Sperm isolation and biochemical analysis of the major sperm protein from Caenorhabditis elegans

DEVELOPMENTAL

BIOLOGY

84,299-312

(1981)

Sperm Isolation and Biochemical Analysis of the Major Sperm Protein from Caenorhabditis elegans MICHAEL Department

of Biology,

University

of Houston,

Received

Houston, University August

R. KLASS’ AND DAVID HIRSH* Texas

77004;

of Colorado,

18, 1980;

accepted

and *Department Boulder, Colorado

in revised form

of Molecular, 80.902 December

Cellular

and Developmental

Biology,

12, 1980

In order to facilitate the biochemical analysis of spermatogenesis in the nematode Caenorhabditis eleguns methods have been developed for obtaining large quantities of males and for the isolation of sperm. Males are isolated by a passive titration method from strains producing high proportions of males and sperm are isolated by physical pressure followed by f&ration and differential centrifugation. Biochemical analyses show that sperm contain a major protein component that represents 17% of the total sperm protein. This protein has a molecular weight of 16,600, an isoelectric pH of 8.6, and exists as a dimer. It is shown by immunocytochemical techniques to be a specific product of spermatogenesis. It is localized in the proximal arm of the male gonad and in the sperm of both the male and hermaphrodite but it is not detected in other tissues of the nematode. It is not a nuclear binding protein. Pulse-labeling studies show that this major sperm protein is tist synthesized in the proximal arm of the male gonad beginning at 39-42 hr after hatching at 20°C. Poly(A) mRNA coding for this protein is first detected in a translatable form just before synthesis of this sperm protein suggesting transcriptional control.

(review by McRorie and Williams, 1974). The temporal appearance of testis-specific isozymes during spermioSpermatogenesis is an intricate process involving the genesis is also being examined (reviewed by Goldberg, morphological and functional differentiation of male ga1977). All of these studies provide examples of the semetes. Understanding the mechanisms involved in this quential biochemical events taking place during spermadifferentiation process is an important part of our undertogenesis. Such biochemical analyses complement our standing of development. Consequently many investigaexisting knowledge of the morphological changes during tors have studied spermatogenesis at various levels in a spermatogenesis. diversity of organisms particularly in mammals (Baccetti Mutational analysis of spermatogenesis is also neceset al., 1976; Courtens et al., 1976; Koehler, 1973; review sary to understand the role of genes in the morphological by Bellve 1979); Drosophila (Meyer, 1969; Kiefer, 1973; and biochemical differentiation of sperm. Although sponHardy et al., 1979); nematodes (Foor, 1968; Beams and taneous mutations affecting germ cell development in Sekhon, 1972; Pasternak and Samoiloff, 1971; Wolf et al., mammals have been found (e.g., T locus, Bennett (1975); 1978; Ward and Miwa, 1978); and many others. A large and Sxr, Cattanach et al. (1971) detailed mutational analnumber of ultrastructural studies of spermatogenesis ysis of spermatogenesis is limited to systems that can be have provided valuable information on the precise and easily manipulated genetically. For example, the ease of extensive morphological changes that occur during genetic manipulation in Drosophila has facilitated the sperm development. isolation and characterization of a large number of YRecently, biochemical analyses have begun playing a linked fertilization defective, autosomal male-sterile, and more important role. For example, O’Brien and Bellve temperature-sensitive male-sterile mutants (Lindsley (1980a, b) have provided a detailed temporal analysis of and the protein constituents of mouse spermatozoa as a basis and Lifschytz, 1972; Kiefer, 1973; Shellenbarger Cross, 1979). Another organism that is amenable to gefor future biochemical analyses of spermatogenesis. netic analysis is the free-living soil nematode CaenorhabPlatz et al. (1975) and Dixon (1972) have studied the reditis elegccns. In addition to its genetic manipulability placement of histones by protamines during the process of sperm maturation in the rat and in the trout, respec- (Brenner, 1974), C. elegctns has a simple, well-charactertively. Extensive studies are being done on specific en- ized anatomy (Sulston and Horvitz, 1977; Depe et al., zymes found in sperm that appear to be involved in the 1978; Kimble and Hirsh, 1979) and it can easily be grown in large quantities for biochemical analysis. fertilization process, e.g., acrosome-associated enzymes These characteristics of C. elegans make it an excel* To whom correspondence should be addressed. lent system for a combined genetic and biochemical analINTRODUCTION

299

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0 1981 by Academic Press, Inc. of reproduction in any form reserved

300

DEVELOPMENTAL

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ysis of spermatogenesis. Furthermore, spermatogenesis in the male nematode has already been examined by light microscopy (Klass et al., 1976) and by electron microscopy (Wolf et al., 1978). The normal function of sperm during the mating and fertilization process has also been studied by Honda (1925), Nigon (1949), and more recently by Ward and Carrel (1979). Many temperaturesensitive mutants that exhibit defects in spermatogenesis and in sperm fertility have been isolated and characterized (Hirsh and Vanderslice, 1976; Ward and Miwa, 1978; Nelson et al., 1978). However, biochemical analysis has been hampered by the difficulty of obtaining large quantities of males and isolated sperm. In this report we describe methods for the enrichment of males and the isolation of sperm. In addition, as part of our initial biochemical analysis of spermatogenesis in the nematode, we describe a major sperm protein, its isolation, characterization, and control of expression.

