P L Sadler et al. (2001) International C. elegans Meeting "Making sperm without APC/C"

P L Sadler, D C Shakes

[International C. elegans Meeting, 2001]

Temperature-sensitive mutations in subunits of the anaphase promoting complex (APC/C) cause metaphase to anaphase transition (mat) defects in C. elegans meiosis. In adult upshifted mat hermaphrodites, the fertilized primary oocytes accumulate at metaphase I and consequently arrest development at the meiotic one cell stage. Likewise, adult upshifted mat males produce primary spermatocytes that accumulate in metaphase of meiosis I. However, despite a strong metaphase I meiotic cell cycle block, the mutant mat primary spermatocytes continue to develop and produce spermatozoa (Golden & Sadler et al., 2000). Like wild type secondary spermatocytes, metaphase I arrested mat primary spermatocytes progress normally through cellular morphogenesis and individualization. All of the events of the budding process occur in the presence of a stable meiosis I metaphase plate and spindle. This analysis of mat spermatogenesis suggests that late APC/C function, specifically the stimulus of M-phase exit, might be dispensable for the budding division in wild type spermatogenesis. To test this hypothesis, we analyzed wild type secondary spermatocytes for indicators of cell cycle exit and found that, like the mat primary spermatocytes, they not only exhibit persistent meiotic spindles but they also lack several key indicators of M-phase exit. Does this new understanding of C. elegans spermatogenesis suggest that it is advantageous for spermatocytes to remain in a M-phase-like state during spermiogenesis? Perhaps C. elegans spermatocytes have evolved a unique way to circumvent the requirement for late APC/C function and thus discovered a faster way to make a sperm cell.

PL Sadler et al. (1999) International C. elegans Meeting "Getting to anaphase: emb-27 is required for chromosome segregation in the germline of C. elegans"

DC Shakes, PL Sadler

[International C. elegans Meeting, 1999]

The mitotic metaphase to anaphase transition in both yeast and vertebrates is driven by a multimeric ubiquitin ligase known as the anaphase promoting complex, APC. While the mitotic functions of APC are being actively investigated, almost nothing is known about APC's meiotic functions, a question which is particularly amenable to analysis in C. elegans. To address this question, we are studying both metaphase to anaphase defective (MAT) mutants and RNAi embryos depleted in maternal APC components (see Abstracts by Golden et al & Wille et al.). Of the five known MAT mutants, emb-27 is our most fully characterized to date. Our analysis indicates that EMB-27 is required for germline chromosome segregation during both meiosis and mitosis. Both emb-27 primary oocytes and spermatocytes mature normally but arrest development in meiotic metaphase I; thus they fail to either segregate homologs or progress to anaphase. Consequences of this metaphase arrest prove different for each type of gamete. emb-27 oocytes arrest development at the meiotic one cell stage. The mutant primary spermatocytes are also blocked in metaphase I; as a result, secondary spermatocytes are never formed. However because this cell cycle block is unlinked to gamete differentiation, anucleate spermatids still bud from the arrested primaries. In addition to their aforementioned meiotic defects, upshifted emb-27 adults also accumulate abnormally high numbers of metaphase figures within their mitotic zones. We believe that these excess metaphase figures arise from mitotic metaphase blockage rather than tumorous overproliferation because the mutants also exhibit abnormally small gonads and variably sized pachytene nuclei. However to further test the requirement for EMB-27 during germline mitosis, we examined the germline proliferation defects in mutants shifted to restrictive temperatures as L1 larvae. Our most severe alleles proved to be GLP. These combined results indicate that EMB-27 is essential for both mitotic germline proliferation and maintenance. Taken together, our results indicate that EMB-27 is specifically required to promote anaphase within the germline. To determine the molecular nature of this critical gene, emb-27 has been physically mapped to a two cosmid region on LGII. Of the potential gene candidates in this region, only APC6(cdc16) yields an RNAi phenotype which mimics the one-cell arrest seen in emb-27 mutants. We are currently sequencing this gene in the mutant alleles. Research funded, in part, by the Jeffress Foundation and NSF.

Sadler PL et al. (1995) International C. elegans Meeting "CENTROSOME RELATED MUTANTS DISRUPT MULTIPLE ASPECTS OF SPERMATOGENESIS IN C. ELEGANS"

Sadler PL, Shakes DC

[International C. elegans Meeting, 1995]

