Recombinant Mouse Sperm-associated antigen 7 (Spag7)

What is Spag7 and what are its primary biological functions?

Spag7 (Sperm-associated antigen 7) is a protein essential for embryonic development and energy homeostasis in mice. Research indicates that Spag7 plays a crucial role in placental development, particularly in the junctional zone of the placenta, which is vital for nutrient transfer to developing fetuses. Additionally, Spag7 is involved in regulating energy expenditure and metabolic function in adult mice. Unlike some other proteins in the SPAG family, Spag7's functions extend beyond reproductive biology to include significant developmental and metabolic roles . This distinguishes it from proteins like SPACA7, which is specifically localized to the acrosome of sperm cells and functions in fertilization .

How is the expression of Spag7 regulated during development?

Expression of Spag7 appears to be developmentally regulated, with critical timing during embryonic development. In some amphibian models, Spag7 has been shown to be activated by thyroid hormone (T3) during metamorphosis via a thyroid hormone response element (TRE) within the first intron . In mice, the protein's expression is essential during embryonic development, as evidenced by the significant developmental abnormalities observed in Spag7 knockout models. The constitutive expression of Spag7 during early development appears to program metabolic function throughout life, as demonstrated by the absence of metabolic phenotypes when Spag7 is knocked out only in adulthood .

What phenotypes are associated with Spag7 deficiency?

Spag7 deficiency produces a complex phenotypic profile characterized by:

• Intrauterine growth restriction (IUGR) due to placental insufficiency

• Reduced birth weight in surviving fetuses

• Development of obesity in adulthood despite normal food intake

• Decreased energy expenditure and reduced locomotor activity

• Impaired glucose tolerance and insulin resistance

• Reduced exercise tolerance and muscle function

• Compromised mitochondrial function in skeletal muscle

These phenotypes collectively suggest that Spag7 deficiency creates a "thrifty phenotype" programmed during embryonic development that manifests as metabolic dysfunction in adulthood .

How does Spag7 deficiency affect embryonic development?

Spag7 deficiency significantly impairs embryonic development through placental insufficiency. In knockout models, researchers observed abnormal development of the placental junctional zone, which is critical for maternal-fetal nutrient exchange. This insufficiency leads to intrauterine growth restriction and increased fetal loss. At embryonic day 18.5 (e18.5), Spag7 knockout fetuses display reduced body weight, altered placental morphology, and metabolic disturbances including reduced blood glucose and plasma insulin levels. Additionally, expression of important growth factors such as IGF1 and IGF2 is altered in Spag7-deficient fetuses . The developmental abnormalities suggest that Spag7 plays a fundamental role in placental formation and function.

What is the relationship between Spag7 and the "thrifty phenotype" hypothesis?

The observed phenotypes in Spag7-deficient mice align closely with the "thrifty phenotype" hypothesis, which proposes that nutritional deprivation during fetal development programs metabolic alterations that persist into adulthood. Spag7 knockout mice experience intrauterine growth restriction due to placental insufficiency, resulting in lower birth weights. These mice subsequently develop obesity and metabolic dysfunction in adulthood despite normal food intake, suggesting metabolic programming during development . This pattern mirrors human epidemiological observations, such as those from the Dutch Hunger Winter, where maternal nutritional deprivation led to low birth weight infants who later developed increased rates of obesity and diabetes as adults. The Spag7 knockout model may therefore serve as a valuable experimental system for studying the molecular mechanisms underlying developmental origins of adult metabolic disease.

How does Spag7 deletion affect energy homeostasis in adult mice?

Spag7 deletion leads to significant alterations in energy homeostasis characterized by:

• Decreased total energy expenditure (15-17% reduction compared to wild-type)

• Reduced locomotor activity (approximately 40% reduction)

• Development of obesity despite normal food intake

• Increased fat mass accumulation

• Impaired glucose tolerance

Importantly, these metabolic disturbances occur without hyperphagia, indicating that the primary defect is in energy expenditure rather than intake. Experimental data shows that at 20 weeks of age, Spag7 knockout mice exhibit a 15-17% increase in body weight compared to wild-type controls across different environmental conditions (room temperature on normal chow, thermoneutrality on normal chow, and room temperature on high-fat diet) . The consistent percentage of weight gain across these conditions suggests that the underlying metabolic programming is robustly established during development.

