Regulation of Hormone Receptors and Gonadal Genes

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25) is a testis-specific member of the DEAD-box family of RNA helicases discovered in our laboratory, which is essential for the completion of spermatogenesis (Dufau & Kavarthapu 2019, Front Endocrinol). It is present in Leydig cells and meiotic (pachytene spermatocytes) and haploid germ cells (round and elongated spermatids). Male null mice lacking GRTH are sterile due to azoospermia resulting from failure of round spermatids to elongate. We demonstrated its participation on the nuclear export/transport of specific mRNAs, the structural integrity of the Chromatoid Body (CB) storage/ processing of relevant mRNAs and their transit/association to the actively translating polyribosomes where it may regulate translational initiation of genes. GRTH is the only family member regulated by hormones. GRTH transcription is stimulated in Leydig cells by LH/cAMP through direct actions of androgen (A)/A receptor (AR) (autocrine), and in germ cells in paracrine fashion through AR in Sertoli cells. The upstream region of the GRTH gene directs its expression in germ cells and downstream in the Leydig cell (LC). Through these regions A/AR exerts its direct (endogenous) regulation of the GRTH gene in LC, and indirectly in germ cells. Functional binding sites for Germ Cell Nuclear Factor (GCNF) present in round spermatids (RS) and spermatocytes (SP) and its regulation by A/AR were identified in the distal region-of the GRTH gene, operative selectively in RS. Current knowledge indicates actions of A on GCNF cell specific regulation of GRTH expression in germ cells (RS). Also, GRTH exerts negative autocrine regulation of GCNF linking A actions to germ cells through GCNF as an A regulated trans-factor that controls transcription/expression of GRTH (Kavarthapu & Dufau, 2015 Mol Endocrinol). These findings provide a connection of androgen action to two relevant germ cell genes (GRTH and GCNF) essential for the progress of spermatogenesis and established their regulatory interrelationship. Our early studies revealed that missense mutation of R to H at aa 242 of GRTH found in 5.8% of patients with complete loss of sperm causes loss of the 61 kDa phospho-species (pGRTH) with preservation of the 52 kDa non-phospho form (Tsai-Morris et al., 2007 Human Reprod). This finding provided an avenue to elucidate the function of pGRTH in spermatogenesis. We generated a humanized mutant GRTH knock-in (KI) mice. Mutant mice are sterile with reduction on testicular size, lack sperm with arrest at step 8 of round spermatids (RS) and complete loss of the pGRTH species with preservation of the nuclear 52 kDa form (Kavarthapu et.al. Front. Cell Dev. Biol. 2019). This model permits to study the biological/biochemical functions of the cytoplasmic pGRTH. In KI mice the nuclear export transport and functions of GRTH are preserved (ie. mRNA export, miRNA regulation) while the cytoplasmic functions including shuttling of messages, storage in the CB and translational events all requiring pGRTH are absent. Marked reduction of the CB size in RS and lack pGRTH in the CBs are observed. Germ cell apoptosis was present in SP and RS. In contrast to KO, KI showed no changes in miRNA biosynthesis excluding participation of pGRTH as transcriptional regulator of the microprocessor complex (Drosha, DCGR) affecting primiRNAs formation and indicative of the participation of non-phospho GRTH in these processes. In KI mice there is loss of chromatin remodeling and related proteins including, TP2, PRM2 and TSSK6. Significant decreases of their mRNA and half-lives indicate that their association with pGRTH in the cytoplasm protect these mRNAs from degradation. Also, our work showed that pGRTH stimulates TP2 translation in a 3'UTR dependent manner. In recent studies we elucidated the GRTH phospho-site at a threonine (T239) structurally adjacent to the mutant site found in patients (R242H). Molecular modelling of the phospho-site based on the RecA domain 1 of the DDX9 crystal structure, pointed to the amino acids that formed the GRTH/PKA interface, solvent accessibility and H-bonding. These include in addition of the core residues T239 and R242 amino acids E165, K240, and D237. The relevance of these residues were demonstrated by disruption of amino acids by mutations which caused reduction or abolition of the pGRTH at T239 (Raju et.al. Sci Rep, 2019). The pGRTH form is the cytoplasmic species which is essential for the progress of spermatogenesis beyond step 8 of round spermatids and for viable sperm formation. It is important to note that the deleterious effects on GRTH phosphorylation caused by the mutations did not result from changes of PKA alpha-catalytic binding affinity but rather to consequential structural changes that can affect PKA catalytic efficiency. Studies based on the abolition of the phospho-form provide the basis for drug design, virtual and throughput screening for discovery of a reversible chemical inhibitor for use as male contraceptive. During this year we determined that cyclic peptides that fit the shallow pocket of the GRTH/DDX5/protein kinase A interphase are preferred compounds to block GRTH phosphorylation and amenable for use in the development of an oral male contraceptive (3). In this regard these cyclic peptides (PEP0, PEP1 and PEP2) have been design and synthesized as promising therapeutic agents. PEP1 and PEP2 revealed by FITC, showed effective internalization in COS-1 cells and seminiferous tubules after 4h treatment. A dose-dependent inhibitory effect on GRTH phosphorylation was observed in COS-1 stably cell line expressing GRTH with significant reduction in pGRTH protein observed after 8-16h treatments. CETSA showed compound binding resulting in thermal stabilization of the soluble non-pGRTH protein when compared to control peptides. Exposure of culture of seminiferous tubules to these compounds resulted in significant inhibition of the pGRTH protein species. Similar results were obtained with the compound which lacks FITC. Taken together, effective internalization and targeted decrease in the expression of pGRTH by cyclic peptides provide a promising angle to develop effective compounds for use as non-hormonal male contraceptive. Also, work in transcriptome analysis of mice germ cells using RNA-Seq technique has provided further insights linking phospho-GRTH to histone ubiquitination and acetylation essential for chromatin compaction and spermatid development during spermiogenesis (Kavarthapu et.al. Hum Mol Gen, 2020). Also, initiated studies on the role of pGRTH in the storage of messages in the CB an organelle present in germ cells prevalent in round-spermatids and found to be a key for the storage of relevance for the progress of spermatogenesis. We observed absence of pGRTH in CB of GRTH KI mice (with insertion of R242H human mutation that abolish GRTH phosphorylation at T239 and spermatogenesis). RNASeq analysis of mRNA isolated from CB revealed 947 decreased genes and 474 genes increased in abundance (1,2). Transcripts related to spermatid development, differentiation and chromatin remodeling were reduced, in contrast those encoding factors involved in RNA transport, regulation, surveillance and transcriptional and translational regulation were increased in the CB of KI mice and these were validated qPCR. Transcripts of several initiation factors (eIF4e, 4ebp2, eIF3l and eIF3m) together with mRNAs related to the 60S subunit (RpL101/RPlp0) were increased and accumulated in CB as mRNAs which could not get transported from the CB to polyribosomes for translation; instead remain stored in the CB in KI mice due to loss of pGRTH. Our studies demonstrated the importance of phospho-GRTH in the maintenance of the structure of CB and its role in the storage and stability of germ cell-specific mRNAs during spermiogenesis.