DNA Synthetic Capabilities of Differentiating Sperm Cells

DNA Syathetic Capabilities of Differentiating Sperm Cells D. L. DAENTL, R. P. ERICKSON* and C. J. BETLACH Departments of Growth and Development and Pediatrics, University of California San Francisco 630S, San Francisco, California 94143, USA Received October 1976/Accepted February 1977

Spermatogenic cells separated by velocity sedimentation were analysed by a micro-procedure for differentiation-associatedchanges in DNA synthetic capabilities. DNA-dependent DNA polymerase activity is maximal in premeiotic and meiotic cells, sequentially declines in progressively more differof the maxientiated spermiogenic cells to a minimum value in testicular spermatozoa which is mum. No further decrease of activity is observed during the subsequent process of sperm cell maturation and, at the end-differentiated state, the potential of sperm cells for DNA synthesis is demonstrated by the presence of substantial activities of thymidine and thymidylate kinases as well as DNA polymerase. Although levels of DNA polymerase activity, as determined by in vitro assay, are negatively correlated with the state of differentiation, the findings support the hypothesis that, in this cell system, DNA synthetic enzymes may not be limiting factors in the control of DNA synthesis.

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Introduction

The presence of both thymidine kinase and DNA polymerase activities in amphibian [l, 21 and mouse [3, 41 oocytes and eggs, might lead to the expectation that the enzymatic machinery necessary for gene replication in the mouse zygote resides solely in the egg. An unexpected finding, therefore, is the presence in mammalian spermatozoa of substantial activities of DNA polymerase [ 5 , 6, 71 even though these highly differentiated cells do not replicate DNA unless fertilization occurs. Such a finding raises an additional question regarding comparative levels of DNA synthetic enzymes at the various stages of spermatogenesis antecedent to the endstate of sperm differentiation. In one previous study 181, this question was pursued by analysis of testes from prepuberal mice at various maturational stages during the first wave of spermatogenesis from days 5 through 42. Interpretation of the results obtained in those experiments is hampered by 1) the existence of several different cell types maturing in parallel at the various developmental stages and by 2) alterations of the pool sizes of the different cell types relative to each other during the course

*

Current address: Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48104, USA Differentiation 8, 159-166 (1977) - 0 by Springer-Verlag 1977

of prepuberal testicular development. For example, on about day 10-12 in the mouse, although the first generation of primary spermatocytes has entered pachytene, division of stem cells simultaneously results in a new population of spermatogonia [91. More conclusive data can be obtained from analysis of the various spermatogenic cell types after separation into homogeneous populations. Although such separations are now possible by velocity sedimentation [lo], a further obstacle is the small numbers of each cell type obtained for analysis after a standard separation procedure. We have been able to solve this problem by adapting microassay techniques previously utilized for mouse eggs [31 to measure total DNA polymerase activities of separation populations of the different types of spermatogenic cells obtained from testes of sexually mature mice. The results of our studies indicate that, although present at all stages of spermatogenesis, DNA polymerase activities are greatest in premeiotic and meiotic cells, decrease during subsequent stages of differentiation and are lowest in spermatozoa. As further indication of DNA synthetic capabilities present even at the end-state of differentiation, we have found that mature spermatozoa also have substantial activities of thymidme and thymidylate kinase.

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Methods Sperm Cells

For each experiment sperm cells were either obtained from all extratesticular reproductive tract structures of one mouse or from the epididymides and ducti deferentes of two mature male mice (Carworth CFW, Portage, MI) by a previously described method [ill. Washing and centrifugation were carried out in Beatty’s medium.

Testicular Cells

To prepare a suspension of testicular cells, seminiferous tubules of testicles from three mice were teased apart, cut into 1 mm pieces and placed in a sterile tube containing 4 ml/lOO mg wet testicular weight of Tyrode solution [ 121 containing 100 u/ml penicillin. This mixture was incubated for 2 h at 30” C with 500 units/100 mg wet testicular weight of collagenase IV (Worthington, Freehold, NJ) and 10 pg/ml deoxyribonuclease I (Worthington, Freehold, NJ). Separation of the cells was aided by pipetting them several times at hourly intervals during the incubation period. At the end of incubation the total number of cells in the extract was 119.5 x lo6. Separation of Testicular Cells

