Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12451-12455, December 1995 Developmental Biology

Premature translation of 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice KEESOOK LEE*t, HARALD S. HAUGENt, CHRISTOPHER H. CLEGGt, AND ROBERT E. BRAUN*§ *University of Washington, Department of Genetics, Box 357360, Seattle, WA 98195-7360; and tBristol-Myers Squibb Pharmaceutical Research, 3005 First Avenue, Seattle, WA 98121 Communicated by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, September 14, 1995

ABSTRACT Translational control is a major form of translational repression, although direct proof that they func- regulating expression during gametogenesis and early tion as translational repressors in vivo has not yet been development in many organisms. We sought to determine demonstrated (14, 15). whether the translational repression of the protamine 1 An important and unanswered question in sperm morpho- (Prml) mRNA is necessary for normal spermatid differenti- genesis is whether the temporal expression of the testis nuclear ation in mice. To accomplish this we generated transgenic basic is actually necessary for normal spermatid animals that carry a Prml transgene lacking its normal 3' differentiation. To address the importance of the translational untranslated region. Premature translation of Prml mRNA repression of Prml mRNA, we generated transgenic mice that caused precocious condensation of spermatid nuclear DNA, fail to delay Prml mRNA translation. We show that premature abnormal head morphogenesis, and incomplete processing of translation of Prml mRNA causes precocious condensation of Prm2 . Premature accumulation of Prml within syn- nuclear DNA and a failure to complete spermatid differenti- cytial spermatids in mice hemizygous for the transgene caused ation, resulting in sterility. dominant male sterility, which in some cases was accompa- nied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prml MATERIALS AND METHODS is necessary for the transition from nucleohistones to nucleo- Plasmid Construction and Transgenic Mice. The 4.5-kb . transgene contains -4.1 kb ofPrml 5'-untranscribed sequence extending 5' of the transcriptional start site to a HindIII site at Haploid spermatid differentiation (spermiogenesis) in the -4.1 kb, the 95-nt noncoding region of exon 1, the complete mouse takes -2 weeks and consists of 16 steps that are coding exons and intronic sequence ofPrml (247 nt) (16), and characterized by changes in nuclear morphology and acrosome the 105-nt 3' noncoding region of the human growth hormone development (1, 2). During spermiogenesis the on the (hGH) gene (17). Previous studies have established that 4.1 kb DNA are gradually replaced by a class of proteins with of Prml 5'-flanking DNA is sufficient to direct spermatid- intermediate basicity called the transition proteins (TPs) (3). specific transcription upon a heterologous gene in transgenic The TPs are thought to participate in the removal of the mice (16). Transgenic mice were generated by pronuclear histones and in the initial condensation of the spermatid injection of fertilized eggs derived from (C57BL/6x SJL)Fl nucleus (4, 5). Later in spermiogenesis the TPs are replaced females mated with identical hybrid males as described (18). with two small arginine-rich proteins called the protamines (6). RNA Analysis. Total RNA was isolated from dissected testes The protamines clearly are not required for initiating sperm using the method of Chomczynski and Sacchi (19). RNA nuclear condensation because condensation begins before samples were separated on a 1.5% agarose/2.2 M formalde- protamine synthesis, but they may have a role in completing it hyde gel, transferred to nitrocellulose membrane, fixed by in later-stage spermatids and in maintaining it in mature baking at 80°C, and hybridized at 45°C in 50% (vol/vol) spermatozoa. formamide/5 x standard saline citrate (SSC)/5 x 50 mM A special feature of sperm morphogenesis is that global NaPO4 (pH 6.5)/single-stranded DNA at 250 ,.tg/ml/1 x Den- transcription from the haploid genome ceases several days hardt's