Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects.

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.

Proteins in the seminal fluid of animals with internal fertilization effect numerous responses in mated females that impact both male and female fertility. Among these proteins is the highly represented class of proteolysis regulators (proteases and their inhibitors). Though proteolysis regulators have now been identified in the seminal fluid of all animals in which proteomic studies of the seminal fluid have been conducted (as well as several other species in which they have not), a unified understanding of the importance of proteolysis to male fertilization success and other reproductive processes has not yet been achieved. In this review, we provide an overview of the identification of proteolysis regulators in the seminal fluid of humans and Drosophila melanogaster, the two species with the most comprehensively known seminal fluid proteomes. We also highlight reports demonstrating the functional significance of specific proteolysis regulators in reproductive and post-mating processes. Finally, we make broad suggestions for the direction of future research into the roles of both active seminal fluid proteolysis regulators and their inactive homologs, another significant class of seminal fluid proteins. We hope that this review aids researchers in pursuing a coordinated study of the functional significance of proteolysis regulators in semen.

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Females and males of sexually reproducing animals must cooperate at the molecular and cellular level for fertilization to succeed, even though some aspects of reproductive molecular biology appear to involve antagonistic interactions. We previously reported the existence of a proteolytic cascade in Drosophila melanogaster seminal fluid that is initiated in the male and ends in the female. This proteolytic cascade, which processes at least two seminal fluid proteins (Sfps), is a useful model for understanding the regulation of Sfp activities, including proteolysis cascades in mammals. Here, we investigated the activation mechanism of the downstream protease in the cascade, the astacin-family metalloprotease Seminal metalloprotease-1 (Semp1, CG11864), focusing on the relative contribution of the male and female to its activation. We identified a naturally occurring semp1 null mutation within the Drosophila Genetic Reference Panel. By expressing mutant forms of Semp1 in males homozygous for the null mutation, we discovered that cleavage is required for the complete activation of Semp1, and we defined at least two sites that are essential for this activational cleavage. These amino acid residues suggest a two-step mechanism for Semp1 activation, involving the action of at least two malederived proteases. Although the cascade's substrates potentially influence both fertility and sperm competition within the mated female, the role of female factors in the activation or activity of Semp1 is unknown. We show here that Semp1 can undergo its activational cleavage in male ejaculates, without female contributions, but that cleavage of Semp1's substrates does not proceed to completion in ejaculates, indicating an essential role for female factors in Semp1's full activity. In addition, we find that expression of Semp1 in virgin females demonstrates that females can activate this protease on their own, resulting in activity that is complete but substantially delayed.

Summary Sorely missing from the “toolkit” for directed differentiation of stem/progenitor cells are agonists of the BMP signaling pathway. Using a high throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1) inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using sgRNA confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/TGFβ signaling pathways, and a useful pharmacological reagent for directed differentiation of stem cells.

Studies of social behavior generally focus on interactions between two or more individual animals. However, these interactions are not simply between whole animals, but also occur between molecules that were produced by the interacting individuals. Such “molecular social interactions” can both influence and be influenced by the organismal-level social interactions. We illustrate this by reviewing the roles played by seminal fluid proteins (Sfps) in molecular social interactions between males and females of the fruit fly Drosophila melanogaster. Sfps, which are produced by males and transferred to females during mating, are involved in inherently social interactions with female-derived molecules, and they influence social interactions between males and females and between a female’s past and potential future mates. Here, we explore four examples of molecular social interactions involving D. melanogaster Sfps: processes that influence mating, sperm storage, ovulation, and ejaculate transfer. We consider the molecular and organismal players involved in each interaction and the consequences of their interplay for the reproductive success of both sexes. We conclude with a discussion of the ways in which Sfps can both shape and be shaped by (in an evolutionary sense) the molecular social interactions in which they are involved.

BackgroundThe genetic basis of postzygotic isolation is a central puzzle in evolutionary biology. Evolutionary forces causing hybrid sterility or inviability act on the responsible genes while they still are polymorphic, thus we have to study these traits as they arise, before isolation is complete.Methodology/Principal FindingsIsofemale strains of D. mojavensis vary significantly in their production of sterile F1 sons when females are crossed to D. arizonae males. We took advantage of the intraspecific polymorphism, in a novel design, to perform quantitative trait locus (QTL) mapping analyses directly on F1 hybrid male sterility itself. We found that the genetic architecture of the polymorphism for hybrid male sterility (HMS) in the F1 is complex, involving multiple QTL, epistasis, and cytoplasmic effects.Conclusions/SignificanceThe role of extensive intraspecific polymorphism, multiple QTL, and epistatic interactions in HMS in this young species pair shows that HMS is arising as a complex trait in this system. Directional selection alone would be unlikely to maintain polymorphism at multiple loci, thus we hypothesize that directional selection is unlikely to be the only evolutionary force influencing postzygotic isolation.