Conservation of progesterone hormone function in invertebrate reproduction

Abstract

Steroids play fundamental roles regulating mammalian reproduction and development. Although sex steroids and their receptors are well characterized in vertebrates and several arthropod invertebrates, little is known about the hormones and receptors regulating reproduction in other invertebrate species. Evolutionary insights into ancient endocrine pathways can be gained by elucidating the hormones and receptors functioning in invertebrate reproduction. Using a combination of genomic analyses, receptor imaging, ligand identification, target elucidation, and exploration of function through receptor knockdown, we now show that comparable progesterone chemoreception exists in the invertebrate monogonont rotifer Brachionus manjavacas, suggesting an ancient origin of the signal transduction systems commonly associated with the development and integration of sexual behavior in mammals.

Fig. 1.

Progesterone (1), and a panel of probes including the IAF-labeled progesterone probe 2, an IAF-labeled control steroid probe 3, and a reactive steroid probe 4.

Fig. 2.

Imaging studies with the rotifer Brachionus manjavacas. (A) Confocal fluorescence microscopic images depicting rotifers treated with progesterone probe 2 or control probe 3. (B) Two-color confocal images showing a female rotifer fed T. suecica then treated with 2; T. suecica autofluoresces in the red channel, and 2 fluoresces in the blue channel. (Scale bars: 100 μm.) Blue and red fluorescence were collected with excitation at 375 ± 10 nm and emission at 458 ± 10 nm and excitation at 488 ± 10 nm and emission at 518 ± 10 nm, respectively. Organelles are noted by: vit, vitellarium (yolk gland); o, ovaries; ov, oviduct; e, egg; sv, seminal vesicle; rg, rudimentary gut; st, stomach; sd, sperm duct.

Fig. 3.

Immunoprecipitation (IP) of a rotifer progesterone receptor (PR). (A) SDS/PAGE analysis depicting the IP of B. manjavacas lysate (L1) in the presence of progesterone probe 2 (L2 and L3). IP of lysates treated with progesterone (1) showed a reduction in the isolated proteins (L4). (B) Reactive probe 4 was designed to form a covalent adduct with a PR by formation of an amide with the side-chain amine of a lysine residue. The resulting covalent adducts can be selectively identified by using an anti-progesterone mAb (anti-P) and can be validated by using an anti-progesterone receptor mAb (anti-PR). (C) Validation of 4 using mammalian MCF-7 cell lysates. Western blot analyses depicting the presence of a 100-kDa protein in MCF-7 cell lysates incubated with 4. Comparable bands corresponding to the MCF-7 PR were identified after incubation with an anti-P mAb in L5 or an established anti-PR mAb in L6. (D) Western blot analysis of B. manjavacas lysate treated with 4 showed a single major band at 82 kDa after treatment with 4 (L8 and L9). This band could be ablated by the cotreatment with 1 (L7). Loading control was conducted with actin (A).

Fig. 4.

RNAi experiments with female rotifers transfected with dsRNA from the rotifer progesterone receptor gene (treatment), dsRNA from the rotifer enlongation factor gene (control for A), or with PBS (control for B). (A) Relative fluorescence intensity of female rotifers incubated with progesterone probe 2 (n = 6, two sample, one-tailed paired Student's t test, P < 0.0001; error bars denote SE). (B) Percent sexual females in first-generation rotifer daughters (F1) and second generation rotifer daughters (F2) (n = 4, two sample, one-tailed paired Student's t test, P = 0.045 for F1; n = 4, two sample, one-tailed paired Student's t test, P = 0.434 for F2; error bars denote SE).