Recombinant Theromyzon tessulatum Egg-laying-like hormone

What is Theromyzon tessulatum Egg-Laying-Like Hormone and how was it first characterized?

Theromyzon tessulatum Egg-Laying-Like Hormone (L-ELH) represents the first biochemically characterized egg-laying hormone in invertebrates other than mollusks. This 38-amino acid peptide (GSGVSNGGTEMIQLSHIRERQRYWAQDNLRRRFLEK-amide) was isolated from the central nervous system (CNS) of the rhynchobdellid leech T. tessulatum through an extensive purification process involving high-performance gel permeation chromatography (HPGPC) and reverse-phase HPLC . The sequence was established through a combination of automated Edman degradation, arginyl-endopeptidase digestion, electrospray mass spectrometry measurement, and carboxypeptidase A treatment . This discovery significantly expanded our understanding of reproductive hormone conservation across invertebrate phyla.

How does L-ELH relate structurally to other known egg-laying hormones?

L-ELH shows variable sequence identity with molluscan egg-laying hormones: 27.8% with Aplysia parvula ELH, 37.2% with Lymnaea stagnalis ELH, and 47.2% with Aplysia californica ELH . Despite these relatively modest sequence similarities, secondary structure prediction analyses revealed a highly conserved segment (positions 29-34) in a strong helicoidal bend that appears critical for receptor recognition and/or activation . This structural conservation suggests functional importance maintained through evolutionary divergence, potentially indicating a fundamental mechanism of action across diverse invertebrate species.

What is the neuroanatomical distribution of L-ELH in Theromyzon tessulatum?

Immunohistochemical studies using antisera specifically directed against Lymnaea stagnalis caudo-dorsal cells egg-laying hormone (CDCH) detected approximately 45 immunoreactive cells in the T. tessulatum brain . Notably, this number fluctuates according to the animal's life cycle stage, reaching maximum cell counts just before egg-laying and decreasing to merely 2-3 cells afterward . Both CDCH and alpha-CDCP epitopes recognized by the respective antisera were localized within neurosecretory granules, confirming the peptide's neurohormonal role in reproductive physiology.

What are the recommended methods for recombinant expression of T. tessulatum L-ELH?

Recombinant expression of T. tessulatum L-ELH typically involves cloning the identified gene sequence into an appropriate expression vector system. Based on protocols used for similar neuropeptides, the most effective approach involves:

• PCR amplification of the L-ELH coding sequence using primers designed from the published sequence

• Insertion into a pET expression vector containing a His-tag for purification

• Transformation into E. coli BL21(DE3) cells for expression

• Induction with IPTG (0.5-1.0 mM) for 3-4 hours at 30°C

• Purification via nickel affinity chromatography followed by reverse-phase HPLC

For optimal bioactivity, post-translational modifications, particularly C-terminal amidation, must be considered, potentially requiring mammalian or insect cell expression systems that possess the necessary enzymatic machinery for these modifications.

How can researchers validate the biological activity of recombinant L-ELH preparations?

Validation of recombinant L-ELH biological activity requires assays that measure reproductive responses. Drawing from approaches used with molluscan ELH, recommended validation methods include:

• In vivo bioassays observing egg-laying behaviors in T. tessulatum following hormone administration

• Quantification of reproductive tissue responses (oocyte maturation, contractions)

• Receptor binding assays using labeled L-ELH and membrane preparations from reproductive tissues

• Calcium mobilization assays in cells expressing putative L-ELH receptors

• Electrophysiological recordings from neurons known to respond to native L-ELH

Similar to methodologies employed for T. pisana ELH bioassays, researchers should administer multiple concentrations (ranging from 10^-12 M to 10^-3 M) of synthetic or recombinant L-ELH while monitoring behavioral and physiological responses at regular intervals (10-minute intervals for the first hour, hourly for the next 5 hours, and then daily for up to 6 days) .

How does the renin-angiotensin system in T. tessulatum interact with the L-ELH pathway?