A 30 ml suspension of cells, (2 x 106/ml) in 0.5% Ficoll-Tyrode solution’ containing penicillin, was then loaded in a Staput chamber over a 1600ml gradient of 1-3% Ficoll in Tyrode solution with penicillin. Velocity sedimentation was performed [ 101 for 10 h at 4’C and the gradient was then collected in 10ml fractions. The number of suspended cells in every second or third fraction was determined by counting an aliquot in a hemocytometer and simultaneously the cellular morphology was noted. Approximately eight cell peaks were obtained: tubes from a single peak were pooled and the cells concentrated by centrifugation and resuspension in 40-70 p1 Tyrode solution’. This resulted in recovery of 30-50% of pooled cells from a single peak. For each peak, lop1 aliquots of the suspended cells were counted and the remainder assayed for DNA polymerase activity (20 pl) and protein content (10-40 pl),

Enzyme Assays

The assays for DNA polymerase, DNA primer activation and thymidine kinase activities were modifications of microprocedures utilized for measurements in preimplantation mouse embryos [131. 1 . DNA Polymerase Assay. A cell pellet of spermatozoa or testicular cells was suspended and rapidly freezethawed in 100 pI of a solution (sterilized by passage through a Millipore fdter) containing 0.05 M ammediol, pH 8.8, 8mM MgCI,, 0.01 M KCI, 0.5 mM EDTA and 1 mM mercaptoethanol. Sixty p1 of this solution, which contained 5-10 pg proteidpl [141, was sonicated and immediately assayed for DNA polymerase. Sonication was accomplished in ice-water with an energy of 200 W at the tip of a standard probe (Brownwill, Biosonik IV) applied to the exterior wall of a tightly stoppered 0.45 ml polypropylene microtube (Beckman, Mountain View, CA) taped to the

’

Glucose omitted

bottom of a 2 1 polypropylene beaker which contained the icewater. The resulting fine dispersion of droplets which was distributed over the interior of the tube after 5 s of sonication was collected at the bottom of the tube by a brief centrifugation in a Beckman Microfuge and the sonication procedure was repeated for an additional 5 s. After again collecting the droplets in the bottom of the tube, a 10 p1 drop of sonicate was transferred to the bottom of a sterile plastic culture tube (Falcon, 2.5 ml, Oxnard, CA) and directly to this was added a 10 pI drop containing a sterile solution of ammediol, pH 8.8 [IS], 10 mM ATP (Sigma, St. Louis, Missouri), 10 mM MgCI,, 100 pM each of dGTP, dATP, and dCTP (Sigma), 10pM ”-dTTP (59.5 C/mmol, Schwartz-Mann, Van Nuys, CA) and 0.2 m g / d “activated” calf thymus DNA (Sigma, St. Louis, Missouri) prepared by the method of Loeb [161. The tubes were capped and, following a 60 min incubation at 37” C, the reaction was terminated by the addition of 0.5 ml of ice cold 5% trichloroacetic acid containing 1% sodium pyrophosphate. After 1 h at 4” C, the precipitates were collected on 2.4 cm glass fibre filters (Whatman GF/C), washed in increments with 50 ml of ice cold 95% ethanol and then suctioned to dryness. The filters were transferred to counting vials and the radioactivity solubilized in 0.5 ml of 0.2 N HCI heated to 80° C for 30 min and cooled. The solutions were dissolved in 10 ml of a 10% Biosolv (Beckman, Mt. View, CA)-Omnifluor; 4 gm/L, New England Nuclear, Boston, M)-toluene mixture and counted in a Beckman liquid scintillation spectrometer at ambient temperature. Measurements were corrected by subtraction of “blanks” consisting of the sonicated enzyme solution heated to 100’ C for 3 min prior to the reaction. When the effect of omitting one or more of the reactants was tested, pooled extratesticular sperm cells were utilized and the procedure was the same as described except for the omission of the indicated compound or compounds from the reaction mixture. Treatment of the radioactive product with DNAse I (after the reaction was stopped by heating to 100’ C for 3 rnin and prior to the addition of 5% TCA-1% sodium pyrophosphate) was accomplished by adding to the 20 ul incubation mixture an equal volume of a solution containing 200 pg/ml, of electrophoretically purified DNAse I (bovine pancreas, Sigma, St. Louis, Mo) in 1.5 mM MgC1,0.1 N sodium phosphate buffer, pH 7.0, prepared with heat-sterilized water. The digestion time was 2 h. The reaction was terminated by the addition of 0.5 ml of ice-cold 5% TCA-1% sodium pyrophosphate; collection of acid-insoluble radioactive precipitates and counting were performed as indicated above. In experiments utilizing initiated homopolymer templates, the same general procedure was utilized as outlined above except that the reaction mixtures contained 0.05 M ammediol, pH 8.8, 0.125 M KCI, 1 mM mercaptoethanol, 0.5 mM MnCI,, 5 mM ATP, 0.0625 pg/pl poly rA (Collaborative Research, Waltham, MA), 0.0625 pg/p1 dT,,-,, (Collaborative Research), and 10 pM W d T T P 1171 or 0.05 M ammediol, pH 8.8, 0.01 M KC1, 0.5 mM MnCI,, 1 mM mercaptoethanol. 4 mM ATP, 0.0625 pg/pl poly dA (Collaborative Research), 0.0625 pg/pl dTIz-,, (Collaborative Research) and 10 pM3H-dTTP [17]. 2. DNA Primer Activation Assay. An adaptation of the procedure of Studzinski and Fischman [181 was utilized. Pooled extratesticular sperm cells were incubated for 1 h under the exact conditions utilized for the DNA polymerase assay above except that the calf thymus DNA was not “activated”. Following this incubation, the reaction mixture was placed in a 60” C water bath for 10 min to inactivate most DNAases and immediately cooled in an ice bath. To the cooled solution were added more sperm cells and substrate nucleotides such as to maintain the original concentrations. The amount of DNA