solution/0.5% SDS/6.25% (wt/vol) dextran sulfate/ before the completion of spermiogenesis and the synthesis of a-32P-labeled DNA probes were prepared by random hexamer the transition proteins and the protamines (7, 8). To accom- labeling. The blot was washed at a final stringency of 0.1 x modate the cessation of transcription, the mRNAs that encode SSC/0.5% SDS at 500C. the TPs and the protamines are synthesized in round sperma- Basic Nuclear Protein Preparation. Crude nuclei were tids (steps 7-9), stored as cytoplasmic ribonucleoprotein par- in ticles for up to a week (steps 7-12), and finally translated in prepared by homogenizing testes 20 mM Tris-HCl, pH elongated spermatids (steps 12-16) (4, 9-11). 7.7/40 mM KCl/17 mM MgCl2/protease inhibitors (aprotinin Transgenic studies have shown that the 3' untranslated at 1 ,ug/ml, 1 mM benzamidine, leupeptin at 0.5 j,g/ml, region (3' UTR) of Prml mRNA is both necessary and pepstatin A at 1 pAg/ml, and 0.5 mM phenylmethylsulfonyl sufficient to mediate translational repression during spermatid fluoride), filtering the homogenates through three layers of differentiation (12, 13). Recent transgenic analysis suggests gauze, and centrifuging at 250 x g for 5 min at 40C. The nuclei that two different regions of the Prml 3' UTR are important pellet was suspended in MKPT [20 mM Tris HCl (pH 7.7)/40 for translational repression and that each is sufficient for mM KCl/10 mM MgCl2/protease inhibitors] supplemented repression independent of the other (unpublished data). Pro- with 1% Triton X-100 and 0.32 M sucrose and further purified teins that bind to different regions of the Prml 3' UTR RNA in vitro have been described and postulated to be involved in Abbreviations: Prml and Prm2, protamine 1 and 2, respectively; TPs, transition proteins; 3' UTR, 3' untranslated region; hGH, human growth hormone. The publication costs of this article were defrayed in part by page charge tPresent address: Fred Hutchinson Cancer Research Center, 11124 payment. This article must therefore be hereby marked "advertisement" in Columbia Street, Seattle, WA 98104. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed.

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by sedimenting through 1.1 M sucrose/MKPT as described overnight at 4°C with the Hup2B (25) antibody diluted in (20). The nuclei pellet was resuspended in cold distilled BPBS. After washing one time for 20 min in BPBS/0.05% water/protease inhibitors and sonicated for three 30-sec Tween 20 and twice in BPBS, the blot was sequentially bursts, each at 60 W. Sonication-resistant spermatid nuclei incubated with biotinylated goat anti-mouse IgG antibody and were recovered by centrifugation at 6000 x g for 5 min at 4°C horseradish peroxidase-conjugated streptavidin. Horseradish from the sonication-sensitive nuclei (supernatant). Basic nu- peroxidase was revealed with the chromogenic substrate clear proteins were isolated as described with some modifica- 4-chloro-1-naphthol (Bio-Rad). tions (21). Briefly, the sonication nuclei were washed twice and dissolved in protamine extraction buffer [6 M guanidine.HCl/ RESULTS 0.5 M Hepes (pH 7.5)/10 mM dithiothreitol] for 1 hr at room temperature. Cysteine residues were alkylated by adding vi- Sterility of Transgenic Mice. To prematurely express Prml nylpyridine to a final concentration of 0.25 M and incubating protein in round spermatids, transgenic animals were gener- the mixture for 1.5 hr at room temperature (22). HCl was ated that carry a chimeric gene containing the promoter, 5' added to a final concentration of 0.9 M and dialyzed overnight noncoding and coding regions of Prml, and the hGH 3' against 0.2 M HCl. Insoluble protein and DNA were removed noncoding region as described in Materials and Methods. We by centrifugation at 12,000 x g for 10 min. The proteins in the expected that the transgene would be transcribed in round supernatant were precipitated with 20% trichloroacetic acid spermatids at the same time as the endogenous Prml gene and and washed with acidified acetone as described (23). Basic that the transgenic mRNA would be