Originally classified as temperature sensitive maternal and paternal effect lethal mutants, emb-27 and emb-30 share highly similar phenotypes. Due to the general rarity of paternal effect phenotypes, we began analyzing gametogenic defects which might underlie the very striking 100 - 400 cell stage arrest of the mutants. Our results show that emb-27 and emb-30 disrupt multiple aspects of spermatogenesis in C. elegans. Spermatids from homozygous mutants show a range of morphological defects in cell size, nuclear distribution, and nuclear position. Under the most restrictive conditions, DNA missegregation is severely affected such that the majority of sperm are anucleate. Analysis of mutant meiotic figures indicates that most of these defects arise during the meiotic segregation of the haploid spermatids from the residual body. In addition, hemizygous males exhibit a wide range of germline defects including a breakdown in overall gonad polarity and DNA degradation in the more distal region of the gonad. Interestingly, synthesis and distribution of the major sperm proteins in both homozygote and hemizygote gonads is normal despite the chromosomal segregation defects. In terms of molecular identity, Tabbish et al (WBG Vol 13,#4,57 and at this meeting) recently described the rescue of emb-30 by a novel gamma tubulin gene. Given the similarities of emb-27 and emb-30 ,we are currently engaged in an analysis of possible centrosome related candidate genes for emb-27. One possibility is a t-complex gene residing on cosmid T05C12. A collaboration with Michel Leroux has been established to test the theory that emb-27 may encode tcp-1.

Sadler PL et al. (1995) International C. elegans Meeting "CENTROSOME RELATED MUTANTS DISRUPT MULTIPLE ASPECTS OF OOGENESIS IN C. ELEGANS"

Sadler PL, Shakes DC

[International C. elegans Meeting, 1995]

emb-27 and emb-30, canonical maternal and paternal lethals (Isenenghi et al., 1983), have now proven to be essential for multiple aspects of gametogenesis including oogenesis. In the mildest maternal effect lethality, embryos arrest as single cells with defects in polar body formation and cytokinesis. At 26oC, oocytes of the homozygous animals exhibit 12 separate chromosomes during diakinetic arrest as if chiasmata failed to form during prophase I. Experiments at intermediate temperatures to determine if recombination rates are altered are in progress. These phenotypes are similar for both emb-27 and emb-30. In hemizygous emb-27 animals at 25oC, we were surprised to find a range of phenotypes depending on the particular hemizygous combination. The "mildest" combination with mnDf91 is only slightly stronger than the homozygotes whereas the stronger combinations exhibit abnormalities in the overall polarity of the gonad and cause DNA degradation in progressively more distal regions. Molecular data from T. Johnson's lab suggests that these deletion breakpoints may all fall within cosmid T05C12 and could thus potentially nibble away at the emb-27 promoter. In emb-27; emb-30 doubles, the 16oC phenotype is similar to that of the singles at 26oC whereas the doubles at 26oC exhibit additional novel defects in germline proliferation. Recent work by Tabish et al, (WBG and this meeting) indicates that emb-30 encodes gamma-tubulin. Thus both emb-27 and emb-30 are likely to be involved in centrosome function and the various mutant defects reflect the importance of this structure throughout oogenesis.

PL Sadler et al. (1999) International C. elegans Meeting "Anucleate C. elegans sperm can crawl, fertilize oocytes, and direct anterior-posterior polarization of the 1-cell embryo."

DC Shakes, PL Sadler

[International C. elegans Meeting, 1999]

Although it has long been appreciated that spermiogenesis, the cellular transformation of spherical haploid spermatids into bipolar, motile spermatozoa, occurs in the absence of new DNA transcription, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack both nuclei and DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they can also crawl towards and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and A-P polarity in the one cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm (Goldstein and Hird, 1996), the sperm nucleus itself is not required. Also unaffected is the completion of oocyte meiosis, the formation of an impermeable eggshell, the process of cytoplasmic rearrangement, and the migration/rotation/centration of the oocyte pronucleus. Our investigation of these mutants confirms that in C. elegans, neither DNA nor a sperm nucleus is required for spermiogenesis, proper egg activation, or the induction of A-P polarity.

Sadler PL et al. (1995) International C. elegans Meeting "ARE NUCLEAR PARTIONING DEFECTS ACCOMPANIED BY GENERAL DEFECTS IN ORGANELLE PARTITIONING IN EMB-27 AND EMB-30?"

Sadler PL, Shakes DC

[International C. elegans Meeting, 1995]

Differential partitioning of cellular organelles occurs at multiple points throughout germ cell development and differentiation in C. elegans. Within the distal region of both male and hermaphrodite gonads, nuclei and nuclear associated organelles are partitioned towards the exterior of the syncytium while the endoplasmic reticulum and ribosomes remain unlocalized. The partitioning event culminates in the budding of a single nucleus and associated organelles to form primary oocytes and primary spermatocytes. Finally, spermatogenesis requires further differential partitioning as haploid spermatids are formed from primary spermatocytes. We are currently engaged in an analysis of a unique set of mutants, emb-27 and emb-30, which show promise as loci required for differential partitioning during germ cell development. Our light microscopic analysis of homozygous and hemizygous gonads indicates obvious defects in at least nuclear partitioning during multiple points of both oocyte and sperm development. Our current focus utilizes transmission electron microscopic studies are designed to evaluate the overall effects of these mutations on differential partitioning of organelles within the C. elegans gonad. The results from our investigation will be included in our poster presentation.

