Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes

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1 Development 106, 25L-261 (1989) Prnted n Great Brtan The Company of Bologsts Lmted Gene expresson durng mammalan oogeness and early embryogeness: quantfcaton of three messenger RNAs abundant n fully grown mouse oocytes R. J. ROLLER 1 *, R. A. KINLOCH 1, B. Y. HIRAOKA 2, S. S.-L. LI 2 and P. M. WASSARMAN 1 'Department of Cell and Developmental Bology, Roche Insttute of Molecular Bology, Roche Research Center, Nutley, New Jersey 07110, USA 2 Laboratory of Genetcs, Natonal Insttute of Envronmental Health Scences, Natonal Insttutes of Health, Research Trangle Park, North Carolna 27709, USA * Present address: Department of Molecular Genetcs and Cell Bology, Unversty of Chcago, Chcago, Illnos 60637, USA Summary Rbonuclease protecton assays have been used to quanttatvely assess changes n steady-state levels of specfc mrnas durng oogeness and early embryogeness n mce. The mrnas encode ZP3 (a glycoproten that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a proten of unknown functon). MOM-1 and LDH-B are expressed n a varety of adult mouse tssues and mdgestaton embryos, whereas ZP3 expresson s restrcted completely to oocytes. All three mrnas are expressed by growng mouse oocytes and accumulate to unusually hgh levels n fully grown oocytes as compared to somatc cells; , and copes mrna per fully grown oocyte for ZP3, LDH-B and MOM-1, respectvely. Steady-state levels of LDH-B and MOM-1 mrna undergo a modest declne (~20-40 %) durng ovulaton when fully grown oocytes become unfertlzed eggs and, n general, mrror the reported change n poly(a) + RNA levels durng ths perod of development. On the other hand, the level of ZP3 mrna declnes dramatcally ( 98%) durng ovulaton, from copes per oocyte to 5000 copes per unfertlzed egg, and ZP3 mrna s undetectable n fertlzed eggs (<1000 copes per fertlzed egg). MOM-1 mrna s expressed at relatvely low levels n morulae ( 2000 copes per embryo) and blastocysts ( 5000 copes per embryo), whereas ZP3 mrna remans undetectable (<1000 copes per embryo) at these stages of premplantaton development. These fndngs are dscussed n the context of overall gene expresson durng oocyte growth, meotc maturaton and early embryogeness n mce. Key words: gene expresson, mouse oocyte, mouse egg, mouse embryo, oogeness, mrna. Introducton To a large extent, unfertlzed mouse eggs can be vewed as end-products of two developmental processes, growth and meotc maturaton (Jones, 1978; Wassarman, 1988a). Durng each reproductve cycle n mce, a small percentage of the female's pool of nongrowng oocytes ( 15 fm n dameter) begn to grow, ncreasng more than 300-fold n volume wthn 2-3 weeks, at whch tme full growth ( 80/xm n dameter) s acheved. Durng ths growth perod, oocytes accumulate large amounts of all classes of RNA, rbosomes, mtochondra, Golg and cortcal granules, as well as a varety of protens, ncludng tubuln, actn and lactate dehydrogenase (Wassarman & Josefowcz, 1978; Wassarman, 1983; Bachvarova, 1985; Schultz, 1986b; Wassarman, 1988a). Growng oocytes also synthesze and secrete glycoprotens that consttute the cell's extracellular coat, or zona pellucda (Wassarman et al. 1985; Wassarman, 1988a,b). Throughout ther growth phase, mouse oocytes are arrested n the dctyate stage of frst meotc prophase (Jones, 1978; Wassarman, 1988a). Dffuse chromosomes, contaned wthn a large nucleus, or germnal vescle (GV), are actvely transcrbed durng ths perod (Bachvarova, 1985). However, at ovulaton, when fully grown oocytes undergo the frst meotc reductve dvson ('meotc maturaton') and become unfertlzed eggs, the nuclear envelope breaks down, chromosomes condense and transcrpton ceases. The maternal genome s not transcrbed agan untl the 2-cell stage of premplantaton development (Johnson, 1981; Flach et al. 1982; Pk6 & Clegg, 1982; Pratt et al ; G. A. Schultz, 1986), about 2-3 days followng ovulaton.

