Proc. Natl. Acad. Sci. USAVol. 88, pp. 10327-10331, November 1991Genetics

Dominant male sterility in mice caused by insertion of a transgene(spermatogenesis/impenetrance)

JEANNE MAGRAM*t AND J. MICHAEL BISHOP*4*The G. W. Hooper Foundation and *Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0552

Contributed by J. Michael Bishop, August 20, 1991

ABSTRACT While examining a series of transgenic mouselines carrying the HCK protooncogene, we encountered one linein which males henmzygous for the transgene were sterile. Thesterile males mated normally but failed to impregnate females.Light and electron microscopy revealed that spermatogenesisproceeds normally until nuclear condensation, which occursbut gives rise to a variety of abnormally shaped nuclei.Expression of the transgene was not detectable. Thus, theinsertion itself probably caused the abnormal phenotype bydisrupting a gene (or genes) important in spermatogenesis. Themutation is genetically dominant, causing an abnormal phe-notype even though the sterile mice carry an ostensibly normalcounterpart of the disrupted locus. The mutant phenotype iscompletely penetrant only in some genetic backgrounds, sug-gesting a modifying influence from a second locus. Junctionsbetween the inserted transgene and adjoining cellular DNAwere cloned, allowing us to confirm the heterozygous nature ofthe genetic disruption and to detect an associated deletion. Wehave designated the mutation Lvs (lacking vigorous sperm) andpresume that it may define a previously undescribed locusimportant in spermatogenesis.

Transgenic mice offer a valuable means with which to eval-uate the control and phenotypic effects ofgene expression (1,2). In addition, insertion of a transgene into the genomeoccasionally disrupts a vital gene, providing a fortuitousmutation that can reveal the physiological function of theaffected gene (3-5). Typically, an insertion mutation causesloss of function, is genetically recessive, and must be bred tohomozygosity before its effects become evident. The virtuein such a mutation is that the presence of the transgeneprovides a molecular "tag" that can facilitate cloning of themutant gene and its normal counterpart.We have produced a series of 10 transgenic mouse lines

with the protooncogene HCK. As the mice were bred, itbecame evident that many males of one line were sterile andthat the sterility arose from a defect in spermatogenesis.Since the sterility segregated with the hemizygous transgeneand occurred in the absence of detectable expression of thetransgene, we concluded that the abnormal phenotype wasdue to mutagenesis by insertion of the transgene. We desig-nated the mutation Lvs for "lacking vigorous sperm."

Spermatogenesis has been well described morphologically,but little is known of the underlying molecular events (6-8).Therefore, we decided to pursue the nature of Lvs and thegene (or genes) that it presumably defines. Here we describethe phenotype and genetic behavior of Lvs and the isolationof DNA from the Lvs locus adjacent to the transgene. Themutation appears to affect spermatogenesis at the time ofnuclear condensation and displays two unexpected proper-ties: genetic dominance and a modifying effect of geneticbackgrounds. We anticipate that characterization of the

genetic locus affected by Lvs will illuminate a previouslyundescribed and important function in spermatogenesis.

MATERIALS AND METHODS

Production of Transgenic Mice. A DNA fragment contain-ing an immunoglobulin enhancer, simian virus 40 (SV40)promoter, a hematopoietic cell kinase (HCK) cDNA (9, 10),and a SV40 poly(A) addition site and splicing signals (referredto as plsh and shown in Fig. 1A) was generated by digestionof its parent plasmid by EcoRI and isolation on an agarose gelfollowed by electroelution (11) and CsCl gradient purification(12). The fragment was microinjected into the pronuclei offertilized (C57BL6/J x SJL/J)F2 embryos, which were usedto generate transgenic mice by standard protocols as de-scribed (12).

