Stanfield, Gillian et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Sperm motility: to Swm or not to Swm?"

Mulia, Amanda, Zhong, Connie, Stanfield, Gillian

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The acquisition of motility is a critical step of sperm development. In C. elegans, sperm become motile during a process termed activation, in which the haploid spermatid undergoes rapid subcellular morphogenesis - fusing intracellular vesicles with the plasma membrane and rearranging its cytoskeleton - to become a polarized spermatozoon. Once activated, sperm are fully competent for directional motility, fertilization of oocytes and sperm competition. Activation is regulated in sex-specific and spatiotemporal ways to maximize fertility, i.e., males and hermaphrodites use distinct mechanisms to activate their sperm and do so at an optimal time. We are investigating the signals that regulate sperm activation, taking advantage of a mutant in which male sperm activate prematurely. Whereas male sperm are normally stored as immotile spermatids , which are activated upon transfer to a hermaphrodite, in swm-1 mutants sperm become activated inside the male and cannot be transferred efficiently. Swm (sperm activation without mating) males are readily distinguishable from those containing non-activated sperm, allowing us to perform a simple visual screen to identify suppressors. So far, the suppressor mutants fall into two categories based on fertility. We have several mutants that are both male- and hermaphrodite-fertile and thus may represent male-specific activation factors. This set includes a protease, try-5, that appears to act as an extracellular activation signal, and an SLC6 family transporter, snf-10, that functions in sperm. The other class of mutants is male-fertile but hermaphrodite self-sterile, like the known spe-8 group. This set includes an allele of spe-8 as well as alleles of two apparently new sperm genes, which we are cloning. We will report on progress with our ongoing screening for and c haracterization of regulators of sperm activation.

Gillian Stanfield (2006) Development & Evolution Meeting "Sperm Activation in C. elegans Males"

Gillian Stanfield

[Development & Evolution Meeting, 2006]

Sperm are highly specialized for their functions of migrating to and delivering their genetic information to an egg. In all animals, sperm must undergo processes of subcellular morphogenesis and maturation to become competent for motility and fertilization. In C. elegans, this competence is conferred during sperm activation, when unpolarized round spermatids are rapidly transformed to polarized, motile, mature spermatozoa. Genetic evidence suggests that there are multiple activation pathways that may be used differentially in males vs. hermaphrodites. We have found that the gene swm-1 is required for proper regulation of sperm activation in males. Whereas wild-type males delay activation of their sperm until after transfer to the hermaphrodite reproductive tract, in swm-1 males sperm become activated precociously within the male gonad. swm-1 encodes a small secreted serine protease inhibitor, suggesting that one method of initiating sperm in vivo involves proteolysis. We are using our swm-1 mutants to identify components of the proteolysis activation pathway by looking for genes for which loss of function suppresses the ectopic activation seen in swm-1 males. We are using RNAi to directly test predicted trypsin-like proteases, as these are good candidates for activation initiation proteases. So far, we have implicated at least one C. elegans trypsin in male sperm activation. We are also doing a chemical mutagenesis screen to search more generally for activation genes. We will report on our progress.

Gillian Stanfield et al. (2004) West Coast Worm Meeting "Sperm activation and male fertility in C. elegans"

Anne Villeneuve, Gillian Stanfield

[West Coast Worm Meeting, 2004]