The presence of both an egg-laying-like hormone system and a renin-angiotensin system in T. tessulatum suggests potential regulatory interactions between reproductive and osmoregulatory processes. T. tessulatum possesses a 32 kDa aspartyl protease characterized as a renin-like enzyme that exhibits 26.5-35.5% sequence identity with mammalian renins . This enzyme hydrolyzes the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide, yielding angiotensin I with a specific activity of 115 μg AI/min/mg (KM 22 μM; Kcat, 2.7) .

The functional relationship between these pathways requires investigation through:

• Co-localization studies of L-ELH and renin-like enzyme expressing cells

• Analysis of L-ELH expression and activity following manipulation of renin-angiotensin components

• Examination of physiological responses when both systems are experimentally activated

• Investigation of shared second messenger pathways in target tissues

This interaction may represent an important regulatory mechanism coordinating reproductive activity with osmotic homeostasis during critical life cycle transitions.

What molecular mechanisms govern L-ELH gene expression throughout the T. tessulatum life cycle?

The dramatic fluctuation in L-ELH immunoreactive cells during the T. tessulatum life cycle suggests sophisticated transcriptional regulation . Based on studies of related neuropeptides, several regulatory mechanisms likely control L-ELH expression:

• Epigenetic modifications (histone acetylation, DNA methylation) of the L-ELH gene promoter

• Transcription factor networks activated by environmental cues (temperature, photoperiod)

• Feedback loops involving hormonal signals that shift during reproductive maturation

• Post-transcriptional regulation through microRNA targeting of L-ELH mRNA

Research approaches to elucidate these mechanisms should include:

• ChIP-seq analysis of histone modifications at the L-ELH locus during different life stages

• Promoter analysis with reporter constructs to identify key regulatory elements

• RNA-seq to identify co-regulated genes during reproductive cycling

• Experimental manipulation of candidate regulatory pathways using RNAi or CRISPR technologies

How do the conserved structural elements of L-ELH contribute to receptor binding and activation?

The identification of a conserved helicoidal bend segment (positions 29-34) in L-ELH suggests functional importance in receptor interaction . Advanced structural studies would illuminate structure-function relationships through:

• NMR or X-ray crystallography of L-ELH alone and in complex with putative receptors

• Molecular dynamics simulations to analyze conformational flexibility

• Alanine-scanning mutagenesis of the conserved segment to identify critical residues

• Photoaffinity cross-linking studies to map receptor contact points

The predicted secondary structure comparison between L-ELH and molluscan ELHs reveals conserved elements despite sequence divergence:

This structural conservation likely represents evolutionary pressure to maintain receptor activation functionality despite sequence divergence.

What does the presence of L-ELH in leeches tell us about the evolution of reproductive hormones across invertebrate phyla?

The characterization of L-ELH in T. tessulatum represents a significant evolutionary finding as it was the first biochemical characterization of an egg-laying hormone outside of mollusks . This discovery suggests that egg-laying hormone systems evolved earlier than previously thought or emerged independently in multiple lineages.

The moderate sequence identity between L-ELH and molluscan ELHs (27.8-47.2%) coupled with the conservation of functionally important structural elements indicates either:

• A common ancestral reproductive hormone predating the divergence of annelids and mollusks

• Convergent evolution driven by similar selective pressures for coordinating reproductive processes

• Horizontal gene transfer between phyla at some point in evolutionary history

Comparative analysis with other Lophotrochozoan reproductive hormones would help distinguish between these possibilities and refine our understanding of reproductive hormone evolution across invertebrate lineages.

How do the multiple hormone systems in T. tessulatum (L-ELH, angiotensin-like, renin-like) interact to coordinate physiological processes?

T. tessulatum possesses multiple peptide hormone systems, including L-ELH , an angiotensin I-like system , and a renin-like enzyme , suggesting sophisticated neuroendocrine integration. These systems likely coordinate reproductive, osmoregulatory, and metabolic processes through:

• Sequential activation during different life stages

• Cross-regulation at the transcriptional level

• Convergence of signaling pathways in target tissues

• Shared or antagonistic effects on physiological processes

The leech angiotensin I-like molecule shares 78.5% homology with the N-terminal part of human angiotensinogen , and its presence alongside a renin-like enzyme capable of angiotensinogen processing indicates remarkable conservation of this system across evolutionary time. Research integrating these pathways would provide insights into the neuroendocrine orchestration of complex physiological responses in invertebrates.