2 252 R. J. Roller and others Meotc maturaton s accompaned by a sgnfcant decrease n levels of total RNA and poly (A) + RNA, as well as by a decrease n rates of synthess of specfc protens (Wassarman, 1983; Bachvarova, 1985; R. M. Schultz, 1986; Wassarman, 1988a). These changes, and those that occur followng fertlzaton and precedng the second cleavage dvson, take place n the absence of new transcrpton (Johnson, 1981; G. A. Schultz, 1986) and, therefore, are programmed durng oogeness. The mechansms by whch these changes are programmed and nsttuted are not clear. Here we descrbe the behavor of specfc mrnas durng oocyte growth, meotc maturaton and early embryogeness n mce. All of these mrnas are abundant n fully grown oocytes. One mrna encodes ZP3, an M r glycoproten ( M r polypeptde chan; Salzmann et al. 1983; Wassarman et al. 1985; Rnguette et al. 1986, 1988; Knloch et al. 1988; Wassarman, 1988b) that serves as the egg's sperm receptor (Blel & Wassarman, 1980a, 1986; Florman & Wassarman, 1985; Wassarman et al. 1985; Wassarman, 1987). A second mrna, called MOM-1, encodes a M t proten (R. Roller & P. Wassarman, unpublshed results) of, as yet, unknown functon. The thrd mrna encodes the M r subunt of heart-type lactate dehydrogenase (LDH-B), a glycolytc enzyme known to accumulate to very hgh levels n fully grown oocytes and eggs (Manga & Epsten, 1975; Manga et al. 1976; Casco & Wassarman, 1982). All three mrnas accumulate throughout oocyte growth, and ther proten counterparts represent relatvely large percentages of total oocyte proten syntheszed durng oogeness. Materals and methods Collecton of mouse oocytes, eggs, and embryos Growng oocytes, fully grown oocytes, unfertlzed eggs and embryos were solated from female, Swss albno mce (CD-I, Charles Rver Breedng Laboratores) as prevously descrbed (Schultz et al. 1919a,b). Purfcaton of oocyte, embryo, and tssue RNA Oocyte and embryo RNA was purfed as descrbed n Knloch et al. (1988). Tssue RNA was purfed as descrbed by Feramnsco et al. (1982) wth some modfcatons. Followng the frst ethanol precptaton, the RNA pellet was dssolved n 0-lM-Trs, ph7-5, 50mM-NaCl, 10mM-EDTA, 0-2% SDS wth 0-5 volumes of the ntal homogenate. Protenase K (100 jg ml" 1 ; Sgma) was added and the soluton ncubated at 37 C for lh. RNA was then extracted twce wth phenol/ chloroform/soamyl alchohol and once wth chloroform/ soamyl alcohol and precptated from 0-3M-sodum acetate, ph5-2, wth 2-5 volumes of ethanol. The fnal step ncluded precptaton from 3 M-sodum acetate. Purfcaton of nuclear DNA Mouse lver nucle were solated by a modfcaton of the procedure descrbed n Busch (1967). Brefly, lvers were excsed, rnsed n cold NaCl (150mM), mnced and homogenzed n 6 volumes of 0-25M-sucrose, 3-3mM-CaCl2 wth a Dounce homogenzer. The homogenate was fltered through cheesecloth and then centrfuged at 600 g for lomn at 4 C to pellet nucle and large debrs. The pellet was resuspended by repeated ppettng n 9 ml of 2-5 M-sucrose, 3-3mM-CaCl2, and centrfuged at 45000g for lh at 4 C n an SW 50-1 rotor (Beckman) to pellet nucle. DNA was purfed from solated nucle by the procedure descrbed by Weeks et al. (1986). Constructon and screenng of cdna and genomc lbrares Total RNA from approxmately growng mouse oocytes was prmed wth olgo-dt and used as a template for cdna synthess. Double-stranded cdna wth EcoBA lnker ends was syntheszed and lgated nto Agtll DNA and packaged nto phage by methods descrbed n Manats et al. (1982), except that cdnas were separated from dgested lnker fragments by several precptatons from 0-3 M-sodum acetate wth 0-6 volumes of sopropanol. The resultng lbrary, contanng recombnants (average nsert sze of 800 bases), was amplfed on E. col LE392. The cdna lbrary was screened by usng a rabbt antserum rased aganst dssolved zonae pellucdae and drected aganst ZP2 and ZP3 (Greve et al. 1982; Salzmann et al. 1983). Inducton of the fuson protens and plaque lfts were carred out as descrbed by Young & Davs (1983). A screen of plaques yelded 2 phage that produced strong, reproducble, IPTG-dependent sgnals, whch proved to carry the same cdna. Constructon of a Sau3A partal-dgest lbrary of CD-I mouse nuclear DNA and solaton of ZP3 genomc clones were carred out as prevously descrbed (Knloch et al. 1988). A mouse LDH-B cdna clone (mb162) was solated from a macrophage cdna lbrary n Agtll (Clontech) by usng a human LDH-B cdna (hb537) as probe (Saka et al. 1987). The cori DNA fragment was subcloned nto M13 mplo phage and ts nucleotde sequence determned from both drectons. Preparaton of RNA probes Hgh-specfc-actvty RNA probes were transcrbed wth T7 RNA polymerase from template sequences cloned nto ether pt7-l or -2 (US Bochemcals) or pgem-3-blue (Promega Botechnology) accordng to a publshed protocol (Promega Botechnology). Reactons contaned 40mM-Trs, ph7-5, 6mM-MgCl2, 2mM-spermdne, lomm-nacl, 10 mm-dthothretol, 500/M each ATP, CTP, GTP, 12HM UTP, l.u./p 1 RNasn, loofc [cr- 32 P]UTP (3000Cmmor l ; Amersham), /g plasmd template, and 40.u. T7 RNA polymerase n a fnal volume of 20fA. Incubatons were at 37 C for lh. Followng synthess of RNA, DNA templates were destroyed by addton of 1 /A of RNase-free DNase (Promega Botechnology) and ncubaton at 37 C for 15 mn. Samples were then extracted wth phenol/chloroform/soamyl alcohol and RNA was precptated wth ethanol. RNA targets were prepared by the same procedure, except that all unlabeled nucleotdes were present at 500 ^tm and 5jtC of [ff- 32 P]UTP or [o-- 35 S]UTP was ncluded. Consequently, the amount of target syntheszed could be calculated from the percentage ncorporaton accordng to the equaton: (ctsmn- 1 ncorponued/ctsmn- 1 added) (4 x KT 8 molntp) (SSOgmoP 1 NTP) = grna syntheszed A synthetc target for MOM-1 probe, used to calbrate the RNase protecton assays, was generated from a plasmd contanng the entre MOM-1 cdna nsert. The plasmd was lnearzed wth Seal such that T7 RNA polymerase yelded a 2-3 kb sense transcrpt. Snce the plasmd nserts used to generate probe and target were dentcal over only 394 nt, the

3 Mouse oocyte abundant mrnas 253 synthetc target protected a 394 nt fragment of the antsense probe n RNase protecton assays. MOM-1 mrna s twce as long as the synthetc target, so that 100 pg of target s equvalent n copy number to 200pg of MOM-1 mrna. Preparaton of olgonucleotde probes Two dfferent 60-mer olgonucleotde probes were used n experments descrbed here. One of these s complementary to ZP3 mrna and was prepared on the bass of the sequence of a ZP3 cdna fragment (Rnguette et al. 1986). The other olgonucleotde s complementary to MOM-1 mrna and was prepared on the bass of the sequence of a MOM-1 cdna fragment. Olgonucleotdes were syntheszed on an Appled Bosystems 380B DNA Syntheszer and purfed by PAGE. Olgonucleotdes were 5' end-labeled wth T4 polynucleotde knase as prevously descrbed (Hay & DePamphls, 1982). Rbonuclease protecton assays RNA preparatons to be examned were combned wth ether lxlo^dsntsmn" 1 (MOM-1 and LDH-B) or 2xl0 6 dsnts mn~' (ZP3) of probe RNA, lyophlzed and dssolved n 10 /jl of hybrdzaton buffer (80% formamde, 40mM-MOPS, ph6-6, 0-4M-NaCl, lmm-edta). RNA was denatured by ncubaton at 85 C for 5 mn and hybrdzed by ncubaton at 45 C C overnght. Followng overnght ncubaton, unhybrdzed RNA was dgested by addton of 200^1 of a mx contanng lomm-trs, ph7-5, 300mM-NaCl, 5ITIM-EDTA, 40 jg ml RNase A, 2,ugml~ t RNase Tl and ncubaton at 37 C for lh. Dgeston was termnated by addton of SDS (0-7%) and Protenase K (150^gml~') and ncubaton at 37 C for an addtonal 15 mn. Protected RNA was purfed by extracton wth phenol/chloroform/soamyl alcohol, followed by ethanol precptaton n the presence of carrer trna (15 fg). Purfed RNA was analyzed on sequencng gels (6% acrylamde/8 M urea; 15 cm long, 0-75 mm thck). Bands were vsualzed by autoradography of dred gels. Quanttatve analyss was carred out by excsng bands from dred gels and subjectng them to lqud scntllaton countng n Aquasol (New England Nuclear). In order to correct for background, each experment ncluded an assay carred out wth E. col rbosomal RNA whch dd not hybrdze to any of the probes used. A band correspondng n sze to protected fragments was cut from the E. colrbosomalrna gel lane, counted and used as background for the experment. Northern blottng Preparaton, runnng and blottng of formaldehyde-agarose gels contanng RNA were performed as descrbed by Manats et al. (1982). Blots were prehybrdzed for 2-3 h at 55 C n 6xSSC, 1-0% SDS, 5xDenhardt's soluton, 20/.tgm\~ l trna, and 50^gml~' denatured salmon sperm DNA. Hybrdzaton was carred out n prehybrdzaton soluton n the presence of 32 P end-labeled olgonucleotdes for 1 h at 55 C. Blots were washed three tmes n 2xSSC, 1-0% SDS at room temperature, for 5mn each, and twce at 55 C C n lxssc, 1-0% SDS for 45 mn. In stu hybrdzaton Thn sectons of embedded ovares from 15-day-old mce were placed on subbed sldes, dred and fxed n 2% paraformaldehyde. Sldes were washed, dred and stored for further use. Hybrdzatons were carred out essentally as descrbed by Lews et al. (1986). After coatng wth photographc emulson (Kodak NTB2 dluted 1:1 wth 0-6 M ammonum acetate), sldes were exposed for 6-14 days n the dark, developed (Kodak D19) and lghtly counter-staned wth toludne blue n sodum borate. Results Genomc and cdna clones used to analyze mouse mrnas Isolaton and characterzaton of a ZP3 genomc clone, desgnated ZP3-G5, s descrbed elsewhere (Knloch et al. 1988). A ZP3-G5 EcoW/Hndlll fragment, 2-6kb n length, was subcloned and used as probe n experments presented here. Ths restrcton fragment contans the two 3'-termnal exons (219 and 137 nt) encodng ZP3 (Knloch et al. 1988). ZP3 probe hybrdzed to a 1-5 kb mrna on Northern blots of total mouse oocyte RNA and to sngle bands n Southern blots of mouse genomc DNA dgested wth a varety of restrcton enzymes. A cdna clone, desgnated MOM-1, was solated as a false-postve durng the screenng of a Agtll-growng oocyte cdna lbrary wth an antserum drected aganst zona pellucda glycoprotens ZP2 and ZP3 (R. Roller and P. Wassarman, unpublshed results). It was shown to represent an abundant oocyte mrna ('major oocyte message-1'; MOM-1) that dd not encode any zona pellucda glycoproten. Ths 558 nt cdna contans 483 nt of open readng frame. A 405 nt EcoRl/Mspl fragment of the cdna was subcloned and used as probe n experments