GH Ovine, Placental

What is the functional relationship between ovine growth hormone and placental lactogen in fetal development?

While oGH regulates growth and metabolism primarily in the postnatal period, oPL appears to function as the predominant growth-promoting hormone during fetal development. In fetal ovine tissues, oPL demonstrates significantly greater metabolic effects than oGH, particularly in stimulating glycogen synthesis and amino acid transport . Experimental evidence indicates that oPL stimulates dose-dependent increases in glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%) in cultured ovine fetal hepatocytes .

Comparative studies show that oPL functions as a "fetal growth hormone," compensating for the relative inactivity of oGH during fetal development . This functional relationship appears to shift during the perinatal transition, when oGH receptor expression increases dramatically in neonatal tissues. Maximal specific binding of oGH to hepatic membranes of neonatal lambs (20.1%) greatly exceeds the binding of oGH to fetal hepatic membranes (0.9-1.5%), indicating a developmental programming of hormone responsiveness .

How do receptor binding affinities differ between oGH and oPL in fetal ovine tissues?

Receptor binding studies demonstrate striking differences in the affinity of fetal tissues for oPL versus oGH. In fetal hepatic membranes, maximal specific binding of radiolabeled oPL reaches 26.3%, while binding of radiolabeled oGH is only 0.9-1.5% . Competitive binding assays show that unlabeled oPL causes dose-dependent inhibition of radiolabeled oPL binding to fetal hepatic membranes, with half-maximal displacement occurring at 5-7 ng/ml .

While oGH and ovine prolactin (oPRL) can competitively displace oPL from its binding sites, their potencies are remarkably lower—only 2% for oGH and 0.1% for oPRL relative to oPL . In cultured fetal ovine fibroblasts, oPL binds with high affinity (EC₅₀ 0.20 nM), and in competitive displacement assays, oPL shows 8-12 times greater potency than oGH . This receptor binding profile explains the differential metabolic effects observed in fetal tissues and supports the concept that oPL acts as the primary growth-promoting hormone in the fetus.

What are the primary metabolic effects of oPL in ovine fetal development?

Ovine placental lactogen exerts multiple metabolic effects in fetal tissues that collectively promote growth and development:

• Glycogen metabolism: oPL stimulates significant dose-dependent increases in glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%) in cultured ovine fetal hepatocytes .

• Amino acid transport: oPL stimulates dose-dependent increases in amino acid uptake in fetal tissues, an effect not observed with oGH or rat growth hormone (rGH) in fetal rat diaphragms .

• Protein synthesis: The stimulation of amino acid transport suggests oPL plays a critical role in promoting protein synthesis in the developing fetus, essential for tissue growth .

• Endometrial effects: oPL binds specifically to endometrial glands and affects endometrial gland number and expression of uterine milk proteins and osteopontin, contributing to the maternal support system for fetal development .

These metabolic effects appear specific to the fetal developmental phase, as the relative potencies of these hormones shift in postnatal tissues.

How does the expression of oGH and oPL receptors change from fetal to neonatal development?

The expression and binding characteristics of receptors for oGH and oPL undergo significant changes during the transition from fetal to neonatal development. In fetal hepatic tissues, oPL binding predominates, with high specific binding (26.3%) . This binding profile changes dramatically after birth, with a substantial increase in oGH receptor expression in neonatal tissues.

The maximal specific binding of oGH to hepatic membranes increases from 0.9-1.5% in fetal tissues to 20.1% in neonatal lambs . Additionally, the potency of oGH in competing for oPL binding sites increases substantially in neonatal liver compared to fetal liver . These changes reflect a programmed developmental transition in hormone responsiveness that coincides with the shift from placental to pituitary regulation of growth and metabolism after birth.

This developmental transition in receptor expression explains why oGH assumes a more prominent role in regulating growth and metabolism in postnatal life, while oPL functions as the primary growth-promoting hormone during fetal development.

What experimental models best demonstrate the differential effects of oPL and oGH on fetal glycogen metabolism?

Several experimental models effectively demonstrate the differential effects of oPL and oGH on fetal glycogen metabolism, each with specific advantages for addressing particular research questions:

• Cultured ovine fetal hepatocytes: This in vitro model allows direct examination of hormone effects on glycogen synthesis. Studies using this model have demonstrated that oPL (150 ng/ml-20 μg/ml) stimulates dose-dependent increases in [14C]glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%), while oGH shows only 12% of oPL's potency . This model permits precise dose-response studies and mechanistic investigations of post-receptor signaling pathways.

