Volume 30, number 2 MOLECULAR & CELLULAR BIOCHEMISTRY April 18, 1980

EXPRESSION OF LACTATE DEHYDROGENASE ISOZYME X IN HAPLOID CELLS OF PIGEON TESTIS*

Jose MORENO, Cristina N. GARDENAL, Agustin AOKP and Antonio BLANCO

C6tedra de Qufmica Bioldgica, Facultad de Ciencias M~dicas and 1Centro de Microscopfa Electr6nica. Universidad Nacional de C6rdoba, 5000 C6rdoba, Argentina.

(Received November 28, 1979)

Summary Electrophoretic patterns of lactate dehydrogen- ase (EC 1.1.1.27) from adult testes show the existence of three different phenotypes for the isozyme X or Ca in populations of racing homer pigeons. These phenotypes are the expression of two different alleles at the Ldh c locus. Analysis of electrophoretic patterns of lactate dehydro- genase from lysates of mature spermatozoa isolated from pigeons heterozygous at the Ldh c locus indicates that the two different alleles are expressed in haploid cells. It is concluded that transcription of the gene coding for isozyme X must occur only in pre-meiotic cells.

Introduction Specific proteins which appear at defined stages of development and show cellular specificity may serve as useful markers of genetic activity. One such protein is the lactate dehydrogenase (EC 1.1.1.27) isozyme found in mature testis and sperm of many species 1,2. This isozyme, originally designated LDH X, is a homotetramer of polypeptide units (C) different to the A and B chains comprising the five molecular forms common to somatic tissues 3.

The C units are synthesized under the control of an additional genetic locus (Ldh c) which shows a striking timing and cellular specificity. LDH X or C4 appears in testis when sper- * This work has been supported, in part, by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) of Argentina and the Fnndaci6n Lucio Cherny (Argentina).

A.A. and A.B. are Career Investigators of the CONICET.

matogenesis start~ and is present in cells of the gametogenic line from primary spermatocytes up to spermatozoa where it constitutes more than 80% of the total lactate dehydrogenase activity 2.

Recently, H~NTZ & GOLDBERG 4, using an immunohistochemical technique, detected the appearance of LDH C4 in mouse testis at the stage of mid-pachytene primary spermatocyte.

One of the questions posed by the finding of LDH X in the testis is whether its synthesis is limited to the diploid primary spermatocyte or if it continues in post-meiotic or haploid elements.

Genetic variants of LDH X found in pigeons provide a useful marker to approach this prob- lem. Natural populations of pigeons present two different alleles at the Ldh c locus. Heterozyg- ous individuals exhibit a set of the five possible tetramers formed by association of two different polypeptides 5. This pattern would only be possi- ble as expression of the genetic complement of diploid cells and it was suggested 6 that the synthesis of LDH X should be limited to the premeiotic stage. However, this assumption was based on patterns obtained with whole testis homogenates which are the result of summation of LDH X allozymes contributed by haploid and diploid cells. The study of pure haploid cells is necessary to confirm this hypothesis.

A recent study by Meistrich et al. 7 demon- strated that the synthesis of LDH X is active not only in midpachytene spermatocytes but also in the more mature cells of the spermatogenic line. It is obvious from these observations that

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands 87

mRNA translation is carried out in pre- and post-meiotic elements. The unsolved problem is whether DNA transcription is limited to the diploid cell or if continues after meiosis.

We have performed the analysis of elec- trophoretic patterns of lactate dehydrogenase from testis and from isolated mature sper- matozoa from pigeons heterozygous for the Ldh c locus.

Results presented in this paper demonstrate that transcription of DNA for LDH X must occur only in premeiotic cells.

Materials and Methods

Adult male racing homer pigeons were obtained from several lofts in C6rdoba (Argentina). Ani- mals were killed by decapitation and laparotomized. The vas deferens were isolated by careful dissection after clamping both ends. Testes, heart and leg muscle were removed, washed in cold saline solution and blotted on filter paper.

Tissue homogenates One part of tissue (testis, heart or muscle) was suspended in nine parts of distilled water (1 : 10 w/v) and homogenized in an all glass Potter-Elvejhem grinder. The suspension was centrifuged 20 min at 25,000 g and 4 °C. Super- natants were used for analyses.

Spermatozoal lysates The sperm contained in both vas deferens from a single animal was collected by retiring the lower clamp and squeezing the tube from the proximal to the distal end. Microscopic control of the material obtained showed that the only cells present were spermatozoa.

Sperm from each bird was washed by sus- pending carefully with a glass rod in about 20 parts of 0.9% NaC1 solution and centrifuging 10 min at 3,000 g. After discarding the super- natant, the spermatozoal pellet was suspended in about five parts of distilled water. Then, the suspension was submitted to three 30-sec bursts of sonication at 100 W in a Faetron (Argentina) equipment. The lysates were centrifuged at 4 °C and 25,000 g for 20 min and the supernatants used for electrophoretic separation.

