The Caenorhabditis Elegans Spe-6 Gene Is Required for Major Sperm

The Caenorhabditis Elegans Spe-6 Gene Is Required for Major Sperm

Copyright 0 1993 by the Genetics Societyof America The Caenorhabditis elegans spe-6Gene Is Required for Major Sperm Protein Assembly and Shows Second Site Non-Complementation With an Unlinked Deficiency Jacob P. Varkey,* Patricia L. Jansma,+Alicia N. Minniti* and Samuel Ward* "Department of Molecular and Cellular Biology and TArizona Research Laboratory, Division of Neurobiology, University of Arizona, Tucson, Arizona 85721 Manuscript received July 23, 1992 Accepted for publication September 1 1, 1992 ABSTRACT Caenorhabditis elegans spermatozoa move by crawling. Their motility requires thin cytoskeletal filaments assembled from a unique cytoskeletal protein, the major sperm protein (MSP). During normal sperm development the MSP is segregated to developing sperm by assembly into filaments that form a paracrystalline array in a transient organelle, the fibrous body-membranous organelle. Mutations in the spe-6 gene cause sterility because they lead to defective primary spermatocytes that do not form spermatids. In these mutant spermatocytes the MSP fails to assemble into fibrous body filaments. Instead, the unassembled MSP distributes throughout the cytoplasm and nucleus. Thus, the spe-6 gene product is necessary for normal MSP localization and assembly during sperm develop- ment. In addition to theirMSP assemblydefect, spe-6 mutant spermatocytes arrest meiosis at diakinesis although their spindle pole bodies still replicate and separate. This results in spermatocytes with four half-spindles surrounding condensed, but unsegregated, chromosomes. All four spe-6 alleles, as well as a chromosome III deficiency that deletes the spe-6 gene, fail to complement two small overlapping chromosome IV deficiencies, eDfl8 and eDfl9. This non-allele-specific second site non-complementa- tion suggests a concentration-dependent interaction between the spe-6 gene product and products of the gene(s) under eDfl8 and eDfl9, which include acluster of sperm-specific genes. Since MSP filament assembly is highly concentration-dependent in vitro, the non-complementation might be expected if the sperm-specific gene products under eDfl8 and eDfl9 were needed together with the spe-6 gene product to promoteMSP assembly. ELLULAR differentiation requires ordered syn- eta1 filaments participating in motility (SEPSENWOL, C thesis and organization of new cell components RIS and ROBERTS1989; ROBERTSand KING 1991). which must be integrated into preexisting structures. Although MSP filament assembly in vitro has been Integration requires mechanisms for targeting pro- recently achieved with Ascaris MSP(KING et al. 1992) teins to specific areas of the cell and assembling them and replicated for Caenorhabditis MSP (SMITH1992), into functional structures. The development of Cae- it is not known how MSP assembly is regulated in vivo norhabditis elegans sperm provides an opportunity to in either nematode species, nor is it known how fila- analyze such mechanisms genetically (WARD, ARGON ment assembly in developing spermatocytes compares and NELSON 1981; WARD 1986). to filament reassembly in the spermatozoa. In this In developing spermatocytes, a number of sperm paper we show that the C. elegans spermatogenesis- specific gene products are localized into a specialized defective gene, spe-6, encodes a product that is nec- transient organelle known as the fibrous body-mem- essary for the properlocalization and assembly ofMSP branous organelle (FB-MO) complex (ROBERTS,PA- filaments in the FB-MO complex. Wealso present VALKO and WARD 1986). These organelles are then genetic evidence suggesting that the spe-6 gene prod- segregatedinto the developingspermatids. The fi- uct may interact with the product(s) of one or more brous bodies contain paracrystalline arrays of 2-4-nm genes defined by a deficiency mapped to a different filaments that are formed of a small basic protein, the chromosome. major sperm protein, MSP (WOLF,HIRSH and MCIN- MATERIALSAND METHODS TOSH 1978;WARD and KLASS 1982; ROBERTS,PA- VALKO and WARD 1986). TheseMSP filaments disas- Strains and genetics: C. elegans var. Bristol, strain N2, semble in mature spermatids and reassemble in sper- was used as wild type. Most strains were first described by matozoa. Analysis of the structure of MSP filaments BRENNER(1974), except as noted below: spe-6(hc49)dpy- I8(e364) 111, spe-6(hc92)unc-32(eI 89) III, spe-6(hcl43)dpy- and their assembly during crawling motility in Ascaris IS(e364) III, spe-6(hcl46)dpy-l8(e364)III, spe-26(hc138ts) IV, spermatozoa shows that they are the major cytoskel- spe-7(mn242) II, spe-4(hc78) I, spe-32(it129ts) IV, dpy-I8(e364) Genetics 133: 79-86 Uanuary, 1993) 80 J. P. Varkey et al. Ill, unc-?2(e189) Ill. The deficiencies used in this analysis The tissue was stained in a moist chamber for 30 min in the wereeDfl8(IV)(H0m~1~ 1986),eDfl9(1V)(H0~~~1~1986) dark with 100 Hg/ml DAPI in PBS. A coverslip was placed and eDf2 (Ill) (ANDERSONand BRENNER1984). The strains on the tissue and pressed gently prior