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beling, the worms were washed and fed nonradioactive E. coli for 15 min followed by a second wash, pelleted, and prepared for electrophoresis as below. For large-scale cultures of synchronous worms, eggs were isolated by dissolving gravid hermaphrodites in 0.5 M NaOH, 1% sodium hypochlorite for 8 min followed by three washes in M9 salt solution. Enriched Mule Cultures

The mutant strain CB879 shows a high incidence of male progeny due to an increase in nondisjunction of the X chromosomes during meiosis (Hodgkin et al., 1979). This strain produces 20% male progeny in each generation and was used to generate mass cultures of worms with a high proportion of males. Mass cultures were grown on NGM agar plates (Brenner, 1974) supplemented with 6 ml of a mixture of 50 ml of H broth and one chicken egg prepared by adding the chicken egg to 50 ml of boiling H broth and mixing for 15 set in a Waring MATERIALS AND METHODS blender (David Baillie, personal communication). After Nematode Strains addition of the chicken egg mixture to the NGM plates they were allowed to dry to a thick paste under a laminar Caenorhabditis elegans var. Bristol was from the Uniflow hood. These plates were then seeded with 0.5 ml of a versity of Colorado, Boulder, stock. Strain designations saturated culture of E. coli strain OP50 (Brenner, 1974) are according to Horvitz et al. (1979). The nondisjunction mutant strain CB879 = him 1 (e879) I was from the MRC and incubated at 20°C for 24 hr prior to use. Plates were (5000 per collection in Cambridge, England, and was described by seeded with either gravid hermaphrodites Hodgkin et al. (1979). Routine culture procedures were plate) or with isolated eggs (100,000 per plate) obtained described in Brenner (1974). The mutant strain DH26 by the sodium hydroxide, sodium hypochlorite method Emmons et al., 1979). carries a temperature-sensitive mutation causing a sper- (Hecht, personal communication; Worms were allowed to grow at 20°C to confluency (apmatogenesis defect. This strain acts as a functional feproximately 5-7 days) at which time they were floated male at the restrictive temperature of 25°C. The strain off the plates by the gentle addition of lo-15 ml of M9 DH402 carries an X-linked mutation causing paralysis. salt solution (Brenner, 1974). Worms were then collected Both DH26 and DH402 are from the University of Coloby washing onto 20-pm nylon-filter screens (Tetko, Inc., rado stock. The strain CB1489 = him 8 (e1489)N was Elmsford, N.Y.) fitted to 7-in.-diameter plastic buchner provided by Dr. Robert Herman, University of Minnefunnels and washed extensively with 5-10 liters of M9 sota. salt solution to remove contaminating bacteria and debris. Washed worms were then layered on 35-pm nylon Growing and Labeling Synchronous Cultures filter screens supported by lo-in.-diameter plastic emWorms were synchronized by the hatching method de- broidery hoops suspended over M9 salt solution. The scribed previously (Hirsh et al., 1976). Newly hatched males (x diameter 140 pm) crawl through the filters paralyzed offspring from a cross between normal males while the hermaphrodites (x diameter 240 pm> do not. and hermaphrodites homozygous for both DH26 and These enriched male cultures were then washed again DH402 were picked. Because the DH402 mutation is Xwith 5-10 liters of M9 salt solution by collecting on a 20linked, all paralyzed progeny from this cross are male, pm nylon filter and were used as a source of sperm for allowing the distinction between male and hermaphrothe following procedure. dite offspring immediately upon hatching. At specific times after hatching synchronous worms were fed Escherichia coli strain OP50 that had been Sperm Isolation grown in M9 low-sulfate medium (M9 salt solution, BrenWorms from enriched male cultures were allowed to ner (1974), supplemented with 1 mM MgC&, 0.1 mM concentrate by settling in a 125-ml conical tube at 0°C MgS04, 0.4% W/V glucose, 2 pg/ml uracil, and 1.2 pug/ and were then squashed between two 6 x lo-in. Plexml vitamin BJ supplemented with 100 pCi/ml iglas plates (80 drops, 1 ml total volume, approximately [35S]sulfate (NEN; sp act, ‘700 mCi/mmole). After la- 50-100,000 worms) in a Carver Laboratory Press at 8000

KLASS AND HIRSH

Isolation and Analysis of Sperm Protein

psi. The plates were then pried apart and rinsed off with lo-20 ml of ice-cold M9 salt solution. (The plates are wiped dry and used repeatedly.) The rinse containing worm carcasses and released sperm was filtered through lo-pm nylon filters fitted to 45mm-diameter Millipore-filter units attached to a 50-ml plastic syringe. The sperm (x diameter 5-6 pm) pass through the lo-pm filters. The sperm were pelleted away from the lighter contaminating particles in a clinical centrifuge at low speed (IEC setting 4 for 10 min), washed in ice-cold M9 salts, and filtered through an S-pm polycarbonate Nucleopore filter to remove larger debris and pelleted a final time in a clinical centrifuge as above. Aliquots of sperm were quick-frozen and stored at - 80°C. Purifiation

of the Major Sperm Protein (Protein 15K)

Sperm were homogenized in a glass Kontes homogenizer in 0.02 M TrisZ-HCl pH 7.6, 0.1% p-mercaptoethanol, 1 mM EDTA at 4°C for 15 min (150 strokes). The homogenate was centrifuged at 100,OOOg for 1 hr and the supernatant (SlOO) was dialyzed overnight at 4°C against 400 vol of 0.035 M p-alanine brought to pH 4.3 with acetic acid (0.8 ml per liter). The precipitate that formed during dialysis was removed by centrifugation at 12,000g for 15 min at 4°C and the resulting supernatant was loaded directly onto a phosphocellulose column at 4°C. The column was washed with 2 column ~010.02 M TrisHCl, pH 7.6, 1 mM EDTA. Protein 15K was eluted with 0.1 M NaCl, 0.02 M Tris-HCl, pH 7.6, 1 mM EDTA and dialyzed overnight at 4°C against 400 vol 0.02 M TrisHCl, pH 7.6, 1 mM EDTA. Aliquots were quick-frozen and stored at -80°C. SDS -Polyacrylamide