presented here. MOM-1 probes hybrdzed to a 4-6 kb mrna on Northern blots of total mouse oocyte RNA and to sngle bands n Southern blots of mouse genomc DNA dgested wth a varety of restrcton enzymes. A cdna clone, desgnated mb162, was solated from a Agtll-mouse macrophage cdna lbrary screened wth a human LDH-B cdna probe (Saka et al.), and ts complete sequence (534 nt; excludng cori lnker sequences) was determned. The deduced amno acd sequence of ts codng regon was confrmed by sequencng tryptc peptdes derved from mouse LDH-B. Clone mb162 contans a partal mouse LDH-B cdna, startng at codon 219 and endng 16 nt after the polyadenylaton sgnal, AATAAA. A 467 nt EcoRl/Sacl fragment of ths cdna was cloned nto plasmd pgem- 3blue to create a plasmd, desgnated pgem/ldh-b, that was used to generate RNA probes used n experments presented here. LDH-B probes hybrdzed to a 1-4 kb mrna on blots of total mouse oocyte RNA. Quantfcaton of mrnas n fully grown mouse oocytes Quantfcaton of absolute amounts of MOM-1, ZP3 and LDH-B transcrpts n fully grown mouse oocytes was carred out by RNase protecton assays, as descrbed n Materals and methods. In such assays, under condtons n whch a radolabeled antsense probe s n large excess over the complementary target sequence, the amount of probe protected from dgeston s proportonal to the amount of target sequence. By usng varyng amounts of a synthetc MOM-1 sense RNA (394 nt) as target, a standard curve was constructed relatng protected MOM-1 antsense probe (ctsmn" 1 ) to target RNA (pg) (Fg. 1). Parallel assays were performed usng both RNA from 250 fully grown

4 254 R. J. Roller and others -Or -Or A. ABCDEFGH JK -394 nt -405 nt nt I 1 I - _» to *. N) Ul O O O Targe/ fpg; nt 8000 a jj T " 4000' c I ' 1QQ pg Target Fg. 1. Calbraton of the RNase protecton assay usng MOM-1 probe and target. RNase protecton assays were carred out as descrbed n Materals and methods, usng a constant amount of MOM-1 antsense transcrpt (lxk^dsntsmn" 1, or ~800pg), transcrbed from EcoRldgested pt7-2/mom-a-mspb by T7 RNA polymerase, and varyng amounts of MOM-1 target transcrpt ( pg), syntheszed as descrbed n Materals and methods. (A) Autoradogram of the gel analyss of RNase protecton assays. The poston of the 394 nt protected MOM-1 fragment s ndcated. Or, orgn. (B) Plot of ctsmn"1 assocated wth the 394 nt protected fragment (panel A) as a functon of pg of target transcrpt added. Bands were excsed from the gel (panel A) and subjected to lqud scntllaton countng, as descrbed n Materals and methods. The lne drawn s derved from a least squares ft of the data. oocytes and varyng amounts of synthetc target RNA (equvalent to 0-80 pg MOM-1 mrna) (Fg. 2). The RNase protecton assay exhbted lnearty usng Fg. 2. Quantfcaton of MOM-1 mrna steady-state levels n fully grown mouse oocytes. Shown s a representatve autoradogram of the gel analyss of RNase protecton assays of MOM-1 calbraton standards (as n Fg. 4) and RNA from fully grown oocytes, carred out as descrbed n Materals and methods. Postons of the 405 nt fragment protected by oocyte MOM-1 mrna, the 394 nt fragment protected by the MOM-1 calbraton target (see Fg. 4), and the 220 nt fragment protected by the recovery target are ndcated. Lane: (A) Undgested probe transcrpt; (B) undgested calbraton target transcrpt; (C) end-labeled sze standards; (D-I) RNase protecton assays carred out wth 0, 2-5, 5, 10, 20, 40, and 80 pg of calbraton target, respectvely; (J) E. col rbosomal-rna plus recovery target; (K) RNA from 250 fully grown oocytes ( 125 ng) plus recovery target. Or, orgn. amounts of the 394 nt target equvalent to nearly 800 pg MOM-1 mrna (Fg. 1). Protected fragments were resolved on denaturng gels, bands excsed and subjected to lqud scntllaton countng. As expected, target RNA protected a 394 nt fragment of probe, whereas authentc MOM-1 mrna protected the entre probe (405 nt) (Fg. 2). To assess recovery of oocyte RNA, a MOM-1 cdna sense transcrpt (220 nt) was added to oocytes pror to extracton of RNA and an equvalent amount of the transcrpt was assayed drectly. The rato of the amount of transcrpt n oocyte

5 Mouse oocyte abundant mrnas RNA preparatons and the amount assayed drectly was used as a correcton factor for recovery. In experments presented here, recovery of oocyte RNA ranged from 70 to 95 %. By usng the procedures just descrbed, the amount of MOM-1 mrna n fully grown oocytes was determned to be 185 ± 15 fg oocyte"1 (4 ndependent experments), or about 7-4xlO4 copes oocyte". The amount of ZP3 and LDH-B mrna per oocyte, relatve to MOM-1 mrna, was determned by performng parallel RNase protecton assays for each message (.e. ZP3 vs MOM-1 and LDH-B vs MOM-1). The amount of ZP3 mrna was calculated from the rato ofctsmn"1 assocated wth the ZP3 protected fragment (219 nt) to ctsmn" 1 assocated wth the MOM-1 protected fragment (405 nt). Ths rato was corrected for dfferences n (a) lengths of protected fragments (MOM-1 fragment s 1-8-tmes longer than ZP3 fragment), (b) compostons of the probes wth respect to the labeled nucleotde (MOM-1 fragment has 1-5-tmes more urdne per unt length than ZP3 fragment), and (c) szes of the mrnas (MOM-1 mrna s three tmes