to microscopic ex- were maintained at appropriate temperatureson Escherichia amination. coli-seededplates, and genetic manipulations were per- Electron microscopy: Adult virgin males were hand dis- formed as described by BRENNER (1974). Anew allele of sected in SM salts, and the testes were transferred to 1% spe-6 was isolated by crossing ethyl methanesulfonate (EMS)- formaldehyde 1% glutaraldehyde inSM in a depression mutagenized N2 males with spe-6(hc49)dpyls(e364) homo- slide. The testes were fixed in a moist chamber for 1 hr at zygotes and screening the Fl outcross progeny for sterile room temperature and then embedded in 1% agar, and hermaphrodites. Similar Fl non-complementation screens small agar blockswith the tissue were incubated in the to chromosome IV deficiencies were done by crossing EMS- fixative overnight at 4". The blocks were rinsed in 10 mg/ mutagenized wild-type males to appropriate deficiency het- ml lysine for 20 min and then rinsed inSM. The blocks erozygous hermaphrodites and screening the F1 outcross were post fixed in 1% oso4in SM. Excess Os04was rinsed progeny for sterile hermaphrodites at 25 O. Instead of using out with water, and theblocks were stained in 0.5% aqueous morphological markers to distinguish the outcross progeny, uranyl acetate. The blocks were then dehydrated in a graded mated hermaphrodites were transferred daily to new plates series of ethanol and embedded in EPON/Araldite resin for and progeny hermaphrodites were selected onlyfrom plates sectioning. Thin sections were examined with a Philips 420 two days after mating that had approximately 50% males, electron microscope operating at 80 kV. ensuring that the hermaphrodites were also outcross. For Immuno-electronmicroscopy: Hand-dissected testes the analysis of the complementation of the chromosome IV were fixed as described above for conventional electron deficiencies eDfl8 and eDfl9 to different spe genes, herma- microscopy except that they were post fixed in 0.1 % OsOl phrodites heterozygous for the deficiency were crossed to in SM for 30 min. The testes were embedded in LR white homozygous males of temperature-sensitive alleles at per- resin following the manufacturer's directions (Polysciences, missive temperature or heterozygous males of noncondi- Warrington, Pennsylvania) without en bloc staining in uranyl tional alleles. acetate. The sections were picked on nickel grids, etched Light microscopy:Spermatogenesis was analyzed in both with sodium metaperiodate, washed thoroughly and then males and hermaphrodites by hand dissecting individual incubated in a 1:200 dilution of anti-MSP serum (BURKE worms in sperm medium (SM) and examining with Nomar- and WARD1983) for 30-60 min. Excess antibody was re- ski differential interference microscopy. SM is 5 mM HEPES moved by washing four times for a total of 20 min in PBS (pH '7.8) with 50 mM NaCI, 25 mM KC], 5 mM CaC12, 1 mM with 1% bovine serum albumin (BSA). These grids were MgS04. then incubated for 30 min in a freshly prepared dilution of Nuclearstaining: Adult virginmales or young adult goat anti-rabbit immunoglobulin conjugated to gold beads hermaphrodites were hand dissected in SM, and the gonads (AuroprobeEM, Janssen Pharmaceuticals). The grids were were transferred to 100 pg/ml 4,6-diamidino-2-phenylin- washed thoroughly in PBS with 1% BSA followed by washes dole (DAPI) (Sigma, St. Louis) in phosphate-buffered saline in 0.05% Tween-20 and distilled water. The grids were (PBS, pH 7.0) and incubated in a moist chamber in the dark stained with uranyl acetate and lead citrate and carbon- for 30 min. The stained gonads were mounted on glass stabilized before examination. slides and examined under the microscope. Cytolocalization of MSP and tubulin: Adult virgin males were hand dissected in SM containing 10% polyvinyl-pyr- RESULTS rolidone (w/v), and the testes were transferred to a poly-^- lysine spotted glass slide. A small drop of 4% paraformal- spe-6 mutant phenotype:All four of the sped alleles dehyde in SM was added to cover the testes, and they were have essentially identical phenotypes, which are un- then incubated in a moist chamber for 30 min at room changed over the deficiency eDf2(III): they produce temperature. A cover glass was placed on top of the testes, no progeny as hermaphrodites, and spermatogenesis and pressure was gently applied. The slide was placed on a arrestswithout formation of spermatids(SHAKES block of dry ice for 5 min, and the coverslip was removed while the tissue was still frozen. The tissue was rinsed in 1988). PBS (pH 7.0) three times for a total of 15 min and then Figure la shows the testis of a heterozygous (spe- rinsed in PBS + 10 mg/ml glycine five times for a total of 6(hc49)/+) male with many spermatids (arrow) which 25 min to block unreacted aldehydes. After permeablizing is indistinguishable fromwild type. Figure lb shows a the tissue with 0.5% Triton X-100 (Sigma) in PBS for 4-5 homozygous see-6 mutant testis that contains defective min, it was rinsed in PBS followed by incubation for 15 min in PBS + 5% goat serum.

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