Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus.

A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein-a component of the flagella central apparatus-was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function.

The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking.

Stimulation of steroid-producing cells of the gonads and adrenals with tropic hormone results in a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system. This process of cholesterol translocation is blocked by inhibitors of protein synthesis, suggesting that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory (StAR) protein appears to be the most likely candidate for the "labile" protein, based on the following observations: 1) Expression of StAR in COS-1 cells engineered to contain the cholesterol side-chain cleavage system substantially augments pregnenolone formation; 2) StAR protein is expressed almost exclusively in steroid-producing cells, except the trophoblast of the human placenta, and its presence is correlated with steroid hormone production; 3) StAR mRNA increases in response to cAMP; 4) StAR is a target for serine phosphorylation mediated by protein kinase A, a process that is essential for maximizing StAR activity; and 5) lack of functional StAR causes the autosomal recessive disease, congenital lipoid adrenal hyperplasia, characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. Studies on the mechanism of action of StAR revealed that import into mitochondria is not essential to its steroidogenesis-enhancing activity and more likely represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations established that the C-terminus of the StAR protein contains the functionally important domains. The demonstration of steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence confirmed that StAR import is not essential for its steroidogenic activity and suggested that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. Evidence that StAR functions as a cholesterol transfer protein raises the possibility that StAR acts directly on lipids of the outer mitochondrial membrane, probably stimulating cholesterol desorption from the sterol-rich outer membrane and its movement to the relatively sterol-poor inner membrane.

cDNA cloning and characterization of a human sperm antigen (SPAG6) with homology to the product of the Chlamydomonas PF16 locus.

Serum from an infertile male with high-titer anti-sperm antibodies was used to identify a novel human sperm antigen by screening of a testis expression library. The clone, initially designated Repro-SA-1 (HUGO-approved symbol SPAG6), was found to encode a sequence highly enriched in testis. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in prostate, ovary, spleen, thymus, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, liver, muscle, kidney, and pancreas. The gene was mapped to chromosome 10p11.2-p12. Antibodies raised against SPAG6 sequences localized the protein to the tails of permeabilized human sperm. Both the Chlamydomonas protein and SPAG6 contain eight contiguous armadillo repeats, which place them in a family of proteins known to mediate protein-protein interactions. The cloning of the human homologue of the Chlamydomonas PF16 locus provides a new avenue to explore the role of the axoneme central pair in human sperm function.

Localization of the steroidogenic acute regulatory protein in human tissues.

The rate-limiting step in steroid hormone production in the adrenal cortex and gonads, the translocation of cholesterol from the outer to the inner mitochondrial membranes, is mediated by the steroidogenic acute regulatory protein (StAR). Heretofore, the localization of StAR in human adult and fetal tissues has not been defined. To this end, expression of StAR was detected in formalin-fixed, paraffin-embedded specimens using a polyclonal antiserum raised against recombinant human StAR. Primordial follicles of adult ovaries did not contain StAR, whereas antral follicles stained intensely in the thecal layer, with occasional staining of granulosa cells. Corpora lutea were intensely stained, but with a patchy distribution. Corpora albicantia did not stain. A luteoma of pregnancy stained with patches of moderate intensity. Ovaries with hyperthecosis contained areas of intense thecal staining. An ovarian Leydig cell tumor stained intensely, whereas granulosa cell tumors were negative. Ovarian adenocarcinomas, borderline tumors, teratomas, cystadenomas, and a Brenner tumor displayed no specific StAR immunostaining. Testicular Leydig cells stained moderately to intensely, as did a testicular Leydig cell tumor. Sertoli cells stained weakly in some specimens. Seminomas and testicular germ cell tumors were negative. There was minimal to moderate staining in the adrenal glomerulosa and faciculata and minimal staining in the reticularis, while the medulla was negative. Adrenal cortical adenomas, hyperplasias, and carcinomas all contained areas of StAR staining. The renal distal tubules stained with moderate to marked intensity. Renal carcinomas had occasional modest staining. No immunostaining was found in the placenta. Fetal ovaries contained sporadic stromal cells displaying intense StAR staining, particularly in the hilar region. Oocytes from a 32-week fetal ovary showed moderate to intense staining. Fetal testes displayed intense Leydig cell staining. The neocortex of the fetal adrenal glands displayed only minimal StAR staining, whereas moderate to intense staining was found in the fetal zone. The fetal kidneys had moderate StAR staining of the distal convoluted tubules. We conclude that StAR is localized to normal and neoplastic cells in the gonads and adrenal cortex, which produce large amounts of pregnenolone. StAR protein was not detected in the placenta, documenting that placental progestin synthesis occurs through StAR-independent mechanisms. The presence of StAR in cells that do not express cholesterol side-chain cleavage enzyme cytochrome P450, including renal distal tubules, Sertoli cells, and fetal oocytes, suggests that StAR has roles in metabolic processes in addition to stimulating pregnenolone synthesis.