16 1

DNA Polymerase in Spermatogenic Cells which had become “activated” as a result of the first incubation was then assayed by incubation for an additional hour at 37” C. The final result was obtained by subtraction of a “blank” run exactly in parallel with the sample except that sperm cells heated to looo C for 3 rnin were utilized for the initial incubation.

Table 1. DNA polymerase activity of mouse spermatozoa

3. Thymidine (and Thymidylate) Kinase Assay. For each experiment, washed sperm cells from two mice were suspended in 180 pl of sterile 0.05 M Tris buffer, pH 8.0 containing 0.02% bovine serum albumin (BSA) and 0.3 mM dithiothreitol (Sigma, St. Louis, MO). After rapid freezethawing, 60 pl of the well-mixed sperm cell suspension were transferred to a microfuge tube and sonicated as described above. The remaining solution was saved for determination of protein content [141. Thymidine and thymidylate kinase activities [19, 201 of 10 pl aliquots of the sonicate were assayed immediately. The reaction was started by the addition of 10 p1 of the sterile Tris-BSA buffer containing 80 pM 3H-methyl thymidine ( 6 C/mmol, Schwartz-Mann, Van Nuys, CA) purified on the day prior to use 1201, 20 mM MgCl,, 0.10 mM ATP, 0.3 mM dithiothreitol, 30 mM K’ phosphoenol pyruvate (Sigma, St. Louis, Mo) and 2.5 x pg pyruvate kinase (Type 11, Sigma, St. Louis, Mo). Following 30 min of incubation at 37” C, the reaction was terminated by placing the tubes in a boiling water bath for 2 min. Radioactive phosphorylated products of the reaction were separated from the radioactive substrate on PEI cellulose thin layer plastic sheets (Brinkman, Burlingame, CA) utilizing the LiCl step-wise elution procedure of Randerath and Randerath [21]. The location of separated products and reactants was determined by UV light absorption of non-radioactive marker compounds which had been spotted with each radioactive mixture. After drying, the chromatograms were cut into 0.5 cm segments in the regions containing phosphorylated nucleotides. The radioactivity eluted from each segment with 0.2 N HC1 was determined by counting in a 10% Bio-Solv-Omnifluor-toluenemixture, utilizing a Beckman liquid scintillation spectrometer at ambient temperature. “Blanks” of the enzyme solution boiled for 2 min prior to addition of the reaction mixture were incubated and chromatographed in parallel. The radioactivity migrating with phosphorylated compounds in the “blanks” was approximately equal to background due to the use of purified isotope. For every radioactive product formed, the values obtained for “blanks” were subtracted from those obtained for samples. The net sum of radioactivities of the dTMP, dTDP and dTTP spots is the value for thymidine kinase activity and the net sum of radioactivities obtained for the dTDP and dTTP spots is a minimal estimate of thymidylate kinase activity. When the effect of dTTP and BrdU on thymidine kinase activity was tested, these compounds were included in the reaction mixtures at the indicated concentrations and the reactions were performed simultaneously with those in which dTTP and BrdU were excluded.