the sze of ZP3 mrna). Accordngly, the amount of ZP3 mrna n fully grown oocytes was determned to be 195 ±20 fg oocyte"1 (4 ndependent experments), or about 2-4X105 copes oocyte"1. The amount of LDH-B mrna n fully grown oocytes was determned just as for ZP3 mrna. As before, correctons were appled for dfferences n LDH-B protected fragment length (460nt), probe composton (26-9% urdne) and message length (l-4kb), as compared to MOM-1. Two ndvdual experments gave a value of 150 ± 15 fg of LDH-B mrna oocyte"1, or about 2xlO5 copes oocyte"1. Assumng a value of 500 pg of total RNA per oocyte, 20% as the fracton of poly(a)+rna per oocyte, and 2000 nt as the average length of poly(a)+rna n mouse oocytes (Clegg & Pk6, 1983 >), t can be calculated that fully grown oocytes contan about 9xlO7 poly(a)+rna molecules. Thus, on a copy-number bass, MOM-1, ZP3 and LDH-B mrna represent approxmately 0-08%, 0-27% and 0-22% of a fully grown oocyte's poly(a)+rna, respectvely (or about 0-6% collectvely; see Dscusson). Specfcty of mrna expresson n mouse tssues Although a great deal s known about LDH-B expresson n mouse tssues (Markert et al. 1975; NadalGnard, 1978), much less s known about expresson of MOM-1 and ZP3. Accordngly, Northern analyses and RNase protecton assays were used to examne the tssue specfcty of MOM-1 and ZP3 mrna n mce. RNA was prepared from fully grown mouse oocytes and 13-day mouse embryos, and from mouse bran, heart, ntestne, kdney, lver, muscle, ovary, tests and uterus. Integrty of RNA n these preparatons was assessed by nspecton of staned formaldehyde-agarose gels, as well as by Northern analyss usng a mouse mtochondral cytochrome b cdna probe. For Northern analyses of ZP3 mrna, an end-labeled, synthetc olgonucleotde (60-mer), DNA probe was employed. For RNase protecton analyses of both ZP3 and MOM- O o O B B H 255 I K L M T U 1-5kb EcOo B C H I K L M O T U 219nt 405nt X 1 405nt Fg. 3. Northern blot analyss and RNase protecton assays of ZP3 and MOM-1 mrna n mouse tssues and mdgestaton embryos. (A) Northern blot analyss usng a 32 P end-labeled olgonucleotde specfc for ZP3 mrna. The oocyte lane contans RNA from 250 fully grown oocytes (~125ng) and all other lanes contan 10/g of RNA. (B) RNase protecton assays usng probe transcrbed wth T7 RNA polymerase from //ndle-dgested pgem/g5/rl-a. The oocyte lane contans RNA from 250 fully grown oocytes and all other lanes contan 10 jg RNA. (C) RNase protecton assays usng probe transcrbed by T7 RNA polymerase from Eco Rl-dgested pt7-2/mom-amspl. The oocyte lane contans RNA from 200 fully grown oocytes (~100ng) and all other lanes contan 100 ng RNA. (D) Same as (C), except that 10 ^g of RNA from mouse tssues, embryos, and E. col, rather than 100 ng, was loaded n each lane. Ec, E. col; Oo, oocytes; B, bran; E, 13-day embryos; H, heart; I, ntestne; K, kdney; L, lver; M, muscle; O, ovary; T, tests; U, uterus. 1 mrna, radolabeled antsense transcrpts of genomc and cdna clones, respectvely, were used. Results shown for ZP3 (Fg. 3) were obtaned usng

6 256 R. J. Roller and others A. b e nt Fg. 4. /n s/fw hybrdzaton and RNase protecton assays of ZP3 and MOM-1 mrna n mouse oocytes and follcle cells. In stu hybrdzatons were carred out usng radolabeled 60-mer olgonucleotdes specfc for ether ZP3 or MOM-1 mrna, as descrbed n Materals and methods. (A and C) Lght mcrographs of ovaran sectons vewed wth brght-feld optcs. The postons of three oocytes, wthn follcles, n each secton are ndcated by arrows. (B and D) Lght mcrographs of autoradograms of the same ovaran sectons seen n (A) and (C), respectvely, but vewed wth dark-feld optcs so that slver grans appear as whte dots on a black background. Also shown are results of RNase protecton experments carred out wth ant-sense probes specfc for ether ZP3 (Top panel, a-c) or MOM-1 mrna (Bottom panel, a-c): Lane (a) E. col rbosomal-rna; (b) RNA from 250 fully grown oocytes; (c) RNA from 100 solated follcles from whch oocytes had been removed (empty follcles), plus recovery target. The protected fragments expected for ZP3 and MOM-1 probes, 219 and 405 nt, respectvely, are seen n lane b (oocytes), but not n lane c (empty follcles). 10j.g of each tssue RNA, or about 100 tmes more RNA than was present n the oocyte samples. However, ZP3 mrna (-1-5 kb) was detected only n RNA prepared from oocytes and ovares. Snce RNAse protecton assays were capable of detectng as lttle as 0-5 % of the amount of ZP3 mrna found n oocytes, t follows that mouse tssues, other than ovary, contaned no more than % the amount of ZP3 mrna found n oocytes. Results shown for MOM-1 (Fg. 3) were obtaned usng 100ng of each RNA preparaton, ncludng oocytes. Although MOM-1 mrna was found to be hghly enrched n oocytes compared to other tssues, t was present n all tssues examned. Estmates of the relatve abundance of MOM-1 mrna were made by carryng out RNase protecton assays wth 10 fg samples of tssue and oocyte RNA (Fg. 3). Excludng oocytes, bran contaned the largest amount of MOM-1 mrna, followed n decreasng order by 13-day embryos, kdney, ovary, lver, tests, uterus, heart, skeletal muscle and ntestne. Based on total RNA levels, bran contaned about 2% and ntestne