Prajapati, Gaurav et al. (2019) International Worm Meeting "Four new members of the sperm activation pathway."

Austin, Sarojani, LaMunyon, Craig, Aldaco, Fermina, Keung, Steven, Prajapati, Gaurav

[International Worm Meeting, 2019]

Becoming active is a critical event in the life of a sperm cell. Failure to activate at exactly the right time dooms a sperm to failure. Here, we describe four genes that add to the growing list of a set involved in determining sperm activation. These genes were identified from mutations recovered in a suppression screen of spe-27(it132ts) IV. Worms homozygous for the spe-27 mutation are normally sterile at 25C, but a mutation in the identified genes results in a small number of sperm that activate prematurely. Normally, sperm remain as inactive spermatids until an extracellular signal interacts with surface receptors on the spermatid. Two parallel activation pathways exist: (i) a TRY-5 signal in male seminal fluid interacting with a SWM-1 receptor, and (ii) an unknown signal in both seminal fluid and in the hermaphrodite reproductive tract interacting with the SPE-8 group receptor complex (SPE-8, -12, -19, -27, -29, and -43). Inside the cell, the signal to activate unleashes a plethora of responses, including zinc, calcium, and pH spikes, and modification of phosphorylation states. The four new genes were identified through one-step whole genome sequencing and SNP-mapping. One of the genes described here is F55F8.7, which was identified from mutation zq9 and is a predicted tyrosine phosphatase. F55F8.7 has 9 paralogs expressed primarily or exclusively in sperm, which is likely the reason that a knockout of the gene has no phenotype. This gene expands the list of kinases/phosphatases involved in sperm activation, including kinases spe-6 and spe-8, and phosphatases gsp-3 and gsp-4. Another gene, preliminarily identified from mutation zq13, is aman-3, a sperm upregulated gene encoding a predicted alpha-mannosidase. Involvement of aman-3 suggests that modification of protein glycosylation is also involved in sperm control of sperm activation. The mutation zq14 was identified as affecting Y73F8A.14, which has no predicted function and has only nematode orthologs. Finally, the hc202 mutation was determined to affect F29B9.7, another gene with no predicted function or orthologs outside nematodes. While the exact role of these gene products remains unclear, they add to the set of genes that have been functionally determined to be involved in sperm activation.

Prajapati, Gaurav et al. (2017) International Worm Meeting "Premature sperm activation is prevented by SPE-6 in the nematode C. elegans."

Sokolich, Thomas, Shakes, Diane, LaMunyon, Craig, Prajapati, Gaurav

[International Worm Meeting, 2017]

Spermatogenesis, pauses only at the penultimate stage, the spermatid. The pause ends when an external signal activates the spermatid into a spermatozoon, activation involves two pathways (i) TRY-5 in seminal fluid interacting with SWM-1 and (ii) an unknown signal both seminal fluid and in the hermaphrodite interacting with the SPE-8 group complex. Mutations in the spe-8 group genes (spe-8, -12, -19, -27, -29) result in spermatids that cannot activate in unmated hermaphrodites. We focus on one likely target of the SPE-8 complex, the SPE-6 serine- threonine kinase. Here, we test the hypothesis that active SPE-6 maintains the spermatid stage, and that activation is achieved through downregulation of SPE-6. A suppressor screen from Sam Ward's lab with spe-27(it132) generated numerous suppressor mutations that restore partial fertility. Our work examines the suppressor mutations recovered in spe-6. Null spe-6 mutations stall spermatogenesis at the transition from primary to secondary spermatocytes. In contrast, the spe-6 suppressor mutations cause premature spermatid activation, bypassing SPE-8 group signaling. The alleles we focus on are: hc166, hc176, hc188, hc190, zq10, zq11, and zq18. These mutations likely reduce function and are scattered across the gene, suggesting that lower of SPE-6 function initiates activation. One mutation, zq11, is an allele specific suppressor of spe27(it132). zq11 does not suppress spe-27(it110), indicating that SPE-6 and SPE-27 interact physically and suggest that SPE-6 is a target of SPE-8 group signaling. Two other spe-6 alleles have intronic mutations. The hc190 and zq18 alleles are G to A substitutions of the 1st and 5th bases, respectively, of the 5' end of the intron a highly-conserved site for splicing. These conditional mutations cause premature sperm activation and partial mRNA loss, although normal splices were present for both mutants at 25 deg C. Thus, loss of SPE-6 function through a simple drop of transcript abundance is sufficient to cause premature activation. Thus, SPE-6 function is necessary to maintain the spermatid stage. We have used CRISPR/Cas9 to induce similar intronic mutations in spe-44. However, neither mutation in spe-44 produces a phenotype. We are now attempting to create both mutations in the same intron.

Prajapati, Gaurav et al. (2017) International Worm Meeting "K01D12.7: a sperm gene identified through its suppression of spe-27 by inducing premature spermatid activation."

