Relevance of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) in the structural integrity of the chromatoid body during spermatogenesis

AbstractGonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)—a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis

Pathophysiological features of the pulsatile secretion of biologically active luteinizing hormone in man

The development of an in vitro bioassay of high specificity, sensitivity and precision for the measurement of low circulating concentrations of biologically active glycoprotein hormones has offered exciting new insights into the in vivo secretion and metabolic clearance of luteinizing hormone (LH) in various pathophysiological states. Moreover, the most recent combined application of the rat interstitial cell testosterone (RICT) bioassay and a novel multiple-parameter deonvolution model has allowed investigators to dissect plasma concentration profiles of bioactive LH into defined secretory bursts, which have numerically explicit amplitudes, locations in time, and durations, and are acted upon by detenninable subject- and study-specific endogenous metabolic clearance rates. Here, we have: (i) reviewed the ability of the endogenous GnRH pulse signal to regulate the in vivo secretion of biologically active LH molecules as assessed in the RICT and by deconvolution mechanics; (ii) demonstrated that low-dose exogenous GnRH pulses effectively mimic spontaneous bioactive LH pulsatility; (iii) investigated the role of endogenous androgen and estrogen in modulating bioactive gonadotropin secretion in men and women; and (iv) described significant alterations in endogenous LH bioactivity in puberty and healthy aging.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27746/1/0000138.pd

Pathophysiology of Male Hypogonadism Associated with Endogenous Hyperestrogenism — Evidence for Dual Defects in the Gonadal Axis

FEMINIZING tumors of the adrenal cortex are associated with symptoms that presumably reflect the combined effects of estrogen excess and androgen deficiency — gynecomastia, diminished libido, attenuated potency, and testicular and prostatic atrophy.1 2 3 4 5 Although such tumors are extremely rare, they provide a unique opportunity to appraise the nature of endogenous estrogen action on the gonadal axis in men. In principle, the pathophysiologic effects of estrogen hypersecretion could be expressed at the level of either the Leydig cell or the hypothalamic–pituitary axis (or both), with consequent suppression of androgen production. In the present studies, we investigated the endocrine consequences of reversible endogenous estrogen excess in a patient with a surgically resectable feminizing adrenal cortical tumor

Repression of the Luteinizing Hormone Receptor Gene Promoter by Cross Talk among EAR3/COUP-TFI, Sp1/Sp3, and TFIIB

Transcription of luteinizing hormone receptor (LHR) gene is activated by Sp1/Sp3 at two Sp1 sites and is repressed by nuclear orphan receptors EAR2 and EAR3 through a direct-repeat (DR) motif. To elucidate the mechanism of the orphan receptor-mediated gene repression, we explored the functional connection between the orphan receptors and Sp1/Sp3 complex, and its impact on the basal transcription machinery. The Sp1(I) site was identified as critical for the repression since its mutation reduced the inhibition by EAR2 and abolished the inhibition by EAR3. Cotransfection analyses in SL2 cells showed that both Sp1 and Sp3 were required for this process since EAR3 displayed a complete Sp1/Sp3-dependent inhibitory effect. Functional cooperation between Sp1 and DR domains was further supported by mutual recruitment of EAR3 and Sp1/Sp3 bound to their cognate sites. Deletion of EAR3 N-terminal and DNA-binding domains that reduced its interaction with Sp1 impaired its inhibitory effect on human LHR (hLHR) gene transcription. Furthermore, we demonstrate interaction of TFIIB with Sp1/Sp3 at the Sp1(I) site besides its association with EAR3 and the TATA-less core promoter region. Such interaction relied on Sp1 site-bound Sp1/Sp3 complex and adaptor protein(s) present in the JAR nuclear extracts. We further demonstrated that EAR3 specifically decreased association of TFIIB to the Sp1(I) site without interfering on its interaction with the hLHR core promoter. The C-terminal region of EAR3, which did not participate in its interaction with Sp1, was required for its inhibitory function and may affect the association of TFIIB with Sp1. Moreover, perturbation of the association of TFIIB with Sp1 by EAR3 was reflected in the reduced recruitment of RNA polymerase II to the promoter. Overexpression of TFIIB counteracted the inhibitory effect of EAR3 and activated hLHR gene transcription in an Sp1 site-dependent manner. These findings therefore indicate that TFIIB is a key component in the regulatory control of EAR3 and Sp1/Sp3 on the initiation complex. Such cross talk among EAR3, TFIIB, and Sp1/Sp3 reveals repression of hLHR gene transcription by nuclear orphan receptors is achieved via perturbation of communication between Sp1/Sp3 at the Sp1-1 site and the basal transcription initiator complex