What are the primary challenges in detecting native L-ELH in tissue samples and how can they be overcome?

Detecting native L-ELH in leech tissues presents several technical challenges:

• Low endogenous concentration, particularly during non-reproductive phases when L-ELH-immunoreactive cell numbers decrease from 45 to 2-3

• Potential cross-reactivity with related neuropeptides

• Variable post-translational modifications affecting antibody recognition

• Limited availability of specific antibodies against leech neuropeptides

To overcome these challenges, researchers should consider:

For optimal detection sensitivity, samples should be collected immediately before the expected egg-laying period when L-ELH immunoreactive cell counts reach their maximum .

What considerations are important when designing experiments to test L-ELH receptor activation in heterologous systems?

When investigating L-ELH receptor activation in heterologous systems, researchers should address several critical factors:

• Receptor identification and cloning

  • Use transcriptomic data from T. tessulatum to identify putative G-protein coupled receptors similar to known peptide hormone receptors

  • Clone full-length receptor candidates using RACE-PCR techniques

• Expression system selection

  • Choose mammalian cell lines (CHO, HEK293) for proper membrane targeting

  • Consider Xenopus oocytes for electrophysiological measurements

• Signaling pathway determination

  • Examine multiple second messenger pathways (cAMP, Ca²⁺, IP₃) as the primary pathway is unknown

  • Employ FRET-based sensors for real-time monitoring of signaling events

• Ligand preparation

  • Ensure proper post-translational modifications, particularly C-terminal amidation

  • Test multiple concentrations (10⁻¹² to 10⁻⁶ M) to establish dose-response relationships

• Controls and validation

  • Include related peptides from other species to test specificity

  • Validate in native tissue with electrophysiological recordings or calcium imaging

What are the most promising applications of recombinant L-ELH in broader invertebrate reproductive research?

Recombinant L-ELH offers several valuable applications for advancing invertebrate reproductive research:

• Comparative endocrinology studies examining the conservation and divergence of reproductive hormone function across lophotrochozoan phyla

• Development of molecular tools for manipulating reproduction in aquaculture or pest management

• Structure-function analyses to identify the minimal peptide motif required for biological activity

• Investigation of receptor evolution and specificity across species boundaries

• Exploration of novel signaling pathways activated by L-ELH and related peptides

The conserved structural elements between L-ELH and molluscan ELHs provide a foundation for understanding fundamental principles of neuropeptide action that may apply broadly across invertebrate taxa, potentially informing reproductive biology studies in economically or ecologically important species.

How might genomic approaches enhance our understanding of L-ELH processing and regulation?

Modern genomic technologies offer powerful means to investigate L-ELH biology beyond traditional biochemical approaches:

• Whole genome sequencing of T. tessulatum would reveal:

  • The complete L-ELH gene structure including promoter elements

  • Related gene family members and potential paralogs

  • Syntenic relationships with other neuropeptide genes

• Single-cell RNA sequencing of the T. tessulatum CNS would:

  • Identify the complete transcriptional profile of L-ELH-producing cells

  • Reveal co-expressed neuropeptides and processing enzymes

  • Characterize receptors expressed in these cells, suggesting autocrine/paracrine regulation

• ATAC-seq and ChIP-seq analyses would:

  • Map regulatory regions controlling L-ELH expression

  • Identify transcription factors governing life-cycle dependent expression

  • Reveal epigenetic modifications associated with reproductive states

• CRISPR-Cas9 genome editing could:

  • Generate L-ELH knockout leeches to definitively establish its function

  • Create reporter lines for real-time monitoring of L-ELH expression

  • Produce modified receptors to study signaling mechanisms

These approaches would significantly advance our understanding of the molecular mechanisms underlying the dramatic fluctuations in L-ELH expression throughout the T. tessulatum life cycle .