translated prematurely in nuclear proteins from sonication-sensitive nuclei (the super- round spermatids because it lacked the Prml 3' UTR. natant fraction) were prepared by extracting with 0.25 M HCI Table 1 summarizes our analysis of seven transgenic founder twice as described (23). Proteins in the pooled extract were males and the F1 male offspring of three transgenic founder precipitated with 20% trichloroacetic acid, resuspended in females. Breeding studies revealed that four of the seven protamine extraction buffer, and processed as those from transgenic founder males were sterile, whereas the remaining sonication-resistant nuclei. Basic proteins were dissolved in 8 three were fertile. The three fertile males were presumably M urea/0.9 M acetic acid/0.1 M 2-mercaptoethanol/1% germ-line mosaics, as none of the animals transmitted the methyl green and separated in polyacrylamide slab gels con- transgene to their offspring. F1 males derived from two of the taining 15% acrylamide, 0.1% bisacrylamide, 6.2 M urea, 0.9 transgenic female founders were also sterile (11135 and M acetic acid by a modification of a described method (24). 11142), whereas male offspring of the remaining founder The gels were prerun at 200 V for 2.5 hr in 0.9 M acetic acid. female (11134) were semifertile. The dominance of the trans- Protein samples were loaded and electrophoresed in fresh gene in the haploid spermatids, only half of which should running buffer at 20-mA constant current for -2 hr. The gels inherit the transgene, is presumably due to sharing the trans- were stained in 0.2% naphthol blue-black/40% (vol/vol) eth- genic mRNA or protein between the haploid spermatids via anol/7% (vol/vol) acetic acid and destained in the same their intercellular bridges (26, 27). Interestingly, males in the solution without dye. semifertile line 11134 appear not to transmit the transgene to Immunoblot Analysis. The proteins separated on an acid- the expected 50% of their offspring, suggesting that although urea gel were electroblotted onto an Immobilon-P filter (Mil- RNA and protein do pass between cells via their connecting lipore) in 0.7% acetic acid at 400 mA for 15 min. Half of the bridges, there may not be equal sharing of all postmeiotic gene blot was stained in 0.25% Coomassie brilliant blue/45% products. methanol/10% (vol/vol) acetic acid and subsequently Among the different sterile founders and transgenic lines, destained in 50% (vol/vol) methanol/10% acetic acid. The histological analysis revealed two clearly distinguishable phe- other half of the blot was blocked with 5% nonfat dry notypes. As detected by light microscopy, the majority of the milk/phosphate-buffered saline (BPBS) and then incubated sterile males contained late-stage spermatid defects charac- Table 1. Summary of mouse lines that prematurely translate Prml mRNA Expression Transmission no./ Line level Fertility total no. offspring Phenotype Founder 11080 High Sterile NA Early spermatid arrest 237 Moderate Sterile NA Late spermatid defects 11085 Moderate Sterile NA Late spermatid defects 11066 Moderate Sterile NA Late spermatid defects 11060 Low Fertile 0/30 Mosaic 11068 Moderate Fertile 0/26 Mosaic 4 Moderate Fertile 0/28 Mosaic F1 11134 Low Semifertile 45/124 ND 11135 High Sterile NA Early spermatid arrest 11142 Moderate Sterile NA Late spermatid defects NA, not applicable; ND, not determined. Fertility of male founders and F, male offspring of female founders was determined by cohabitation with multiple females for at least 1 mo. Semifertility in the 11134 line means that some Fi males are fertile and some are sterile. Expression level of the transgene refers to a qualitative assessment of transgenic mRNA levels as determined by Northern blotting (Fig. 3). Transmission refers to the fraction of F1 animals that inherit the transgene from a fertile transgenic male. Phenotype refers to the histological phenotype. In the early spermatid-arrest phenotype all seminiferous tubules show a uniform arrest at the round-spermatid stage (Fig. 1 C and F). The late-stage spermatid- defect phenotype refers to abnormal head