Gel Electrophoresis

Whole worms, whole cells, or cell extracts were prepared for SDS-gel electrophoresis by boiling for 5 min in sample buffer containing 10% (w/v) glycerol, 5% (v/v) /3mercaptoethanol, 2.3% (w/v> SDS, 0.0625 M Tris-HCl, pH 6.8, and subjected to electrophoresis on SDS-polyacrylamide slab gels after the procedure of Laemmli (1970). Biochemical Characterization

of Protein 15K

Molecular weight determination. The molecular weight of protein 15K purified by phosphocellulose chromatography was determined by SDS-gel electrophoresis by the procedure of Laemmli (1970) in a slab gel apparatus using molecular weight markers from Bio-Rad Laboratories. * Abbreviations used: SDS, sodium dodecyl sulfate; TEMED, N,N,N’, N’-tetramethylethylenediamine; TCA, trichloroacetic acid; EDTA, ethylenediaminetetraacetic acid; Tris, hydroxymethylaminomethane.

301

The molecular weight of protein 15K under nondenaturing conditions was determined by chromatography on Sephadex G-100 and Bio-Gel P-30. A 25 x l-cm column (using either Sephadex G-100 or Bio-Gel P-30) was calibrated with molecular weight markers (0.1 mg each) in column buffer (0.05 M Tris-HCl, pH 7.5, 1 mM EDTA) with a flow rate of 0.5 ml per minute at 4°C. After calibration samples of protein 15K (0.1 mg) were loaded in 0.1 ml column buffer and the elution peak volume was determined by O.D. at 280 nm. Isoelectric focusing. The isoelectric point of protein 15K was determined by isoelectric focusing in polyacrylamide slab gels. Briefly, a solution composed of 2% Biolytes (Biolyte 3/10 and 7/10 mixed 60:40; Bio-Rad Laboratories) 5% acrylamide, 0.14% N&V’-methylenebisacrylamide was polymerized between two glass plates (4 x 8 in.) by the addition of ammonium persulfate (fmal cone, 0.03%, w/v) and TEMED (final cone, 0.003%, v/v>. Protein samples (l-5 pg) were focused for 7 hr at lOO300 V. Gels were then ilxed in 12.5% TCA for 30 min and stained in 0.2% Coomassie blue in water:ethanol:acetic acid, 45:45: 10, for 20 min, and destained overnight in ethanol:acetic acid:water, 25: 10:65. The pH was determined either by using a Beckman pH surface electrode or by cutting out 0.5 x 0.5~cm strips of the gel and soaking them for 24 hr in 1 ml of degassed distilled water and then measuring the pH of the solution. Amino acid analysis of protein l5K. For total amino acid analysis, 0.35-mg samples of purified protein 15K were subjected to hydrolysis in 1.5 ml of 6 N HCl at 110°C for 24 hr in an evacuated, sealed ampule. Protein hydrolysates were dried in a desiccator, redissolved in 1.0 ml of water, and aliquots applied to a Beckman Model 120C amino acid analyzer. The tryptophan content of 15K was determined calorimetrically by the use of p-dimethylaminobenzaldehyde according to Spies and Chambers (1949). Antibody

Preparations

New Zealand white rabbits were stimulated on Day 1 by injection of 0.125 ml of Freund’s complete adjutant into the popliteal lymph nodes. This was followed on Day 7 by injection into the sensitized lymph nodes of 0.125 ml of Freund’s complete adjuvant mixed with 50-75 pg purified protein 15K. Rabbits were boosted on Days 25 and 35 with 0.125 ml of Freund’s incomplete adjuvant and 50-75 pg of protein 15K. Initial injections were performed with 50-75 pg of protein 15K purified by centrifugation and phosphocellulose binding. All subsequent boosting was done by using 50-75 pg of protein 15K purified by SDS-gel electrophoresis and eluting the protein as a single band from the gel. After a positive test bleed on Day 45, serum was col-

302

DEVELOPMENTAL

FIG. 1. Light micrograph of C. eleguns the procedure described under Materials contrast optics. Mag. 500X. (Approximate

sperm. Sperm was isolated and Methods. Interferenceyield 50%).

BIOLOGY

by

lected and subjected to ammonium sulfate precipitation. The IgG fraction was precipitated by the addition of an equal volume of saturated ammonium sulfate, pH 7.4, at room temperature for 30 min followed by centrifugation at 12,000g for 15 min. The pellet was resuspended in onefifth the original volume in 0.15 M NaCl, 0.02 M TrisHCl, pH 7.4, and dialyzed against the same buffer at 4°C for 2-4 hr followed by a second ammonium sulfate precipitation as before. The final pellet was dissolved in 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.4, to 10 mg/ml and dialyzed extensively against the same buffer. The serum was further purified by immunoaffinity chromatography (Oroszland et al., 1975). Protein 15K was eluted from a single band of an SDS-gel and covalently bound to agarose by reaction with an N-hydroxysuccinimide ester of a succinylated aminoalkyl Bio-Gel A support (Affi Gel 10, Bio-Rad). Of 1.4 mg of SDS-denatured protein 15K, 95% bound in 0.1 M NaHCO, , pH 8.3, in 4 hr at 4°C to 2 ml of Affi Gel 10. After washing the agarose with 0.1 M NaHCO,, pH 8.3, followed by 1 M ethanolamine, and then 1 M propionic acid, and finally 0.1 M NaHCO, , pH 8.3, serum was then added and allowed to bind for 2 hr. After extensive washing with 0.1 M NaHC03, pH 8.3, bound antibody was eluted with 1 M propionic acid and collected into a tube containing 0.5 g of NaHCO,. The eluted antibody was dialyzed immediately against several changes of 0.02 M Tris, pH 7.6, 0.15 M NaCl. This antibody preparation was then concentrated to l-5 mg/ml by ammonium sulfate precipitation, resuspended in 0.02 M Tris, pH 7.6, 0.15 M NaCl, and used in the following procedures. I~~unocytoche~~cul

