Regulation of Hormone Receptors and Gonadal Genes

促性腺激素调节的睾丸RNA解旋酶（GRTH/DDX25）是我们实验室发现的DEAD-box RNA解旋酶家族的睾丸特异性成员，它对精子发生的完成至关重要（Dufau & Kavarthapu 2019, Front Endocrinol）。它存在于间质细胞、减数分裂（粗线精母细胞）和单倍体生殖细胞（圆形和细长的精母细胞）中。缺乏GRTH的雄性不育小鼠由于圆形精子无法伸长而导致无精子症而不育。我们证明了它参与特定mrna的核输出/运输，相关mrna的染色质体（CB）储存/加工的结构完整性，以及它们与主动翻译的多核糖体的转运/关联，在那里它可能调节基因的翻译起始。GRTH是唯一受激素调节的家族成员。在间质细胞中，LH/cAMP通过雄激素(A)/A受体（AR）（自分泌）的直接作用刺激GRTH转录，而在生殖细胞中，则通过支持细胞中的AR以旁分泌方式刺激GRTH转录。GRTH基因的上游调控生殖细胞的表达，下游调控间质细胞（LC）的表达。通过这些区域，A/AR在LC中直接（内源性）调控GRTH基因，并间接调控生殖细胞。在圆形精子（RS）和精母细胞（SP）中发现了生殖细胞核因子（GCNF）的功能结合位点，并通过A/AR在GRTH基因的远端区域进行调控，在RS中选择性地起作用。目前的研究表明，A通过GCNF细胞特异性调控生殖细胞（RS）中GRTH的表达。此外，GRTH对GCNF施加负自分泌调节，通过GCNF作为A调节的反式因子，将A作用与生殖细胞联系起来，控制GRTH的转录/表达（Kavarthapu & Dufau, 2015 Mol Endocrinol）。这些发现提供了雄激素作用与精子发生过程中必不可少的两个相关生殖细胞基因（GRTH和GCNF）之间的联系，并建立了它们之间的调节相互关系。我们的早期研究表明，在5.8%的精子完全丧失患者中发现，GRTH的aa 242位点的R到H错义突变导致61 kDa的磷酸化形式（pGRTH）丢失，而52 kDa的非磷酸化形式保留（Tsai-Morris et al., 2007 Human Reprod）。这一发现为阐明pGRTH在精子发生中的作用提供了途径。我们产生了一种人源化突变的GRTH敲入（KI）小鼠。突变小鼠不育，睾丸大小减小，缺乏精子，在圆形精子（RS）的第8步停止，pGRTH物种完全丧失，细胞核52kda形式保留（Kavarthapu等）。前面。Cell Dev. Biol. 2019)。该模型允许研究细胞质pGRTH的生物学/生化功能。在KI小鼠中，核输出、转运和GRTH功能得以保留。mRNA输出，miRNA调节)，而细胞质功能，包括信息穿梭，在CB中的储存和翻译事件都需要pGRTH。观察到RS中CB大小明显减小，CB中缺乏pGRTH。SP和RS均存在生殖细胞凋亡。与KO相比，KI没有显示miRNA生物合成的变化，除了pGRTH作为微处理器复合物的转录调节因子（Drosha， DCGR）影响primirna的形成，表明非磷酸化GRTH参与了这些过程。在KI小鼠中，染色质重塑和相关蛋白包括TP2、PRM2和TSSK6的缺失。它们的mRNA和半衰期的显著减少表明它们与细胞质中pGRTH的关联保护了这些mRNA免受降解。此外，我们的工作表明，pGRTH以3'UTR依赖的方式刺激TP2的翻译。在最近的研究中，我们阐明了在苏氨酸（T239）上的GRTH磷酸化位点在结构上与在患者中发现的突变位点（R242H）相邻。基于DDX9晶体结构的RecA结构域1对磷酸位点进行分子建模，指出了形成GRTH/PKA界面的氨基酸、溶剂可及性和氢键。这些包括核心残基T239和R242氨基酸E165， K240和D237。这些残基的相关性通过突变导致T239处pGRTH的减少或取消的氨基酸破坏得到了证明（Raju等）。科学代表，2019)。pGRTH形式是细胞质种，对于圆形精子的第8步之后的精子发生过程和活精子的形成至关重要。值得注意的是，突变对GRTH磷酸化的有害影响不是由PKA α -催化结合亲和力的变化引起的，而是由影响PKA催化效率的相应结构变化引起的。基于磷酸形式的消除研究为药物设计、虚拟筛选和通量筛选提供了基础，以发现一种可逆性化学抑制剂用于男性避孕药。在这一年中，我们确定适合GRTH/DDX5/蛋白激酶A间期浅袋的环肽是阻断GRTH磷酸化的首选化合物，可用于开发口服男性避孕药(3)。在这方面，这些环状肽（PEP0， PEP1和PEP2）已被设计和合成为有前途的治疗剂。FITC显示，处理4h后，PEP1和PEP2在COS-1细胞和精管中有效内化。在表达GRTH的COS-1稳定细胞系中，观察到对GRTH磷酸化的剂量依赖性抑制作用，在处理8-16h后观察到pGRTH蛋白显著降低。与对照肽相比，CETSA显示化合物结合导致可溶性非pgrth蛋白的热稳定。将培养的精管暴露于这些化合物中导致pGRTH蛋白物种的显著抑制。不含FITC的化合物得到了类似的结果。综上所述，环肽有效内化和靶向降低pGRTH的表达为开发有效的非激素男性避孕药化合物提供了一个有希望的角度。此外，利用RNA-Seq技术对小鼠生殖细胞进行转录组分析，进一步揭示了磷酸化- grth与组蛋白泛素化和乙酰化之间的联系，这对精子发生过程中染色质压实和精子发育至关重要（Kavarthapu等）。Hum Mol Gen, 2020)。此外，启动了pGRTH在圆形精子中普遍存在的生殖细胞细胞器CB中信息存储中的作用的研究，并发现pGRTH是与精子发生过程相关的存储的关键。我们在GRTH KI小鼠的CB中观察到pGRTH的缺失（插入R242H人类突变，该突变在T239和精子发生时消除了GRTH的磷酸化）。对CB分离的mRNA进行RNASeq分析，发现947个基因丰度降低，474个基因丰度增加（1,2）。在KI小鼠的CB中，与精子发育、分化和染色质重塑相关的转录本减少，而与RNA转运、调控、监视以及转录和翻译调控相关的编码因子增加，这些都得到了qPCR的验证。几个起始因子（eIF4e、4ebp2、eIF3l和eIF3m）的转录本以及与60S亚基相关的mrna （RpL101/RPlp0）在CB中增加和积累，这些mrna不能从CB转运到多核糖体进行翻译；而是由于pGRTH的缺失而储存在KI小鼠的CB中。我们的研究证明了phospho-GRTH在维持CB结构中的重要性，以及它在精子发生过程中生殖细胞特异性mrna的储存和稳定性中的作用。

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.