Identification of Transgenic Mice. High molecular weightDNA from a small piece of mouse tail was isolated asdescribed (12). DNA was digested with HindIII and thenanalyzed by Southern blot analysis (13) using the SV40 earlyregion as a probe. Hybridization was in 0.5 M Na2HPO4, pH7.2/7% SDS/1 mM EDTA at 650C overnight, followed by twowashes of 20 min each at 650C in 0.5 M Na2HPO4, pH 7.2/1%SDS/1 mM EDTA.Genomic Cloning. Genomic DNA from a transgenic mouse

was digested to completion with EcoRI and electrophoresedon a 0.9o agarose gel. DNA ranging in size from 4.5 to 7kilobases (kb) was electroeluted (11) and used to construct apartial genomic library in the phage vector AgtlO. The librarywas screened using the SV40 early region as a probe.Subcloning and other standard molecular biological tech-niques were performed as described (11).

Microscopic Examination. For analysis by light micros-copy, testes were dissected and then fixed in 4% formalin,dehydrated in a gradient of ethanol, cleared with toluene, andembedded in paraffin, followed by preparation of thin sec-tions and staining with hematoxylin and eosin. For analysisby electron microscopy, mice were perfused through theheart with phosphate-buffered saline containing heparin (10units/ml) followed by perfusion with 1.5% glutaraldehyde in0.1 M sodium cacodylate buffer. Testes were removed andplaced in additional fixative. The tissue was then further fixedin 1% osmium tetroxide in acetate vernanal buffer for 1 hr at4°C followed by 1 hr in 1% tannic acid at room temperatureand then 1 hr at 37°C in Kellenburger buffer. The tissue wasthen dehydrated in a gradient of ethanol and embedded inEpox; ultrathin sections were then prepared.

Screening for Fertility. Males were tested for fertility bymating with FVB/N females (mating was identified by thepresence of a vaginal plug in the female after copulation) andthen dissecting the females 7-10 days later to determine

Abbreviation: SV40, simian virus 40.tTo whom reprint requests should be addressed at: G. W. HooperFoundation, Box 0552, University of California, San Francisco, CA94143-0552.

10327

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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10328 Genetics: Magram and Bishop

whether the female was pregnant and, if so, the number ofpups present.

RESULTSIdentification of Sterile Males Among Transgenic Mice. Ten

founder mice were generated by microinjection of fertilizedeggs with the plsh construct (Fig. 1A). The founder female oftransgenic line pIsh 3 was mated to a (C57BL/6J x SJL/J)F1male (Fig. 1B). A transgenic female from the first cross wassubsequently mated to a different (C57BL/6J x BALB/cByJ)Fl male due to the inability of the regular supplier toprovide the starting (C57BL/6J x SJL/J)F1 mice. Femalesfrom the resulting generation were also mated to (C57BL/6Jx BALB/cByJ)Fl males. Male progeny were tested forfertility. Because normal plugs were obtained with the ex-pected frequency, it appeared that males exhibited appropri-ate sexual behavior and copulated normally. Transgenicmales fell into three different phenotypic classes. First, themajority (2/3) were completely sterile: no pregnancy oc-curred in a minimum of five matings. The second class ofmales had very limited fertility: no more than one femalebecame pregnant in a minimum of six matings and that femalehad no more than five pups (generally, one to three). Thethird class of males had apparently normal fertility. Incontrast, all nontransgenic males displayed normal fertility,demonstrating cosegregation of the sterile phenotype withthe presence of the transgene.

Effect of Genetic Background on Penetrance of the Pheno-type. One possible explanation for the appearance of variousphenotypic classes was differences in genetic background.To test this hypothesis, transgenic females were backcrossedto males of four different inbred strains and resultant femaleprogeny were used to continue backcrossing onto the sameinbred strain. Male progeny were tested for fertility. Incontrast to transgenic males on both the C57BL/6J and

A

Table 1. Effect of genetic background on penetrance of sterilityBALB/

C57BL/6J cByJ SJL/J FVB/NF1 60% (6/10) 57% (4/7) 75% (3/4)* 100o (3/3)F2 40% (2/5) 0%1 (0/6) 100lo (4/4) 10%to (4/4)F3 0% (0/3) 0%o (0/5) 100%o (4/4) 100%o (4/4)

Percentage of sterile transgenic male progeny derived from back-crossing two different transgenic females with males of the indicatedinbred strain. Data were pooled for preparation of the table. Num-bers in parentheses indicate sterile males of total number tested. Findicates the backcross generation number.*The one male that was not sterile was of very limited fertility; onlyone female of seven was pregnant and with only five pups.

