To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive

The ligand binding domain (LBD) and the amino-terminal, transactivation domain (TAD) of the androgen receptor (AR) were separately linked to the GAL4 DNA binding domain (DBD) and to the GAL4(TAD). Resulting constructs were tested in the yeast two-hybrid system for protein-protein interactions. In the presence of androgen [methyltrienolone (R1881) or dihydrotestosterone (DHT)] a transcriptionally active complex was formed, reflecting an association between the AR(LBD) and the AR(TAD). No interactions were found in the presence of low-affinity ligands like estradiol (E2), promegestone (R5020), or progesterone (Pg). Use of the Thr-868-Ala mutated AR(LBD) in the assay resulted not only in a clear AR TAD-LBD interaction in the presence of R1881 and DHT but also in the presence of E2, Pg, and R5020, corresponding to the alteration in ligand specificity induced by the mutation. Coexpression of the fusion protein Gal4(DBD)AR(LBD) and the separate AR(TAD) also gave rise to the formation of a transcriptionally active complex. No interactions were found between two AR LBDs at the low-expression level of the two components. However, LBD-LBD interaction was detectable by application of a high-expression vector for GAL4(TAD)AR(LBD), albeit at high ligand concentrations. To substantiate the observation of the AR LBD-TAD interaction, CHO cells were cotransfected with expression plasmids for a truncated AR, which lacks the TAD [AR(DBD)(LBD)], and for the separate AR(TAD). This resulted in stimulation of a MMTV-LUC reporter gene in the presence of R1881 but not in the absence of hormone. This finding indicates that, like in the yeast system, in mammalian cells, TAD-LBD interactions are of importance for AR activation. In the mammalian system, a maximal AR TAD-LBD interaction was obtained at approximately 10-fold higher ligand concentrations than required for full-length AR activation. In the presence of low-affinity ligands, the AR TAD-LBD interaction as measured by transcriptional activation was considerably weaker than the activity of the full-length AR. From the present results a concept of hormone-dependent AR activation is proposed, which requires a functional, direct or indirect intramolecular interaction between the TAD and the LBD.

Limited proteolysis of in vitro produced human androgen receptor was used to probe the different conformations of the receptor after binding of androgens and several antiandrogens. The results provide evidence for five different conformations of the receptor, as detected by the formation of proteolysis resisting fragments: 1) an initial conformation of the unoccupied receptor not resisting proteolytic attack; and receptor conformations characterized by 2) a 35-kDa proteolysis resisting fragment spanning the ligand binding domain and part of the hinge region, obtained with most antagonists, and in an initial step after agonist binding; 3) a 29-kDa proteolysis resisting fragment spanning the ligand binding domain, obtained in the presence of agonists after an activation process; 4 and 5) 30-and 25-kDa fragments, derived from 2 and 3, but missing part of the C terminus, obtained with RU486 (RU486 has antiandrogenic properties, besides its effects as an antiprogestagen/antiglucocorticoid). Concomitantly with the change from 2 to 3 (and of 4 to 5 for RU486), dissociation of the 8 S complex of receptor with associated proteins occurred. With a mutant receptor (LNCaP cell mutation in C-terminal region), some antagonists activated transcription analogous to agonists, and induced the activated receptor conformation 3. A mutant lacking the C-terminal 12 amino acids bound RU486 but not androgens, and formed with RU486 conformation 5. These data imply that, after the initial rapid binding of ligand, androgens induce a conformational change of the receptor, a process that also involves release of associated proteins. RU486 induces an inappropriate conformation of the C-terminal end, similar as found for its effect on the progesterone receptor. In contrast, the other antiandrogens act at a different step in the mechanism of action: they do not induce an abnormal conformation, but act earlier and prevent a conformation change by stabilizing a complex with associated proteins.The biological effects of androgens and other steroid hormones are mediated through intracellular receptors, belonging to the steroid and thyroid hormone receptor superfamily (1). Upon activation by the hormone, steroid receptors interact with specific DNA sequences, located in the flanking regions of target genes, resulting in modulation of the expression of these genes (2-4). Steroid hormone receptor antagonists inhibit the biological effects of agonists, and are frequently used in the treatment of hormone-based dysfunctions in human. Furthermore, the synthetic antagonists are important tools to define the molecular mechanism of transactivation by steroid hormones (5, 6).Agonists and antagonists may change the spatial structure of the receptor in distinct ways, as was first indicated by gel retardation experiments: antagonist-and agonist-receptor-DNA complexes showed slightly different mobilities (7-10). Recently, limited proteolysis of progesterone, estrogen, and glucocorticoid receptors pinpointed the distinction in conformation between hormone-and ant...

The Slc26 family is a conserved family of anion transporters. In the human, their physiological relevance was highlighted with the discovery of pathogenic mutations in several Slc26 transporters that lead to distinctive clinical disorders (Pendred syndrome, deafness, diastrophic dysplasia, congenital chloride diarrhoea) that are related to the specific distribution of these genes. We previously identified TAT1 as a new family member (Slc26A8), very specifically expressed in male germ cells in both the human and the mouse. To investigate Tat1 function in the male germline, we generated mice with a targeted disruption of the Tat1 gene. Heterozygous and homozygous Tat1 mutant mice were indistinguishable from wild-type littermates concerning survival rate, general appearance and gross behaviour; however, Tat1 null males were sterile due to complete lack of sperm motility and reduced sperm fertilization potential. Ultra-structural analysis revealed defects in flagellar differentiation leading to an abnormal annulus, disorganization of the midpiece-principal piece junction, hairpin bending of the sperm tail with disruption of the axial structures, and abnormal mitochondrial sheath assembly. While ATP levels were normal, ATP consumption was strongly reduced in Tat1 null spermatozoa. Interestingly, Tat1 is located at the annulus, a septin-based circular structure connecting the midpiece to the principal piece. Altogether, our results indicate that Tat1 is a critical component of the sperm annulus that is essential for proper sperm tail differentiation and motility.

Androgen receptor mRNA was translated in vitro, and androgen-and antiandrogen-bound receptor complexes were studied using limited proteotytic digestion by trypsin. Partial proteolysis of androgen-Lund receptor proteiu resulted in a 29-kDa proteolysis-resisting fragment, whereas antiandrogen binding stabilised a 3%kDa fragment. Both fragments contain the entire ligand binding domain, and the 35kDa fragment extended into the hinge region of the receptor. Several antiandrogens show agonistic properties for a mutated androgen receptor (LNCaP cell variant); trypsin digestion of antiandrogen-bound mutated receptor also resulted in a 29-kDa fragment. Our results point to an important difference between antiandrogens and antagonists of other steroid hormone receptors. Antiandrogens result in protection of both the hinge region and C-terminus of the androgen receptor against proteolytic attack, whereas other studies showed that ~tiestrogens and antiprogestagens expose the C-terminal end of the ligand binding domain of their respective receptors to protease. Differences in conformation of the hinge region distinguish androgen-bound from antiandrogen-bound receptor complexes, which represents au important feature of antiandrogen action.