Sperm cells from head of epididymis (4) Sperm cells from vas deferens (4) Pooled extratesticular sperm cells (4)

Results

.DIVA Polymerase Activity of Sperm Cells Initial experiments studied the DNA polymerizing reaction in sperm cells. Table 1 summarizes the results obtained for total DNA polymerase activity using an “activated” calf thymus DNA template and 3H-dTTP as the radioactive substrate. Formation of the acid precipitable

Pmol 3H-dTMP incorporated/mg protein/h 28.5 k 2.1 25.3 k 6.7 24.8 k 6.8

Reaction mixture minus dATP (1) dGTP, dCTP (1) dCTP (1) dGTP (1) dGTP, dCTP, dATP (1) MgC4 (1) “activated” DNA (1)

0.4 k 0.03 0 0 0 0 0.6 k 0.04 0.5 k 0.04

Product DNAse treated (2)

0.6 k 0.04

Values are the mean k SEM of triplicate samples from the number of separate experiments indicated in parenthesis

product was linear for greater than 60 min and template, Mg2+ and all four deoxynucleoside triphosphates were required for maximal activity. The magnitude of incorporation of radioactivity was directly proportional to protein concentration in the reaction mixture over the range of protein concentrations utilized. For this crude reaction mixture pH 8.8 was optimal and there was 70% inhibition of activity by 1 mM NEM (N-ethyl-maleimide) and 85% inhibition by 10mM NEM. In the presence of 0.15 M NaC1, the overall activity was reduced by 25%. Incubation of the radioactive product formed with RNAse-free DNAse I resulted in almost complete loss of radioactivity; however, this did not occur when purified RNAse, protease, or trypsin was used. Polymerase activities observed for sperm cells from the epididymis were comparable to those for sperm cells from the vas deferens and to pooled total extratesticular sperm cells, suggesting that no net loss of activity occurs during the process of sperm maturation. No purification steps were carried out prior to the DNA polymerase assay; therefore, endogenous DNAse activity could result in values which are either higher or lower than actual. To obtain some estimate of the potential error produced by endogenous nuclease activity of the type causing breaks of phosphodiester bonds to liberate 3’OH termini, the extent of activation of the DNA template by the sonicate was measured under the exact conditions of the polymerization reaction. After incubation at 37” C for one hour at pH 8.8, DNA template activation amounted to 0.6 i-0.1 pmoles 3H-dTMP incorporated/h/mg protein. This represented only 2% of

162

D. L. Daentl et al.:

Table 2. hitiated homopolymer replication activity of mouse sper-

matozoa Homopolymer

Pmol 3H-dTMP incorporated/rng proteidh

Poly rA/dT,,-,, Reaction mixture complete (2) minus Poly rA (2) minus dT,,-,, (1)

2.17 k 0.06 0.17 k 0.002 0.76 k 0.05

Product DNAse I treated (2)

0.11

Poly dA/dT,,-,, Reaction mixture complete ( 2 ) minus Poly dA (2) minus dT,,-,, (1)

0.06

9.23 0.63 0.17 & 0.002 0.29 k 0.03

Product DNAse I treated (2)

0.07 & 0.07

Values are the mean k SEM activities of duplicate samples from two experiments

--SPERMAlOCYlfS

ROUND SPERMATIDS

the value obtained for DNA polymerase activity, suggesting that under the conditions of the DNA polymerase assay, the error caused by endogenous nuclease activity of this specific type was probably negligible. The effects of endogenous nuclease activity of the type causing hydrolysis of phosphodiester bonds with liberation of 3‘ phosphoryl groups were not assessed. Alkaline phosphatase could also have reduced the incorporation of thymidylate in the polymerase reaction. However, there was a large excess of ATP and non-radioactive triphosphate substrates relative to the 3H-dTTP concentration in the reaction mixture, and this probably protected against significant error due to that cause. When initiated homopolymers were utiliied as templates for replication rather than “activated” DNA, considerable net incorporation of 3H-dTTP was noted (see Table 2). Both ribose (poly rA) and deoxyribose (poly dA) templates were active and in each case the presence of 0.5 mM MnCl,, the single-stranded polymer and the oligomer initiator were all required for maximal enzyme activity. Regardless of which template was utilized, the radioactive products formed using the oligodeoxyribose initiator (dTlz-18)were susceptible to digestion with DNAse I.

