Biomedical subjects

M F Wolfner

Publications and source records attributed to M F Wolfner.

At least 19 recordsLinked to original sources

PubMed2005

Cross-species comparison of Drosophila male accessory gland protein genes.

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.

Animals↗

PubMed2005

Fates and targets of male accessory gland proteins in mated female Drosophila melanogaster.

Male accessory gland proteins (Acps) in Drosophila are components of the seminal fluid and are transferred to females during copulation. In mated females, Acps enhance egg production, augment sperm storage, induce refractory mating behaviors, and affect the female's longevity. To address the functions of eight previously uncharacterized Acps and further analyze five others, we determined the tissues to which they target after transfer to females. Each Acp has multiple targets and is unique in its pattern of localization. Within the reproductive tract, Acps target to the uterus, oviduct, sperm storage organs, ovary and oocytes. Some Acps also leave the reproductive tract, to enter the hemolymph. Some Acps are detected on the surface of eggs laid by mated females but were not detectable within those eggs. Our results can help to identify the likely functions of these Acps as well as to create models for the mechanism of action of Acps.

Animals↗

PubMed2002

The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila.

During mating, males transfer seminal proteins and peptides, along with sperm, to their mates. In Drosophila melanogaster, seminal proteins made in the male's accessory gland stimulate females' egg production and ovulation, reduce their receptivity to mating, mediate sperm storage, cause part of the survival cost of mating to females, and may protect reproductive tracts or gametes from microbial attack. The physiological functions of these proteins indicate that males provide their mates with molecules that initiate important reproductive responses in females. A new comprehensive EST screen, in conjunction with earlier screens, has identified approximately 90% of the predicted secreted accessory gland proteins (Acps). Most Acps are novel proteins and many appear to be secreted peptides or prohormones. Acps also include modification enzymes such as proteases and their inhibitors, and lipases. An apparent prohormonal Acp, ovulin (Acp26Aa) stimulates ovulation in mated Drosophila females. Another male-derived protein, the large glycoprotein Acp36DE, is needed for sperm storage in the mated female and through this action can also affect sperm precedence, indirectly. A third seminal protein, the protease inhibitor Acp62F, is a candidate for contributing to the survival cost of mating, given its toxicity in ectopic expression assays. That male-derived molecules manipulate females in these ways can result in a molecular conflict between the sexes that can drive the rapid evolution of Acps. Supporting this hypothesis, an unusually high fraction of Acps show signs consistent with their being targets of positive Darwinian selection.

Animals↗

PubMed2001

The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster.

Drosophila melanogaster male accessory gland proteins (Acps) that are transferred in the ejaculate with sperm mediate post-mating competition for fertilizations between males. The actions of Acps include effects on oviposition and ovulation, receptivity and sperm storage. Two Acps that modulate egg production are Acp26Aa (ovulin) and Acp70A (the sex peptide). Acp26Aa acts specifically on the process of ovulation (the release of mature eggs from the ovaries), which is initiated 1.5 h after mating. In contrast, sperm storage can take as long as 6-9 h to complete. Initial ovulations after matings by virgin females will therefore occur before all sperm are fully stored and the extra eggs initially laid as a result of Acp26Aa transfer are expected to be inefficiently fertilized. Acp26Aa-mediated release of existing eggs should not cause a significant energetic cost or lead to a decrease in female lifespan assuming, as seems likely, that the energetic cost of egg laying comes from de novo egg synthesis (oogenesis) rather than from ovulation. We tested these predictions using Acp26Aa(1) mutant males that lack Acp26Aa but are normal for other Acps and Acp26Aa(2) males that transfer a truncated but fully functional Acp26Aa protein. Females mating with Acp26Aa(2) (truncation) males that received functional Acp26Aa produced significantly more eggs following their first matings than did mates of Acp26Aa(1) (null) males. However, as predicted above, these extra eggs, which were laid as a result of Acp26Aa transfer to virgin females, showed significantly lower egg hatchability. Control experiments indicated that this lower hatchability was due to lower rates of fertilization at early post-mating times. There was no drop in egg hatchability in subsequent non-virgin matings. In addition, as predicted above, females that did or did not receive Acp26Aa did not differ in survival, lifetime fecundity or lifetime progeny, indicating that Acp26Aa transfer does not represent a significant energetic cost for females and does not contribute to the survival cost of mating. Acp26Aa appears to remove a block to oogenesis by causing the clearing out of existing mature eggs and, thus, indirectly allowing oogenesis to be initiated immediately after mating. The results show that subtle processes coordinate the stimulation of egg production and sperm storage in mating pairs.