Does Spag7 deficiency affect thermogenesis and adaptive responses to different diets?

Research indicates that Spag7 deficiency does not primarily affect thermogenesis. When wild-type and Spag7 knockout mice were housed at thermoneutrality (conditions that minimize thermogenic requirements), the metabolic phenotypes persisted without significant changes. Specifically, Spag7 knockout mice still exhibited increased body weight, dramatically increased fat mass, unchanged food intake, and decreased total energy expenditure compared to wild-type controls .

Similarly, when challenged with a high-fat diet (60% HFD), Spag7-deficient mice maintained the same pattern of increased body weight and fat mass without increased food intake. The percent body weight difference between knockouts and wild-types remained consistent (15-17%) across standard chow at room temperature, chow at thermoneutrality, and high-fat diet at room temperature . This consistency suggests that Spag7 deficiency creates a fundamental alteration in energy homeostasis that is largely independent of diet composition or environmental temperature.

What is the relationship between Spag7, mitochondrial function, and muscle performance?

Spag7 deficiency leads to impaired skeletal muscle function characterized by:

• Decreased exercise endurance

• Reduced force generation

• Altered oxidative capacity

• Compromised mitochondrial function

These deficits contribute to the decreased locomotor activity observed in knockout animals, which appears to be a primary driver of reduced energy expenditure and subsequent obesity. Mitochondrial dysfunction in Spag7-deficient muscle likely stems from developmental programming, as conditional knockout of Spag7 in adult animals does not reproduce these phenotypes . The precise molecular mechanisms by which developmental Spag7 deficiency impairs adult mitochondrial function remain to be fully elucidated but may involve persistent alterations in mitochondrial biogenesis, dynamics, or respiratory chain function.

What animal models are available for studying Spag7 function?

Several mouse models have been developed to study Spag7 function:

• Constitutive Spag7 knockout (KO) model: Created through CRISPR/Cas9 technology with complete deletion of Spag7 throughout development and adulthood. This model displays the full spectrum of phenotypes including IUGR, reduced birth weight, adult obesity, glucose intolerance, and reduced energy expenditure .

• Conditional Spag7 knockout (cKO) model: Generated by inserting two LoxP sites flanking exon 2 of the Spag7 gene using CRISPR/Cas9. One LoxP site is positioned 316 bp upstream of exon 2, and the other 97 bp downstream. This model allows for tissue-specific or temporally-controlled deletion of Spag7 when crossed with appropriate Cre-expressing lines .

• Inducible Spag7 knockout model: Created by combining the conditional Spag7 allele with a tamoxifen-inducible Cre system, allowing for deletion of Spag7 specifically in adulthood. This model importantly demonstrates that adult-specific deletion does not reproduce the metabolic phenotypes seen in the constitutive knockout .

For genotyping these models, researchers have utilized genomic PCR with primers external to the 5' and 3' homology arms (5'-TCACATCACGGTCCATCATC and 5'-TCATGACATAGCGACAGTCA, respectively) .

What techniques are most effective for measuring metabolic parameters in Spag7-deficient mice?

Several complementary techniques have proven effective for comprehensive metabolic phenotyping of Spag7-deficient mice:

• Comprehensive Lab Animal Monitoring System (CLAMS): This metabolic cage system provides continuous measurement of oxygen consumption, carbon dioxide production, energy expenditure, locomotor activity, and feeding behavior. In Spag7 studies, CLAMS revealed significant decreases in total energy expenditure and locomotor activity in knockout animals .

• BioDaq Food and Water Intake Monitoring System: This automated system allows precise measurement of food intake patterns, showing that Spag7-deficient mice do not exhibit hyperphagia despite developing obesity .

• Body composition analysis: Techniques such as EchoMRI or DEXA scanning quantify fat mass, lean mass, and other body composition parameters, revealing increased fat accumulation in Spag7 knockouts .

• Glucose tolerance testing: Intraperitoneal or oral glucose tolerance tests assess glucose handling, demonstrating impaired glucose tolerance in Spag7-deficient mice .