Sadler PL et al. (1997) International C. elegans Meeting "ANUCLEAR SPERMATOZOA CAN ACTIVATE OOCYTES BUT CAUSE PATERNAL EFFECT LETHALITY IN C. ELEGANS."

Sadler PL, Johnson N, Shakes DC

[International C. elegans Meeting, 1997]

The minimal but critical functions of sperm in C. elegans are to reach and fertilize an oocyte and to contribute both a haploid genome set and a competent MTOC to the newly formed zygote. In C. elegans, more than 60 genes have been described which effect the ability of the sperm to form or function properly. In a subset of spermatogenesis mutants known as paternal effect mutations, the sperm is competent to fertilize oocytes but the resulting embryos fail to develop properly (L'Hernault, C. elegans II). In our study (a continuation of initial observations by R. Cassada and B. Fiebich), we investigate the mechanism responsible for the paternal effect lethality associated with emb-27(g48ts) and emb-30(g53ts). Our phenotypic analysis of mutant emb-27(g48) and emb-30(g53) gonads reveals that the mutant sperm develop without a nucleus in both hermaphrodites and males due to temperature dependent chromosome loss during meiosis. Despite abnormalities in both size and nuclear content, the mutant sperm can undergo spermiogenesis and directional navigation towards the spermatheca. Furthermore, both emb-27(g48) and emb-30(g53) anucleate sperm can fertilize and activate oocytes. The resulting haploid embryos have abnormalities in the formation in the early blastomeres but nevertheless develop into multicellular embryos. These affected embryos undergo limited cellular differentiation but fail to undergo morphogenesis. Thus in C. elegans, while there are definite cleavage defects associated with fertilization by anucleate emb-27(g48) or emb-30(g53) sperm, the sperm themselves do not require a nucleus for cellular morphogenesis, directional locomotion or fertilization.

Minniti AN et al. (1996) Genetics "Genetic and molecular analysis of spe-27, a gene required for spermiogenesis in Caenorhabditis elegans hermaphrodites."

Sadler C, Ward S, Minniti AN

[Genetics, 1996]

Hermaphrodites with mutations in the spe-27 gene are self-sterile, laying only unfertilized eggs; mutant males are fertile. Hermaphrodites make spermatids that fail to activate to crawling spermatozoa so passing oocytes sweep them out of the spermatheca. These spermatids do activate and produce self-progeny if young mutant hermaphrodites are mated by fertile (or sterile) males. Spermatids isolated from either mutant males or hermaphrodites initiate activation in vitro when treated with proteases, but then arrest with spiky membrane projections that resemble those of a normal intermediate in pseudopod formation. These phenotypes are identical to spe-8 and spe-12 mutants. They can be explained if males and hermaphrodites have distinct pathways for spermatid activation, and these three genes are necessary only for the hermaphrodite pathway. Consistent with this model, when spe-27 mutant male spermatids without seminal fluid are artificially inseminated into hermaphrodites, they fail to activate. The spe-27 gene has been isolated, sequenced and its regulatory regions identified. The sequence predicts a 131 amino acid polypeptide that has no striking structural motifs and no resemblance to known proteins. Two of the mutations in spe-27 alter mRNA splicing; a third mutation is a temperature-sensitive missense mutation.

Nance JF et al. (1999) Genetics "spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans."

Sadler C, Ward S, Nance JF, Minniti AN

[Genetics, 1999]

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.

Minniti AN et al. (1995) International C. elegans Meeting "THE HERMAPHRODITE-INITIATED SPERMIOGENESIS PATHWAY."

Minniti AN, Nance JF, Sadler C, Ward S

[International C. elegans Meeting, 1995]

During spermiogenesis, round nonmotile spermatids are rapidly transformed into asymmetrical crawling spermatozoa. In addition to spe-8 and spe-12, we found two new genes, spe-27 and spe-29, that when mutated, disrupt spermiogenesis in an identical manner: mutant hermaphrodites are self-sterile, while mutant males are fertile. Sperm from both sexes activate abnormally in vitro, suggesting that these gene products are expressed in both sperm, but only hermaphrodites require them for activation. The hermaphrodite's spermatids, however, can be activated by mating. This phenotype has been explained by a model that has two distinct pathways of activation, one for males and one for hermaphrodites (Shakes and Ward, 1989). The four genes are needed only for the hermaphrodite pathway. spe-27 and spe-12 have been cloned. Their mRNA is found exclusively in the germline during spermatogenesis. Their predicted protein sequences show no similarities to known proteins. We are currently cloning spe-29. To further understand how these genes act during spermiogenesis we are determining the subcellular localization of their gene products. In addition, suppressor analysis is under way (see abstract by Paul Muhlrad and Samuel Ward) to look for other genes in this pathway and for interactions between these genes.