Reconsidering reproductive patterns in a model dissociated species, the red-sided garter snake: Sex-specific and seasonal changes in gonadal steroidogenic gene expression

Abstract

Sex steroid hormones are powerful regulators of reproductive behavior and physiology in vertebrates, and steroidogenesis has distinct sex- and season-specific patterns ultimately dictated by the expression of key enzymes. Most comparative endocrinology studies, however, focus only on circulating levels of sex steroids to determine their temporal association with life-history events in what are termed associated reproductive patterns. The red-sided garter snake (Thamnophis sirtalis parietalis) is a notable exception; this species exhibits maximal sex behavior decoupled from maximal sex steroid production and gametogenesis in what is termed a dissociated reproductive pattern. And while this is true for male red-sided garter snakes and their production of testosterone, females have maximal estradiol production during peak breeding (spring) but only immediately after mating. Here, we demonstrate that expression of ovarian aromatase (conversion of androgens to estrogens) matches the established seasonal hormone pattern in females. Additionally, steroidogenic gene expression in the ovary is broadly reduced if not suppressed compared to the testis throughout the active year. Bizarrely, male red-sided garter snakes demonstrate an unexplained pattern of steroidogenic gene expression in the testis. StAR (import of cholesterol to steroidogenesis) is maximally expressed in spring, yet Hsd17b3 expression (conversion of androstenedione to testosterone) is highest in summer, with the latter matching the established summer peak in male testosterone. The function of elevated StAR in spring is unknown, but our results suggest a decoupling between maximal StAR expression and testosterone biosynthesis (Hsd17b3 expression). We also purport that the reproductive pattern binary should be reassessed given its lack of fit for many vertebrate species that demonstrate seasonal, mixed patterns of (a)synchrony between circulating sex hormones and reproductive behavior.

Keywords: garter snake; gene expression; ovary; reproductive pattern; season; steroidogeneis; testis.

Figure 1

Dissociated (A) vs. associated reproductive patterns (B) based on the (a) synchrony of behavior with gonadal activity. Redrawn from (9).

Figure 2

Simplified vertebrate steroidogenesis pathway focused on principle sex steroids (modified from (41)). Steroid hormones and precursors are in gray, enzyme gene names are in italics, and enzymes in bold indicate target genes for which primers could be designed. Genes are defined below pathway.

Figure 3

All primers were validated in traditional PCR using tissue-specific cDNAs from red-sided garter snakes. (A) Primer validation for StAR (steroidogenic acute regulatory protein), Cyp17a1 (17α-hydroxylase), and Hsd17b3 (17β-hydroxysteroid dehydrogenase); small amplicons (left) and large amplicons (right); pooled testis cDNA. (B) Primer validation for control genes (Gapdh, TATA box binding protein) and aromatase (Cyp19a1); small amplicons; pooled ovarian cDNA. (C) Primer valdiation for control genes and aromatase; large amplicons; pooled female liver cDNA. In all images, numbers following gene name indicate PCR product length (base pairs).

Figure 4

Normalized steroidogenic gene expression in testes from male red-sided garter snakes across three seasons (spring, summer, and fall). Genes: (A) StAR, (B) Cyp17a1, (C) Hsd17b3, (D) aromatase (Cyp19a1). Uppercase letters indicate significant differences (p<0.05; lowercase marginal, 0.05<p<0.1). Bars are means (+S.E.M., -95% C.I.). Sample sizes: spring (n=10), summer (n=10), fall (n=10).

Figure 5

Normalized steroidogenic gene expression in ovarian tissue from female red-sided garter snakes across three seasons (spring, summer, and fall). Genes: (A) StAR, (B) Cyp17a1, (C) Hsd17b3, (D) aromatase (Cyp19a1). Uppercase letters indicate significant differences (p<0.05; lowercase marginal, 0.05<p<0.1). Bars are means (+S.E.M., -95% C.I.). Sample sizes: spring (n=10), summer (n=9), fall (n=10).

Figure 6

Sex differences in steroidogenic gene expression in the gonads of red-sided garter snakess across three seasons (spring, summer, and fall). Genes: (A) StAR, (B) Cyp17a1, (C) Hsd17b3, (D) aromatase (Cyp19a1). Asterisks indicate significant differences (p<0.001). Bars are means (+95% C.I., -S.E.M.). Sample sizes for each bar are n=10 except for summer females (n=9).

Figure 7

Some steroidogenic genes showed positive correlations in expression depending on gonad type and season. (A) Cyp17a1 and Hsd17b3; summer testis. (B) StAR and aromatase; summer testis. (C) StAR and aromatase; spring ovary. (D) Cyp17a1 and aromatase; fall ovary. Target gene expression was normalized (log₁₀[2^-ΔCt]).