ELONGAIED SPERMATIDS

l,perm.bgoni.l

DNA Polymerase Activity of Separated Spermatogenic Cells

t t 1

.6

L

MIGRATION RATE mm/hr 3?4

I

0

3p4 3?3

3 9 3 2?3

I

I

20

2fZ I

40

60

2!2

\

IF2 I

80

1$0

.3

122

100

FRACTION NUMBER

Fig. 1. DNA polymerase activity of separated testicular cells. Enzyme activities were comparable when expressed either on a cell or mg protein basis. Also indicated in the figure are the cell numbers, migration rates and cytologic features of the fractioned cells. The three major fractions each contained > 70% of the indicated major cell type (as noted by phase contrast microscopy). Results of a single experiment are depicted. Similar results were obtained when the experiment was repeated

During mouse spermatogenesis, the last major DNA synthesis occurs in preleptotene primary spermatocytes [22, 231. A small amount of DNA synthesis may also take place during meiotic prophase [24,251 and may be related to repair of chromosome breaks at the time of crossing over. It is therefore of interest to compare the DNA polymerase activity obtained for mature sperm cells with that obtained for cells at the various stages of sperm formation. DNA polymerase activity was assayed in separated fractions of spermatogenic cells after velocity sedimentation on a 1-3% linear gradient of bovine serum albumin. Both the migration pattern of the various types of testicular cells and the values obtained for DNA polymerase in the various fractions are plotted in Figure 1. As might be expected, a sharp peak of activity is present in fractions containing a mixture of primary and secondary spermatocytes (premeiotic and meiotic cells). Subsequent stages, corresponding to a progressive loss of cytoplasm, show a considerable decline in activity (round spermatids and elongated sperof the maximum, a value comparable to matids) to that obtained for mature spermatozoa. Some of the premeiotic cells which undergo mitosis (spermatogonia)

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163

DNA Polymerase in Spermatogenic Cells Table 3. Thymidine kinase and minimal thymidylate kinase activities of mouse spermatozoa: Inhibition by dTTP and BrdU Thymidine kinase activity

Minimal thymidylate kinase activity

Pmol "-dTMP + 3H-dTDP + 3H-dTTP formed/mg proteidh

Pmol 3H-dTDP + 3H-dTTP formed/mg proteidh

Control 370 & 25 + 0.1 mM dTTP 58.9 ? 3.9 (16%) + 0.04 mM BrdU 16.2 & 1.0 (4%)

49.1

t 9.4

2.01 T 0.46 1.29 & 0.15

Values are expressed as the mean k SEM of duplicate samples from two different experiments

separated together with elongated spermatids and may account for the shoulder of polymerase activity seen in the last portion of the plotted curve. Thymidine Kinase Activity of Sperm

To assess further the capability of sperm cells to synthesize DNA, the thymidine kinase activity of these cells was determined by a micro-procedure. Radioactive phosphorylated products of the reaction were identified and quantitated after separation on PEI cellulose thin layers. The reaction was linear for 60 min and a 30-min incubation period was utilized for all experiments. Table 3 indicates that considerable thymidine kinase activity is present in spermatozoa and the value obtained is ten-fold greater than the value for DNA polymerase activity. Under the conditions of the reaction, radioactive dTDP and dTTP were formed in addition to radioactive dTMP, indicating the presence also of thymidylate kinase. In the table a minimal estimate of thymidylate kinase activity of sperm cells is given; however, an assay in which 3H-dTMP substrate was initially saturating was not performed. Distal end product allosteric inhibition of thymidine kinase by dTTP is a property which has been observed in a variety of cells [191. In agreement with these results and those obtained for preimplantation mouse embryos [201, 84% inhibition of activity was noted when 0.1 mM dTTP was present in the sperm reaction mixture. Further similarity of sperm thymidine kinase to the enzyme found in other tissues [261 and in preimplantation mouse embryos 1201 was discovered when competition of bromodeoxyuridine (BrdU) for thymidine as substrate was tested. The presence of 40 pM non-radioactive BrdU in the reaction mixture together with 80 pM radioactive

thymidine resulted in a value for radioactive phosphorylated product which was 4% of that obtained when BrdU was absent from the reaction mixture. Discussion

