Investigating the role of glycoprotein hormone GPA2/GPB5 signaling in reproduction in adult female Rhodnius prolixus

Abstract

Glycoprotein hormones are essential for regulating various physiological activities in vertebrates and invertebrates. In vertebrates, the classical glycoprotein hormones include follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG), which have crucial roles in growth, development, metabolism, and reproduction. In female mammals, FSH stimulates egg production in the ovaries, whereas LH and CG act as the triggers for follicular ovulation. The more recently discovered heterodimeric glycoprotein hormone GPA2/GPB5 (called thyrostimulin in vertebrates) is suggested to be involved in reproductive processes in arthropods. Here, we focus on understanding the role of GPA2/GPB5 and its receptor, LGR1, in the reproductive success of adult female Rhodnius prolixus, a vector of Chagas disease. qPCR was used to monitor the expression of GPA2 and GPB5 transcripts and their receptor in different tissues. Immunohistochemistry was used to show the distribution of GPB5 in the nervous system and reproductive system, and RNA interference was used to disrupt the glycoprotein hormone signaling pathway. Both subunit transcripts, GPA2 and GPB5, are present in a variety of tissues, with the greatest expression in the central nervous system; whereas the LGR1 transcript is present in peripheral tissues, including the fat body and the reproductive system of adult females. In the adult female, GPB5-like immunoreactive axonal projections are present in the trunk nerves extending onto the reproductive tissues, with processes overlaying the ovaries, oviducts, spermatheca, and bursa, indicating the possibility of neural control by neurons containing GPA2/GPB5. In addition, GPB5-like immunostaining is present in muscles encircling the ovarioles, and in the cytoplasm of trophocytes (nurse cells) located in the tropharium. GPB5-like immunoreactive processes and blebs are also localized to the previtellogenic follicles, suggesting an involvement of this glycoprotein hormone signaling in oocyte development. LGR1 transcript expression increases in the adult female reproductive system post-feeding, a stimulus that initiates reproductive development, adding further support to an involvement in reproduction. We have investigated the effect of LGR1 downregulation on reproductive processes, monitoring the number and the quality of eggs laid, hatching ratio, and production of vitellogenin (Vg), the major yolk protein for developing eggs. Downregulation of LGR1 leads to increases in transcript expression of vitellogenin, RhoprVg1, in the fat body and the vitellogenin receptor, RhoprVgR, in the ovaries. Total protein in the fat body and hemolymph of dsLGR1-injected insects increased compared to controls and associated with this effect was a significant increase in vitellogenin in these tissues. dsLGR1-injection leads to accelerated oogenesis, an increase in the number of eggs produced and laid, an increase in egg size and a reduction in hatching rate. Our results indicate that GPA2/GPB5 signaling acts to delay egg production in adult female R. prolixus.

Keywords: egg production; immunohistochemistry; kissing bug; qPCR; reproduction; transcript; triatomine.

Figure 1

GPB5-like immunoreactivity associated with the trunk nerves and reproductive tissues of adult female R. prolixus. (A) Mesothoracic ganglionic mass (MTGM) with attached abdominal nerves displaying GPB5-like immunoreactive processes in the trunk nerves (TN) (arrows). (B) GPB5-like immunoreactive processes projecting from the trunk nerve to the reproductive tissues (arrows). (C) Common oviduct (CO) and lateral oviduct (LO) containing GPB5-like immunoreactive processes (arrows) extending along the LO to the calyx (CL) shown in (D) (arrows). (E) GPB5-like immunoreactive processes and blebs are present in the previtellogenic follicles of the ovarioles (inset). GPB5-like staining is also found in muscle fibers encircling the ovariole forming a criss-cross pattern (thin arrow). (F) GPB5-like immunoreactive axons are present in nerves that project to the bursa (BU), as indicated by arrows. (G) Enlargement of boxed area in F showing GPB5-like immunoreactive processes in the nerves (arrows). (H) GPB5-like immunoreactive processes in the spermatheca (SP) (arrows). These are representative images obtained from 10 preparations. Scale bars: (A, B) are 50 μm, (C–E) and (H) are 100 μm, (F–G) 20 μm.

Figure 2

Immunostaining of GPB5-like staining in the ovariole in adult female R. prolixus. (A) GPB5-like staining (in green) is observed in the processes over the follicle cells as well as in the cytoplasm of trophocytes (nurse cells). GPB5-like staining is brightest in Zone 3 of the tropharium where nuclear aggregates are peripherally arranged around the central core of the cytoplasm (encircled in white) (B–D) DAPI staining (in blue) shows GPB5-like staining in the cytoplasm around the large nuclei in the tropharium. TRP, tropharium; α FOL, alpha follicle (terminal follicle); β FOL, beta follicle (terminal follicle); Z1, zone 1; Z2, zone 2; Z3, Zone 3. Similar results were obtained in 10 preparations. Scale bars: (A–D) 100 μm.