• Fetal hepatic membrane preparations: This approach allows quantitative assessment of hormone receptor binding. Using radiolabeled hormones, researchers have demonstrated that maximal specific binding of [125I]oPL reaches 26.3% in fetal hepatic membranes, while binding of [125I]oGH is only 0.9-1.5% . Scatchard analysis of these binding studies indicates that oPL binds to a single class of receptors with a dissociation constant of 1.1 × 10⁻¹⁰ M .

• In vivo intrauterine hormone infusion: This model most closely approximates physiological conditions. Research shows that intrauterine infusion of oPL and/or oGH following interferon τ in ovariectomized ewes treated with progesterone differentially affects endometrial gland number and protein expression . This model allows investigation of complex tissue interactions and maternal-fetal endocrine relationships.

When designing experiments to compare oPL and oGH effects on glycogen metabolism, researchers should consider using multiple complementary models to establish both direct cellular effects and their physiological relevance.

How do the mechanistic pathways of oPL-mediated amino acid transport differ from those of oGH in fetal tissues?

The mechanistic pathways underlying oPL-mediated amino acid transport in fetal tissues differ fundamentally from those of oGH, explaining their differential effects on fetal metabolism and growth:

Understanding these mechanistic differences is essential for interpreting experimental results and developing hypotheses about the developmental regulation of growth.

What techniques are most effective for studying the competitive binding of oPL, oPRL, and oGH to fetal receptors?

Several specialized techniques have proven effective for investigating the competitive binding characteristics of oPL, oPRL, and oGH to fetal receptors:

• Radioligand binding assays: Using radiolabeled hormones (e.g., [125I]oPL) with various concentrations of unlabeled competitors (oPL, oGH, oPRL) allows quantification of relative binding affinities. These studies have demonstrated that unlabeled oPL causes dose-dependent inhibition of [125I]oPL binding to fetal hepatic membranes, with oGH and oPRL showing only 2% and 0.1% of oPL's potency, respectively .

• Chimeric protein binding assays: A novel approach using chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL (SEAP-oPL) has allowed visualization and characterization of binding sites in frozen tissue sections. This technique has revealed specific binding of SEAP-oPL to endometrial glandular epithelium throughout pregnancy, with differential displacement by oPL, oPRL, and oGH .

• Recombinant receptor domain studies: Using the extracellular domain of the oPRLR has enabled determination that iodinated oPL binding sites can be competed for by oPRL and oPL but not by oGH . This approach helps identify specific receptor domains involved in hormone recognition.

• Fluorescence resonance energy transfer (FRET) microscopy: This emerging technique allows visualization of hormone-receptor interactions in living cells, providing spatial and temporal information about receptor dynamics that cannot be obtained from binding assays using membrane preparations .

When selecting techniques for competitive binding studies, researchers should consider combining multiple approaches to overcome the limitations of any single method and to establish both the quantitative binding parameters and their functional significance.

How does heterologous receptor dimerization affect signaling pathways in oPL and oGH action?

Heterologous receptor dimerization represents a critical mechanism in the differential signaling of oPL and oGH, with significant implications for their biological effects:

• Receptor sharing and specificity: The biological actions of oPL in vitro are mediated by both homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor . This receptor flexibility enables oPL to trigger multiple signaling pathways and explains its broader range of biological activities compared to oGH in fetal tissues.

• Developmental regulation of dimerization: The capacity for receptor heterodimerization appears to be developmentally regulated. The ability of oGH to compete for [125I]oPL binding sites increases substantially from fetal to neonatal tissues , suggesting changes in the relative abundance or conformation of receptors that affect their dimerization properties.

• Signal transduction consequences: Different receptor dimers activate distinct downstream signaling pathways. Although both oPL and oGH can stimulate glycogen synthesis in fetal hepatocytes, their dose-response curves are parallel and their maximal effects identical, suggesting they ultimately converge on common signaling pathways despite different receptor interactions .

• Tissue-specific dimerization patterns: The patterns of receptor dimerization vary between tissues. In endometrial glands, oPL binding can be displaced completely by oPL and oPRL but only partially by oGH , indicating tissue-specific differences in receptor composition or conformation that affect dimerization potential.

Understanding how heterologous receptor dimerization influences hormone action is essential for interpreting the complex and sometimes contradictory effects of these hormones in different experimental systems.