Localization

Immunofluorescence using rhodamine-conjugated goat anti-rabbit IgG was carried out on isolated sperm, dis-

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84, 1981

sected tissues, and sections. Tissue fixation was in 1.5% paraformaldehyde in PBS (2.5 mM KCl, 1.5 mM 0.5 mM KHzPOl, 150 mM NaCl, 8 mM N+HPO,, MgCl,). Material was affixed to gelatinized slides (Klass et al., 1976), dehydrated, acetone treated at 4°C for 1 hr, rehydrated in PBS, and subjected to the following treatments: (1) undiluted goat serum, 37°C for 30 min; (2) immunoafiinity-purified rabbit antibody or nonimmune serum diluted 1:20 in 10% goat serum in PBS at 37°C for 30 min; (3) rinsed in 10% goat serum in PBS; (4) goat anti-rabbit IgG rhodamine-conjugated antiserum diluted 1:60 in 10% goat serum in PBS at 37°C for 30 min; (5) rinsed in PBS and mounted in an aqueous mounting medium. The peroxidase-anti-peroxidase method for sectioned worms was essentially that of Mackenzie et al. (1978) except that 0.5~pm-thick sections were used. All antibody sera were from Miles Biochemicals unless otherwise specified. Precipitation

of IgG by Staphylococcus

aureus

Staphylococcus aureus was prepared and used to precipitate IgG according to the procedure described by Kessler (1975). Formaldehyde-fixed, heat-killed S. aureus was added to protein solutions previously challenged with primary rabbit antibody and allowed to bind at 4°C for 10 min. After binding, the Staphylococcus cells bound to IgG were removed by centrifugation, washed extensively in a solution of 1% SDS, 1% Triton X-100, 1% deoxycholate, and then pelleted. IgG was released by boiling in SDS sample buffer and separated from the Staphylococcus cells by centrifugation. The remaining supernatant containing IgG and antigen was then subjected to SDS electrophoresis (Laemmli, 1970) as described above.

FIG. 2. Isolated sperm stained with fluorescent nuclear dye. Isolated sperm were tlxed in 1.5% paraformaldehyde, washed in PBS, affixed to gelatinized slides, and stained with Hoechst 33258 (100 pgiml in 95% ETOH for 10 min at room temperature). Fluorescence was observed using a Leitz fluorescence microscope with filter system HZ for wide blue light excitation. Mag. 500 X .

KLASS AND HIRSH

PO&(A)

mRNA

Isolation

and

303

Analysis of Sperm Protein

Zsolation and in Vitro Translation

Isolation of nucleic acid from nematodes was done by the proteinase K-SDS method described previously (Emmons et aZ., 1979). After ethanol precipitation the poly(A) RNA was selected by binding to oligo(dT)-cellulose (Collaborative Research) according to Aviv and Leder (1972). The material binding to oligo(dT)-cellulose was used in an in vitro translation system to detect the presence of mRNA species coding for protein 15K. Either the rabbit reticulocyte translation system of Pelham and Jackson (1976) or one purchased from New England Nuclear was used. RESULTS

Enriched

Male Cultures

A

Utilizing a mutant strain CB879 to produce a population of approximately 20% males and a passive filtration method it was possible to routinely obtain cultures of l5 x lo6 worms with 70-80% males. More recently another strain, CB1489, that produces cultures of 36% males has been used to obtain enriched cultures of 90% males. Passive filtration through the use of nylon screens with specific pore sizes avoids the use of density gradient techniques.

B

FIG. 4. Synthesis of protein 15K in adult males. (A) An electrophoretic pattern of whole-worm extract from adult males labeled by feeding YS-labeled E. coli as described under Materials and Methods and run on a 12.5% SDS-polyacrylamide gel. *, Protein 15K. (B) An electrophoretic pattern of whole-worm extract from adult hermaphrodites labeled in the same manner as above. Hermaphrodite labeling was done after spermatogenesis had been completed. Minor bands at same molecular weight are not 15 K as determined by 20 gel electrophoresis (Klass, unpublished observations).

Sperm Isolation 94 68

c-7 0

A

B

FIG. 3. A major sperm protein in isolated sperm and its molecular weight. (A) An electrophoretic pattern of whole sperm extract isolated as described under Materials and Methods electrophoresed on a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and stained in 0.1% Coomassie blue in 50% TCA for 20 min and destained in 7% acetic acid overnight. *, Protein 15K. (B) Protein 15K was isolated as described under Materials and Methods and was run on a 12.5% SDS gel and stained with Coomassie blue as above. The arrows indicate the positions of molecular weight markers. 94,000, Phosphorylase A; 68,000, bovine serum albumin, 43,000, ovalbumin; 30,000, carbonic anhydrase; 21,000, soybean trypsin inhibitor; 14,300, lysozyme (Bio-Rad Laboratories).

Properly applied physical pressure causes the release of sperm by the males via the cloaca at the tail. Sperm appear to be the majority of free-floating cells liberated by this process. Approximately 1 x 108 sperm are obtained from 1 x lo6 males using strain CB1489. Figure 1 shows an interference-contrast photomicrograph taken of sperm isolated by the above procedure and suspended in M9 salt solution. The final sperm preparations appear to be relatively free of contaminating cellular debris by microscopical examination. Pseudopodial movement is readily visible if sperm are isolated in a phosphate-free buffer (unpublished results). Previous attempts at isolating sperm by more conventional biochemical methods of homogenization of males and subsequent density gradients gave less favorable results due to the presence of large amounts of cellular debris in all fractions. To determine the degree of contamination present in our sperm preparations, isolated sperm were stained with the DNA-specific dye Hoechst 33258. The tightly condensed nuclei characteristic of sperm represent 95% of the Hoechst-positive particles present in these preparations (Fig. 2). In five separately prepared samples an average of 5% of the nuclei were not tightly condensed. These contaminating nuclei are likely to be primary and secondary spermatocytes and other gonial nuclei observed to be isolated by these procedures.