C. elegans comes in two sexes: hermaphrodites, which make both eggs and sperm, and males, which make sperm that they can transfer to hermaphrodites. Hermaphrodite sperm are stored within the spermatheca, and as oocytes are made, they are ovulated into and fertilized within the spermatheca. Hermaphrodites normally use their own sperm to fertilize their eggs. If male sperm is available, it moves to the spermatheca and displaces resident hermaphrodite sperm; it then preferentially fertilizes the hermaphrodite's eggs. To characterize the cellular interactions involved in C. elegans reproduction, we have screened genetically for factors specifically important for male reproduction as assayed by a loss of male sperm precedence in crosses to wild-type hermaphrodites. One mutant from the screen, swm-1 , exhibits an unexpected defect in regulating the timing of sperm activation. In C. elegans , sperm move by amoeboid motility (i.e., crawl) using a pseudopod that forms during activation (spermiogenesis). Hermaphrodite sperm are activated rapidly after they complete the meiotic divisions, but male sperm are activated after transfer to a hermaphrodite. By contrast, swm-1 mutant sperm become activated within the male gonad (swm, s perm activation w ithout m ating). swm-1 males rarely transfer sperm to hermaphrodites, thus they rarely produce progeny. However, swm-1 male sperm can crawl to the spermatheca if they are transferred and can also move around inside the male gonad, suggesting that the fertility defect results from the transfer defect and not from a secondary defect in motility. We have cloned swm-1 and found that it encodes a small serine protease inhibitor. Others have shown that sperm can be activated in vitro by treatment with Pronase, a complex protease mixture, and our data may provide a link to this in vitro activation data. We suggest a model in which male sperm are activated by proteolysis upon transfer to the hermaphrodite and SWM-1 prevents this activation from occurring early. The genes spe-8 , -12 , -27 , and -29 are known to be required for hermaphrodite (but not male) sperm activation, but precisely how they function to activate sperm is not clear. Currently, we are testing whether premature activation in swm-1 mutants is dependent on these genes, and we are also trying to determine where ( i.e. , germ line or soma) SWM-1 is produced and where it functions to inhibit sperm activation.

Gillian M Stanfield et al. (2003) International Worm Meeting "Male fertility in C. elegans"

Anne M Villeneuve, Gillian M Stanfield

[International Worm Meeting, 2003]

Hermaphrodites do not need males to reproduce and may encounter them rarely, so it is advantageous for males to ensure that any rendezvous with a hermaphrodite results in reproductive success. When a male mates with a hermaphrodite, he transfers spermatids, which become activated and crawl to the spermatheca where they displace self sperm and preferentially fertilize the hermaphrodite's eggs. How do male sperm win? We are interested in how sperm functions (e.g., activation, motility, or interactions with the hermaphrodite reproductive tract and oocytes) are modulated to enhance males' ability to reproduce.We are screening for mutant males that fail to show precedence in crosses to hermaphrodites. We cross males from mutagenized him-5 lines to spe-8; dpy-4 tester hermaphrodites, which cannot activate their own spermatids and thus produce no progeny prior to mating. Matings with wild-type males result in nearly 100% cross progeny; we select lines for which testers produce an increased fraction of self progeny. Using this screen, we can identify both mutations that affect male sperm precedence and mutations that affect spermatogenesis specifically in males.For one class of mutants, male sperm appear morphologically normal but are used inefficiently. These mutants may define factors important for sperm competition. For the me69 mutant, hermaphrodites have normal brood sizes, suggesting that me69 hermaphrodite sperm are used efficiently in the absence of competition. We are using mitotracker dye to label male sperm so that their behavior within hermaphrodites can be tracked. Using this assay, it appears that me69 sperm accumulate more slowly in spermathecae than do wild-type sperm. Although this delay may not fully explain the precedence defect, these data do indicate that me69 may be involved in some aspect of sperm motility or targeting to the spermathecae.A second class of mutants activates sperm inappropriately within the male gonad. Whereas wild-type virgin males accumulate unactivated spermatids, me66 males contain activated sperm, which can crawl throughout the gonad. me66 males mate at normal rates but most fail to transfer sperm. Occasionally, sperm are transferred, and these migrate to the spermatheca. Our results suggest that reproductive success in males depends on inhibiting sperm activation until after transfer. We are testing whether premature activation in me66 is dependent on other spermiogenesis genes. We have obtained cosmid rescue of the me66 mutant and hope to report on the molecular identity of the me66 gene at the meeting.

