MOLECULAR REPRODUCTION AND DEVELOPMENT 30154-158 (1991)

Analysis of Protamines Isolated From Alcohol Preserved Epididymides CATHERINE LEE, JOE MAZRIMAS, AND ROD BALHORN Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California

ABSTRACT Protamines 1 and 2 have been isolated from the sperm of frozen and isopropanol pre- served Syrian hamster (Mesocricetus auratus) epidid- ymides and analyzed by gel electrophoresis, high-perfor-. mance liquid chromatography (HPLC), and amino acid analysis and sequencing. The results show that alcohol preservation does not alter the primary structure of the two sperm nuclear proteins and that the preservation of mammalian reproductive organs in alcohol is a viable alternative to freezing tissues collected in the field. Sperm were isolated from tissues fixed in isopropanol for as long as 7 months without detectable adverse effects on either the isolation of sperm or the primary structure of the protamines.

Key Words: Sperm, Preserved tissue, Hamster, Me- socricetus auratus, Is0 p ro pa no1

INTRODUCTION The sperm of eutherian mammals has been shown to

contain two small, basic proteins that package its DNA in a genetically inactive, maximally condensed state (Kolk and Samuel, 1975; Calvin, 1976; Balhorn et al., 1977; McKay et al., 1986; Corzett et al., 1987; Bellve et al., 1988; Balhorn, 1989; Pirhonen et al., 1989). Prota- mine 1, the smaller of the two proteins, has been detected in the sperm of every mammalian species examined (Balhorn, 1989). Protamine 2, in contrast, is only present in the sperm of selected species (Balhorn, 1989).

Amino acid and primary sequence analyses of the protamine 2 molecules isolated from various species of mammals (Corzett et al., 1987; Balhorn, 1989) have shown it to be extremely rich in histidine (12-21%). Protamine 1, by comparison, usually contains 5% or less histidine (Corzett et al., 1987; Balhorn, 1989). Protamine 2 also differs from protamine 1 in that i t is synthesized as a larger precursor protein (Yelick et al., 1987) that is processed through a series of specific proteolytic cleavages after being incorporated into the sperm chromatin complex (Green et al., 1987; Balhorn, 1989). These differences in structure and metabolism, coupled with observations that the presence of prota- mine 2 in sperm facilitates sperm decondensation (Per- reault et al., 1988) and its deficiency may be correlated with male infertility in certain species (Balhorn et al., 19881, suggest that protamine 1 and protamine 2 may

0 1991 WILEY-LISS, INC.

perform different functions in sperm chromatin orga- nization.

As part of a study investigating the extent to which the protamine 2 gene is expressed in different mam- mals and the final stoichiometry of the two protamines in the mature sperm chromatin complex, we have begun to analyze the protamines present in the sperm of a wide variety of species. Recent difficulties associ- ated with the storage and shipment of frozen testicular material obtained from rodents and other mammals collected in remote areas have led us to seek an alternate approach for tissue preservation that will not alter the structure or extractability of the protamines from sperm. Using the Syrian hamster, Mesocricetus auratus, as a test system, we have shown that the preservation of epididymides in 70% isopropanol is not detrimental to the isolation or analysis of protamine structure by gel electrophoresis high-performance liq- uid chromatography (HPLC), amino acid analysis or protein sequencing.

MATERIALS AND METHODS The Syrian hamsters used in this study were bred in

our own colony. The original animals used to establish the colony were obtained from Simonsen Laboratories (Gilroy, CAI.

Tissue Fixation Immediately after removing the epididymides from

adult male hamsters 16-24 weeks of age, the organs were placed in scintillation vials containing 70% iso- propyl alcohol or 70% ethanol sufficient to cover them completely (10-15 ml). Because tissue preservation was effective using both minced and intact organs, all experiments were performed using whole organs. The epididymides were stored in the alcohol at room tem- perature (21°C) for intervals ranging from 1 day to 7 months.

Protamine Isolation Two methods were used to isolate sperm from alcohol

preserved epididymides. One involved mincing the

Received February 11, 1991; accepted June 3, 1991. Address reprint requests to Rod Balhorn, Biomedical Sciences Divi- sion, Lawrence Livermore National Laboratory, Livermore, CA 94550.