morphogenesis in early elongating spermatids and the failure to release spermatids from the seminiferous epithelium (Fig. 1 B and E). Mosaic refers to a mixed histology, where either whole sections of seminiferous tubules or portions of tubules showing the late- stage spermatid-defect phenotype are present along with regions of normal histology (data not shown). Downloaded by guest on September 24, 2021 Developmental Biology: Lee et al. Proc. Natl. Acad. Sci. USA 92 (1995) 12453 terized by abnormal head morphogenesis and a failure to complete the final stages of spermiogenesis (Fig. 1 B and E). A second phenotype, seen in founder animal 11080 and line 11135, was a complete developmental arrest at the round spermatid stage observed in every seminiferous tubule examined (Fig. 1 C and F). In some instances spermatids formed multinu- cleated cells, by relaxation of intercellular bridges and the con- vergence of cytoplasm (Fig. 1F). In both phenotypes no mature spermatozoa were detected in the epididymis (Fig. 1 H and 1). Synthesis and Premature Translation of Prml mRNA. On the basis of previous experiments (12), we expected that replacement of the 3' UTR of Prml with the 3' UTR of hGH would lead to premature translation of the transgenic Prm- hGH mRNA. We confirmed that Prml protein was synthe- sized early by using immunocytochemistry. Treatment of testis sections from males in lines 11142 and 11135 (Fig. 2A and B, FIG. 2. Immunocytochemical detection of premature synthesis of Prml protein. Localization of Prml protein in nuclei of elongating respectively), with anti-Prml antibody showed that Prml was spermatids in an F1 male from line 11142 with late-stage spermatid- synthesized prematurely and that it was localized to the defect phenotype (A) and in nuclei of round spermatids in an F1 male nucleus. We detected Prml in the testis from males in line from line 11135 with early-stage spermatid-arrest phenotype (B). 11142 in round and elongating spermatids (steps 7-12) and in Prml protein was detected with a mouse monoclonal antibody, line 11135 in round spermatids (steps 7-8), whereas Prml HuplN, raised against human PRM1 (25). The antibody crossreacts protein is normally first detected in step 12 (data not shown). with mouse Prml but not with Prm2 (supplied by A. Wyrobek, To determine whether the different histological phenotypes Lawrence Livermore Laboratory). The primary antibody was detected correlated with the level of transgene expression, Northern with a biotinylated rabbit anti-mouse secondary antibody and strepta- vidin-conjugated horseradish peroxidase with aminoethylcarbazole blot analysis was performed on RNA isolated from the testes substrate (Zymed and Vector Laboratories). Sections were counter- of the transgenic mice (Fig. 3). Highest levels of transgene stained with hematoxylin. The arrows point to immunolocalization of expression (founder 11080 and line 11135) correlated with the Prml protein in the nucleus. Photographs were taken by using early spermatid-arrest phenotype, moderate levels of trans- differential interference-contrast microscopy. (Bar = 8 Jim.) genic mRNA with late-stage spermatid-defect phenotype, and extremely low levels (line 11134) with semifertility. Theoret- does not affect spermatid differentiation, but its time of ically, the spermatogenic phenotypes we observed could be synthesis does. due solely to increased amounts of Prml protein and not to its Expression of TPs and Endogenous Protamines. Given that altered temporal expression. However, >40 lines of transgenic premature expression ofPrml protein occurs when the haploid mice have been established that contain as much as three times genome is still engaged in the transcription of required the normal amount of Prml synthesized at the appropriate for completion of spermiogenesis, one explanation for the time during spermiogenesis (16, 28, 29), and in no case has this observed phenotypes is that Prml protein disrupts the tran- led to fertility defects. Thus, overexpression of Prml protein scription of other postmeiotically expressed genes. To test this B~~~~~~~~i. . '-.