Gillian M Stanfield et al. (2001) International C. elegans Meeting "Screening for sperm competition mutants"

Gillian M Stanfield, Anne M Villeneuve

[International C. elegans Meeting, 2001]

C. elegans hermaphrodites produce a supply of self sperm, which are normally used with extremely high efficiency (>99%). However, if a hermaphrodite mates with a male, its self sperm must compete with any transferred male sperm to fertilize oocytes. In this situation, sperm provided by the male are used preferentially(1). Male sperm precedence requires sperm motility(2) and involves displacement of self sperm from the hermaphrodite spermatheca. However, successive mating experiments have shown that sperm from different males do not exhibit any precedence relationship, indicating that sperm usage is not dictated by a "last in" mechanism of placement within the spermatheca and that there is a bona fide difference between hermaphrodite sperm and male sperm(1,3). Male sperm precedence is nearly complete: after mating, very few if any self progeny are produced until a mated hermaphrodite runs out of transferred male sperm. The strength of this effect provides a robust assay for alterations in the normal pattern of sperm usage. To identify factors that confer male sperm advantage, we are screening for mutants that do not show the normal precedence of male sperm. Specifically, we are assaying males from mutagenized him-5 lines for lack of precedence in crosses to spe-8; dpy-4 hermaphrodites. spe-8 hermaphrodites are defective in sperm activation, but spe-8 self sperm can be trans - activated upon mating to a male; dpy-4 provides a readily-scored marker for self vs. cross progeny. We expect that sperm precedence-defective males should produce sperm that are capable of fertilizing oocytes but do not suppress the production of hermaphrodite self progeny. Matings to such males should result in a mixture of self and cross progeny, and the relative numbers of self and cross progeny produced should be dependent on the numbers of each sperm type present within the hermaphrodite spermatheca. Sperm precedence mutants should be distinct from the previously described spe and fer mutants, which show defects in various aspects of spermatogenesis and/or fertilization(4). 1. Ward, S. and J.S. Carrel, Dev Biol 73 : 304-321, 1979. 2. Singson, A., K.L. Hill and S.W. L’Hernault, Genetics 152 : 201-208, 1999. 3. LaMunyon, C.W. and S. Ward, Experientia 51 :817-823, 1995. 4. L’Hernault, S.W., Spermatogenesis (p.271-294) in C. elegans II , 1997.

Gillian M Stanfield et al. (2002) West Coast Worm Meeting "Sperm competition and reproductive interactions in C. elegans"

Gillian M Stanfield, Anne M Villeneuve

[West Coast Worm Meeting, 2002]

The success of sexual reproduction is influenced by interactions between a sperm cell and the female reproductive tract, the egg, and other sperm. We are using C. elegans to identify and characterize male factors that are involved in these interactions. C. elegans comes in two sexes, self-fertilizing hermaphrodites and males. Early in reproductive development, a hermaphrodite generates and stores a supply of sperm; she then switches permanently to making oocytes, which are fertilized by her "self" sperm. However, if a male mates with the hermaphrodite, his sperm must compete with her self sperm to fertilize eggs. In C. elegans, males always win: male sperm are used preferentially, and self sperm are not used until the supply of male sperm is exhausted. It is clearly advantageous for a male to ensure that his sperm is used whenever mating occurs, since in wild populations this opportunity is not always assured.

Chavez, Daniela et al. (2011) International Worm Meeting "A sperm competition mutant with defects in sperm motility."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2011]

Although males are not necessary for reproduction in C. elegans, if mating occurs then male sperm win: cross progeny are produced to the exclusion of self progeny. We would like to understand the mechanistic basis for male sperm precedence. It has been shown previously that precedence is due to an intrinsic characteristic of male sperm cells, that it requires motility, and that it correlates with the larger size of male sperm. Using a genetic screen for males with reduced precedence, we identified a mutant, me69, that has defects in sperm usage under conditions of competition with wild-type sperm. me69 male sperm show reduced precedence when they compete with wild-type hermaphrodite sperm. However, me69 hermaphrodites have normal self fertility, arguing against a generalized spermatogenesis defect. Interestingly, me69 sperm show a normal size distribution. me69 male sperm show motility defects; after transfer to the hermaphrodite, sperm accumulate poorly in the spermatheca. We are analyzing their migration in more detail to determine whether this difference arises from poor targeting, poor retention, or some other alteration in motility. We have identified a candidate gene encoding a divergent kinase that is expressed in sperm. A deletion mutant shows migration and precedence defects similar to those of me69. We are determining the localization of this kinase and investigating the possibility that either its levels or localization may be different in males vs. hermaphrodites.