about 0-02% of the amount of MOM-1 mrna found n fully grown oocytes. The relatve amounts of ZP3 and MOM-1 mrna found n solated oocytes and ovary suggested that these messages were expressed exclusvely n oocytes wthn the ovary. Ths possblty was examned further by usng n stu hybrdzaton to localze transcrpts wthn the ovary (Fg. 4) and RNase protecton assays wth solated ovaran follcles from whch oocytes had been removed (Fg. 4). Results of both analyses suggested that ZP3 and MOM-1 mrna were found only n oocytes, not n surroundng follcle cells. Quantfcaton of mrnas n growng mouse oocytes, ovulated eggs, and early embryos Snce MOM-1, ZP3, and LDH-B mrnas proved to be abundant transcrpts n fully grown mouse oocytes, ther steady-state levels durng oogeness and early embryogeness were determned by usng RNase pro-

7 Mouse oocyte abundant mrnas a b c d e f 9 h k j k nt B. a b c d e f 9 h I m n o 405nt c 460nt W. Fg. 5. Quantfcaton of ZP3, MOM-1 and LDH-B mrna steady-state levels durng oocyte growth, meotc maturaton and early embryogeness n mce. Shown are representatve autoradograms of RNase protecton assays for ZP3 (A), MOM-1 (B), and LDH-B (C) mrna, carred out as descrbed n Materals and methods. (A) In lanes b-, RNA from 500 oocytes or ovulated eggs was assayed. Lane (a) E. col RNA; (b) 12-20/an oocytes; (c) 30-40/zm oocytes; (d) 50-60nm oocytes; (e) 60-70/an oocytes; (f and h) /m\ (fully grown) oocytes; (g and ) ovulated eggs. Lane (j) RNA from approxmately cell embryos. Lane (k) RNA from approxmately 500 empty follcles. The poston of the ZP3 protected fragment, 219 nt, s ndcated. (B) In lanes b-o, RNA from 350 oocytes, eggs or embryos was assayed. Lanes (a and h) E. col RNA; (b) (m oocytes; (c) 30-40/m oocytes; (d) /m oocytes; (e) 50-60,um oocytes; (f) ^m oocytes; (g and ) urn (fully grown) oocytes; (j) ovulated eggs; (k) 1-cell embryos; (1) 2-cell embryos; (m) 4- to 8-cell embryos; (n) morulae; (o) blastocysts. The poston of the MOM-1 protected fragment, 405 nt, s ndcated. It should be noted that a very fant band at 405 nt was seen n lanes (n) and (o) of the orgnal autoradogram. (C) Lanes (a and d), E. col RNA; (b) RNA from /an oocytes; (c and e) RNA from /an (fully grown) oocytes; (f) RNA from 250 ovulated eggs. The poston of the LDH-B protected fragment, 460 nt, s ndcated. tecton assays. In each experment, RNA was prepared from equvalent numbers ( ) of growng oocytes, fully grown oocytes, ovulated eggs and early embryos. RNase protecton assays were carred out usng as probes the antsense transcrpts of MOM-1 cdna, ZP3 genomc clone or LDH-B cdna descrbed above. As before, a synthetc MOM-1 sense-strand target was added to each preparaton n order to determne a correcton factor for RNA recovery. Some examples of the autoradographc results of these experments are presented n Fg. 5. As seen n Table 1, MOM-1 and ZP3 mrnas were detected by RNase protecton assays at the earlest stages of oocyte growth (~20 /m dameter oocytes) and contnued to accumulate to about the md-to-late stages of oocyte growth (~65/tm dameter oocytes). The steady-state levels of MOM-1 and ZP3 mrnas dd not ncrease durng the fnal stages of oocyte growth (from 65 /jm to 80/m dameter oocytes). Rather, the level of MOM-1 mrna remaned nearly constant, whereas the level of ZP3 mrna fell by about 22 %. On the other hand, the steady-state level of LDH-B mrna exhbted a dfferent pattern, ncreasng by about 20% durng the fnal stages of oocyte growth. The steady-state levels of MOM-1 and ZP3 mrnas changed markedly durng ovulaton (Table 1), when fully grown oocytes underwent the frst meotc reducton and became unfertlzed eggs. As compared to fully grown oocytes, MOM-1 and ZP3 mrna levels fell by approxmately 50% (to - W f g e g g " 1 ) and 98% (to ~4fgegg~ 1 ), respectvely. The level of LDH-B mrna also fell durng ovulaton (~20 % ), but not to the extent seen wth MOM-1 and ZP3 mrnas. ZP3 mrna was vrtually undetectable n 2-cell and later stage embryos, whereas MOM-1 mrna, present n 2-cell embryos at about 1 % the level found n fully grown oocytes, became undetectable n 4- to 8-cell embryos, and then reappeared n morulae and ncreased n blastocysts to approxmately 6% the level ( 11 fg embryo" 1 ) found n fully grown oocytes (Table 1). Dscusson As mouse oocytes grow durng a 2- to 3-week perod, MOM-1, ZP3 and LDH-B mrna accumulate to unusually hgh levels (Table 1). Durng ths perod, the absolute rate of total proten synthess ncreases about 40-fold (Schultz et al. I919a,b; Wassarman, 1988a). However, the three mrnas behave dfferently durng the fnal stages of oocyte growth, as oocytes ncrease n