BALB/cByJ genetic backgrounds, transgenic males onSJL/J or FVB/N genetic background were sterile (Table 1).The differences in genetic background were apparent after asingle backcross and were completely established after threebackcrosses. These data suggest that the impenetrance of theLvs mutation was due to the influence ofgenetic background.

Testicular Hstology of Sterile Lvs Males. The testes ofnontransgenic and mutant adult males were fixed and pre-pared for light microscopy. Although overall organizationand numbers of germ cells within the tubules appearednormal in sterile mice, mature spermatids appeared to bemore darkly stained and misshapen (Fig. 2). Spermatids didnot appear to have the normal elongated shape but ratherwere short and wide. The morphologies of both Sertoli andLeydig cells were indistinguishable between sterile trans-genic testes and nontransgenic testes.

Electron Microscopy of Spermatids from Sterile Lvs Males.Further characterization by electron microscopy revealedthe absence of normally shaped mature spermatids in thetestes of sterile males. Although there appeared to be normalnumbers of spermatids and development appeared normal

Eco RI Eco RI

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FIG. 1. The transgene and mu-tant pedigree. (A) pIsh consists ofan immunoglobulin enhancer(IgE), SV40 promoter (SVp),cDNA for human HCK (hckcDNA), and a SV40 poly(A) addi-tion site and splicing signals(SVpA/sp; see ref. 14). An acti-vating mutation was introducedinto the HCK cDNA, which re-sulted in a substitution of phenyl-alanine for tyrosine at position 501and enabled the encoded proteinto transform NIH 3T3 cells (15).(B) Pedigree for line 3 of pIshtransgenic mice. The founder fe-male was mated to a (C57BL/6J xSJL/J)F1 male, whereas F1 and F2female progeny were mated to(C57BL/6J x BALB/cByJ)F1males due to the unavailability ofthe starting strain. Shaded boxesrepresent transgenic animals,which fall into three classes offertility as defined in the text:completely sterile (solid boxes),very limited fertility (stippled box-es), and normal fertility (hatchedboxes). All nontransgenic animals

ec display normal fertility. nt, Nottested for the presence of thetransgene; *, only one pup presentin the observed pregnancy.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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Genetics: Magram and Bishop

throughout most of spermatogenesis, no mature spermatidshad normal morphology. Instead, spermatids from steriletransgenic males had abnormally shaped nuclei, although theacrosome and tail remain normal (Fig. 3).

Cloning of Junction Fragments Containing DNA Flankingthe Transgene Insertion Site. In an effort to identify thegenetic defect in Lvs mice, junction fragments betweenmouse genomic DNA and transgene DNA were cloned.These fragments were determined to be 6.0- and 6.5-kbEcoRI fragments by Southern blot analysis of transgenicmouse DNA using the SV40 portion of the transgene as aprobe (data not shown). EcoRI-digested transgenicDNA wassize fractionated and used to construct a library in phageAgtlO. The library was screened using the same SV40 probeand three clones were identified as positive. Two inserts weresubcloned into the plasmid Bluescript KS+ (the third positiveclone did not digest with EcoPJ and was therefore discarded)and shown to have the correct insert sizes. The regionsindicated in Fig. 4A as Xba and Hinfl indicate portions ofthegenomic DNA associated with these junction fragments thatwere used as probes on Southern blots to recognize EcoRIpolymorphisms (Fig. 4 B and C). These polymorphisms were

Proc. Natl. Acad. Sci. USA 88 (1991) 10329

generated by insertion of the transgene and serve as i markerfor the disrupted locus. A transgenic male and female dis-played the same pattern of EcoRI-generated bands indicatingthe presence of both the normal and disrupted alleles (Fig.4C). Therefore, insertion is not into the X chromosome sincethat would predict the absence of the normal allele in sterilemales. In addition, ifthere was not a deletion at the target siteupon integration, then cellularDNA adjacent to the two endsof the transgene would be linked in the normal locus- (Fig.4A). Since both junction probes do not recognize the simeEcoRI fragment associated with the normal allele, there mustbe a deletion or rearrangement of DNA associated with theinsertion event. Initial efforts to detect an RNA transcript intestes by using the junction probes described here have beenunsuccessful (data not shown).

