulcerative colitis. Patients with Crohn's disease, celiac disease, or with rheumatoid arthritis were found to have significantly decreased urinary excretion of PEG 400. The results of this study indicate that there is no identifiable increase in intestinal permeation as measured by PEG 400 excretion during periods of active inflammatory disease.

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.