8 258 R. J. Roller and others Table 1. Steady-state levels of ZP3, MOM-1 and LDH-B mrna durng oogeness and early embryogeness Growng oocytes 10-20/an 30-40/an 50-60/an /m Fully grown oocytes Unfertlzed eggs Fertlzed eggs Embryos 2-cell 4- to 8-cell Morula Blastocyst ~ZP3 MOM-1 LDH-B Poly(A) + RNA (no. copes, (no. copes mrna, xlo 3 ) nd (0-27 %)t (0-08%) (0-22%) 5 (0-01%) (0-08%) (0-22%) 26 nd (<0-003%) (0-07%) * Calculated by usng data presented n Bachvarova & DeLeon (1980), Bachvarova (1985), Pk6 & Clegg (1982), and Clegg & Pk6 (1983ft). t Values n parentheses represent mrna levels as a percentage of total poly(a) + RNA. I Ths value represents the lower lmt of detecton of mrna by RNase protecton assays n experments descrbed here (.e. mrna was undetectable). nd, not determned. dameter from about 65 urn to 80/an. Durng ths perod, levels of MOM-1, ZP3 and LDH-B mrna reman constant, decrease (~23%) and ncrease (-20%), respectvely (Table 1). Levels of all three mrnas fall durng ovulaton (10-12 h), when the absolute rate of total proten synthess falls by about 20% (Schultz et al. 1978, 1979a; Wassarman, 1988a) and RNA synthess falls to undetectable levels (Wassarman & Letourneau, 1976; Rodman & Bachvarova, 1976; Bachvarova, 1985). Durng meotc maturaton steady-state levels of MOM-1 and LDH-B mrna fall by about 50 % and 20 %, respectvely, whereas the level of ZP3 mrna decreases by nearly 98% (Table 1). By the 2-cell stage of embryogeness, ZP3 mrna s undetectable and MOM-1 mrna s present at about 1% the level found n fully grown oocytes (Table 1). Although transcrpton of the embryonc genome takes place at the late 2-cell stage of development (G. A. Schultz, 1986a), ZP3 mrna s undetectable n blastocysts, whereas MOM-1 mrna reappears n morulae and s present n blastocysts at about 6% the level found n fully grown oocytes (Table 2). Thus, transcrpton of the ZP3 gene occurs exclusvely n oocytes, whereas the MOM-1 gene s also expressed n premplantaton embryos. The fact that ZP3 s expressed only n growng oocytes suggests the presence of specfc factors that are nvolved n regulatng the gene. In vew of the extreme stablty of many transcrpton Table 2. Summary ofzp3, MOM-1 and LDH-B expresson n fully grown oocytes ZP3 MOM-1 LDH-B mrna % Poly(A) + % Polysomal % Proten (no. copes)* RNAt poly(a) + RNAt syntheszed} 2-4X X OxlO Determned by usng RNase protecton assays, as descrbed here (see Table 1). t Calculated by usng data presented here and n Bachvarova & DeLeon (1980), DeLeon et al. (1983), and Bachvarova (1985). t Estmated by usng data presented n Blel & Wassarman (1980>) and Casco & Wassarman (1982), and from unpublshed results (R. Roller, G. Salzmann and P. Wassarman). complexes (Davdson etal. 1983; Fre et al. 1984; Klen et al. 1985; Sassone-Cors et al. 1985; Wang & Calame, 1986), these actvaton factors need not be syntheszed contnuously durng oocyte growth (2-3 weeks). Rather, they may be syntheszed smultaneously wth, or for a bref perod just subsequent to, the onset of oocyte growth when ZP3 synthess s ntated. Changes n ZP3 mrna descrbed here parallel changes n ZP3 synthess reported prevously (Blel & Wassarman, 1980ft; Salzmann et al. 1983; Wassarman et al. 1985; Wassarman, 1988ft), and support and extend results of n stu hybrdzaton experments (Phlpott et al. 1987). ZP3 synthess and secreton are frst detected when oocytes begn to grow and acqure a zona pellucda. The zona pellucda ncreases n thckness as oocytes ncrease n dameter, reachng a fnal wdth of about 7 an. Durng ths perod, ZP3 synthess represents 1-5% to 2-5% of total proten synthess n growng oocytes. As oocytes grow from about 65 fm n dameter onward, there s a small (~20%) decrease n the relatve rate of ZP3 synthess (consstent wth changes n ZP3 mrna levels), however, synthess contnues even n fully grown oocytes. Only after ovulaton of unfertlzed eggs does ZP3 synthess fall to vrtually undetectable levels. The decrease n ZP3 mrna steady-state levels (~20 %) seen durng late stages of oocyte growth, when poly(a) + RNA levels ncrease slghtly (Table 1), may ndcate that ZP3 transcrpts are relatvely unstable compared to other oocyte mrnas. Ths possblty s supported by thefndng that about 98 % of ZP3 mrna present n fully grown oocytes s lost durng ovulaton (10-12 h) when transcrpton s termnated and about 50% of poly(a) + RNA s lost (Table 1; Bachvarova & DeLeon, 1980; DeLeon et al. 1983; Bachvarova et al. 1985). The extensve loss of ZP3 mrna durng ovulaton, as compared to other transcrpts, could reflect sequence-specfc degradaton of the mrna. Regulaton of mrna abundance by such a mechansm has been reported for several mrnas (Shapro et al. 1987) and, n some cases, the sequences that medate selectve degradaton, often located n the 3'-untranslated regon, have been characterzed (Alterman et al. 1985; Smcox et al. 1985; Shaw & Kamen, 1986; Graves et al. 1987; Capasso et al. 1987). In ths context, t should be ~3

9 Mouse oocyte abundant mrnas 259 noted that ZP3 mrna has an unusually short 3'- untranslated regon (16 nt; Knloch et al. 1988) that provdes very lttle recognton sequence for a specfc degradaton mechansm. Of course, such determnants could be located elsewhere n the mrna. Fnally, the behavor of ZP3 mrna durng ovulaton could be explaned were the mrna nherently unstable. Accordngly, hgh rates of transcrpton durng oocyte growth would be requred to accumulate ZP3 mrna, whereas n eggs, n the absence of transcrpton, levels of ZP3 mrna would fall dramatcally. However, ths explanaton s unlkely n vew of the enormously hgh rates of ZP3 transcrpton that would be requred to produce the steady-state levels of ZP3 mrna found n oocytes (see Footnote* for calculatons). Thus, ZP3 mrna must be much more unstable