Cloning and expression of primate Daxx cDNAs and mapping of the human gene to chromosome 6p21.3 in the MHC region.

Murine Daxx, a protein that binds to Fas and enhances Fas-mediated apoptosis through a signal transduction pathway involving the Jun amino-terminal kinase, was recently described. Here we report the cloning of human and monkey Daxx cDNAs, the widespread expression of Daxx mRNA in human tissues, and the mapping of the human Daxx gene to 6p21.3 in the major histocompatibility complex (MHC) region. The location of the Daxx gene, which is implicated in the pathway for deletion of autoreactive lymphocytes, in the MHC region may shed light on the genetic basis of autoimmune diseases.

Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5'-flanking region are required for maximal promoter activity and cyclic AMP responsiveness.

A proximal element from the human StAR gene promoter, containing the sequence (-105)TATCCTTGAC(-95), was shown to confer responsiveness to 8-Br-cAMP in the presence of steroidogenic factor 1 (SF-1) when placed behind a minimal thymidine kinase promoter or an SV40 promoter and transfected into BeWo cells which normally lack StAR and SF-1. This element was also transactivated by SF-1 in a yeast one-hybrid system. The -105 to -95 sequence was protected by SF-1 in footprint analysis and a double-stranded oligonucleotide containing the element bound SF-1 specifically in electrophoretic mobility shift assays. Another SF-1-binding sequence 35 bp upstream of the transcription start site ((-42)CAGCCTTC(-35)) was identified in the DNase 1 footprint analysis and, when mutated, markedly reduced SF-1-dependent and 8-Br-cAMP-stimulated StAR promoter activity in BeWo cells. The two proximal SF-1 response elements were shown to be critical for StAR promoter function in human granulosalutein cells, which express SF-1 and respond to cAMP with increased transcription of the StAR gene. Mutation of either element substantially reduced basal and forskolin-stimulated promoter activity, although mutation of the -105 to -95 element had more pronounced effects. Mutation of a third, more distal, SF-1-binding site at -926 to -918 also reduced basal but not forskolin-stimulated promoter activity in the granulosa-lutein cells. These findings demonstrate that multiple SF-1 response elements are required for maximal StAR promoter activity and regulation by cAMP.

Structural and functional changes in mitochondria associated with trophoblast differentiation: methods to isolate enriched preparations of syncytiotrophoblast mitochondria.