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FIG. 1. Histological analysis of testes from sterile transgenic males. Low (A) and high (D) magnification of sections through control testis. The arrow points to the head of a normal elongating spermatid(es). Low (B) and high (E) magnifications of sections through testis of F1 male from 11142 line with the late-stage spermatid-defect phenotype. Tubules in B are highly disorganized; they contain numerous abnormal elongating spermatids and few late-stage elongated spermatids. The arrowheads in E point to a collection of elongating spermatids that have grossly abnormal heads. Low (C) and high (F) magnifications of testis sections from F1 male from 11135 line with early-stage spermatid-arrest phenotype. Note the absence of any elongating spermatids and the many uniformly staged round spermatids (rs). Seminiferous tubules in D, E, and F are all at the same developmental stage (XII) as characterized by the presence of spermatocytes in metaphase (m). G, H, and I show sections through the epididymis of a control, 11142, and 11135 male, respectively. Arrows point to the lumen of tubules that are; packed with sperm (G), completely devoid of sperm (H), or contain a few round spermatids, apparently sloughed from the seminiferous epithelium (I). Testes were fixed in Bouins' solution, embedded in paraffin, sectioned, and stained with hematoxylin and periodic acid/Schiff reagent. Bar = 25 ,um (A), 8 ,um (D), and 50 ,um (G). Downloaded by guest on September 24, 2021 12454 Developmental Biology: Lee et al. Proc. Natl. Acad. Sci. USA 92 (1995) A second hypothesis to explain the sterility phenotype is that premature synthesis of Prml affects the synthesis of the TPs and the endogenous protamines. To analyze synthesis of the various testis basic nuclear proteins, total testis basic proteins A+°pX~~0 were isolated from sonication-sensitive and sonication- resistant nuclei and separated by acid-urea gel electrophoresis. Spermatid nuclei normally become sonication-resistant in wild-type mice after deposition of the TPs and protamines Prm-hGH ** , iIh (Fig. 4A, lanes 1 and 2) (3). In animals with the late-stage spermatid-defect phenotype (Fig. 4A, lanes 6, 8, 10, and 16), we detected the presence of Prml, transition protein 2 (TP2), Prml9a9199 #_* and the precursor of Prm2, in sonication-resistant nuclei only. The absence of Prml in sonication-sensitive nuclei (Fig. 4A, lanes 5, 7, 9, and 15) suggests that accumulation of Prml leads Prm2 **PO99@, 3*4 to sonication resistance. In no animals did we observe levels of Prml protein in excess of that seen in normal control males, again supporting the conclusion that it is the time of Prml Actin *,*P0S U,W expression and not the absolute amount of Prml protein that ,. 3**" 0 **' is responsible for the spermiogenic phenotypes. 1 2 3 4 5 6 7 8 9 10111213 By protein analysis we cannot distinguish between Prml protein expressed from the transgene and from the endoge- FIG. 3. Northern blot analysis of RNA from founder and F1 nous Prml gene. However, translation of endogenous Prml transgenic males. Lanes 1-9 contain total testis RNA isolated from mRNA is normally accompanied by significant shortening of control or transgenic founders, and lanes 10-13 contain RNA isolated its poly(A) tail, resulting in a heterogeneous population of from control and F1 transgenic offspring. Each lane contains 15 ,ug transcripts ranging in size between -400 and 560 nt (9). A of total RNA isolated from the testes of sexually mature males. The and Prml RNA is blot was hybridized sequentially with 32P-labeled DNA probes specific shortened, presumably deadenylated, seen for each of the mRNAs. The Prm-hGH mRNA was detected with a in the control nontransgenic samples and in RNA samples probe prepared from a DNA fragment containing the hGH 3' UTR. from all transgenic animals that show the late-stage spermatid- The endogenous Prml mRNA was detected with a probe prepared