n ovulated eggs than n growng oocytes. Durng meotc maturaton of mouse oocytes, the steady-state level of LDH-B mrna falls by only 20 % (Table 1), whle the relatve rate of LDH-B synthess decreases from about 1-8% to 0-25% of total proten synthess (Casco & Wassarman, 1982). A smlar dscrepancy has been noted for changes n /3-actn synthess and mrna levels durng meotc maturaton of mouse oocytes, and has been attrbuted to deadenylaton of /J-actn mrna (Bachvarova et al. 1985; Paynton et al. 1988). Conversely, polyadenylaton of tssuetype plasmnogen actvator mrna durng meotc maturaton of mouse oocytes s thought to account for actvaton of translaton of the mrna durng ths perod (Huarte et al. 1987). Results reported for a varety of eukaryotc systems strongly suggest that the 3'-poly(A) sequence affects both mrna stablty and translatonal effcency (e.g. Palatnk et al. 1984; Drummond et al. 1985; Gall et al. 1988). In ths context, we have found that the sze of LDH-B mrna decreases from 1-4 (fully grown oocytes) to 1-3 kb (unfertlzed eggs) durng meotc maturaton (R. Roller & P. Wassarman, unpublshed results), suggestng that the sharp declne n LDH-B synthess durng ovulaton may be due to deadenylaton of LDH-B mrna. Thus, synthess of ZP3 and LDH-B durng meotc maturaton may be subject to dfferent types of regulatory mechansms. In the former case, decreased synthess s probably due to mrna degradaton and, n the latter, * ZP3 mrna has a T/ 2 of ~4 h durng ovulaton, assumng frst-order knetcs of degradaton. If ZP3 mrna had the same T x / 2 n jan growng oocytes, where the steadystate level of ZP3 mrna s -SxlO 5 copes oocyte" 1, then ~l-5xlo 5 copes would have to be transcrbed every 4h to mantan the level of ZP3 mrna. Snce the ZP3 locus s about 9 kb (Knloch et al. 1988), ths would requre synthess of ~2-4xl0 4 nts~' copy" 1. The rate of elongaton by RNA polymerase II s estmated at about 50nts~ L (Manley, 1984). Thus, the ZP3 gene would need ~480 polymerase molecules per 9 kb, or about one polymerase molecule every 19 nt, an extremely unlkely stuaton. For comparson, t has been estmated that RNA polymerase molecules are located at nt ntervals along amphban lampbrush chromosomes (Hll, 1979). decreased synthess may be due to deadenylaton of mrna. Although the translatonal effcency of mrnas may vary several-fold, steady-state levels of MOM-1, ZP3 and LDH-B mrna n mouse oocytes are underrepresented n the poly(a) + RNA populaton by about a factor of ten, wth respect to ther contrbuton to proten synthess (Table 2). Ths suggests that the mrnas may be represented solely n the oocyte's translated, poly(a) + RNA populaton. Assumng that 15 % of total poly(a) + RNA s assocated wth polysomes n fully grown oocytes (DeLeon et al. 1983), and that the three mrnas are restrcted to ths populaton, ther representaton n that populaton becomes 0-6 %, 1-8% and 1-5% for MOM-1, ZP3 and LDH-B, respectvely. These values are good approxmatons of the contrbutons of the mrnas to proten synthess n oocytes (Table 2). In ths context, more than 90% of /J-actn mrna n fully grown oocytes s assocated wth polysomes (Bachvarova et al. 1986). Results of prelmnary experments suggest that an analogous stuaton apples to ZP3 and MOM-1 mrna n growng and fully grown oocytes (R. Ren & P. Wassarman, unpublshed results). To carry the suggeston just made a step further, evdence that the pool of untranslated poly(a) + RNA n mouse oocytes s, n fact, untranslatable, s provded by results of n vtro translaton of RNA purfed from oocytes and eggs. It can be argued that the proporton of total proten synthess drected by a specfc mrna n vtro should be roughly proportonal to ts representaton n the total mrna populaton, regardless of the n vvo stuaton. In support of ths argument, t should be noted that RNA purfed from cells that exhbt hghly selectve translatonal control n vvo, such as sea urchn (Jenkns et al. 1978; Moon et al. 1982) and surf clam (Rosenthal et al. 1980) oocytes and embryos, do not exhbt such selectvty n vtro. Accordngly, were all mouse oocyte poly(a) RNA translatable mrna, t would follow that translaton of LDH-B and /3-actn mrna would be 6- to 10-fold lower n vtro than n vvo. However, such s not the case for ether proten. Actn synthess represents about 0-3% of total proten synthess n solated fully grown oocytes and about 0-4% of synthess usng RNA prepared from fully grown oocytes (G. Salzmann & P. Wassarman, unpublshed results). Smlarly, oocyte LDH-B synthess represents about 1-8% of total proten synthess both n vvo and n vtro (Casco & Wassarman, 1983). These fndngs suggest that the pool of translatable mrna n mouse oocytes s represented largely by polysomal poly(a) + RNA. Therefore, bona fde mrna probably consttutes only about 3 % of total RNA, or about 15 % of poly(a) + RNA n mouse oocytes. An analogous stuaton exsts n sea urchn and amphban oocytes, where bona fde mrna consttutes about 30% of total poly(a) + RNA (Davdson, 1986). We are grateful to all the members of our laboratory for valuable advce and constructve crtcsm throughout the course of ths research. In partcular, we thank Karen Wenk-

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