Chavez, Daniela et al. (2015) International Worm Meeting "Regulation of C. elegans Sperm Motility By Protease Signaling."

Chavez, Daniela, Stanfield, Gillian

[International Worm Meeting, 2015]

C. elegans sperm undergo rapid cellular morphogenesis to develop into motile, fertilization-competent cells. During this process, termed sperm activation, cells reorganize their cytoskeleton to generate a pseudopod used to crawl towards oocytes. Sperm activation is regulated by the serine protease, TRY-5, and the serine protease inhibitor, SWM-1. TRY-5 is a seminal fluid protease transferred to hermaphrodites during mating and has the characteristics of a male sperm activation factor. In swm-1 mutant males, sperm become prematurely activated and are inefficiently transferred to hermaphrodites during mating, resulting in reduced male fertility. These previous studies suggest a model in which SWM-1 delays sperm activation by inhibiting TRY-5 protease activity from cleaving sperm cell membrane factors to initiate activation. To test this model, we have sought to determine the relationship between SWM-1 and TRY-5. It was previously shown that TRY-5 is expressed in the male somatic gonad in cells of the seminal vesicle, valve and vas deferens. We now have shown that swm-1 is co-expressed with try-5 in a subset of vas deferens cuboidal cells and that SWM-1::mCherry and TRY-5::GFP co-localize in vesicles within these cells. We also have found that swm-1 is expressed in extra-gonadal somatic cells near the seminal vesicle, and is not expressed in the valve. These data are consistent with SWM-1's inhibiting TRY-5 activity, but also suggest an alternative model where SWM-1 indirectly inhibits TRY-5, perhaps by regulating the release of TRY-5 vesicles. To further dissect the relationship of SWM-1 and TRY-5, we are analyzing the functional domains of the proteins. SWM-1 is predicted to have two trypsin inhibitor-like (TIL) domains, and previous studies have suggested that these domains may have distinct functions. To directly test the role of SWM-1 TIL domains in regulating sperm activation, we are generating precise endogenous gene edits of the predicted reactive site residues of SWM-1. Similarly, we are testing the predicted catalytic residues of TRY-5 for their role in activation. In ongoing studies, we are testing our models for SWM-1 regulation of TRY-5 activity or release by expressing swm-1 and try-5 in specific tissues and visualizing the dynamics of vesicle release from the vas deferens during mating.

Chavez, Daniela et al. (2017) International Worm Meeting "Sex, Secretion, Muscle, and Males."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2017]

