Polymorphism of Rat Seminal Vesicle Secretory ...

Biochemical Genetics, Vol. 22, Nos. 5/6, 1984

Polymorphism of Rat Seminal Vesicle Secretory Proteins: Characterization of svp-1 and svp-2 and Their Identification with the Major Secretory Proteins IV and V A. Maffei, 1 G. Paonessa, 2 P. Abrescia, 3 S. Metafora, 2 and J. Guardiola ~'4

Received 16 Sept. 1983--Final 10 Feb. 1984

The proteins secreted by the rat seminal vesicle can be separated into five major fractions (namely, RSV-I through I7) by gel electrophoresis in denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and R S V - V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant o f RSV-IVthat can be expressed only in rats homozygous for a given allele at the s v p - 2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology o f R S V - I V and R S V - V and of their corresponding gene.

This work was supported by a CNR grant from Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie genetiche. International Institute of Genetics and Biophysics, CNR, via Marconi 10, 1-80125, Naples, Italy. z Institute of Molecular Embryology, CNR, Arco Felice, Naples, Italy. 3 Istituto di Chimica Organica e Biologica, Facoltfi di Scienze, Universitfi di Napoli, Naples, Italy. 4 To whom correspondence should be addressed. 567 0006-2928/84/0600-0567503.50/0

© 1984 Plenum Publishing Corporation

568

Maffei, Paonessa, Abrescia, Metafora, and Guardiola

KEYWORDS:polymorphism;rat seminal vesicle;secretoryproteins.

INTRODUCTION The seminal vesicles are male accessory sex glands whose secretions contribute a large portion of the seminal plasma in many animals. The proteins secreted by these glands have been classified as five major fractions (I through V) by gel electrophoresis under denaturing conditions (Ostrowski et al., 1979). Fraction IV, also named RSV-IV or SVS-IV, and fraction V, also named RSV-V or SVS-V, have been studied in some detail; they have no biological function that can be tested. Their amino acid compositions and sequences have been reported (Kistler et al., 1981; Mansson et al., 1982; Abrescia et al., 1982). The mRNA sequence of RSV-IV has been inferred from the corresponding cloned cDNA. Some information on the genomic organization of the gene(s) for protein IV and the purification of genomic clones has been published (Abrescia et al., 1982; Higgins et al., 1983; Kandala et al., 1983). Other authors (Platz and Wolfe, 1969; Gasser, 1972; van Zutphen et al., 1981) described electrophoretic polymorphisms related to two secretory proteins in the rat and in the mouse, namely, svp-1 and svp-2; the svp-I locus maps on chromosome 2 in the mouse (Platz and Wolfe, 1969) and has been assigned to the fourth linkage group in the rat (Gasser, 1972). These two proteins were defined on the basis of their mobility in a nondenaturing gel system; consequently no conclusion can be drawn as to their correspondence to one of the five major proteins observed in denaturing conditions. The aim of the present work is to bring together all these data by showing that the svp-1 protein is indeed the RSV-V (or SVS-V) protein and that the svp-2 protein is a variant of RSV-IV.

MATERIALS AND METHODS Commercial stocks of Fisher and Wistar rats were obtained through a local breeder (Parrella-Lisbo, Brusciano, Naples). Matings were established between rats of the two stocks, each of which proved to be monomorphic for a different svp-1 allele; inter- and intrastock crosses were performed in our animal house under controlled conditions. Polyacrylamide gel electrophoresis was carried out under previously described conditions either in Tris-glycine buffer, pH 9.5 (Platz and Wolfe, 1969), or in SDS-containing buffer (Abrescia et al., 1982). Protein elution from unstained gels was performed according to a slight modification of published procedures (Higgins et al., 1976). Automated amino acid composition determinations were made as described by Higgins et al. (1976).