LaMunyon, Craig, Prajapati, Gaurav, Sokolich, Thomas, Lynn, Nicole, Keung, Steven, Aldaco, Fermina

[International Worm Meeting, 2017]

A critical decision in the life of a male gamete is when to become active. The timing of this seminal event normally co-occurs with ejaculation for males. Failure to activate in a timely manner dooms the sperm to the Darwinian junk heap. In most species, sperm activation occurs after the nuclear material is ultra-condensed and expressionless. Thus, the genesis of motility is orchestrated through preexisting gene products, which in C. elegans either prevent or promote activation. An external signal promotes activation by interfacing with surface receptors on the spermatid. Interestingly, two parallel activation pathways exist: (i) a TRY-5 signal in male seminal fluid interacting with a SWM-1 receptor, and (ii) an unknown signal in both seminal fluid and in the hermaphrodite reproductive tract interacting with the SPE-8 group receptor complex (SPE-8, -12, -19, -27, and -29). Here, we focus on the SPE-8 group complex, which awaits the activation signal bound to the plasma membrane. Upon encountering the signal, SPE-8 is released to the cell's interior, where this tyrosine kinase presumably phosphorylates targets. Mutations in any of the spe-8 group genes results in spermatids that cannot activate in unmated hermaphrodites. A suppressor screen of spe-27(it132ts) recovered numerous mutations that restore partial fertility. Our laboratory has identified a number of these spe-27 suppressor mutations. In most cases, these mutations result in premature activation, bypassing the signaling pathways altogether. We have learned that SPE-6 prevents activation until being down regulated, SPE-4, SPE-46, and SPE-47 all can be mutated to cause premature sperm activation, and that SPE-6 and SPE-27 interact directly during activation. Here, we describe the K01D12.7(hc201)V suppressor mutation. Like the other suppressors, hc201 bypasses the need for signaling and causes premature sperm activation. The K01D12.7 protein is sperm specific and tiny, has 4 sperm specific paralogs, and resembles absolutely nothing outside of its nematode paralogs and homologs. A reporter construct indicates plasma membrane localization but does not rule out localization to other cellular compartments. The role of this gene product in spermatogenesis is still not clear.

LaMunyon, Craig et al. (2019) International Worm Meeting "Two sperm gene paralogs that are not functionally redundant."

Kang, Joon, Keung, Steven, Austin, Sarojani, LaMunyon, Craig, Aldaco, Fermina, Prajapati, Gaurav

[International Worm Meeting, 2019]

Our laboratory earlier identified the sperm gene spe-47 as being involved in sperm development and activation. There is one paralog of spe-47 in the C. elegans genome: Y48B6A.5. Here, we examine this gene to determine if it is functionally redundant to spe-47. Using the MosSCI technique, translational reporters of both genes were created. Expression for the two genes is very similar: they colocalize, appear in late pachytene and associate with the forming Fibrous Body-Membranous Organelles (FB-MOs). By the time the spermatids form both proteins have disappeared in a way that suggests they are actively degraded. Knockout mutations in both genes have little effect on spermatogenesis, either separately or in combination. Thespe-47 gene was originally identified from the hc198 mutation that causes premature sperm activation. We created an equivalent mutation in Y48B6.5, but this mutation had no effect on sperm activation. Our results indicate that while the sequences are very similar, and their proteins colocalize in space and time, we cannot support the hypothesis that the two genes are functionally redundant.

De La Cruz, Aubrie et al. (2015) International Worm Meeting "How to remain inactive: maintaining the spermatid stage requires inhibition and properly formed FB-MOs."

De La Cruz, Aubrie, Sullivan, Nicholas, Huang, Haifen, Christensen, Matthew, Prajapati, Gaurav, Clark, Jessica, LaMunyon, Craig, Sokolich, Thomas

[International Worm Meeting, 2015]

After their production, spermatids remain quiescent until they receive a signal to activate, stimulating a wholesale reorganization resulting in the crawling spermatozoon. Activation is signaled by the protease TRY-5 in males and by zinc in both hermaphrodites and males. Here we focus on zinc activation, which triggers a signal transduction pathway involving products of the spe-8 group genes (spe-8, spe-12, spe-19, spe-27, and spe-29). Mutations in any of the spe-8 group genes prevent spermiogenesis, and this group likely forms the receptor complex that interacts with extracellular zinc. SPE-12 and SPE-19 possess extracellular domains, and we have shown that SPE-8 (a tyrosine kinase) is localized to the membrane by other SPE-8 group members, but it moves to the interior during activation. A suppressor screen of spe-27(it132ts)I identified numerous mutations that restore fertility to spe-27 mutants. More than 20 suppressor mutations were recovered in spe-6. These spe-6 suppressors induce a partial loss of function, and most cause premature sperm activation. Thus, SPE-6 appears to inhibit spermatid activation. However, spe-6(zq11), is an allele specific suppressor that does not induce rampant premature activation, indicating that SPE-6 and SPE-27 interact physically. The remaining suppressor mutations all bypass the sperm activation signal with no two mutations affecting the same gene. One mutation affects spe-4, which encodes a Presenilin-1 homolog that localizes to the fibrous body-membranous organelle (FB-MO) membrane and is involved in loading MSP (major sperm protein) in the FB. Another mutation affected spe-47, whose product appears to associate with the FB-MO during its formation. SPE-47 is then degraded in secondary spermatocytes, long before spermatids are formed. A paralog of spe-47, Y48B6A.5, has a similar pattern of expression and may have redundant function with SPE-47. A fourth gene affected by a suppressor mutation is spe-46, whose role in spermatogenesis is unknown, but the suppressor mutation also causes a host of defects, in addition to premature sperm activation. Finally, a small gene, K01D12.7, also harbors a suppressor mutation. The function of K01D12.7 is unknown, but it resembles a signaling molecule. Our working hypothesis is that functional SPE-6 inhibits activation and interacts with SPE-27 during activation. Further, spermatids cannot refrain from activating when mutations cause errors in the formation of the FB-MO. Thus, both inhibition and properly formed FB-MOs are necessary to maintain the inactive spermatid.