Animals↗

PubMed2001

Ovulation triggers activation of Drosophila oocytes.

Drosophila melanogaster mature oocytes in ovaries are arrested at metaphase I of meiosis. Eggs that have reached the uterus have released this arrest. It was not known where in the female reproductive tract egg activation occurs and what triggers it. We investigated when and where the egg is activated in Drosophila in vivo and at what meiotic stage the egg is fertilized. We found that changes in the egg's envelope's permeability, one feature of activation, initiate during ovulation, even while most of the egg is still within the ovary. The egg becomes impermeable as it proceeds down the oviducts; the process is complete by the time the egg is in the uterus. Cross-linking of vitelline membrane protein sV23 also increases progressively as the egg moves through the oviducts and the uterus. Activation also triggers meiosis to resume before the egg reaches the uterus, such that the earliest eggs that reach the uterus are in anaphase I. We discuss models for Drosophila egg activation in vivo.

Animals↗

PubMed2001

Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila.

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen ( approximately 11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that approximately 90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.

Analysis of Variance↗

PubMed2001

Identification and characterization of the major Drosophila melanogaster mating plug protein.

In many insects, semen coagulates into a mating plug at the distal part of the female's genital tract. Mating plugs have been proposed to facilitate sperm movement or to prevent subsequent matings or sperm loss. The molecular constituents of insect mating plugs have not previously been characterized. Here we report that an abundant autofluorescent protein made by the Drosophila melanogaster male's ejaculatory bulb is a major constituent of the posterior region of the mating plug. Identities in size, chromosomal location and expression pattern indicate that the autofluorescent protein is PEB-me, an abundant ejaculatory bulb protein reported by Ludwig et al. [Biochem. Genet. 29 (1991) 215]. We cloned and sequenced the RNA encoding this protein. The transcript, which is male-specific and expressed only in the ejaculatory bulb, encodes a 377 a.a. predicted secreted protein with PGG repeats similar to those in homopolymer-forming proteins found in spider silk.

Amino Acid Sequence↗

PubMed2001

Positive Darwinian selection drives the evolution of several female reproductive proteins in mammals.

Rapid evolution driven by positive Darwinian selection is a recurrent theme in male reproductive protein evolution. In contrast, positive selection has never been demonstrated for female reproductive proteins. Here, we perform phylogeny-based tests on three female mammalian fertilization proteins and demonstrate positive selection promoting their divergence. Two of these female fertilization proteins, the zona pellucida glycoproteins ZP2 and ZP3, are part of the mammalian egg coat. Several sites identified in ZP3 as likely to be under positive selection are located in a region previously demonstrated to be involved in species-specific sperm-egg interaction, suggesting the selective pressure is related to male-female interaction. The results provide long-sought evidence for two evolutionary hypotheses: sperm competition and sexual conflict.

Animals↗

PubMed2001

Male contributions to egg production: the role of accessory gland products and sperm in Drosophila melanogaster.

Drosophila melanogatser seminal fluid components, accessory gland proteins (Acps) and sperm, induce females to deposit high numbers of fertilized eggs for about 11 days. This high and sustained level of egg deposition requires that oogenesis be stimulated to provide the necessary mature oocytes. To investigate the relative timing and contributions of Acps and sperm in the egg-production process, we examined the rates of oogenic progression and egg deposition in females mated to genetically altered males that have seminal fluid deficient in Acps and/or sperm, and subjected these data to path analysis. We found that Acps and sperm are complementary stimuli necessary for inducing high rates of oogenic progression and rapid egg deposition. While egg deposition and oogenic progression can be induced by Acps alone, both Acps and sperm are required for maximum stimulation of oogenic progression and egg deposition immediately after mating.

Animals↗

PubMed2001

The nuclear envelope: emerging roles in development and disease.