• Exercise capacity testing: Treadmill testing with incremental protocols evaluates exercise endurance, showing reduced exercise tolerance in knockout animals .

When applying these techniques, it's important to control for potential confounding variables by including appropriate littermate controls and conducting measurements at consistent times of day to account for circadian variations.

What are the key experimental considerations when working with recombinant Spag7 protein?

When working with recombinant Spag7 protein, researchers should consider:

• Expression system selection: Bacterial systems may not provide appropriate post-translational modifications. Mammalian or insect cell expression systems are often preferable for functional studies.

• Purification strategy: Since Spag7 appears to interact with multiple cellular components, purification conditions should be optimized to maintain native conformation and interaction capabilities.

• Functional assays: Based on known functions, assays should focus on:

  • Mitochondrial function assessments

  • Protein-protein interaction studies to identify binding partners

  • Cell-based assays examining metabolic parameters

• Storage considerations: Purified recombinant Spag7 should be tested for stability under various storage conditions, as protein degradation could significantly impact experimental outcomes.

• Validation of activity: Functional validation using knockout cell systems with recombinant protein complementation can confirm biological activity of the recombinant protein.

How does developmental timing affect the metabolic impact of Spag7 deficiency?

Developmental timing is critical in determining the metabolic consequences of Spag7 deficiency. The research demonstrates a striking contrast between constitutive knockout (throughout development) and adult-induced knockout models:

This stark difference indicates that Spag7's critical metabolic programming occurs during embryonic development, likely through its effects on placental function and subsequent fetal nutrition . This aligns with the developmental origins of health and disease (DOHaD) hypothesis, suggesting that metabolic programming established during critical developmental windows has lifelong consequences that cannot be replicated by gene deletion in adulthood.

What is the relationship between Spag7 and fetal/placental IGF signaling?

Spag7 deficiency disrupts IGF signaling in the developing fetus. Analysis of e18.5 Spag7 knockout fetuses revealed:

• Significantly reduced IGF1 protein levels in fetal liver (approximately 50% reduction compared to wild-type)

• Reduced IGF2 protein levels in fetal liver

• Altered expression of IGF1 and IGF2 genes

These changes likely contribute to the intrauterine growth restriction observed in Spag7-deficient fetuses. The IGF signaling pathway is a critical regulator of fetal growth, and impairments in this pathway can lead to reduced birth weight and altered metabolic programming . The observed reduction in both IGF1 and IGF2 suggests that Spag7 may function upstream of these growth factors, potentially affecting their transcription, translation, or post-translational regulation. The precise molecular mechanism by which Spag7 influences IGF signaling remains an important area for future research, as it may provide insight into the developmental origins of metabolic disease.

How might Spag7 research inform our understanding of human metabolic disease programming?

Research on Spag7 provides valuable insights for understanding human metabolic programming in several ways:

• Mechanistic model for IUGR-associated metabolic disease: Spag7 knockout mice represent a genetically defined model of intrauterine growth restriction leading to adult metabolic dysfunction, paralleling human epidemiological observations such as those from the Dutch Hunger Winter studies .

• Placental development and function: The placental abnormalities in Spag7-deficient mice may inform understanding of human placental insufficiency syndromes, which affect approximately 10% of pregnancies and are associated with increased risk of later-life metabolic disorders.

• Developmental windows for metabolic programming: The striking contrast between developmental and adult Spag7 deletion emphasizes the importance of specific developmental windows in metabolic programming, which has implications for timing interventions in human pregnancies complicated by growth restriction.

• Molecular basis for the "thrifty phenotype": Spag7 research may help elucidate the molecular mechanisms underlying the thrifty phenotype hypothesis, potentially identifying biomarkers or therapeutic targets for intervention.

• Exercise intolerance and mitochondrial dysfunction: The muscle and mitochondrial phenotypes in Spag7-deficient mice may provide insights into exercise intolerance and metabolic inflexibility observed in some human metabolic disorders.

Translation of these findings to human health will require investigation of SPAG7 function in human tissues and cells, as well as examination of potential SPAG7 variants or expression changes in human cohorts with intrauterine growth restriction and later metabolic disease.