To successfully complete the journey to fertilize an oocyte, sperm rely on extracellular cues to direct their development and migration. In the final stages of maturation, during a process called sperm activation, sperm become polarized and develop a pseudopod used to crawl towards oocytes. Establishment of an extracellular signaling environment that spatially and temporally regulates activation is critical to fertility, as males with prematurely activated sperm are infertile. Key components of this regulation are the serine protease TRY-5 and a protease inhibitor, SWM-1. TRY-5 is a seminal fluid protein that is stored in the vas deferens and transferred to hermaphrodites, where it activates sperm. In the absence of SWM-1, TRY-5 spreads into the seminal vesicle, where sperm are stored, resulting in premature activation. We are investigating how TRY-5 is regulated by SWM-1 to ensure that sperm gain motility at the right place and time. We have discovered that somatic tissues create the sperm signaling environment that precisely controls the location and timing of activation. By analyzing a swm-1 transcriptional reporter and a SWM-1::mCherry knock-in, we determined that swm-1 is expressed by body wall muscle cells and cuboidal cells in the vas deferens while SWM-1 protein is present not only in the vas deferens but also in the seminal vesicle, surrounding sperm. SWM-1 is also present in the pseudocoelom, where it is taken up into coelomocytes. Surprisingly, tissue-specific expression suggests that muscle-derived SWM-1 is sufficient to rescue the premature activation of swm-1 mutants, but that vas deferens-derived SWM-1 fails to rescue. Our data suggest a model in which SWM-1 is secreted from muscle into the pseudocoelom and then taken up by the gonad where it attenuates TRY-5. Indeed, removing the secretion signal from SWM-1 caused it to accumulate in muscle, where it no longer rescues activation. These data show that we have identified a soma-to-germline signal critical to fertility and have determined that muscle is a key regulator of sperm cell success. Interestingly, uptake of proteins from the pseudocoelom is not specific to SWM-1, as extragonadal sources of mCherry and GFP also enter the gonad. Furthermore, vas deferens-specific mCherry is found in the pseudocoelom, thus, exchange of proteins into and out of the gonad is apparently not limited to SWM-1, and could be a general phenomenon. In ongoing work, we seek to determine whether swm-1 plays a second role within the hermaphrodite to promote reproductive success. SWM-1 is present in hermaphrodites, where it has a small but measurable effect on sperm activation. Additional SWM-1 is transferred in seminal fluid, but its role in this context is unknown. We are analyzing whether absence of SWM-1, in either the hermaphrodite or seminal fluid, affects sperm functions including migration, fertilization, or competition.

Fenker, Kristin et al. (2015) International Worm Meeting "SNF-10, a Solute Carrier 6 family protein, connects male-derived protease signals to the onset of sperm motility."

Stanfield, Gillian, Fenker, Kristin

[International Worm Meeting, 2015]

For sexually reproducing animals, motile sperm are critical for fertilization. This motility is tightly regulated, and cells must be able to sense and respond to environmental cues to ensure they initiate movement at the proper time and place. In C. elegans, sperm gain motility during a process termed activation, which is initiated in response to specific environmental signals. We are interested in the signals that regulate male sperm activation, and have identified the trypsin-like serine protease TRY-5 and the Solute Carrier 6 (SLC6) family protein SNF-10 as key components of the male-derived sperm activation pathway. In previous work, we demonstrated TRY-5 is present in seminal fluid and positively regulates activation, while SNF-10 is present in the sperm themselves, and is required for sperm to activate in response to the signal conferred by TRY-5.SNF-10 provides the first known link between protease signaling and the onset of sperm motility, and we are currently investigating both the mechanism of SNF-10's function and how the protein is regulated. SNF-10 localizes to the plasma membrane of spermatids and may also be present on membranous organelles (MOs), which are located at the cell periphery and fuse with the plasma membrane during activation. In spermatozoa, SNF-10 is restricted to the cell body plasma membrane and is absent from the pseudopod. We hypothesize that this localization, particularly the protein's presence on the sperm plasma membrane, puts SNF-10 in an ideal position for transducing extracellular signals into sperm. One way SNF-10 may do this is by importing specific cargo molecules into sperm, through a mechanism similar to other SLC6 proteins. We are currently testing if SNF-10 has such activity. Additionally, because SNF-10 localizes to the plasma membrane, we hypothesize it may be cleaved by TRY-5 as a means of positive regulation. Therefore, we are currently determining if cleavage of SNF-10 occurs when sperm activate, and if cleavage is necessary and/or sufficient for activation. Finally, preliminary data indicates that SNF-10 may promote activation through interactions with additional sperm proteins. We have found SNF-10 mislocalizes in mutant sperm with defects in either the formation or fusion of MOs, as well as in sperm with defects in the presenilin protease SPE-4, which localizes to the MOs. Experiments are in progress to further test the relationship of SNF-10 with these and other candidate MO proteins.