defect phenotype (Fig. 3, lanes 1, 3-11, and 13), suggesting that from a DNA fragment containing the Prml 3' UTR. The Prm2 and actin the endogenous Prml mRNA is translated in those animals. mRNAs were detected with probes prepared from their full-length Prm2 is normally synthesized as a precursor protein of 106 cDNAs, respectively. A low level of Prm-hGH transcript was detected in amino acids and is processed through a series of proteolytic founder 11060 (lane 6) upon longer exposures (data not shown). cleavages to a final size of 63 amino acids (30, 31). Interest- ingly, animals that displayed the late-stage spermatid-defect hypothesis Northern blot analysis was used to assess the phenotype only contained the largest of the unprocessed forms mRNA levels from the endogenous Prml and Prm2 genes. Fig. of Prm2 (Fig. 4A, lanes 6, 8, 10, and 16). To verify the identity 3 shows that, relative to actin RNA, the endogenous Prml and of precursor, partially processed, and mature Prm2, we ana- Prm2 transcripts are present in normal amounts in animals lyzed the basic proteins prepared from a normal testis, and with both phenotypes, suggesting that a general block in global from a founder and F1 male with the late-stage spermatid- transcription is not responsible for the phenotypes observed. defect phenotype, by immunoblot analysis. In the control A B Coomassie blue Immunoblot

e,§~ ~ ~ C C >\d C`C v coo m

O co cm n cn U)acncr cn cnonn cnoCo cnCcn c cn cn0 cn c o CD z

...... _R TP2 g I S_ .. -- TP2- Prm2 = - Prm2[4 Prml -Prml 7---

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 FIG. 4. (A) Analysis of total testis basic proteins from sonication-sensitive and sonication-resistant spermatid nuclei. Lanes 1-10 contain protein isolated from control and transgenic founders, and lanes 11-16 contain protein isolated from control and F1 transgenic offspring. Total basic testis proteins were prepared from sonication-sensitive (SS) and sonication-resistant (SR) nuclei, fractionated by acid-urea polyacrylamide gel electrophoresis, and detected by stainingwith 0.2% naphthol blue-black, as described. Positions ofTP1; Prml; and the precursor, partially processed, and mature forms of Prm2 are labeled. (B) Immunoblot analysis of precursor and processed forms of Prm2. Sonication-resistant testis nuclear basic proteins were prepared from a nontransgenic male, a transgenic founder animal with the late-stage spermatid defect (11066), and a transgenic F1 animal with the late-stage spermatid defect (11142-28). Proteins were fractionated by acid-urea/polyacrylamide gel electrophoresis, transferred to Immobilon-P filter (Millipore), and either stained with Coomassie blue (lanes 1-3) or incubated with an anti-Prm2 mouse monoclonal antibody (Hup2B) that was raised against human PRM2 (lanes 4-6). This antibody crossreacts with mouse Prm2 but does not crossreact with mouse Prml. The primary antibody was detected with a biotinylated goat anti-mouse secondary antibody and streptavidin-conjugated horseradish peroxidase (Zymed and Vector Laboratories) with the chromogenic substrate 4-chloro-1-naphthol (Bio-Rad). Positions of Prml; TP2; and the precursor, processed, and mature forms of Prm2, are indicated. Downloaded by guest on September 24, 2021 Developmental Biology: Lee et al. Proc. Natl. Acad. Sci. USA 92 (1995) 12455 sample we detected several forms of Prm2 (Fig. 4B, lanes 1 and data suggest that the failure to complete spermatid differen- 4), whereas in animals 11066 and 11142-28 only the precursor tiation is due to a failure to complete the transition from and the largest of the partially processed forms were detected nucleohistones to nucleoprotamines and demonstrate that (Fig. 4B, lanes 2, 3, 5, and 6). Thus, mice with the late-stage condensation depends on the correct temporal spermatid-defect phenotype fail to make mature Prm2 protein, synthesis of the TPs and the protamines. suggesting that transition from nucleohistones to protamines