What are the implications of differential oPL and oGH binding in developmental programming?

The differential binding of oPL and oGH in fetal and neonatal tissues has profound implications for developmental programming and the transition from fetal to postnatal growth regulation:

• Metabolic programming: The predominance of oPL binding in fetal tissues suggests it plays a key role in establishing metabolic pathways during critical developmental windows. This may program long-term metabolic responsiveness, as the early metabolic environment influences adult physiology .

• Growth transition mechanisms: The developmental switch from predominant oPL binding in fetal tissues to increased oGH binding in neonatal tissues represents a critical transition point in growth regulation. Understanding this transition may provide insights into growth disorders with developmental origins.

• Placental adaptation: Since placental hormones have evolved differently across species but with convergent functions , their specific binding patterns likely reflect adaptive mechanisms that optimize maternal-fetal resource allocation. The specific binding of oPL to endometrial glands throughout pregnancy (Days 30, 60, 90, and 120) indicates its role in regulating maternal adaptations to support fetal growth.

• Immune tolerance implications: Placental hormones also influence maternal immune adaptations during pregnancy. Human chorionic gonadotropin, for example, induces proliferation of uterine natural killer cells that play a key role in maternal-fetal interactions . The binding patterns of placental hormones may thus have implications for immune tolerance of the fetal semiallograft.

• Evolutionary perspective: The differential binding properties of oPL and oGH reflect separate evolutionary trajectories. Placental lactogens occur in primates, rodents, and ruminants but evolved through duplication of different genes , suggesting their binding properties and developmental roles coevolved with placentation strategies.

Research into these developmental programming implications requires longitudinal studies that examine both immediate hormone actions and their long-term consequences for growth and metabolism.

What tissue culture techniques are most appropriate for studying oGH and oPL effects?

The selection of appropriate tissue culture techniques is critical for investigating the differential effects of oGH and oPL. Several approaches have proven valuable, each with specific advantages for particular research questions:

• Primary ovine fetal hepatocyte cultures: These cultures have been instrumental in demonstrating the glycogenic effects of oPL and oGH. Researchers have successfully used these cultures to show that oPL (150 ng/ml-20 μg/ml) stimulates dose-dependent increases in glucose incorporation into glycogen and cellular glycogen content . When establishing these cultures, careful attention to isolation procedures, culture media composition, and maintenance of differentiated hepatocyte functions is essential.

• Fetal ovine fibroblast cultures: These have been effectively used to study hormone binding characteristics and metabolic responses. Cultured skin fibroblasts from midgestational fetal lambs bind oPL specifically and with high affinity (EC₅₀ 0.20 nM) . These cultures are relatively easy to establish and maintain, making them suitable for large-scale screening studies.

• Explant cultures of fetal tissues: This approach maintains tissue architecture and cell-cell interactions, potentially providing more physiologically relevant results. Explant cultures of fetal diaphragm have been used to demonstrate that oPL, but not oGH, stimulates amino acid transport in fetal tissues .

• Co-culture systems: These allow investigation of interactions between different cell types, such as placental and endometrial cells, which may be particularly important for understanding the integrated physiology of placental hormone actions.

When designing tissue culture experiments, researchers should consider:

• Using physiologically relevant hormone concentrations based on circulating levels during pregnancy

• Including appropriate positive controls (e.g., insulin for metabolic studies)

• Validating culture conditions to ensure they maintain cell differentiation and hormone responsiveness

• Incorporating time-course studies to capture both rapid and delayed hormone effects

How should receptor binding assays be optimized for distinguishing oPL and oGH activity?

Optimizing receptor binding assays is crucial for accurately distinguishing oPL and oGH activity. Based on the literature, several technical considerations emerge as particularly important:

• Selection of appropriate tissue preparations: Hepatic membranes from fetal and neonatal lambs have successfully demonstrated developmental differences in binding profiles . Membrane preparation techniques should preserve receptor integrity while minimizing non-specific binding.

• Radioligand selection and purification: High-specific-activity radiolabeled hormones ([125I]oPL, [125I]oGH) are essential for detecting low-abundance receptors. The purity of radioligands should be verified, as contamination can confound binding data.

• Binding conditions optimization: Parameters such as incubation time, temperature, and buffer composition significantly affect binding kinetics. Studies have shown that [125I]oPL binding is saturable and reversible and varies with incubation time and temperature .

