Placental Lactogen Ovine

What is the biochemical structure and physical properties of ovine placental lactogen?

Ovine placental lactogen (oPL) is a protein hormone that has been purified approximately 1,000-fold from sheep cotyledons using conventional protein purification procedures. The molecular weight of oPL is approximately 22,000 daltons as determined by gel filtration on Sephadex G-100. Its isoelectric point, determined by isoelectric focusing, is 8.8. The protein exhibits both growth hormone-like and prolactin-like activities, with the ratio of GH-activity to PRL-activity being approximately 1:2. These properties make oPL a unique member of the somatotropin/prolactin family of hormones with dual biological functions in sheep pregnancy .

How is oPL produced and secreted in the sheep placenta?

oPL is produced by binucleate trophoblast cells (BNCs) within the sheep placenta. These non-proliferative BNCs comprise about 15% of the trophectoderm and are formed from adjacent uninucleate trophectoderm cells through nuclear division without cytokinesis. The BNCs develop a rounded appearance and produce granules containing oPL and other protein products. Subsequently, they migrate from the trophectoderm layer and fuse with maternal epithelial cells to form a maternal-fetal hybrid syncytium. Following fusion, the oPL-containing granules translocate to the basal plasmalemma of the syncytium, and oPL is released into the maternal compartment by exocytosis. This process of BNC migration and fusion occurs from as early as day 16 of pregnancy and is essential for both placental differentiation and placental lactogen secretion .

What are the most effective methods for purifying oPL from sheep placental tissue?

The purification of oPL from sheep cotyledons typically involves a series of conventional protein purification steps. The process begins with homogenization of the placental tissue followed by ammonium sulfate precipitation to isolate the protein fraction containing oPL. Subsequent steps include ion-exchange chromatography, gel filtration on Sephadex G-100, and potentially isoelectric focusing. Throughout the purification process, researchers commonly monitor oPL activity using radioreceptor assays (RRAs) with rabbit liver particulate fractions for growth hormone activity (RRA-GH) and rabbit mammary gland particulate fractions for prolactin activity (RRA-PRL). This combined approach allows for approximately 1,000-fold purification of oPL from sheep cotyledons while maintaining its biological activities .

What detection methods can be used to quantify oPL in biological samples?

Several methods are available for detecting and quantifying oPL in biological samples:

• Radioimmunoassay (RIA): A homologous RIA procedure using a highly specific anti-oPL antibody (non-cross-reactive with ovine GH or ovine prolactin) is effective for measuring oPL concentrations in plasma or serum. The assay sensitivity is typically around 100 pg/tube, with a practical detection limit of 250 pg/ml when using 400 μl of serum. Depending on gestational age, serum volumes between 10 and 400 μl may be used, with consistent detection observed at 100-150 pg/ml.

• Radioreceptor Assays (RRAs): Both growth hormone and prolactin radioreceptor assays can be employed to measure the biological activities of oPL.

• Immunohistochemistry: For tissue samples, immunohistochemical staining using monoclonal rat anti-oPL antibodies can visualize oPL within placental tissues. Quantification can be performed using computer-aided image analysis, typically measuring over multiple fields of view taken randomly across placental sections.

• Real-time PCR: For measuring oPL gene expression, ovine-specific primers can be used in quantitative PCR, with transcript abundance typically normalized to housekeeping genes such as cyclophilin .

How can researchers effectively design experiments to study oPL's dual biological activities?

To study the dual biological activities of oPL (growth hormone-like and prolactin-like), researchers should implement the following experimental approaches:

• Receptor Binding Studies: Utilize radioreceptor assays with rabbit liver particulate fractions for growth hormone activity and rabbit mammary gland particulate fractions for prolactin activity. These assays can help quantify the relative potencies of oPL's dual functions.

• Growth Promotion Assays: Employ body weight gain assays using hypophysectomized rats to assess the growth-promoting potency of oPL (approximately 1.3 U/mg).

• Lactogenic Assays: Use rabbit mammary explant cultures to evaluate oPL's ability to stimulate casein synthesis, which reflects its prolactin-like activity.

• Comparative Design: Include appropriate controls such as ovine growth hormone (oGH), ovine prolactin (oPRL), and human placental lactogen (hPL) in parallel experiments to establish relative potencies. This is particularly important given that oPL is approximately 20 times more active than hPL in the growth hormone receptor assay, but 5 times less active than oGH .