The chromosomes of eukaryotic cells are separated from the cytoplasm by the nuclear envelope. The nuclear envelope includes two riveted membranes, plus embedded pore complexes that mediate nuclear import and export. In this sense, the nuclear envelope is truly a border zone. However, the envelope also links directly to chromosomes, and anchors two major infrastructures--the nuclear lamina and Tpr filaments--to the nuclear perimeter. Proteins of the nuclear envelope mediate a variety of fundamental activities, including DNA replication, gene expression and silencing, chromatin organization, cell division, apoptosis, sperm nuclear remodeling, the behavior of pronuclei, cell fate determination, nuclear migration and cell polarity. Furthermore, mutations in nuclear lamins and lamin-binding proteins cause tissue-specific inherited diseases. This special issue of Cell and Molecular Life Sciences is devoted to recent major advances in the characterization of nuclear envelope proteins and their roles. We offer here an overview of the topics covered in this issue of CMLS, and also discuss the emerging recognition that the nuclear envelope is an organelle critical for a wide range of genetic and developmental activity in multicellular organisms.

Animals↗

PubMed2000

The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster.

A crucial factor determining sperm fertilization success in multiply mated Drosophila melanogaster females is the efficiency with which sperm are stored. This process is modulated by the accessory gland protein Acp36DE. In this study, we show that the effect of Acp36DE on sperm storage itself alters the outcome of sperm competition. As second-mating males, Acp36DE1 (null) males had significantly lower P2-values than Acp36DE2 (truncation) or Acp36DE+ (control) males, as might be expected as the null males' sperm are poorly stored. We used spermless males, which are null for Acp36DE, to show that, in the absence of sperm co-transfer, Acp36DE itself could not displace first-male sperm. The results therefore suggest that males null for Acp36DE suffer in sperm displacement because fewer sperm are stored or retained, not because Acp36DE itself displaces sperm. Acp36DE1 (null) males also gained significantly fewer fertilizations than controls when they were the first males to mate. Using spermless males, we also showed that significantly more second-male offspring were produced following the transfer of Acp36DE by spermless first-mating males. This implies that the transfer of Acp36DE itself by the first male facilitated the storage or use of the second male's sperm and that co-transfer with sperm is not necessary for Acp36DE effects on second-male sperm storage. Acp36DE may persist in the reproductive tract and aid the storage of any sperm including those of later-mating males or prime the female for future efficient sperm storage. Our results indicate that mutations in genes that affect sperm storage can drastically affect the outcome of sperm competition.

Animals↗

PubMed2000

The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary.

Mating stimulates the rate of egg-laying by female insects. In Drosophila melanogaster this stimulation is initially caused by seminal fluid molecules transferred from the male (Acps or accessory gland proteins; reviewed in [1] [2] [3]). Egg-laying is a multi-step process. It begins with oocyte release by the ovaries, followed by egg movement down the oviducts and the deposition of eggs onto the substratum. Although two Acps are known to stimulate egg-laying [4] [5], they were detected by assays that do not discriminate between the steps of this process or allow examination of its earliest changes [4] [5] [6] [7]. To determine how egg-laying is regulated, we developed a generally applicable assay to separate the process into quantifiable steps, allowing us to assess the ovulation pattern and rate of egg movement. As the steps are interdependent yet potentially subject to independent controls, we determined the contribution of each step and effector independent of the others. We used a statistical method [8] [9] that separately considers and quantifies each 'path' to a common end. We found that the prohormone-like molecule Acp26Aa [5] [10] stimulates the first step in egg-laying - release of oocytes by the ovary. During mating, Acp26Aa begins to accumulate at the base of the ovaries, a position consistent with action on the ovarian musculature to mediate oocyte release. Understanding how individual Acps regulate egg-laying in fruitflies will help provide a full molecular picture of insects' prodigious fertility, of reproductive hormones, and of the roles of these rapidly evolving proteins [11] [12].

Animals↗

PubMed2000

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins.

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.

Animals↗

PubMed1999

Nuclear entry of the Drosophila melanogaster nuclear lamina protein YA correlates with developmentally regulated changes in its phosphorylation state.