Polymorphismof svp-1 and svp-2 Proteins

569

RESULTS svp Polymorphisms of the Rat Stocks Employed

Commercial stocks of rats were obtained through a local breeder; outbred Fisher male rats and outbred Wistar male rats were mated with outbred Fisher females under controlled conditions. The male offspring of Fisher x Fisher crosses were sacrificed together with their male parents, their seminal vesicle was excised, and their seminal vesicle fluid was collected and analyzed by gel electrophoresis in Tris-glycine buffer, pH 9.5. All the animals were found to be homozygous for the svp-1 a (Gasser, 1972) allele (2/2 male parents and 18/18 male progeny; see Fig. 1, lane b). Two males from the Wistar stock were killed after fertile crosses with Fisher females. Upon analysis of their seminal vesicle fluids, they turned out to be svp-1 b homozygotes (Fig. 1, lane a); their male offspring generated by matings with Fisher females were s v p - l a / s v p - 1 b heterozygotes (4/4) (see Fig. 1, lanes c and d). We then examined the segregation of the svp-2 allele. According to van Zutphen et al. (1981) an individual whose seminal vesicle secretion shows the presence of the svp-2 protein when analyzed under nondenaturing electrophoretie conditions can be classified as a svp-2 a homozygote; on the contrary, failure to exhibit the protein band relative to svp-2 is characteristic of svp-2 b hornozygotes or of heterozygotes. In our case, some Fisher x Wistar male offspring expressed the svp-2 protein band (see Fig. 1, lane d) and were thus homozygous for the svp-2 a allele (2/4). Conversely, and in analogy with their parents, all Fisher x Fisher male progeny (18/18) failed to exhibit the svp-2 protein band, thus suggesting that they were svp-2 b homozygotes (see Fig. 1, lane c). The appearance of s v p - 2 a homozygotes among the male progeny of Wistar x Fisher crosses can be best explained by assuming that the Fisher females and the Wistar males were heterozygotes, while the Fisher male carried only the svp-2 ~ allele (see Table I). Electrophoretic Analysis and Amino Acid Composition Determinations

The proteins, svp-1 and svp-2, have been defined solely on the basis of their electrophoretic pattern in nondenaturing gels (Platz and Wolfe, 1969; Gasser, 1972; van Zutphen et al., 1981). The major secretory proteins of the seminal vesicle, however, have also been separated into five homogeneous fractions by electrophoresis under denaturing conditions (Ostrowski et al., 1979); fraction IV (RSV-IV) and fraction V (RSV-V), because of their basic character, can be suspected to migrate as svp-1 and/or svp-2 under nondenaturing conditions. In order to establish whether svp-1 and svp-2 actually correspond to

570


a

b

c

•-

svp

-

sv

-

svp

- I b p - 2 a

- I a

d

Fig. 1. Total seminal vesicle secretory protein pattern after polyacrylamide (7%) gel electrophoresis in Tris-glycine, p H 9.5, buffer. Lane a: (Wistar × Wistar)F1. Lane b: (Fisher × Fisher)F1. Lane c: (Fisher × Wistar)F 1. Lane d: (Fisher × Wistar)F1 homozygous for the s v p - 2 ~ allele.

Polymorphism of svp-1 and svp-2 Proteins

571

Table I. Geotypes and Phenotypes of the Progeny of Crosses Among Rats Carrying Different svp-2 Allelesa i

i

svp-2 genotype of parents

Cross 2

¢

~

Fisher

× Fisher

b/b

a/b

Wistar

x Fisher

a/b

a/b

Genotype of the progeny

Presence of the svp-2 protein band

a/b b/b a/a a/b b/b

+

i

aThe number of offspring exhibiting a given phenotype is referred to in the text (see Results).

RSV-1V and/or RSV-V, we obtained the seminal vesicle secretion from rats of different genotype; the proteins contained in such secretions were analyzed by electrophoresis under nondenaturing conditions (Fig. 2A, lanes a, c, e, and f); the svp-1 ~ and svp-1 b proteins were eluted from the gel and tested in nondenaturing gels for their purity (Fig. 2A, lanes b and d). The same secretions were analyzed under denaturing conditions (Fig. 2B, lanes A, B, D, and F). The proteins at 18K and 15K are, according to Ostrowski et al. (1979), respectively, RSV-IV and RSV-V; the position of RSV-IV in this type of gel was independently determined by running native RSV-IV protein purified according to Ostrowski et al. (1979) (not shown). As shown in Fig. 2B, lanes C and E, the svp-1 variants have the same electrophoretic mobility as RSV-V. As an additional test, the amino acid compositions of both svp-1 forms were determined and compared with the amino acid composition of RSV-V (Ostrowski et al., 1979). This analysis confirms that svp-1 and RSV-V are the same protein (see Table II). The svp-2" protein was also eluted from an unstained gel such as that in Fig. 2B, lane F. The protein content of such a svp-2 preparation was analyzed in SDS gels (Fig. 3); it was shown that svp-2 (lane C) is distinct from both forms of svp-1 (Fig. 3, lanes B and D). A comparison of the apparent molecular weights of svp-1 and svp-2 with purified RSV-IV and RSV-V apparent molecular weights is shown in Fig. 3, lane E. The svp-2 protein migrates slightly ahead of RSV-IV; an additional protein band is in fact observed when the secretory proteins of a s v p - 2 a homozygote are analyzed under denaturing conditions (compare Fig. 2B, lanes A and B). The amino acid composition of svp-2 was determined and compared with the amino acid composition of purified RSV-IV (Ostrowski et al., 1979). The amino acid composition of svp-2 turned out to be strikingly similar to that of RSV-IV (see Table II). This strongly suggests that svp-2 is a variant of RSV-IV; we tentatively indicate it RSV-IV'.