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VOLUME 84. 1981

weight of 15,600. The average MW from five different gels was 15,600 -+ 600 (95% CIM). Densitometer tracing shows the relative abundance of this 15K protein to be approximately 17% of the total sperm protein (data not shown). As shown in Fig. 4A, this low-molecular-weight protein represents a significant fraction of the protein synthesized by adult males and can easily be visualized by SDS-gel electrophoresis of radioactively labeled adult males. In contrast, adult gravid hermaphrodites labeled after the completion of spermatogenesis did not show a major band after SDS electrophoresis (Fig. 4B), suggesting that protein 15K was a specific product of spermatogenesis. Protein 15K can be detected if hermaphrodites are labeled during spermatogenesis (data not shown). Protein

FIG. 5. Molecular weight of nondenatured protein 15K. Protein 15K, purified by phosphocellulose binding as described under Materials and Methods was chromatographed on Sephadex G-100. Sephadex G-100 calibrated with molecular weight markers: cytochrome c, 12,500; chymotrypsinogen, 25,000; ovalbumin, 43,ooO; bovine serum albumin, 68,000 (Schwarz/Mann). (---I Elution peak of 15K. The x MW from five determinations was 30,600 2 2100 (95% CIM). (A) Peak fraction of protein 15K. Fraction number is expressed in ml.

Spew. Proteins As part of our initial biochemical analysis, isolated sperm were fractionated on SDS -polyacrylamide gels as described under Materials and Methods. Figure 3A is a photograph of the Coomassie blue-stained protein pattern obtained by SDS electrophoresis of isolated sperm. The major sperm protein seen in Fig. 3B has a molecular

1 o.o‘,/’

PH

9.0.

, 7’ -; z ,.

8.0.

‘.

L

Distance

FIG. 6. Protein 15K is a basic protein. Isolated protein 15K was subjected to isoelectric focusing in polyacrylamide slab gels as described under Materials and Methods. The a isoelectric pH from five isoelectric focusing gels was 8.66 2 0.15 (95% CIM).

15K Isolation

and Characterization

The major protein of C. elegans sperm can be readily purified by gentle homogenization in a low-salt buffer and phosphocellulose chromatography (Fig. 3B). Approximately 2 mg of pure 15K can be obtained from 1 x 10’ sperm (approximate yield 40-50% of total 15K). Whereas the major sperm protein has a molecular weight of 15,600 on SDS gels (Fig. 3B), nondenatured TABLE 1 AMINO ACID COMPOSITION OF PROTEIN 15K Amino acid LYS His NHs Arg Asx Thr Ser Glx Pl-0

GUY Ala Half-cystinec Val Met Ise Leu TF Phe TWd

Residues/15,600-MW

monomer

7.9” (8y 1.9 (2) 17 (17) 8.0 (8) 17.7 (18) 8.0 (8) 3.9 (4) 10.7 (11) 9.1 (9) 11.9 (12) 9.9 (10) Trace 8.3 (8) 2.2 (2) 7.4 (7) 5.0 (5) 3.9 (4) 4.6 (5) 1.7 (2)

’ Values represent the average value from two separate analyses and are calculated using a monomer molecular weight of 15,600. b Nearest integer. e 0.35 mg Protein 15K was hydrolyzed in 6 N HCI for 24 hr at 110°C and the hydrolysate analyzed on a Beekman amino acid analyzer. Under these conditions, cysteine was partially converted to cysteic acid. Only trace levels of cysteic acid were detected. ’ Determined calorimetrically as described under Materials and Methods.

KLASS AND HIRSH

Isolation and Analysis of Sperm Protein

305

purified protein 15K has a molecular weight of 30,600 -+ 2100 as determined by both Sephadex G-100 and Bio-Gel P-30 (Fig. 5). Crude homogenates of sperm prepared by taking the 100,OOOg supernatant from homogenized sperm also gave a molecular weight of 30,600 for protein 15K, (data not shown) indicating the isolation procedure does not cause denaturation of 15K. Therefore, 15K appears to be a dimer in its native form. Protein 15K has an isoelectric pH of 8.6 determined by isoelectric focusing (Fig. 6). Only a single protein band is observed when phosphocellulose-purified 15K is focused on slab gels.

A

A

B

C

FIG. 8. Specificity of antibody to protein 15K. Whole sperm were subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel. Proteins were then transferred to nitrocellulose by the method of Towbin et al. (1979). Part of the filter containing the transferred protein was incubated with affinity-purified antibody to 15K followed by reaction with horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:60 in PBS). For the color reaction, blots were incubated in 0.02% 3,3’-diaminobenzidine, 0.01% H*O,. (A) nitrocellulose filter after protein transfer stained with 0.1% amino black (Towbin et al., 1979); (B) filter reacted with antibody to 15K and peroxidase-conjugated goat anti-rabbit IgG; (C) nonimmune control. Minor bands sometimes observed and often seen in nonimmune controls may be due to nonspecific binding or a small degree of crossreactivity.