DISCUSSIONLvs as an Insertional Mutation. The mutation designated

Lvs was encountered during the initial breeding of a singleline ofmice bearing a HCKprotooncogene as a transgene. Onfurther analysis, it became apparent that the male sterilitycharacteristic of Lvs segregated with the transgene,-although

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FIG. 2. Light microscopy oftestes from normal and sterilemales. Low-power micrograph oftestes from a normal adult male(A) and a sterile transgenic male(B) showing representative semi,niferous tubules. Overall testicu-lar organization and approximatenumbers of germ cells appearidentical. Higher-power micro-graph of a seminiferous tubulefrom a normal adult male (C) anda sterile transgenic male (D). Al-though germ cell numbers in thesterile mouse are normal, maturespermatids appear misshapen andmore heavily stained. Sectionshave been stained with hematox-ylin and eosin. (A and B, x200; Cand D, x400.)

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10330 Genetics: Magram and Bishop

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FIG. 3. Electron microscopy of spermatids from normal and sterile males. (A) Representative spermatids from a normal adult male. (B-D)Examples of spermatids from a sterile transgenic male. A variety of spermatid shapes were observed, but there was no consistent representativeshape. Instead, all spermatids appeared to have misshapen nuclei, although the acrosomes and tails appeared normal. (x 18,000.)

penetrance of the mutation varied as a function of geneticbackground (see below). Thus, we attribute the occurrence ofLvs to the transgene itself rather than to a coincidentalmutation at another locus. In principle, the mutant phenotypecould be due to either ectopic expression ofHCK or disrup-tion of an intrinsic gene by insertion of the transgene. Sincewe could detect no expression of the transgene, we con-cluded that Lvs represents an insertional mutation.

Genetic Dominance ofLvs. Insertional mutations caused bytransgenes typically result in a loss of function and, thus,become apparent only when bred to homozygosity (3, 5).Why then is Lvs genetically dominant? A variety of expla-nations are possible. First, the transgene might be located onthe X chromosome and create a complete deficiency for thedisrupted gene in males. This is demonstrably not the case forLvs (see below). Second, Lvs may indeed represent a het-erozygous loss of function, leading to a haploinsufficiency ofa gene product. Haploinsufficiency has not been a commonlyencountered genetic lesion in mammals, but it is well de-scribed in invertebrates and microbes. Third, insertion of thetransgene might truncate or otherwise alter the product of agene, resulting in a protein with an anomalous function. Thiscould be a gain of function mutation or an alteration of thegene product such that it results in transdominant inactiva-tion of the normal counterpart, a "dominant negative" effect(16). Fourth, the presence of the transgene might activate agene that is not normally expressed in testicular tissue,although the transcriptional controls used in the transgenethat engendered Lvs are not expected to be active in testes(immunoglobulin enhancer and SV40 promoter; see refs. 17and 18), and the transgene itself is not detectably expressedin testes or other tissues. Fifth, insertion of the transgenemight inactivate one allele at a locus that does not express theother allele due to genomic imprinting, resulting in a mutation

that is effectively null. The correct answer will probably beknown only when the mutation responsible for Lvs has beencharacterized molecularly.

Variable Penetrance of Lvs. In the initial breedings thatrevealed Lvs, the phenotype proved to be only partiallypenetrant. Some males in the lineage were sterile, somedisplayed only very limited fertility, and some were fullyfertile. One possible explanation for the variation was that itwas due to the influence ofgenetic background. Backcrossingonto different inbred strains confirmed this explanation andLvs became fully penetrant on particular genetic back-grounds within one to three generations (see Table 1). Thus,expression of the Lvs phenotype is apparently subject toepistatic modification and the rapidity with which full pene-trance can be established suggests that a single gene may beresponsible. Since we do not yet understand the nature of themutation responsible for Lvs (see above), it is impossible toanticipate the mechanism of modification. Methylation ofDNA and genomic imprinting are obvious possibilities,should the impact of the Lvs mutation rely upon expressionof the mutant locus (see above).