Figure 3

Tissue distribution of GPA2, GPB5, and LGR1 transcripts in unfed adult female Rhodnius prolixus at 10-12 d post ecdysis. The transcript levels in each tissue were quantified relative to the expression in the CNS (value ~ 1) using qPCR and the 2^-ΔΔCt method. The y axes represent the relative expression obtained via geometric averaging using Rp49 and actin as reference genes. The results are shown as the mean ± SEM (n = 5-6, where each n represents a pool of tissues from 3 insects). Statistical analysis was performed using a one-way ANOVA and Tukey’s test for post-hoc analysis. Significance of P< 0.05 is denoted using letters to indicate bars that are significantly different from others. CNS, central nervous system.

Figure 4

Temporal expression levels of LGR1 in the fat body and reproductive tissues of adult female R. prolixus at 10-12 d post ecdysis. Fat body, reproductive tissues (tropharium, follicles, oviducts, and bursa) were dissected and analyzed for LGR1 during different nutritional states (unfed and fed) and different time points (unfed; 1 to 6 days post blood meal (PBM). Expression levels of LGR1 were quantified using RT-qPCR and the 2^{− ΔΔCt} method. Results are shown as mean ± SEM (n = 4-5, where each n represents a pool of tissues from 2 insects). Statistical analysis was performed using a one-way ANOVA test with Tukey's multiple comparisons. Significance of P < 0.05 is denoted using different letters to indicate bars that are significantly different from others.

Figure 5

Effects of LGR1 mRNA knockdown on egg laying.(A) Representative images showing the reproductive system from dsARG and dsLGR1-injected insects 4 d PBM and 6 d PBM. Note that dsLGR1-injected insects have less eggs in the ovaries at 6 d PBM since they have an accelerated rate of egg laying when compared with dsARG-injected insects. (B) The average eggs laid per female each day (error bars indicates ± SEM; n = 17-21). (C) The cumulative average of eggs laid per female throughout the 16 d PBM. The significance of differences between linear regression slopes of dsLGR1-injected and dsARG-injected groups was determined using an F-test (P = 0.0001). (C′) the cumulative average of eggs laid per female by 16 d PBM. Statistically significant differences were determined by t-test (****p < 0.0001).

Figure 6

Effects of LGR1 mRNA knockdown on egg phenotype from recently laid eggs. Representative images on the left panel displaying the eggs 1-2 days post egg-laying from dsLGR1-injected insects and dsARG-injected insects. Graphs on the right panel show the measurements of the length, width, and volume of eggs. Results are shown as mean ± SEM, n = 15 eggs. Statistically significant differences were determined by Student's t-test (****p < 0.0001).

Figure 7

Effects of LGR1 knockdown on vitellogenesis at 6d post blood meal (PBM). (A) Experimental scheme. (B) RhoprVg1 and RhoprVgR mRNA expression in the fat body and ovaries of fed adult females at 6 d PBM after dsLGR1 injection. Transcript levels were quantified using RT-qPCR and analyzed by the 2^-ΔΔCt method. The y axes represent the fold change in expression relative to control (dsARG, value ~ 1) obtained via geometric averaging using Rp49 and actin as reference genes. The results are shown as the mean ± SEM (n = 5-6, where each n represents an individual tissue from 1 insect). (C) Protein content in the fat body and hemolymph (he) of dsRNA-injected females at 6 d PBM. The results are shown as the mean ± SEM (n = 5-6, where each n represents the fat body or hemolymph from 1 insect). The SDS-PAGE analysis of hemolymph (1 μL) after downregulation of LGR1. Image representative of 3 independent experiments. (D) Quantification of vitellogenin in the fat body and hemolymph (he) of dsRNA-injected females measured by ELISA. The results are shown as the mean ± SEM (n = 5-6, where each n represents the fat body or hemolymph from 1 insect). Western blot image showing vitellogenin in the hemolymph of dsRNA-injected females (1 μL of he at a 1:20 dilution). Image representative of 3 independent experiments. Statistically significant differences were determined by student’s t-test. ns, not significant, i.e. p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8

Effect of dsRNA treatment on hatching. (A) Egg phenotype at 10-15 days post egg laying (PEL) for dsARG-injected insects, (A) shows a 1^st instar from a hatched egg. Representative image from n = 10 insects. (B) Egg phenotype at 10 - 15 days PEL from dsLGR1-injected insects, (B) shows a 1^st instar from a hatched egg. Representative image from n = 10 insects. (C) Percentage of hatching from eggs laid for dsARG- injected and dsLGR1-injected insects (475 eggs from dsARG treated and 550 from dsLGR1 treated insects). (D) Displays unsuccessful hatching at 20-25 days post egg laying for dsLGR1-injected insects. Scale bars: 1 mm for all images.