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Electrophoresis Electrophoresis of extracts was carried out in starch gel prepared with Tris-borate-EDTA buffer as indicated by MARKERT and FAULHABER 8. Migration was obtained by apply- ing a voltage gradient of 7 Volts/cm during 15 h at 4 °C.

After migration, lactate dehydrogenase activ- ity was revealed on the gel by staining with the method described by BLANCO et al.

Morphological studies Adult pigeons were anesthesized by i.v. injec- tion of chloral hydrate and the testes exposed through a wide laparotomy. The testicular pedi- cle was clamped and the testes removed and fixed by vascular perfusion with diluted Kar- novsky fixative 9 containing 1.5% distilled glutaraldehyde and 1.5% formaldehyde in 0.1 M phosphate buffer pH 7.3.

The fixed testis were cut into small blocks and then treated overnight with 1% osmium tetrox- ide. Embedding was carried out in araldite. Thin sections were cut and examined by elec- tron microscopy after double staining with uranyl acetate and lead citrate. From the same plastic embedded blocks, serial l-micron sec- tions were obtained for light microscopical studies.

Spermatogenic cells isolation The animals were decapitated and allowed to bleed. Testes were removed, decapsulated and excised in small fragments which were incubated at 31 °C for 15 to 30 min in Krebs-Ringer solution containing 2.5 mg of trypsin per ml. Incubation was carried out in a Dubnoff shaker working at 70 strokes/rain. The digestion was stopped by addition of soybean trypsin inhibitor in a final concentration of 2.5 mg per ml.

With this treatment, the seminiferous tubules disassembled and germ cells detached free into the medium. Portions of partially disarranged tubules and isolated spermatogenic cells were gently transferred to a glass microscope slide and studied with phase contrast and Normaski optics.

Results

Electrophoretic patterns Three phenotypes for LDH X o r C 4 were observed in the sample of pigeon testes

analyzed. These phenotypes were the same as those already described in natural populations and arbitrarily designated as types I, II and IIP. Although the number of bands and relative distribution of activity for each of the phenotypes were identical to those described previously, there were some differences in mo- bility of the fractions because a different buffer system was used in the present study.

Type I testes showed an electrophoretic pat- tern with six bands of lactate dehydrogenase activity; four of them migrate as the isozymes 1 through 4 common to other tissues. The other two were unique to testis extracts and could not be demonstrated in preparations of somatic tissues; they migrated toward the cathode. The fastest band was the most prominent of the pattern and the second was a minor fraction, migrating in a position intermediate between the origin and the major band (Fig. 1, H).

Fig. 1. Starch gel electrophoretic patterns of lactate dehyd- rogenase from extracts of pigeon tissues and spermatozoa. Numbers, X and X' indicate the position of the correspond- ing isozymes. A: leg muscle, B: mixture of type I+type III sperm, C: type III sperm, D: type III testis, E: type II sperm, F: type II testis, G: type I sperm, H: type I testis, I: heart and J: leg muscle.

Type III testis presented a pattern showing four bands with the mobility of the common isozymes i to 4. The fraction in the position of L D H 4, close to the origin, on the cathodal side, was the most intensely stained (Fig. 1, D).

Patterns of type I! testes exhibited eight bands. Five of these migrated toward the cathode and showed a binomial distribution of staining intensity. The fastest band of this complex presented the same mobility as that of the principal fraction of type I phenotype, while the slowest band was close to the origin, in the same position as the main fraction of type III patterns. The other three bands, migrating toward the anode, were identical to the com- mon isozymes 1, 2 and 3 (Fig. 1, F).

Sperm lysates presented electrophoretic pat- terns which matched those observed for testis from the same animal, except that the common isozymes were much fainter; only traces of L D H 1 and 2 were detected (Fig. 1, C, E and G).

Sperm from animals showing type II phenotype in testis exhibited five bands whose intensity of staining followed a binomial dis- tribution (Fig. 1, E). Type I sperm patterns did not show the minor additional fraction migrat- ing behind the principal band (Fig. 1, G).

When a mixture of equal amounts of Type I and III sperm was processed as indicated to obtain the lysates, the main band from each of the original preparations remained unmodified; there was no recombination of polypeptide units to form intermediate fractions (Fig. 1, B).

Morphological studies Light and electron microscopic observations revealed seasonal variations of spermatogenic activity with periods of rest. Our studies were carried out in animals showing active sper- matogenesis, with abundant production of sperm.

A thorough examination was performed searching for the occurrence of the intercellular bridges which afford protoplasmic continuity between differentiating spermatogenic cells and, hence, exchange of cytoplasmic components and genetic information.

In pigeon testis, as it was described for many other species, the cytokinesis of the last sper- matogonial division is incomplete, giving rise to conjoined primary spermatocytes. The com- munication gaps diminish, in size as well as in

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number, in more mature elements. In early spermatids, bridges were much less prominent and were not found in later stages of sper- miogenesis.