The amino acid composition of 15K was determined and is summarized in Table 1. Only trace levels of cysteic acid were detected. Cell SpeciIity

FIG. 7. Antibody detection of protein 15K in crude homogenates. (A) Double-diffusion tests were done of crude homogenates (15,000g supernatant after 3 min of sonication) from 36% male cultures at different developmental stages using affinity-purified antibody to 15K. Approximately 1 mg of protein was loaded in each well for Ll, L2, L3, L4, and adults. Ll, first-stage larvae immediately after hatching; L2, secondstage larvae 21 hr posthatch, 20°C; L3, third-stage larvae 29 hr posthatch, 20°C; L4, fourth-stage larvae 39 hr posthatch, 20°C; A, adults, 82 hours posthatch, 20°C; 15K, approximately 10 pg of purified protein 15K. (B) Double-diffusion test of antibody to 15K with nonimmune control. 15K, purified protein 15K; Ab, affinity-purified antibody to protein 15K; Non-imm, nonimmune serum.

and Localization

of Protein

15K

To determine the specificity of the antibody preparation and to detect the presence of protein 15K in males at different stages in development, the double-immunodiffusion tests shown in Fig. 7 were done. A single precipitation band was observed between purified protein 15K and its antibody (Figs. 7A and B). Controls with nonimmune serum failed to show any antibody precipitation (Fig. 7B). Crude homogenates from 36% male cultures taken prior to spermatogenesis at the first, second, third, and fourth larval stages (Ll-L4) also failed to show any precipitation bands when tested with the affinity-purified antibody. The crude homogenates from adult cultures

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DEVELOPMENTALBIOLOGY

FIG. 9. Cross-reactivity of the major sperm protein from Ascaris with antibody to protein 15K of C. eleguns. Protein 15K isolated from A. lumbricoides (15K,) was tested against antibody to 15K from C. eleyans (15K Ab) by double diffusion. 15K,,, protein 15K from C. elegans.

(36% males) did show a single precipitation band corresponding to protein 15K (Fig. 7A). Crude homogenates from L4 cultures of 36% males failed to show a positive precipitation reaction in the double-diffusion test. This result is most likely due to the fact that the worms used were in the early L4 stage (approximately 39-42 hr old, 20°C) and were only beginning to synthesize protein 15K. The controls of nonimmune serum and crude extracts from immature worms not bearing sperm indicate the specificity of the purified antibody. Furthermore, binding of affinity-purified antibody to total sperm protein separated by SDS-gel electrophoresis and transferred to nitrocellulose indicates binding to only a single protein corresponding to protein 15K (Fig. 8). To determine if protein 15K was unique to C. elegans, sperm from another nematode Ascaris lumbricoides was isolated by dissecting the vas deferens from adult males and withdrawing the sperm. Using an identical isolation procedure as that described for C. elegans protein 15K, a major 15,000-MW protein was isolated and tested for cross-reactivity to antibody to C. elegans protein 15K. Figure 9 shows that 15K from A. lumbricoides crossreacts to affinity-purified antibody to protein 15K from

VOLUME 84, 1981

sected tissues and sections of whole animals. The results are depicted in Figs. 11 and 12. Figure 11 is a photomicrograph of a dissected hermaphrodite gonad with the spermatheca labeled in the same manner as in Fig. 10. Only sperm are fluorescently labeled. No somatic tissue examined, i.e., intestine, muscle, nerve, or hypodermal, was observed to have positive fluorescence. Mature oocytes and the hermaphrodite gonad likewise do not show positive fluorescence. Some fluorescence seen in Fig. 11B not corresponding directly to sperm shown in Fig. 11A is from sperm located at different focal planes. To avoid the complication of antibody permeability, worms were sectioned in polyethylene glycol 4000. Figure 12 shows sections of males and hermaphrodites labeled by the rabbit peroxidase anti-peroxidase method. Figure 12B is a longitudinal section through an adult male showing the 180” bend in the gonad with distal and proximal arms. The distal tip does not show a positive reaction indicating the absence of protein 15K. However, approximately one-fourth of the distance past the loop region in the proximal arm there is a positive reaction indicating the presence of this protein. In this region the gonadal nuclei are in the mid- to late pachytene stage of meiosis and remain connected to a central core of cytoplasm (Klass et al., 1976). The presence of 15K in the proximal arm is also evident in the cross sections in Figs. 12A and C. Again, no other tissue examined indicated the presence of protein 15K. Figure 12D is a longitudinal section through the spermatheca of a mature hermaphrodite. The only positive reaction is seen in the sperm in the spermatheca. Time of Synthesis of Protein 15K

We performed pulse-labeling experiments to determine when protein 15K was first synthesized. Figure 13 shows the results of pulse-labeling young developing males at 3-hr intervals after hatching. Protein 15K begins to be synthesized between 39 and 42 hr after hatching at 20°C. Its synthesis continues through the’ C. elegans. adult stage. The mean length of the males was 638 ? 14 Immunocytochemical localization. The positive reac- pm (+ 95% CIM) and inspection with the light microtion of the antibody in isolated sperm is seen in Fig. 10. scope revealed that primary spermatocytes were not yet In Fig. 10 isolated sperm were incubated in either non- present at this stage. immune or immune serum and then allowed to react with To determine the presence of messenger RNA coding rhodamine-labeled goat anti-rabbit antibody. Sperm are for protein 15K, total poly(A) mRNA was extracted from cultures of 70-80% males. A large proportion of the in highly fluorescent when incubated in the immune serum, but show no fluorescence when incubated in the nonim- vitro synthesized protein is in the 15,600-MW band. Anmune control. Controls incubated with anti-rabbit anti- tibody binding and precipitation by S. aureus shows that body alone also showed no fluorescence (Figs. 10D and this protein is the sperm-specific protein 15K (Fig. 14). Total poly(A) mRNA was isolated from synchronous E). Figure 1OC is a higher magnification of a fluorescently labeled sperm. cultures of worms (36% males) at specific times during Localization of protein 15K in the whole animal was development and translated to determine when during done by reacting the affinity-purified antibody to dis- development poly(A) mRNA coding for protein 15K was