The Drosophila melanogaster YA protein is a maternally provided nuclear lamina component that is essential during the transition from meiosis to mitosis at the beginning of embryogenesis. Localization of YA to the nuclear envelope is required for its function; this localization is cell cycle-dependent during embryogenesis. Here we show that the ability of YA to enter nuclei is modulated during development. In developing egg chambers, YA protein is made but excluded from nuclei of nurse cells and oocytes; upon egg activation, YA acquires the ability to enter nuclei and becomes incorporated into the nuclear lamina in unfertilized eggs and embryos. This localization switch correlates with changes in the phosphorylation state of YA. YA in ovaries is hyperphosphorylated relative to YA in unfertilized eggs and embryos. Through site-directed mutagenesis, we identified 443T, a potential phosphorylation site for both cyclin-dependent protein kinase and mitogen-activated-protein kinase, as one of the sites likely involved in this developmental control. Our results suggest that phosphorylation plays a role in modulating the localization of YA during development. A model for developmental regulation of the nuclear entry of YA is proposed and implications for understanding Drosophila egg activation are discussed.

Animals↗

PubMed1999

Wise, winsome, or weird? Mechanisms of sperm storage in female animals.

Female sperm storage is an integral part of the reproductive pattern of many species. In the female, sperm become sequestered in specialized storage organs or reservoirs, where they may remain for several days, weeks, months, or years before being used to fertilize eggs. Several different but interrelated mechanisms are used by animals to target the sperm to the portion of the female genital tract adapted for sperm storage. Both males and females influence this process. This review describes themes among the mechanisms and molecules necessary for sperm to become efficiently stored in females and the roles that the female storage organs play in the nourishment, protection, and release of stored sperm.

Animals↗

PubMed1999

Drosophila seminal fluid proteins enter the circulatory system of the mated female fly by crossing the posterior vaginal wall.

Seminal fluid proteins from males of many insect species affect the behavior and physiology of their mates. In some cases, these effects result from entry of the proteins into the female's circulatory system. In the fruit fly Drosophila melanogaster, some seminal fluid proteins enter the female's circulatory system after transfer from the male while others remain confined within the reproductive tract. To address where and how seminal fluid proteins enter the hemolymph of the mated female, we compared the kinetics of transfer and localization in mated females of two seminal fluid proteins that enter the hemolymph (Acp26Aa and Acp62F) and one that does not (Acp36DE). We also generated transgenic flies that produce Acp26Aa tagged with Aequorea victoria green fluorescent protein (GFP) to monitor its transfer in vivo. We report that Acps enter the female circulatory system from the posterior vagina immediately after insemination. The ability of Acps to enter the female hemolymph correlates with their ability to cross the intima that lines the posterior vagina. The ventral posterior vagina is structurally unlike other parts of the female reproductive tract in that it lacks muscles. We hypothesize that it has higher permeability thus affording access to the female's circulatory system.

Animals↗

PubMed1999

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.

Animals↗

PubMed1999

Mated Drosophila melanogaster females require a seminal fluid protein, Acp36DE, to store sperm efficiently.

Mated females of many animal species store sperm. Sperm storage profoundly influences the number, timing, and paternity of the female's progeny. To investigate mechanisms for sperm storage in Drosophila melanogaster, we generated and analyzed mutations in Acp36DE. Acp36DE is a male seminal fluid protein whose localization in mated females suggested a role in sperm storage. We report that male-derived Acp36DE is essential for efficient sperm storage by females. Acp36DE(1) (null) mutant males produced and transferred normal amounts of sperm and seminal fluid proteins. However, mates of Acp36DE(1) males stored only 15% as many sperm and produced 10% as many adult progeny as control-mated females. Moreover, without Acp36DE, mated females failed to maintain an elevated egg-laying rate and decreased receptivity, behaviors whose persistence (but not initiation) normally depends on the presence of stored sperm. Previous studies suggested that a barrier in the oviduct confines sperm and Acp36DE to a limited area near the storage organs. We show that Acp36DE is not required for barrier formation, but both Acp36DE and the barrier are required for maximal sperm storage. Acp36DE associates tightly with sperm. Our results indicate that Acp36DE is essential for the initial storage of sperm, and that it may also influence the arrangement and retention of stored sperm.

Animals↗