ANALYSIS OF PROTAMINES 155

tissue in cold 0.15 M sodium chloride, 0.01 M Tris buffer, pH 8 (Tris saline), and aspirating the suspension until the sperm were released. The second method involved placing the fixed epididymides and Tris saline in a Black and Decker “Handy Chopper” model HC20 (Black and Decker Inc., Shelton, CT) and processing the sample until the slurry appeared homogeneous. The suspension was sonicated with a W-220 sonicator (Heat Systems-Ultrasonics, Farmingdale, NY) at level 5 (using the microtip) for several 15 sec intervals, filtered through cheesecloth, and refiltered through a fine silk filter (No. 11 Standard Swiss Silk Doufour; Abbe Engineering Co., Brooklyn, NY). The sperm suspen- sions were centrifuged a t 4,OOOg for 5 min and the unmodified protamines were extracted from the sperm pellets as described previously (Balhorn et al., 1977).

Gel Electrophoresis and Protamine Quantification by Microdensitometry

Equivalent amounts of protamine isolated from un- treated and isopropanol-fixed tissues were dissolved in 0.9 N acetic acid, 20% sucrose, 0.5 M mercaptoethanol, and analyzed by electrophoresis in 10 cm acid urea gels (Balhorn et al., 1977). The electrophoretic separation of protamine 1 and protamine 2 was facilitated by first aminoethylating the protein samples with aziridine prior to electrophoresis (Balhorn et al., 1977). The gels were stained in 0.9 N acetic acid, 20% ethanol, 0.1% naphthol blue black overnight and subsequently de- stained electrophoretically. The gels were scanned at 600 nm using a Shimadzu CS9000 Flying-Spot micro- densitometer (Shimadzu Scientific Instruments, Co- lumbia, MD) to quantitate the amount of protein present in each stained band.

Amino Acid Analysis and Amino Terminal Sequencing

The HPLC-purified protamines were carboxymethy- lated with sodium iodoacetate and desalted by chroma- tography on Sephadex G-25 as described previously (Balhorn et al., 1977). The carboxymethylated prota- mines were hydrolyzed in 6 N HC1 for 22 hr at 105”C, the hydrolysates were lyophilized, and the amino acid compositions were determined by analysis on a Beck- man 6300 analyzer. The amino terminal sequences of the HPLC-purified protamines were determined using an Applied Biosystems 477A sequencer.

HPLC Fractionation of Protamine The protamines were reduced with dithiothreitol (10

mg/mg protamine) in 6 M guanidine hydrochloride and separated by HPLC on a Nucleosil RP-C18 column (7.5 mm x 300 mm) using a linear acetonitrile gradient and the ion-pairing agent trif luoroacetic acid (TFA). The gradient, 40-80% buffer B, was run at a rate of 2 ml/min over a period of 50 min. Buffer A = 0.1% TFA; buffer B = 30% acetonitrile, 0.1% TFA.

RESULTS Isolation of Protamine From

Preserved Epididymides Pairs of epididymides from mature Syrian hamsters

were either frozen a t -20°C or preserved in 70% isopropanol for intervals ranging from 1 day to 7 months. Both intact and minced organs appeared to be preserved equally well in isopropanol. Two different approaches were tested for releasing sperm from the fixed epididymides. The technique used routinely for isolating sperm from fresh or frozen tissues, mincing the organs in Tris saline and aspirating the tissue suspension to release the sperm, gave poor yields from the fixed organs. This process also proved to be much more tedious with preserved tissue; the tested organs became hard and rubbery, and teasing did not ade- quately release the sperm stored in the lumen of the ductus epididymis. The method that worked best em- ployed a combination of tissue homogenization and sonication. After filtering the tissue suspension through cheesecloth and a 70 pm silk filter, sperm yields were improved by resuspending the tissue re- maining in the filter in additional Tris saline, sonicat- ing the suspension, and passing it again through the filter.

Analyses of Protamines Isolated From Fixed Tissues

Hamster protamines were extracted from the iso- lated epididymal sperm and compared by electrophore- sis, HPLC, amino acid analysis, and amino terminal sequencing. The electrophoretic pattern of basic nu- clear proteins isolated from frozen and isopropanol fixed sperm, shown in Figure 1, are identical. The process of fixation did not alter the charge density or apparent molecular weight of the protamine, nor did it generate multimeric protamine complexes. Further- more, the modification of protamine cysteines by ami- noethylation with aziridine and the electrophoretic separation of the modified P1 and P2 variants were unaffected by exposure to isopropanol (Fig. 1). The aziridine-modified P1 and P2 separated normally and the relative proportion of P1 and P2 as determined by microdensitometry of napthol blue black-stained gels was comparable to samples isolated from frozen epi- didymides (Table 1) and within the limits of variability observed between individual animals and individual isolations.

