Subunits of Cyclic Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase Show Differential and Distinct Expression Patterns during Germ Cell Differentiation: Alternative Polyadenylation in Germ Cells Gives Rise to Unique Smaller-Sized mRNA Species

BIOLOGY OF 43, 46-54 REPRODUCTION (1990) Subunits of Cyclic Adenosine 3’, 5’-Monophosphate-Dependent Protein Kinase Differential and Distinct Expression Patterns during Germ Cell Differentiation: Alternative Polyadenylation in Germ Cells Gives Rise to Unique Smaller-Sized mRNA Species1 OLE 0YEN,2’3’4 FRODE MYKLEBUST,3 JOHN VIDAR Institute of Pathology3 D. Rikshospitalet, University of Physiology Department of Pharmacology6 Oslo of and University and Oslo, GARY TORE 1, Norway, of Ca4fornia Irvine, University G. STANLEY McKNIGHT,6 of Medical Biochemistry4 of Biological Chemistry5 3, Norway Oslo and CADD,6 Institute Blindrn, Biophysics, G. JAHNSEN3’4 Department Irvine, California of Washington, 92717 Seattle, Washington 98195 ABSTRACT Cyclic AMP spermatozoal ument the and cAMP-dependent functions, such as motility, presence demonstrate to be The The expression patterns at premeiotic study, furthermore, Specific, to the use of alternative for the delayed this to be due may be late spermatid and 5 different PKA for during involved in the acrosome (RI,., RIB, R.IL, spermatogenesis. IUIB, Messenger RNA5 whereas mRNAs for RI! (RU,, and R1IB) are functions in developing germ cells and mature demonstrates the presence RIIB, and C,. mRNAs site signals. polyadenylation The selection of shorter at haploid sperm. cells germ cells. mRNA species, compared Our with suggest higher studies on PKAs, primarily involving have revealed an in the regulation of highly specialized spermatozoal functions such as motility, epididymal maturation, capacitation, plicity in isoforms different regulatory representing subunits and Tash subunits these the gene/mRNA et al., 1983), RI the and acrosome Means, that reaction 1983). protein kinase differentiation presence of significant umented dependent exists Accepted February Received August V.H.), Kopf, 1980; to elucidate in sperm its effector (PKA). Also (spermatocytes, levels has effector ‘This work wegian Research (Tj., and have focused on enzyme, cAMP- of growth 16, 28, been obscure, a general and differentiation Foundation Promotion of Science (Tj., been identified, rise to type II. In addition, of cAMPstages of for a third catalytic from testis, and substanPKA as a expression for this subunit In previous investigations Norgrant V.H.), Nordic 0027 Oslo 1. human 46 demonstrated at least at distinction selective with the possible decrease progress has been in type of spermatid found to be the major II was complex pool form gating cDNA spermatids (Conti et al., 1983). The probes for the different subunits abled arately us to investigate at the mRNA expression recently C.5, (Beebe et al., 1990). regarding PKAs in germ and the only and type II. demonstrated A have designated a testis-specific the enzyme one homogenous cell-specific Norway. we subunit, (and PICAS in general), less been considered type clonmulti- et al., 1986; Levy et al., 1988), C,, (Uhler et (Showers and Maurer, 1986; Uhler et al., give rise to type I PKA, whereas JUl give Society (Tj., V.H.), (TJ., V.H), Torsteds from the NIH (G.S.M.). of Pathology, Rikshospitalet, now cDNA 1978). cDNA unexpected These have been designated RI,, (Lee et a!., 1988), RIl,, (Scott et al., 1987), isolated and for the (Jahnsen PKA have level. (Clegg subunits 1990. Insulin Foundation (T.J., V.H.) and grants 2Reprint requests: Ole Oyen. Institute for doc- (Russell, by the Norwegian Cancer Science and the Humanities (C) of the firmly although role for R1I 1989. was supported Council for AndersJahres remained suggesting at translation different gene products. Four (R) and two different catalytic al., 1986a), and C 1986b). RI subunits at earlier stages spermatids), of PKA has ing is involved (Conti et al., 1983). However, the role regulatory mechanisms during earlier spermatogenesis tial evidence positive (cAMP) investigations functions cAMP and dependent germ cell AMP (Garbers Numerous diverse and complex the role played by cyclic for stability, sequencing, established of mRNA with data and is well doc- of transcription. INTRODUCTION It may and and expressed first in germ in the and RiB) for RI (Ri,, Recent cessation This mRNAs to be selected cells levels after in spermatids. smaller-sized appear we in germ certain stages, observed unique C,,) study, ensure essential translation of of essential In this and stages, of RI,,, RI!,,, regulation reaction. specific forms cell subunits subunits to be the convey may cells. believed capacitation, for these germ subunits truncated levels (PKAs) kinases epididymal mRNA PKA are maturation, protein significant induced individual present somatic of differential C,. appear stages. (cAMP) the expression level. We have of mRNAs or between type I I PKA has been development, present in elon- newly developed of PKA have en- of each previously for cells has more of molecules, subunit reported septhe Pd,, (previously de- Department SCOTF,5 HANSSON,4 Show UNIQUE noted RI), RII (previously ferent testicular cell al., 1987). Recently, et al., R1I51), OF PKA and C,, in dif- the (#{248}yenet high-level cDNA mRNA species for Ril,, at late stages during spermatid elongation (#{248}yen 1988b). We have now the expression all known subunits of PKA at the mRNA highly specific cDNA (and oligonucleotide) level by means of probes. In the present patterns study, been we able report spermatogenesis the presence adenylation mechanism to investigate characteristic for several of a unique in germ of expres- subunits, alternative cells that of and favors we poly- smaller species. ORE, AND of various tissues and isolated taken from rat liver, Preparation cells. Tissue samples ovary (hypophysectomized [Jahnsen et al., (nonpathologic) patient. and and samples Cultured Sertoli dibutyryl-cAMP), mM were at -75#{176}Cfor later frozen kb) tumor tissue, and ages were prepared were isolated old rats. from A cell seminiferous quence on 0.5 a 1.5% kb stimulated cells, and lung, SSC rats of different by #{248}yenet al. Germ of 32- and obtained by isolated by the StaPut using a BSA gradient, in 44-day-old rats) were sedimentation then method tially as described by Grootegoed et al. (1977). The cells were examined both by phase-contrast microscopy by regular light microscopy after fixation and staining. purities of these germ cell fractions were evaluated 44-day- Preparation RNA of total RNA extraction from 1988) containing 0.7 kb SAII-BglII was a 1.5 kb EcoRI the entire fragment fragment ORE. The (Scott et a!., rat (...truncated)