Spermatid nuclear shaping begins several days before syn- in these animals is incomplete. thesis of the TPs and protamines. Spermatid nuclear morphol- ogy is under genetic control (32), and the extranuclear forces that impose this shaping process are mediated, in part, by a DISCUSSION microtubular array called the manchette (33). On the basis of Premature translation of Prml mRNA in transgenic mice the abnormal nuclear morphology in animals with the late- resulted in a failure to produce mature sperm. In animals that stage spermatid-defect phenotype, our data support the hy- expressed the highest levels of Prml protein, pothesis that intranuclear forces, mediated by the process of was arrested uniformly at the round-spermatid stage. In ani- chromatin condensation, may also contribute to the shape of mals that expressed lesser amounts of Prml protein, spermat- the spermatid nucleus (34). ogenesis continued beyond the round-spermatid stage but was highly abnormal. Both spermatogenic phenotypes are due to We thank R. Palmiter and L. Hartwell for critical reading of the premature translation of Prml mRNA and not to its overex- manuscript, A. Wyrobek and R. Balhorn (Lawrence Livermore Lab- pression, as previous studies have clearly shown that overex- oratories, Livermore, CA) for the HuplN and Hup2B antibodies, M. pression of Prml protein at its normal time of synthesis does Meistrich for providing his interpretation of the histological pheno- not adversely affect spermatogenesis (16, 28, 29). Further- types, and K Clegg for expert technical assistance. This work was more, in none of the transgenic animals was the amount of supported by research grants to R.E.B. from the March ofDimes (Basil Prml protein higher than the endogenous Prml protein level O'Connor) and from the National Institutes of Health (HD27215). in control animals. Therefore, translational regulation of Prml 1. Oakberg, E. F. (1956) Am. J. Anat. 99, 391-413. mRNA is absolutely required for normal spermatid differen- 2. Russell, L. D., Ettlin, R. A., SinhaHikim, A. P. & Clegg, E. D. (1990) tiation, and premature translation of Prml mRNA leads to Histological and Histopathological Evaluation of the Testis (Cache River dominant male Press, Clearwater, FL). sterility. 3. Meistrich, M. L. (1989) in and Basic Nuclear Protein Transitions in Spermatid nuclear condensation normally begins in the Mammalian Spermatogenesis, eds. Hnilica, G., Stein, G. & Stein, J. (CRC, step-12 elongating spermatid and is thought to be mediated by Orlando, FL), pp. 165-182. the TPs (3). On the basis of the relative timing of events, it has 4. Balhorn, R., Weston, S., Thomas, C. & Wyrobek, A. J. (1984) Exp. Cell Res. been that the TPs initiate nuclear and 150, 298-308. proposed condensation 5. Loir, M. & Lanneau, M. (1984) J. Ultrastruct. Res. 86, 262-272. that the protamines complete the process. Significantly, how- 6. Bellve, A. R. (1979) in The MolecularBiology ofMammalian Spermatogen- ever, spermatids in animals with the early spermatid-arrest esis, ed. Finn, C. A. (Oxford Univ. Press, London), Vol. 1, pp. 159-261. phenotype failed to make TPs but had become sonication- 7. Kierszenbaum, A. L. & Tres, I. L. (1975) J. Cell Biol. 65, 258-270. that Prml is for the 8. Monesi, V. (1964) J. Cell Biol. 22, 521-532. resistant, suggesting protein responsible 9. Kleene, K C., Distel, R. J. & Hecht, N. B. (1984) Dev. Biol. 105, 71-79. sonication resistance. In addition, we were unable to detect 10. Kleene, K. C. & Flynn, J. (1987) Dev. Biol. 123, 125-135. Prml protein in sonication-sensitive spermatid nuclei in ani- 11. Yelick, P. C., Kwon, Y. H., Flynn, J. F., Borzorgzadeh, A., Kleene, K. C. & mals with the early-stage spermatid-arrest phenotype or the Hecht, N. B. (1989) Mol. Reprod. Dev. 1, 193-200. Together these exper- 12. Braun, R. E., Peschon, J. J., Behringer, R. R., Brinster, R. L. & Palmiter, late-stage spermatid-defect phenotype. R. D. (1989) Genes Dev. 3, 793-802. iments provide a direct demonstration that Prml protein medi- 13. Braun, R. E. (1990) Enzyme 44, 120-128. ates the precocious nuclear condensation that leads to sonica- 14. Fajardo, M. A., Butner, K. A., Lee, K. & Braun, R. E. (1994) Dev. Biol. 166, tion resistance; however, this process is probably abnormal. 