The HPLC fractionations of total protamine isolated from frozen and 3 month isopropanol-preserved epi- didymides are shown in Figure 2. Both the P1 and P2 protamine peaks are clearly resolved, and the retention times are essentially identical. Additional peaks sug- gestive of altered or complexed proteins are not de- tected, and the amino terminal sequences for the two protamines were found to be identical (Fig. 3). Amino acid analyses of the carboxymethylated P1 and P2 proteins isolated by HPLC also show similar composi-

156 C. LEE ET AL.

UNTREATED

Protamine P I & P2

+ 1 - FROZEN

ISOPROPANOL

AZlRlDlNE MODIFIED

+

FROZEN

1 WEEK

1 MONTH

7 MONTHS

P2

Fig. 1. Electrophoretic pattern of mouse sperm basic nuclear proteins isolated from frozen and isopropanol preserved tissues. Electrophoresis was performed in 10 cm acid urea gels at 130 V, 20 mA for 1 hr 45 min. Top: Untreated basic nuclear proteins. Bottom: Aziridine modified basic nuclear proteins.

tions for the protamines isolated from frozen and fixed epididymides (Table 2).

DISCUSSION As studies of protamine structure and gene expres-

sion are extended to a larger variety of species, sample collection, storage, and transport problems occur more frequently. Presently, the only method available for preserving testes and epididymides for future biochem- ical studies is freezing. This often proves to be difficult, if not impossible, in extended field trips into remote areas. Tissue shipments into the United States must also be certified disease-free by the Department of Agriculture, and this can often be a problem with frozen tissue.

To overcome these difficulties, we have investigated

the feasibility of using alternative methods for preserv- ing epididymides under conditions that will not hinder our analyses of the sperm basic nuclear proteins. Alcohols and aldehydes have been used for decades as preservatives for foods, tissues, cosmetics, pharmaceu- ticals, and other materials for medical and biological studies (Brother and McKinney, 1938; Von Fenyes, 1970; Bandelin, 1977; Lipinski and Wolfhard, 1983). While formaldehyde and glutaraldehyde have been shown to generate protein-protein and DNA-protein cross-links (Avrameas and Ternynck, 1969; Hancock, 1970; Doenecke, 1978) that severely limit the useful- ness of fixed cells for many biochemical studies, alco- hols have been used successfully to preserve tissues for future DNA isolations (Rake, 1972).

Isopropanol was chosen because it is readily avail-

ANALYSIS OF PROTAMINES 157

TABLE 1. Relative Proportion ofprotarnine P1 and P2 in Hamster Sperm Isolated From Frozen and Isopropanol-Fixed Epididymides (expressed as a

percent of total protarnine) Tissue Percent protamine 2

Frozen 34.0 k 0.7 Isopropanol

1 Week 31.7 k 1.3 1 Month 34.0 + 1.9 7 Months 35.5 f 0.2

10 30 50

ELUTION TIME (MIN)

P1

n

70

I I I I I I I I 1 a I I

10 30 50 70

ELUTION TIME (MIN)

Fig. 2. HPLC fractionation of basic nuclear proteins isolated from sperm of frozen tissues (A) and tissues preserved for 3 months in 70% isopropanol (B). Conditions used for chromatography are described in Materials and Methods.

able throughout the world, i t is relatively nontoxic (all alcohols, including ethanol, have been listed as toxic by OSHA), and i t can be shipped with a minimum hazard. The preservation of biological tissues in this alcohol should also minimize or eliminate concerns about the

Frozen

P1: Ala arg tyr arg cys cys arg......

P2: Arg gly gln his his his arg......

lsopropanol

P1: Ala arg tyr arg cys cys arg......

P2: Arg gly gln his his his arg......

Fig. 3. Amino terminal sequences of protamine 1 and protamine 2 isolated from sperm of frozen epididymides and epididymides pre- served in 70% isopropanol.

potential contamination of tissues with disease organ- isms.