KLASS

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and Analysis

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Protein

G FIG. 10. Antibody reaction to 15K in sperm by immunofluorescence. Paraformaldehyde-lixed, acetone-treated sperm were treated with aftlnitypurified antibody or nonimmune serum followed by incubation with rhodamine-conjugated goat anti-rabbit IgG (Rh-GAR) as described under Materials and Methods. (A) Interference-contrast optics, mag. 500x, 15K Ab, Rh-GAR; (B) same as (A) but with fluorescence optics; (C) same treatment as (A) but higher magnification, mag. 1200x; (D) interference-contrast optics, mag. 500x, Rh-GAR only; (E) same as (D) but with fluorescence optics; (F) interference-contrast optics, mag. 500x, nonimmune serum, Rh-GAR; (G) same as (F) but with fluorescence optics.

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FIG. 11. Tissue specificity of protein 15K in whole dissected tissue. A dissected hermaphrodite gonad showing the spermatheca and spermathecal constriction fixed and treated as in Fig. 10A. Int, intestine; 0, oocyte; S, sperm. (A) Interference-contrast optics; (B) fluorescence picture after reaction with antibody to 15K plus Rh-GAR.

first made. Translation of these mRNA fractions and subsequent antibody precipitation of the in vitro translation products (Fig. 15) indicates that poly(A) mRNA coding for 15K is first present in a translatable form at 39 hr after hatching, approximately the same time that the synthesis of protein 15K is first detected. DISCUSSION

To facilitate the biochemical analysis of spermatogenesis in the nematode C. elegans, we have developed methods for obtaining large quantities of males and for the isolation of sperm using a passive filtration method

and simple physical pressure. The male enrichment procedure can serve as an important tool for future biochemical studies on sexual dimorphism, dosage compensation, spermatogenesis, and analyses requiring large quantities of males. The sperm isolation technique enables the biochemical analysis of sperm in C. elegans. Our initial biochemical fractionation of isolated sperm has demonstrated that sperm cells contain a major protein which we call 15K. The 15K protein is not detectable in other cells by immunological methods. It is developmentally regulated, being synthesized at a specific time and in a specific region of the male gonad. Protein 15K is synthesized prior to the morphological

KLASS

AND HIRSH

FIG. 12. Tissue specificity of protein 15K in thin rabbit peroxidase anti-peroxidase method. (A and longitudinal section of an adult male showing the maphrodite through the spermatheca showing an (Int).

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sections. Sections (0.5 pm) of adult worms were treated with antibody to 15K and labeled by the C) Cross sections of an adult male showing the proximal (P) and distal (D) arm of the gonad; (B) loop of the gonad and the proximal and distal arms; (D) a longitudinal section of an adult heroocyte (0) lying in the spermatheca surrounded by sperm (S). Section also shows the intestine

differentiation of primary spermatocytes at the time that corresponds to the last mitotic divisions of the somatic cells of the developing gonad (Kimble and Hirsh, 1979). This chronology suggests that 15K may be a necessary precursor to sperm cell differentiation. Identification and analysis of mutants defective in 15K will allow a more precise determination of the role of 15K in spermatogenesis. Protein 15K begins to appear in a specific region of the male gonad, approximately one-fourth of the distance past the loop region. In this region the nuclei of the gonad are in the pachytene stage of meiosis and are arranged around a common central core of cytoplasm (Klass et al., 1976). Individual spermatocytes are not yet formed. This region has been identified as one in which the formation of sperm-specific organelles is first observed at the EM level (Wolf et al., 1978). The formation of specific Golgi-associated membrane vesicles and large fibrous bodies occurs in this region. Large fibrous bodies containing 48 f 11 A fibers have been described in the sperm of C. elegans and other nematodes (Wolf et al., 1978; Pasternak and Samoiloff, 1971; Beams and Sekhon, 1972). During the maturation process in C. elegans these fibrous bodies fuse with the Golgi and seem to empty out their fibrous contents into the cytoplasm (Wolf et al., 1978). The function of the fibrous bodies is not known. Antibody localization with the electron microscope will allow determination of the presence of 15K in specific

structures and determine presumably whether 15K is associated with the fibrous bodies or the other organelles. Poly(A) RNA isolated from males contains a signifi-

A

B

c

FIG. 13. Kinetics of in vitro synthesis of protein 15K. Electrophoresis pattern from males pulse-labeled at various times after hatching as described under Materials and Methods. Labeling time: (A) 39-42, (B) 36-39, and (C) 33-36 hr after hatching at 20°C. *, Protein 15K, a, actin.

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ABCD FIG. 14. The major protein synthesized from male poly(A) mRNA is protein 15K. (A) In ritro translation products from poly(A) mRNA isolated from 70% male cultures; (B and C) antibody precipitation of in vitro translation products from (A) with antibody to 15K and Staphylococcus aureue; (D) nonimmune control. The gel was prepared for fluorography as described by Bonner and Laskey (1974) and exposed to flashed fdm at - 70°C according to Laskey and Mills (1974). *, Protein 15K; 12.5% SDS-polyacrylamide gel.

