Drosophila Insulin-Like Peptide 8 (DILP8) in Ovarian Follicle Cells Regulates Ovulation and Metabolism

Abstract

In Drosophila melanogaster eight insulin-like peptides (DILP1-8) are encoded on separate genes. These DILPs are characterized by unique spatial and temporal expression patterns during the lifecycle. Whereas, functions of several of the DILPs have been extensively investigated at different developmental stages, the role of DILP8 signaling is primarily known from larvae and pupae where it couples organ growth and developmental transitions. In adult female flies, a study showed that a specific set of neurons that express the DILP8 receptor, Lgr3, is involved in regulation of reproductive behavior. Here, we further investigated the expression of dilp8/DILP8 and Lgr3 in adult female flies and the functional role of DILP8 signaling. The only site where we found both dilp8 expression and DILP8 immunolabeling was in follicle cells around mature eggs. Lgr3 expression was detected in numerous neurons in the brain and ventral nerve cord, a small set of peripheral neurons innervating the abdominal heart, as well as in a set of follicle cells close to the oviduct. Ovulation was affected in dilp8 mutants as well as after dilp8-RNAi using dilp8 and follicle cell Gal4 drivers. More eggs were retained in the ovaries and fewer were laid, indicating that DILP8 is important for ovulation. Our data suggest that DILP8 signals locally to Lgr3 expressing follicle cells as well as systemically to Lgr3 expressing efferent neurons in abdominal ganglia that innervate oviduct muscle. Thus, DILP8 may act at two targets to regulate ovulation: follicle cell rupture and oviduct contractions. Furthermore, we could show that manipulations of dilp8 expression affect starvation resistance suggesting effects on metabolism. Possibly this reflects a feedback signaling between ovaries and the CNS that ensures nutrients for ovary development. In summary, it seems that DILP8 signaling in regulation of reproduction is an ancient function, conserved in relaxin signaling in mammals.

Arthropod IGF, relaxin and gonadulin, putative orthologs of Drosophila insulin-like peptides 6, 7 and 8, likely originated from an ancient gene triplication

“…Insulin and related peptides typically stimulate growth and reproduction, so its presence in the ovaries and testes suggests a function in reproduction. Its presence in unfecundated eggs of Blattella suggests that within the ovary it are the oocytes themselves that express gonadulin, likely the follicle cells that in Drosophila have been shown to express Drosophila ilp 8 ( Liao & Nässel, 2020 ). In the crab Portunus trituberculatus gonadulin expression is on occasion very high in the gonads ( Veenstra, 2020 ), but the very variable degree of expression makes it difficult to see this hormone as merely stimulating reproduction.…”

“…All these ilps are believed to activate the single Drosophila insulin RTK, while Drosophila ilps 7 and 8 are either known (ilp 8) or suspected (ilp 7) to activate Drosophila LGRs 3 and 4 respectively ( Vallejo et al, 2015 ; Gontijo & Garelli, 2018 ; Veenstra, Rombauts & Grbic, 2012 ). Drosophila ilp 7 is expressed by neurons in the abdominal neuromeres in a sex specific manner ( Miguel-Aliaga, Thor & Gould, 2008 ; Yang et al, 2008 ; Castellanos, Tang & Allan, 2013 ), while ilp 8 is expressed by the imaginal disks as well as the ovary and testes as shown by flyatlas ( Gontijo & Garelli, 2018 ; Liao & Nässel, 2020 ).…”

“…When it is no longer released this is used by the brain as a signal to initiate metamorphosis ( Garelli et al, 2012 ; Colombani, Andersen & Léopold, 2012 ; Jaszczak et al, 2016 ). Drosophila ilp 8 is furthermore produced by the testes and ovaries ( Liao & Nässel, 2020 ) and since imaginal disks are only present in holometabolous insects, it is tempting to speculate that the gonads are the original site of expression of orthologs of this peptide. I had previously suggested that the crustacean androgenic insulin-like peptide that stimulates premature sexual maturation in male crustaceans and can induce sex reversal in females, might be an ortholog of Drosophila ilp 8 ( Veenstra, 2016b ).…”

Identification of Gonadulin and Insulin-Like Growth Factor From Migratory Locusts and Their Importance in Reproduction in Locusta migratoria

“…The expression of LGR3 in the central nervous system may thus be consistent with gonadulin signaling a need for more food. It may also indirectly exert its effect on vitellogenin synthesis by controlling the production or release of other insect hormones, as was proposed for D. melanogaster (19), while its expression in the ovary itself may reflect an autocrine stimulation of vitellogenin uptake.…”

“…A very well conserved peptide across insect species is dilp 7, which has also been called relaxin, and influences lipid synthesis and regulates egg laying decisions ( 15 , 16 ). Interestingly, dilp 8 is expressed in imaginal discs and coordinates aspects of metamorphosis ( 17 ), and is also expressed in both the testis and the ovary ( 18 , 19 ). Interestingly, although the sequence of this ILP is poorly conserved across species, putative orthologs of dilp8 were recently identified from a variety of arthropods, including the brown marmorated stink bug, Halyomorpha halys , the southeastern field cricket, Gryllus rubens and R. prolixus , and since their transcripts are highly expressed in the ovaries, have been called gonadulins ( 3 , 20 ).…”