How does maternal nutrition influence oPL expression and secretion during pregnancy?

Maternal nutrition significantly affects circulating oPL levels during pregnancy, but through mechanisms that appear independent of placental oPL gene expression. In studies of adolescent ewes with singleton pregnancies fed either high (H) or moderate (M) nutrient intake diets, the following effects were observed:

• High nutrient intake resulted in reduced circulating oPL concentrations at both mid-gestation (day 81) and late gestation (day 130).

• The gestational rise in oPL above the minimal detection threshold was delayed by at least 7 days in high-intake animals.

• Despite these dramatic reductions in systemic oPL, there was no corresponding reduction in placental oPL mRNA or protein at either day 81 or 130 of gestation.

• By late gestation, maternal oPL concentrations were reduced by approximately 69% (days 111-146) in high-intake animals, while term placental size was reduced by about 43%.

These findings suggest that reduced circulating oPL likely reflects both reduced placental mass and impaired trophoblast migration affecting hormone release following BNC fusion, rather than changes in gene expression. The delayed rise in oPL may relate to developmental alterations in BNC migration and fusion, potentially linked to an altered proliferation/apoptosis balance in the placenta of high-intake animals .

What is the relationship between oPL and intrauterine growth restriction (IUGR) in sheep?

The relationship between oPL and intrauterine growth restriction (IUGR) in sheep is complex:

• In adolescent ewes on high-nutrient diets, reduced circulating oPL levels correlate with smaller placental and fetal size at term, suggesting a potential link between oPL and fetal growth.

• Similar findings occur in a different sheep model of fetal IUGR induced by maternal hyperthermia, where circulating oPL concentrations are reduced but placental oPL mRNA levels remain unchanged.

• The mechanism is thought to reflect altered placental differentiation affecting oPL release rather than changes in gene expression.

• At mid-gestation (day 81), reduced oPL occurs without changes in placental or fetal size, indicating that altered oPL secretion may precede growth restriction.

• While there is evidence that oPL infused directly into late gestation ovine fetuses has subtle effects on some major organ systems, a direct causative role for reduced oPL in the induction of fetal IUGR requires further investigation.

• Day 80 pregnant adolescent sheep on high nutrient intake diets exhibit reduced proliferative activity of the fetal trophectoderm with increased expression of pro-apoptotic proteins, suggesting an altered proliferation/apoptosis balance that could affect trophoblast cell migration, fusion, and consequently, oPL release .

How does oPL compare with human placental lactogen (hPL) in terms of structure and function?

Ovine placental lactogen (oPL) and human placental lactogen (hPL) show important similarities and differences:

These similarities make sheep an excellent animal model for studying placental lactogen physiology relevant to human conditions, though the structural differences must be considered when interpreting results .

What experimental models using oPL can inform human placental lactogen research?

Several experimental models using oPL can provide valuable insights for human placental lactogen research:

• Nutritional Models: Studies in adolescent pregnant ewes with altered nutritional status demonstrate how maternal nutrition affects placental lactogen secretion and fetal growth, which may have parallels in human pregnancy complications such as IUGR.

• Hyperthermia-Induced IUGR Model: Pregnant adult ewes exposed to high ambient temperatures develop placental insufficiency and fetal IUGR with reduced circulating oPL, providing a model for environmental influences on placental function.

• Receptor Binding Studies: Comparative analyses of oPL and hPL binding to growth hormone and prolactin receptors can illuminate the evolutionary conservation of hormone-receptor interactions.

• Placental Developmental Models: Research on binucleate trophoblast cell migration and fusion in sheep provides insights into trophoblast differentiation and hormone secretion mechanisms that may have parallels in human syncytiotrophoblast formation.

• Hormone Supplementation Studies: Experiments involving direct oPL infusion into ovine fetuses can help elucidate the physiological effects of placental lactogens on fetal development, with potential implications for understanding hPL functions .

How can we resolve contradictions in data regarding oPL expression versus secretion?

Research data shows an apparent contradiction between oPL expression and secretion, particularly in models of maternal nutritional manipulation and hyperthermia. To resolve these contradictions, researchers should consider the following approaches:

• Comprehensive Methodology: Employ multiple complementary techniques (mRNA quantification, protein detection, and hormone assays) to assess oPL at different levels of biological organization.