These investigations suggest that differentiating sperm cells undergo a sequential decline in total cellular deoxynucleotide polymerizing activity from a maximum in primary and secondary spermatocytes to a minimum value which is one-fourteenth of the maximum at the endstate of testicular sperm cell formation. If DNA polymerase activity were in some way correlated with the amount of DNA, the initial rapid loss of activity observed for postspermatocyte fractions might be understood in terms of alterations in DNA content per cell - a decrease from 4n to n with completion of meiosis. Taking this decrement of cellular DNA content into consideration, the 14-fold decrease in specific activity calculated on a per cell basis would then amount to a 3'/,-fold decrease when based on DNA content. As seen in Figure 1, the loss of specific activity is more rapid on a per cell basis than on a per mg protein basis - a finding consistent with the fact that the major protein loss does not occur until sperm release. This result suggests that there is a continued slow decrease of polymerase activity antecedent to sperm release. No further decrease of activity is apparent during sperm cell maturation and extratesticular sperm cells possess substantial amounts of both DNA polymerase and thymidine kinase activities. This suggests that DNA synthetic enzymes may not be limiting factors in the control of DNA synthesis in these cells. The possible importance of template restriction in this control has been suggested by studies of Heston et al. 1271, who have shown that treatment of rabbit spermatozoa with disulfide reducing reagents, followed by acidic polymers such as heparin or polyxanthylic acid, allows the endogenous DNA to serve as template for DNA synthesis by exogenous E. coli DNA polymerase. Analogous to the observations in differentiating sperm cells, a sequential decrease in DNA polymerase activity to low but not totally absent levels has been observed in other differentiating systems such as brain [28,291, skeletal muscle [301 and cardiac muscle [311, although in cardiac muscle cytodifferentiation and cellular proliferation are not mutually exclusive events. Our studies have been conducted on cells from mature mouse testes separated by velocity sedimentation. Because of the small numbers of cells obtained in each fraction, analysis of DNA polymerase activities of subcellular fractions was not performed. When other mam-

164 malian cells have been separated into subcellular fractions by aqueous techniques, at least four DNA polymerases have been identified. A high molecular weight (6-8 S) soluble fraction enzyme, DNA polymerase a, predominates in rapidly dividing cells, has a pH optimum at neutral pH and is sensitive to high ionic strength and to N-ethyl-maleimide (NEM) 1301. A low molecular weight (3.4 S) enzyme, DNA polymerase p, is found in both nuclear and cytoplasmic fractions and appears to vary little with the proliferative state of the cell [321. It is stimulated by high ionic strength, resistant to NEM, and its pH optimum is well into the alkaline range. Two other cellular DNA polymerases, proportionally small in amount, are mitochondrial [33, 341 and DNA polymerase y [351. The latter enzyme utilizes polyribonucleotide templates; however the ability to utilize poly rA has also been attributed to ,B polymerase [321. Analogously to these findings, in aqueous extracts of subcellular fractions of whole testis Hecht has distinguished at least four DNA-dependent DNA polymerase activities [361: a low molecular weight DNA polymerase in the nuclear fraction corresponding in properties to DNA polymerase p, two DNA polymerases in the soluble fraction which are probably DNA polymerases a and an aggregated form of p, and mitochondrial DNA polymerase 151. However in mouse spermatogenic cells and spermatozoa, a nuclear location of DNA polymerase activity appeared to predominate in the cytochemical studies of Chevaillier and Pillippe [61, suggesting the presence of DNA polymerase a and/or p. Their studies revealed that sperm enzyme activity was sensitive to high KCl concentrations, but insensitive to NEM. We detected a limited decrease of total sperm DNA polymerase activity in the presence of 0.15 M NaC1, but contrary to the results of Chevaillier and Philippe, we found an alkaline pH optimum, 70% inhibition of DNA polymerase activity in the presence of 1 mM NEM and enhanced activity in the presence of mercaptoethanol. However, in neither our study nor that of Chevaillier and Philippe were the enzymes purified. When purified enzymes from nuclear and cytosol fractions of whole testes of 5-10 day-old mice were analyzed, sensitivity to 200mM KC1 and NEM was evident, especially in one cytosol enzyme corresponding in pH optimum and chromatographic behaviour on phosphocellulose to DNA polymerase a 181. As had been found in mature mouse testes, a nuclear enzyme corresponding to DNA polymerase J’/ had only a 50% reduction of activity in 0.3 mM NEM and was resistant to the effects of 200 mM KC1. Similar properties were present in a cytosol enzyme which may have been an aggregated form of DNA polymerase p. In Hecht’s studies of prepuberal