Cloning the Disrupted Locus ofLvs. The transgene provideda molecular probe for the cloning of adjoining DNA. Ensuinganalysis of the disrupted locus revealed a rearrangement ordeletion, the nature of which remains undetermined. Rear-rangements and deletions at the site oftransgene insertion area common occurrence (5). Since a deletion could remove allor part of the affected gene, the cloned DNA adjacent to theHCK transgene may not represent the disrupted gene itself.Efforts to detect a mRNA with the junction probes have sofar failed.

Analysis of Southern blots revealed that both the normaland disrupted versions of the Lvs locus are present in sterilemales. Thus, the locus is not situated on the X chromosome.

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Proc. Natl. Acad. Sci. USA 88 (1991) 10331

ARI-6.5kb -R Fl Fl l R6.0 kb F

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Hinfl probe Xba probe

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FIG. 4. Identification of junction fragments containing DNAflanking the transgenic insertion site. (A) Map of transgenic insertionsite containing -12 copies ofthe transgene (open boxes). Solid boxesrepresent adjacent mouse cellular DNA. RI, EcoRI restriction site.(B and C) Restriction fragments designated as Hinfi probe and Xbaprobe represent the regions of mouse DNA used as probes onSouthern blots to detect EcoRI polymorphisms in transgenic mouseDNA. Also note the same pattern ofrestriction fragments in male andfemale transgenic DNA, indicating that the transgene (tg) is not onthe X chromosome.

In work to be reported elsewhere, we have mapped the locusto the proximal region of mouse chromosome 9 (J.M., N.Jenkins, N. Copeland, and J.M.B., unpublished data).Lvs and Spermatogenesis. Spermatogenesis occurs in an

ordered and synchronous manner within the seminiferoustubules of the testes (6-8). The sequence of events appearsnormal in Lvs until a relatively late stage marked by nuclearcondensation, which takes place but results in a variety ofatypical products. We hypothesize that the lesion responsiblefor Lvs arrests the maturation ofsperm at the point of nuclearcondensation, causing the sperm to be unable to developfurther. Thus, the sterility that defines Lvs apparently resultsfrom a failure of sterile males to produce mature sperm. Theability to fertilize in vitro cannot be directly tested becausespermatozoa cannot be recovered from the epididymis ofLvsmales.The defect associated with Lvs appears to affect only the

germ cells. No abnormalities of other tissues or cell typeswere found in sterile males; the mice appeared healthy at allstages of life and survived to the expected age. The Lvsphenotype is apparent in developing spermatids. It remainspossible, however, that the physiological abnormality re-

sponsible for the phenotype is not intrinsic to spermatids butresides instead in surrounding somatic tissue, such as theLeydig or Sertoli cells.

Little is known about the molecular basis of geneticallydetermined male sterility (7, 19). There is one previousexample of male sterility produced by insertion of the trans-gene (20). In contrast to Lvs, however, the lesion wasgenetically recessive and affected an earlier stage in germ celldevelopment. Further study of Lvs may illuminate a previ-ously undescribed and important function in spermatogene-sis.

We thank Nancy Quintrell for the human HCK cDNA, RudiGrosschedl for the immunoglobulin enhancer, Cheryl Pedula andPaul Goldsmith for histology, Ivy Jacques and Dan Friend forelectron microscopy, and Kiran Chada and Richard Lang for reviewof the manuscript. We would also like to thank Bob Paulson for helpwith naming the mutation. The work reported here was supported bya grant from the National Institutes ofHealth (CA 44338), funds fromthe G. W. Hooper Foundation, and fellowships to J.M. from theAnna Fuller Fund and the American Cancer Society.

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