572


a

a

b

c

d

e

f

-

_ svp-1 _

svp-

b 2 a

" svp- la

Fig. 2. Purification of svp-1 variants by preparative polyacrylamide (7%) gel electrophoresis in Tris-glycine, pH 9,5, buffer. (A) Seminal vesicle secretory protein pattern after gel electrophoresis in Tris-glycine, pH 9.5, buffer. Lane a: (Wistar x Wistar) Fi, total seminal vesicle secretory proteins. Lane b: purified svp-lb variant. Lane c: (Fisher x Fisher)Fl, total seminal vesicle secretory proteins. Lane d: purified svp-la variant, Lane e: (Fisher x Wistar)F~, total seminal vesicle secretory proteins. Lane f: (Fisher x Wistar)Fl (homozygous for the svp-2" allele), total seminal vesicle secretory proteins. (B) Lower region crf a seminal vesicle secretory protein pattern after gel electrophoresis in SDS-containing buffer. Lane A: (Fisher x Wistar)Fl (homozygous for the svp-2" allele), total seminal vesicle secretory proteins. Lane B: (Fisher x Wistar)F~, total seminal vesicle secretory proteins. Lane C: purified svp-1" variant. Lane D: (Fisher x Fisher)F1 total seminal vesicle secretory proteins. Lane E: purified svp-1 bvariant. Lane F: (Wistar x Wistar)F1, total seminal vesicle secretory proteins.

Polymorphism of svp-1 and svp-2 Proteins

A

b

RSV-IV RSV-

V

B

573

C

D

E

F

-

-18K

-

--15K

Fig. 2. Continued.

DISCUSSION The secretion of the rat seminal vesicle contains five major proteins (indicated by Roman numerals) which can be separated by electrophoresis in SDS gels (Ostrowski et al., 1979); fraction IV (or RSV-IV) and fraction V (or RSV-V) each compose a homogeneous basic protein. Tile amino acid compositions of protein IV and V have been determined. No biological activity has been assigned to either of these proteins; their identification is thus based exclusively on molecular weight and amino acid composition. A small degree of heterogeneity might, however, exist and several forms of RSV-IV and RSV-V may be present (Higgins and Fuller, 1981). The secretion of the rat seminal vesicle has also been analyzed by electrophoresis under nondenaturing conditions (Gasser, 1972; van Zutphen et al., 1981). Under this condition a polymorphism related to a major secretory protein, svp-1, has been demonstrated. Although svp-1, on account of its abundance, should correspond to one of the five fractions observed under denaturing conditions and, most likely, because of its basic character, to either protein IV or V. No data are presently available in this respect. We have simply purified the svp-1 protein under nondenaturing conditions and determined its electrophoretic mobility under denaturing conditions; the apparent molecular weight of svp-I under these conditions corresponds to that of RSV-V. To confirm that RSV-V and

574

Maffei, Paonessa, Abrescia, Metafora, and Guardiola Table II. Amino Acid Compositionof Secretory Proteins from the Rat Seminal Vesicle Composition (nmol/100 nmol recovered)a Amino acid residue

RSV-Vb

svp-P

svp-1b

svp-2

RSV-IVc

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Trp Cys

9.72 3.50 14.67 15.15 3.49 8.13 6.0 3.45 3.43 3.31 3.36 0.88 4.80 1.43 9.26 8.37 0.0 1.05

10.27 3.52 15.27 16.02 3.61 7.14 6.34 2.08 2.80 3.27 2.78 1.14 5.23 1.45 9.82 9.28 ndd nd

10.62 3.54 15.49 15.63 3.73 6.85 6.20 3.06 2.77 3.18 2.40 1.03 4.97 1.35 9.90 9.25 nd nd

7.88 0.99 19.31 15.93 3.97 11.96 4.61 6.07 1.52 3.03 3.09 1.46 3.85 1.46 7.93 6.94 nd nd