• Competitive displacement design: To accurately assess relative binding affinities, competitive displacement studies should use a wide concentration range of unlabeled hormones. Studies showing that oGH and oPRL caused parallel displacement of [125I]oPL with potencies only 2% and 0.1% that of oPL used concentrations from 1 ng/ml to 5 μg/ml of unlabeled hormones .

• Data analysis approaches: Scatchard analysis of dose-response curves has successfully identified a single class of oPL receptors with a dissociation constant of 1.1 × 10⁻¹⁰ M . Other analytical approaches, including kinetic analyses and thermodynamic evaluations, may provide additional insights into binding mechanisms.

• Alternative binding detection methods: Novel approaches such as the SEAP-oPL chimeric protein binding assay have allowed visualization of binding sites in tissue sections . This approach complements traditional binding assays by providing spatial information about receptor distribution.

By carefully optimizing these parameters, researchers can develop binding assays that reliably distinguish between oPL and oGH activity, providing insights into their differential roles in development.

What in vivo models are most suitable for investigating developmental endocrinology of placental hormones?

Several in vivo models have proven valuable for investigating the developmental endocrinology of placental hormones, each offering distinct advantages:

• Intrauterine hormone infusion models: These models allow direct manipulation of the fetal environment. Studies have used intrauterine infusion of oPL and/or oGH following interferon τ in ovariectomized ewes treated with progesterone to examine effects on endometrial gland development and function . This approach allows precise control of hormone exposure while maintaining the complex maternal-fetal interface.

• Pregnant sheep models: The ovine model has been particularly valuable for placental hormone research due to similarities in placental structure and function with humans, relatively large fetal size facilitating sampling, and well-characterized endocrinology. Studies examining binding of placental lactogen and growth hormone to fetal sheep fibroblasts have provided insights into their differential activities .

• Hormone replacement in ovariectomized pregnant models: This approach allows manipulation of the maternal hormonal environment to isolate effects of specific hormones. Studies have used ovariectomized ewes with hormone replacement to investigate the hormonal servomechanism regulating endometrial glandular epithelium differentiation and function .

• Genetic models with receptor modifications: While not yet widely used in sheep, models with modified hormone receptors could provide valuable insights into specific receptor-mediated effects. These approaches could include receptor knockdown or knockout technologies.

When designing in vivo studies, researchers should consider:

• The developmental timing of interventions relative to critical windows in placental and fetal development

• Appropriate sampling protocols to capture both immediate and delayed hormone effects

• Ethical considerations and animal welfare

• Translation of findings between species, recognizing that placental hormones have evolved differently across species

What techniques are most reliable for quantifying metabolic effects of placental hormones?

Several techniques have proven reliable for quantifying the metabolic effects of placental hormones, particularly in measuring glycogen synthesis, amino acid transport, and related metabolic processes:

• Radiotracer incorporation assays: These have been successfully used to measure [14C]glucose incorporation into glycogen, demonstrating that oPL stimulates dose-dependent increases (18-167%) in fetal hepatocytes . Similarly, radiolabeled amino acid (e.g., α-aminoisobutyric acid, AIB) uptake assays have shown that oPL, but not oGH, stimulates amino acid transport in fetal tissues .

• Direct biochemical measurements: Total cellular glycogen content can be quantified using enzymatic or colorimetric methods, showing oPL-stimulated increases (10-69%) in fetal hepatocytes . These direct measurements complement radiotracer studies by confirming net changes in metabolite pools.

• Protein synthesis assays: Since amino acid transport is closely linked to protein synthesis, techniques measuring incorporation of labeled amino acids into proteins provide insights into the functional consequences of hormone-stimulated transport.

• Enzyme activity assays: Measuring activities of key metabolic enzymes (e.g., glycogen synthase, glycogen phosphorylase) helps elucidate the mechanisms underlying hormone effects on metabolic pathways.

• Gene expression analysis: Quantifying mRNA levels of metabolic enzymes and transporters using quantitative PCR or RNA sequencing provides insights into transcriptional regulation by placental hormones.

• Metabolomic approaches: Comprehensive analysis of metabolite profiles using mass spectrometry or nuclear magnetic resonance spectroscopy can reveal broad metabolic effects that might be missed by targeted assays.

When selecting techniques for quantifying metabolic effects, researchers should consider:

This multi-technique approach provides a more comprehensive understanding of how placental hormones regulate fetal metabolism and growth.