D. L. Daentl et al.:

testes, a six to seven-fold decrease in the specific activity of the cytosol DNA polymerase activity occurred between five days and sexual maturity while over the same period, there was a two to three-fold increase in enzyme activity of the nuclear fraction [81. However, location in cytosol or nuclear fractions may only be an artefact of the aqueous subcellular fractionation procedure and, considering the low relative contribution of DNA polymerase mt and y , total DNA polymerase activity may be a more meaningful measurement in physiological terms. Recent investigations have provided support for this possibility, DNA polymerase a activity was largely associated with the nuclear fraction when nonaqueous isolation procedures were used [371. Also, greater than 85% of total DNA polymerase a activity was located in nucleated cell fragments when enucleation was induced in mouse L 929 cells with cytochalasin B [381. This suggests that the enzyme is either nuclear of perinuclear. Taking all these studies into consideration, it is possible that both DNA polymerase a! and p activities were measured in our studies of spermatogenic cells and spermatozoa. DNA polymerase a is the same enzyme type which progressively decreased during terminal differentiation of skeletal muscle 1301, cardiac muscle 1311 and brain 1281. Similarly, of the two types of thymidine kinase, soluble and particle-bound 1391, it is the activity of the soluble enzyme in this salvage pathway to production of dTTP which varies directly with the proliferative state of the cell [401. However, unlike DNA polymerase a, its activity is cytosol-associated in cytochalasin B enucleation studies [381. What is the physiological function of thymidine kinase and DNA polymerase in terminally differentiated sperm cells? Mitochondria1 enzymes might be used for turnover of mitochondrial DNA - since this is reportedly high [36, 41, 421 - however activity in the nucleus has been demonstrated [61. “Repair” DNA synthesis would seem to be a potential function; however, the studies of a number of investigators have shown that repair synthesis following UV [43,441 or X-irradiation 1451 or treatment with a chemical mutagen 1461 does not occur in mature sperm cells, although it does occur at earlier stages of maturation and during spermatogenesis. (This artificially stimulated type of repair synthesis may or may not be identical to the type of meiotic repair synthesis that has been observed as a distinctive activity of pachytene cells presumably related to crossing over [471). In our studies, the peak of polymerase activity observed in the separated cells seems to correspond to spermatocytes which have already completed the major DNA replication; but these spermatocytes could be engaged in pairing synthesis. Progressive failure to repair

DNA Polymerase in Spermatogenic Cells

damaged DNA has been observed in another differentiating system: Muscle cells, as they differentiate, become less capable of repairing damaged DNA induced by univalent alkalating agents [481 and have a diminished ability to engage in non-semiconservative DNA synthesis following UV irradiation [49, 501. It is possible that DNA polymerase and thymidine kinase serve no function in spermatozoa and are simply proteins which are coincidentally stabilized, perhaps through tight binding to chromatin and the template restriction caused by nucleoprotein disulphide bond formation which occurs during sperm maturation b1I. If this is the case, an additional question can be raised: can these paternal enzymes persist during fertilization and participate in initial replication of the embryonic genome? Acknowledgements: We thank Mrs. Katalin Nyiredy, Miss Susan Martin and Miss Linda Schmidt for excellent technical assistance. This work was supported by grants BC-134 from the American Cancer Society and HD 07061 from the National Institutes of Health.

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