The syncytiotrophoblast of the human placenta is derived from the fusion of cytotrophoblast cells. The syncytiotrophoblast and cytotrophoblast cells have different functional properties. Here, we document that syncytiotrophoblast mitochondria have a distinct phenotype that differs from that of the mitochondria ofcytotrophoblast cells. Syncytiotrophoblast mitochondria are small and have a dense matrix and vesicular cristae. They contain the machinery to convert cholesterol into pregnenolone. The larger cytotrophoblast mitochondria have lamellar cristae and do not have detectable P450scc. These observations imply that trophoblast mitochondria undergo morphological and functional changes as cytotrophoblast cells differentiate into syncytiotrophoblast. Structural changes in mitochondria and accumulation of P450scc were induced in a clonal line of BeWo choriocarcinoma cells by treatment with 8-Br-cAMP, which promotes formation of syncytial structures in these cultures. We conclude that the terminal differentiation program of trophoblast cells includes major changes in the architecture and function of mitochondria. Based on the unique features of syncytiotrophoblast mitochondria, we developed a method to prepare highly enriched syncytiotrophoblast mitochondria from term placenta using differential centrifugation and density gradient centrifugation.

Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene: a central role for steroidogenic factor 1.

Steroidogenic acute regulatory protein (StAR) plays a critical role in regulating the rate-limiting step in steroid hormone synthesis, cholesterol side-chain cleavage. StAR gene expression is transcriptionally controlled in the gonads by gonadotropic hormones via a cAMP second message. We have begun to analyze factors responsible for the transcriptional activation of the StAR gene. The human StAR gene promoter has at least two cis elements that govern basal and cAMP-regulated gene expression. One of these elements (the distal element) is a consensus binding sequence for the orphan nuclear receptor transcription factor, steroidogenic factor 1 (SF-1); the other (the proximal element) is a related motif. The human StAR promoter is not active in BeWo choriocarcinoma cells, but is functional and cAMP-responsive in murine Y1 adrenal cortical tumor cells. Cotransfection of a plasmid expressing SF-1 allows a StAR promoter construct to function in BeWo cells. Other orphan nuclear transcription factors do not support StAR promoter function in BeWo cell hosts. Deletion or mutation of the distal and proximal cis elements individually substantially reduces SF-1-supported StAR promoter activity. The distal site binds SF-1 with high affinity, whereas the proximal site binds SF-1 with lower affinities. These findings demonstrate a requirement for SF-1 for human StAR gene expression.

Expression of steroidogenic acute regulatory protein (StAR) in the human ovary.

Expression of steroidogenic acute regulatory protein (StAR) messenger ribonucleic acid (mRNA) in human ovary and cultured proliferating granulosa-lutein cells, theca interna cells, and luteinized granulosa cells was examined. The StAR transcripts were restricted in situ to theca of preovulatory follicles and luteinized granulosa and thecal cells of the corpus luteum. The cyclic nucleotide analog, 8-bromo-cAMP (8-Br-cAMP), increased StAR mRNA in all cell types studied by a process requiring on-going RNA and protein synthesis. Phorbol myristate acetate prevented the stimulatory effects of 8-Br-cAMP. In proliferating granulosa-lutein cells, 8-Br-cAMP increased StAR gene transcription and did not significantly affect StAR mRNA stability. Forskolin treatment was also found to increase the expression of a human StAR proximal promoter-luciferase fusion gene transfected into the proliferating granulosa-lutein cells. We conclude that 1) the StAR gene is expressed in the most steroidogenic compartments of the human ovary; 2) induction of StAR gene transcription by cAMP, produced in response to the LH surge, accounts for the appearance of StAR transcripts in luteinized granulosa cells; and 3) the effects of cAMP are antagonized by activators of protein kinase C.

Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene.

Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.

Placental steroid hormone synthesis: unique features and unanswered questions.

Despite the amazing diversity of placental architecture across species, a number of common elements can be found, including the ability of all placentae to synthesize and metabolize steroid hormones; the assignment of steroidogenic activities to specific trophoblast phenotypes; the use of novel mechanisms to control expression of steroidogenic enzyme genes, which differ from those employed in the adrenal cortex and gonads; and interactions with the maternal and fetal compartments encompassing supply of steroid hormone precursors as well as regulatory influences of maternal ovarian and pituitary hormones and fetal adrenal cortical steroids.