• Cellular Process Focus: Investigate specific cellular processes that might disconnect expression from secretion, particularly:

  • BNC formation and migration rates

  • Fusion efficiency with maternal epithelium

  • Exocytosis mechanisms for oPL-containing granules

  • Post-translational modifications affecting hormone bioactivity

• Temporal Resolution: Design studies with frequent sampling to capture dynamic changes in oPL expression and secretion throughout gestation, especially around critical developmental windows.

• Placental Heterogeneity Analysis: Examine regional differences within the placenta, as reduced secretion might reflect altered differentiation in specific placental regions rather than global expression changes.

• Metabolic Clearance Consideration: Measure maternal metabolic clearance rates of oPL to determine whether altered circulating levels reflect changes in production, clearance, or both .

What are the methodological challenges in measuring the true biological activity of oPL in vivo?

Measuring the true biological activity of oPL in vivo presents several methodological challenges:

• Dual Biological Activities: oPL exhibits both growth hormone-like and prolactin-like activities with a ratio of approximately 1:2, making it difficult to design assays that accurately capture both functionalities simultaneously.

• Receptor Cross-Reactivity: oPL interacts with both GH and prolactin receptors, potentially activating multiple signaling pathways with different downstream effects.

• Species-Specific Responses: The biological responses to oPL may vary between species, complicating the interpretation of results from animal models in relation to sheep physiology.

• Developmental Changes: The physiological significance of oPL may change throughout gestation, requiring temporal consideration in experimental design.

• Integration with Other Hormones: oPL functions within a complex hormonal milieu, and its effects may be modulated by interactions with other pregnancy-related hormones.

• Maternal versus Fetal Effects: Distinguishing between the direct effects of oPL on maternal tissues and its indirect effects on fetal development through altered maternal physiology requires sophisticated experimental approaches, such as selective hormone infusion into either maternal or fetal compartments .

What novel approaches could advance our understanding of oPL's role in placental development?

Novel approaches to advance our understanding of oPL's role in placental development include:

• Single-Cell RNA Sequencing: Apply single-cell transcriptomics to characterize the developmental trajectory of BNCs and identify factors regulating oPL expression at the cellular level.

• CRISPR-Cas9 Gene Editing: Develop gene editing strategies to modulate oPL expression in ovine trophoblast cells, potentially creating in vitro models with altered oPL production.

• Organoid Models: Establish sheep placental organoid systems to study the regulation of oPL expression and secretion in a controlled environment that mimics the three-dimensional structure of the placenta.

• In vivo Imaging: Develop methods for real-time tracking of BNC migration and fusion in living placental tissues using fluorescent protein tagging of oPL or BNC-specific markers.

• Systems Biology Approaches: Integrate transcriptomic, proteomic, and metabolomic data to create comprehensive models of the regulatory networks controlling oPL expression and function during placental development.

• Receptor Signaling Analysis: Investigate the distinct intracellular signaling pathways activated by oPL binding to GH versus prolactin receptors to better understand its dual biological functions .

How might research on oPL inform therapeutic strategies for pregnancy complications?

Research on oPL could inform therapeutic strategies for pregnancy complications in several ways:

• Biomarker Development: Changes in maternal oPL levels correlate with placental development and function, suggesting potential use as a biomarker for early detection of placental insufficiency or IUGR.

• Nutritional Interventions: Understanding how maternal nutrition affects oPL secretion could guide the development of targeted nutritional interventions for high-risk pregnancies.

• Hormone Supplementation: Knowledge of oPL's biological activities might inform the development of recombinant placental lactogen therapies for pregnancy complications associated with hormonal deficiencies.

• Placental Transport Modulation: Insights into how oPL affects placental nutrient transport could lead to strategies for enhancing fetal nutrient delivery in growth-restricted pregnancies.

• Prevention of Trophoblast Dysfunction: Understanding the mechanisms of BNC formation, migration, and fusion could help develop approaches to prevent or correct trophoblast dysfunction in pathological pregnancies.

• Comparative Studies with Human Conditions: The striking homologies between oPL and hPL make sheep an excellent model for studying placental lactogen physiology relevant to human pregnancy complications .