In this study, we used epididymides isolated from the Syrian hamster to demonstrate that 70% isopropanol can be used to preserve epididymides from which sperm are isolated for biochemical studies involving the two sperm chromosomal proteins protamine P1 and P2. Sperm were successfully isolated from the fixed organs using a combination of homogenization and sonication.

Sperm isolated from tissue preserved for extended periods of time (up to 7 months) could not be distin- guished, by biochemical means, from those isolated from frozen tissue. Longer periods of fixation (up to 1 year) were also tested and were found to yield similar results, but the protamines were not analyzed as com- pletely as those fixed for the shorter periods of time. The efficiency of sperm release from the fixed tubules appears to be the only step in the procedure adversely affected by alcohol fixation. The removal of the tail, acrosome, and nuclear membranes using mixed alkyl- trimethylammonium bromide, the solublization of chromatin with GuC1, and extraction of protamine were not detectably altered by prior fixation of the tissues in isopropanol.

Differences in the protamines extracted from the sperm of frozen and isopropanol-fixed organs could not be detected by either gel electrophoresis or HPLC. Similar experiments performed with ethanol-fixed tis- sues (data not shown) provided identical results. Nei- ther the electrophoretic mobilities of the untreated and aziridine-modified protamines nor the retention times of P1 and P2 on a C-18 column were altered by the isopropanol. The efficiency of extraction of P1 and P2 was also unaffected by the alcohol. The protamine 2 contents (expressed as percent of total protamine in the sperm) of sperm isolated from frozen epididymides or epididymides fixed €or 1 week, 1 month, and 7 months in isopropanol fell within the limits observed for frozen epididymides obtained from different individuals (un- published data). Amino terminal sequence analyses of P1 and P2 isolated from frozen and isopropanol samples were also found to be indistinguishable.

158 C. LEE ET AL.

TABLE 2. Amino Acid Composition of Hamster Protamines P1 and P2 Isolated From Frozen and

Isopropanol-Fixed Epididymides* Frozen Ispropanol

P1 P2 P1 P2

Arginine 62.4 58.4 62.2 58.4 Histidine 0.7 14.4 0.9 13.5 Lysine 4.2 1.8 4.2 1.9 Serine 5.7 6.0 6.1 5.9 Threonine 3.5 0.4 3.8 0.6 G 1 y c i n e 0 3.2 1.9 3.8 Alanine 1.6 0.1 2.0 0.3 Leucine 1.9 1.8 2.2 1.8 Isoleucine 0 1.4 0.1 1.3 Tyrosine 5.4 1.8 5.3 1.9 Cysteine 14.5 9.2 10.8 9.0 Glu/gln 0 1.6 0.4 1.6 Asp/asn 0 0 0 0 Valine 0 0 0 0 Proline 0 0 0 0 No. of analyses 2 2 3 2

*Cysteine values are obtained as carboxymethylcysteine from hydrolysates of protein samples carboxymethylated with iodoacetate. This reaction is usually inefficient in modifying the cysteines residues of protamine.

Only the cysteine and glycine contents appear to differ in the amino acid compositions of protamine 1. The quantitation of cysteine in proteins as carboxy- methylcysteine is characteristically variable, as a re- sult of both the inefficient conversion of cysteine to carboxymethylcysteine and the instability of the deriv- ative to acid hydrolysis. In this study, attempts were not made to recover cysteine quantitatively. The amino acid sequence of protamine 1, on the other hand, does not contain glycine (Corzett et al., 19871, and the presence of glycine in P1 fractions obtained from the sperm of isopropanol-treated epididymides must repre- sent a contaminant. This is consistent with the indi- vidual analyses (the value for glycine in isopropanol treated P1 in Table 2 is the average value for glycine obtained from analyses of three separate P1 prepara- tions). Two of the three P1 samples contained only minimal levels of glycine (0.2-0.4 glycines/molecule). The third sample contained the equivalent of two glycines per molecule.

ACKNOWLEDGMENTS We thank Michele Corzett for performing the prota-

mine aminoethylations. This work was performed un- der the auspices of the U.S. Department of Energy a t Lawrence Livermore National Laboratory under con-

tract W-7405-ENG-48 and supported in part by an Associated Western Universities fellowship to C.L.

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