6.0 1.23 20.31 15.87 3.50 10.79 4.96 5.62 1.09 2.46 2.38 3.43 3.47 1.32 10.29 7.70 0.0 0.0

aThe proteins of saline-soluble rat seminal vesicle secretion were separated on preparative 7% polyacrylamide gels in Tris-glycine buffer, pH 9.5; the bands correspondingto svp-1 and svp-2 were eluted and hydrolyzed in 6 M HC1 at 110°C for 20 or for 72 hr. The amino acids in hydrolysates were separated and measured by the use of an automated analyzer. Values for aspartic acid and glutamic acid include those for asparagine and glutamine, respectively.The value shown refer to 20 hr of hydrolysis.Estimates of the decompositionof threonine and serine and the incomplete release of valine and isoleucineduring this period were made by reference to the hydrolysisvalues at different times. bValues from Higgins et al. (1976). CValues from Ostrowski et al. (1979). dNot determined.

svp-1 are the same protein, we also compared their a m i n o acid compositions; the latter t u r n e d out to be identical, within experimental error. This finding brings together i n f o r m a t i o n on the genetics of the s v p - I locus (Platz and Wolfe, 1969; Gasser, 1972; van Z u t p h e n et al., 1981) and a n u m b e r of data on the molecular biology of the R S V - V protein a n d of its corresponding gene (Kistler et al., 1981), thus increasing the usefulness of this locus as a m a r k e r for chromosome m a p p i n g in the rat and probably in the mouse. W e were also able to confirm the existence of the svp-2 protein, which seems subject to a different control p a t t e r n in different male individuals (van Z u t p h e n et al., 1981). T h e expression of the svp-2 protein or its lack of expression seems to depend more on an allelism in a regulatory gene rather then on true p o l y m o r p h i s m (van Z u t p h e n et al., 1982); lack of expression in a

Polymorphism of svp-I and svp-2 Proteins

A

RSV-IV RSV RSV-

B

575 C

D

~o

E

,~

-

18K

-

15K

- IV' V

-

~

Fig. 3. Detail of seminal vesicle secretory protein pattern after gel electrophoresis under denaturing conditions. Lane A: total seminal vesicle secretory proteins from a svp-2 ~ homozygous rat. Lane B: purified svp-I b variant. Lane C: purified svp-2a variant. Lane D: a mixture of svp- 1~and svp-2".

so-called svp-2a/svp-2 b heterozygote suggests that the expression of the svp-2 protein may be negatively controlled by this regulatory gene. Our data show that svp-2 is most likely a variant of RSV-IV, another major secretory protein present in the rat seminal vesicle fluid. ACKNOWLEDGMENT

Wethank Ms.C. Migliaccio~rherskill~ltechnicalhelp. REFERENCES Abrescia, P., Guardiola, J., Felsani, A., and Metafora, S. (1982). Expression in males and genomic organization of the gene(s) coding for a major protein secreted by the rat seminal vesicle epithelium. Nucleic Acids Res. 10:159. Gasser, D. L. (1972). Seminal vesicle protein in rats: A gene in the fourth linkage group determining electrophoretic variants. Biochem. Genet. 6:61. Higgins, S. J., and Fuller, F. M. (I 981 ). Effects of testosterone on protein synthesis in rat seminal vesicles analysed by two-dimensional gel electrophoresis. Mol. Cell. Endocrinol. 24:85. Higgins, S. J., Burchell, J. M., and Mainwaring, I. P. (1976). Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle. Biochem. J. 158:271. Kandala, J. C., Kistler, M. K., Lawther, R. P., and Kistler, W. S. (1983). Characterization of a genomic clone for rat seminal vesicle secretory protein IV. Nucleic Acids Res. 11:3169.

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Kistler, M. K., Taylor, R. E., Kandala, J. C., and Kistler, W. S. (1981). Isolation of recombinant plasmids containing structural gene sequences for rat seminal vesicle secretory proteins IV and V. Biochem. Biophys. Res. Commun. 99:1161. McDonald, C., Williams, L , McTurk, P., Fuller, F., Mclntosh, E., and Higgins, S. (1983). Isolation and characterization of genes for androgen-responsive secretory proteins of rat seminal vesicles. Nucleic Acids Res. 11:917. Mansson, P. E., Sugino, A., and Harris, S. E. (1981). Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: The effect of testosterone in gene expression. Nucleic Acids Res. 9:935. Ostrowski, M. C., Kistler, M. K., and Kistler, W. S. (1979). Purification and cell-free synthesis of a major protein from rat seminal vesicle secretion. A potential marker for androgen action. J. Biol. Chem. 254:383. Platz, R. D., and Wolfe, H. G. (1969). Mouse seminal vesicle proteins. The inheritance of electrophoretic variants. J. Hered. 60:187. van Zutphen, L. M. F., Lagerwerf, A., Bouw, J., and den Bieman, M. G. C. W. (1981). Biochemical polymorphism in the rat: Genetics of three electrophoretic variants and characterization of inbred strains. Biochem. Genet. 19:173.