In isolated cells, the opening up of the intercellular bridges leads to the formation of multinucleated giant cells, acquiring the appear- ance of syncitial clusters of germ cells. By far, the most common aggregations are constituted by spermatocytes. Spermatids are rarely seen forming giant cells, but free after a short incubation in the trypsin medium.

Discussion

As demonstrated in a previous study s, the three phenotypes of lactate dehydrogenase X ob- served in pigeon testes result from the existence of two different alleles at the locus controlling the synthesis of that enzyme. Type I and III patterns correspond to animals homozygous at the L d h c locus. The main fraction in those patterns are homotetramers, designated C4 for type I and C; for type III. The C~ band happens to have the same mobility as the common isozyme 4. The second additional fraction in type I pattern (X' in Fig. 1) is a recombinant of C and B chains whose structure was proposed to be C3B1 s.

Type II zymograms correspond to heterozy- gous individuals which synthesize both polypep- tide units, C and C'. These chains are able to combine and form the five possible tetrameric associations C4, C3C~, C2C~, C1C; and C4. The expected ratio of the five forms when equal amounts of C and C' are associated is 1 : 4 : 6 : 4 : 1, which is roughly the relative dis- tribution observed in the electrophoretic pat- terns. These patterns reflect the transcription of a diploid genotype.

As the appearance of LDH X is rNated to initiation of spermatogenic actii, ity 2"1°'H, the demonstration of allozymes provides a useful model to study genetic expression during the process of maturation of the sperm cell.

It has been demonstrated that RNA synthesis occurs in post-meiotic cells ~2. This fact suggests the possibility of continued DNA transcription throughout pre- and post-meiotic elements. This appears to be the case for certain proteins 13.

Studies of MEISTmCH et al. 7 ifi the mouse

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demonstrated that LDH X or C4 is synthesized along the different stages of spermatogenesis from primary spermatocytes (diploid cells) up to spermatids (haploid cells). Whether this synth- esis involves mRNA produced de novo at each stage or mRNA produced in pre-meiotic cells and stored in more mature elements could not be ascertained from those studies.

Genetic information of haploid cells could only originate one class of polypeptide C units. If DNA transcription would occur at postmeio- tic cells, one homotetramer would be present in each individual cell. In this case, the pattern of spermatozoal lysates from animals heterozygous at the L d h c locus should show the two major bands C4 and C4 as observed with mixtures of equal parts of type I and III sperm (Fig. 1, B). Instead, patterns of sperm from heterozygous birds show that two different units are being produced simultaneously in the same cell and that they are able to associate at random.

The existence of a syncitium with wide bridges in spermatogenic cells could account for a homogeneous distribution of mRNA and proteins among all elements of the syncitium. Our observations with sections and isolated cells demonstrated broad communications at early or premeiotic stages of differentiation. The bridges are much less obvious in more mature elements. In late spermatids, where LDH synthesis occurs actively 7, the intercellular junctions are uncon- spicuous or absent. It seems very unlikely that a uniform distribution of genetic information or cytoplasmic content could occur after meiosis.

Evidence presented here indicate that trans- cription of the L d h c gene must be limited to the primary spermatocytes and, if the synthesis of the enzyme is carried out at later stages, as in the mouse, it must utilize mRNA produced in those diploid elements and stored in succeeding cells. This would be a case similar to that for protamine mRNA in trout testes, reported by IATROU and D1XON 14.

Other findings worth to mention are the disappearance of the heterotetramer C3B1 in type I spermatozoa and the marked reduction of activity of common isozymes in sperm cells of the three phenotypes.

In a study of ontogeny of LDH X in pigeon testis, it was found that the minor additional band X' (C3B1) of type I testis appears earlier than the major homotetramer C45. It seems

tha t , at t he in i t ia t ion of L D H X synthesis , at l eas t C and B p o l y p e p t i d e s a re p r o d u c e d s imul- t a n e o u s l y in t he s ame cell and are f ree to combine . E l e c t r o p h o r e t i c p a t t e r n s of s p e r m ex-

t rac t s sugges t t ha t the synthes is of A and B p o l y p e p t i d e s mus t b e t u r n e d off in m o r e m a t u r e

e l emen t s . Spe r rna togenes i s is a comp lex d e v e l o p m e n t a l

p rocess which mus t b e finely r e g u l a t e d at the gene t ic level . S tud ies of gene t ic express ion of loci r e l a t e d to specific p ro t e in s l ike those p r e - s e n t e d here , m a y con t r i bu t e to a b e t t e r u n d e r - s t and ing of tha t p rocess and afford useful m a r k e r s to assess t he effect of fac tors invo lved in t he sequen t i a l even t s compr i s ing the d i f feren- t i a t ion of gamet i c cells in test is .

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