cant fraction of message coding for Protein 15K. These results indicate that this protein is not a degradation product found in sperm cells. Furthermore, translation of poly(A) mRNA isolated at different times during development indicates that mRNA coding for 15K is first present in a translatable form at the time that the synthesis of protein 15K can be detected by pulse-labeling, implying a transcriptional control. However, by the methods employed we cannot rule out the possibility that mRNA for 15K is transcribed at an earlier time and is present in a nontranslatable or nonpolyadenylated form until synthesis of protein 15K. Purified fractions of poly(A) mRNA are being used to select recombinant DNA molecules in order to determine the presence of RNA coding for protein 15K by hybridization techniques. These recombinant molecules will be used as in situ or in vitro hybridization probes to assay where and when the RNA transcript for protein 15K is present. Our initial analyses have not determined a specific function for this abundant sperm protein. The fact that it is also present in a large amount in the parasitic nematode A. lumbricoides implies that it is not unique to the free-living genus and may be important generally in nematode sperm function. The abundance of 15K would

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seem to imply a structural role. Sperm cells often contain specialized low-molecular-weight basic proteins in large amounts. For example, on a weight basis there is as much histone or protamine as DNA (Dixon, 19’72; Dixon and Smith, 1968; Candid0 and Dixon, 1972). The antibody localization and the amino acid content of protein 15K, however, indicate that this protein is not a histone or protamine-like nuclear-binding protein. Furthermore, protein 15K is a very soluble protein liberated by gentle lysis in contrast to the extraction methods used for protamines and histones (Mirsky and Silverman, 1973; Murray, 1966). Although the abundance of protein 15K suggests a structural role, enzymatic function of this basic protein cannot be ruled out. Basic proteins in sperm have been isolated that exhibit enzyme activity, most notably the proteolytic enzyme acrosin which in some species occurs as a dimer (Polakoski et al., 1972). Other enzymes unique to sperm have also been described. Hyaluronidase is another prevalent enzyme that occurs as a tetramer with a monomer molecular weight of 14,000 and is important for sperm function (Khorlin et al., 1973). Other enzymes have been detected in sperm from many species but none are as abundant as 15K. Many of these are from the acrosome having been derived from the Golgi complex and are hypothesized to be important in fertilization (e.g., Dott and Dingle, 1968; review by McRorie and Williams, 1974). Golgi-derived special membrane vesicles are located around the perimeter of C. elegans sperm (Wolf et L1

L2

L3

L4

A

6

C

D

ADULT

E

FIG. 15. Time of appearance of poly(A) mRNA coding for protein 15K. This figure is a fluorogram of the antibody-Staphylococcus precipitation of the in vitro translation products from poly(A) mRNA isolated from synchronous 36% male cuhures at specific times during development. One microgram of poly(A) mRNA from each sample was translated in a rabbit reticulocyte in vitro translation system. (A) First-stage larvae, immediately after hatching, ..? length 230 pm; (B) second stage, 21 hr posthatch, x length 330 nm; (C) third stage, 29 hr posthatch, 2O”C, 8 length 520 pm; (D) precipitation products from poly(A) mRNA from fourth-stage larvae 39 hr posthatch, 2O”C, x length at time of mRNA extraction, 660 pm; (E) precipitation products from poly(A) mRNA from adult males, 82 hr posthatch, 20°C.

KLASS AND HIRSH

Isolation

al., 1978). The pattern of immunofluorescence seen in the light microscope supports the presence of 15K in vesicles around the membrane. C. elegans sperm and that of other nematodes move by means of pseudopodia (Foor, 1968; Foor et al., 1971; Wolf et al., 19’78; Ward and Carrel, 1979). It is possible that protein 15K is important for pseudopodial formation and/or movement. In our earlier studies we described the selection and characterization of spermatogenesis-defective mutants (Hirsh and Vanderslice, 1976). Using the techniques described in the present study it will now be possible to analyze biochemically these mutants to detect genes affecting specific sperm proteins and to study their interrelationships. For example, using the antibody to protein 15K it should be possible to screen the spermatogenesis mutants presently available by the immunocytochemical localization methods described here for whole worms. Earlier works (Hirsh and Vanderslice, 1976; Ward and Miwa, 1978) have shown that sperm function is dispensible to hermaphrodites so it should be possible to obtain viable null mutants even if protein 15K is essential for spermatogenesis. Finally, it is important to point out that 95% of the Hoechst-positive particles in these sperm preparations are sperm nuclei. We had recently shown by comparing restriction fragments of DNA from germ line and somatic tissue that very little, if any, rearrangement occurs in the DNA of C. elegans during development (Emmons et al., 1979). The source of germ line DNA was sperm isolated by the procedure described in the present study. Even if the non-sperm-like nuclei were somatic, the DNA isolated from such preparations would still have been 90% germ line and only 10% somatic DNA. This work was initially supported by U.S. Public Health Service Research Grants GM 19851 and AGO0 310 and later supported under U.S. Public Health Service Research Grants GM 26808 and AG 02115. We would like to express our appreciation to Barbara Carson and Dyan Holden for technical assistance, John Izant and Dr. John MacKenzie for assistanee with immunocytochemistry, Dr. Eugene Foor for providing samples of Ascaris lumbricoides, and to Dr. Kathleen Matthews for use of the amino acid analyzer. REFERENCES Av~v, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose. Proc. Nat. Acad. Sci. USA 69, 1408-1412. BACCETTI, B., PALLINI, V., and BURRINI, A. G. (1976). The accessory fibers of the sperm tail. III. High-sulfur and low-sulfur components in mammals and cephalopods. J. Ultrastruct. Res. 57, 289-398. BEAMS, H. W., and SEKHON, S. S. (1972). Cytodifferentiation during spermiogenesis in Rhabditis pellio. J. Ultrastruct. Res. 38, 511-527. BELL&, A. R. (1979). The molecular biology of mammalian spermatogenesis. In “Oxford Reviews of Reproductive Biology” (C. A. Finn, ed.), Vol. 1, pp. 159-261. Oxford Univ. Press (Clarendon), London/New York. BENNETT, D. (1975). The T-locus of the mouse. Cell 6, 441-454.

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