643-653. In one founder and in all males in one 15. Kwon, Y. K & Hecht, N. B. (1991) Proc. Natl. Acad. Sci. USA 88, transgenic male, 3584-3588. transgenic line, premature expression of Prml protein resulted 16. Peschon, J. J., Behringer, R. R., Brinster, R. L. & Palmiter, R. D. (1987) in a uniform arrest of spermiogenesis at the round-spermatid Proc. Natl. Acad. Sci. USA 84, 5316-5319. stage. Northern analysis suggests that the premature synthesis 17. DeNoto, F. M., Moore, D. D. & Goodman, H. M. (1981) NucleicAcids Res. of Prml protein does not block global transcription; however, 9, 3719-3730. 18. Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K. & Palmiter, our data do not rule out the possibility that synthesis of specific R. D. (1985) Proc. Natl. Acad. Sci. USA 82, 4438-4442. mRNAs is affected by Prml synthesis and that it is the absence 19. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. of these mRNAs that cause the developmental arrest. Unifor- 20. Bellve, A. R. (1993) Methods Enzymol. 225, 84-113. mity of the developmental block suggests that if transcriptional 21. Meistrich, M. L., Reid, B. 0. & Barcellona, W. J. (1976) Exp. Cell Res. 99, 72-78. defects are responsible for the arrest, genes that regulate 22. McKay, D. J., Renaux, B. S. & Dixon, G. H. (1985) Biosci. Rep. 5, 383-391. spermiogenesis are the ones affected. 23. Platz, R. D., Meistrich, M. L. & Grimes, S. R., Jr. (1977) Methods Cell Biol. In most transgenic animals, premature expression of Prml 16, 297-316. protein caused abnormal nuclear shaping, precocious nuclear 24. Panyim, S. & Chaildey, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 25. Stanker, L. H., Wyrobek, A., McKeown, C. & Balhorn, R. (1993) Mol. DNA condensation, and a general failure to complete the Immunol. 30, 1633-1638. latter stages of spermiogenesis. Again, Northern analysis sug- 26. Caldwell, K A. & Handel, M. A. (1991) Proc. Natl. Acad. Sci. USA 88, gests that global transcription is not affected and is, therefore, 2407-2411. not responsible for the phenotype observed. Unlike the ani- 27. Braun, R. E., Behringer, R. R., Peschon, J. J., Brinster, R. L. & Palmiter, R. D. (1989) Nature (London) 337, 373-376. mals with the early spermatid-arrest phenotype, animals with 28. Peschon, J. J. (1988) in Regulation of Protamine 1 Expression During the late-stage spermatid defects synthesize the TPs and the Spermatogenesis in Transgenic Mice (Univ. of Washington, Seattle), p. 99. endogenous protamines. Significantly, however, processing of 29. Zambrowicz, B. P., Harendza, C. J., Zimmermann, J. W., Brinster, R. L. & the Prm2 precursor protein is incomplete, and no mature Prm2 Palmiter, R. D. (1993) Proc. Natl. Acad. Sci. USA 90, 5071-5075. 30. Carre Eusebe, D., Lederer, F., Le, K H. & Elsevier, S. M. (1991) Biochem. protein is produced, suggesting that the processing of Prm2 is J. 277, 39-45. coupled with its normal codeposition with Prml protein. The 31. Chauviere, M., Martinage, A., Debarle, M., Alimi, E., Sautiere, P. & deposition of Prml protein on chromatin before synthesis of Chevaillier, P. (1991) C. R Acad. Sci. Ser. 3 313, 107-112. the TPs demonstrates that removal of the TPs is not required 32. Illison, L. (1969) Austr. J. Biol. Sci. 22, 947-963. 33. Meistrich, M. L. (1993) in Nuclear Morphogenesis During Spermiogenesis, for Prml deposition; however, our data suggest that the ed. Kretser, D. M. (Academic, San Diego), pp. 67-97. deposition of Prml protein is abnormal and that it interferes 34. Fawcett, D. W., Anderson, W. A. & Phillips, D. M. (1971) Dev. Biol. 26, with the normal events of spermiogenesis. In summary, these 220-251. Downloaded by guest on September 24, 2021