Analysis of the hotfoot (ho) locus by creation of an insertional mutation in a transgenic mouse

DEVELOPMENTAL

BIOLOGY

137,849-358

(1990)

Analysis of the Hotfoot (ho) Locus by Creation of an Insertional Mutation in a Transgenic Mouse JON W. GoRDoN,*~~~~ JOAN UEHLINGER,§ NOOSHINE DAYANI,* BETH E. TALANSKY,$ MILDRED GORDON,~~GREG S. RUDOMEN/ AND PAUL E. NEUMANN# *Brookdale Center for Molecular Biology, TDepartment of Geriatrics and Adult Development, *Department of Obstetrics, Gynecology, and Reproductive Science, and ODepatiment of Medicine, Mt. Sinai School of Medicine, New York, New York 10029;flDepartment of Anatomical Sciences, CUNY Medical School, New York, New York 10031;and #Departments of Neurology, Children’s Hospital and Harvard Medical School, Massachusetts 02115 Accepted September 22, 1989 Hotfoot (ho) mutation is a recessive trait in mice, characterized by motor disorder and male sterility, that maps to chromosome 6. We have identified a transgenic mouse pedigree with a similar trait. Using genetic and molecular approaches, we have demonstrated that the foreign DNA element is located in or near the ho locus. This new allele, designated hdwOand presumably created by insertional mutagenesis, should make it possible to clone the ho gene. Male infertility in hdug male homozygotes was determined to be due to inability of sperm to penetrate the zona pellucida. This was demonstrated by rescuing mutant males by a new technique of gamete micromanipulation, zona pellucida drilling. These findings show that zona drilling is useful both for analysis and preservation of animals with reduced IKtk fertility. 0 1990 Academic Press, Inc. INTRODUCTION

The transfer of foreign DNA into the mouse germ line by retroviral infection (Jaenisch, 1976) or microinjection (Gordon et al., 1980) creates the potential for insertional disruption of host genetic loci by integration of foreign genetic material. These insertional mutations are particularly useful, because tagging of the host locus with a defined DNA element allows for cloning of the disrupted gene by use of probes homologous to the exogenous sequence. The best-studied example of insertional mutagenesis resulting from retrovirus-mediated gene transfer involved a mutation sustained at the al(I) collagen gene. Cloning of the interrupted gene allowed its identification and the determination that proviral DNA integration altered the pattern of DNAseI-hypersensitive sites around the collagen gene and blocked initiation of transcription (Jaenisch et al., 1983; Schnieke et al., 1983; Breindl et al., 1984; Hartung et al., 1986). Several insertional mutations affecting complex programs of cell differentiation and organogenesis have also been produced by microinjection. Mutations involving postmeiotic sperm differentiation (Palmiter et ah, 1983), limb deformity (Woychik et aL, 1985, Overbeek et al, 1986; McNeish et al., 1988), dystonia muscularis (Kothary et al., 1988), and Purkinje cell degeneration i To whom correspondence should be addressed at Box 1175, Mt. Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029.

(Kruwlewski et al., 1989) have been reported. These findings demonstrate that the most intricate developmental mechanisms may be rendered accessible to molecular analysis through the creation of insertional mutations. An intriguing group of mouse mutations which has thus far resisted detailed scrutiny consists of several loci which affect both neurologic development and spermatogenesis. Within this group are quaking (qk) which maps to chromosome 17 and causes reduced myelination and degeneration of spermatids (Sidman et al., 1964; Bennett et al, 1971); Purkinje cell degeneration (pcd), located on chromosome 13, which results in loss of Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptors, and is associated with sperm immotility (Mullen and LaVail, 1975; Mullen et al, 1976; Southard and Either, 1977); and hotfoot (ho), situated on chromosome 6, which induces an ill-defined motor disorder and male infertility (Dickie, 1966). In some of the pcd and ho alleles, the male-sterile phenotype is incompletely penetrant. In fact, the current existing allele of ho (ho4”> is fertile, which suggests that the role of this locus in male reproductive function may never be elucidated from analysis of this allele. We have identified a transgenic mouse line with an insertional mutation allelic with ho. In the new allele created by transgene insertion, hoJwQ, the male-sterile phenotype is fully penetrant. In this communication evidence is presented that hoJwQ is an insertional mutation. In addition, a new technique of gamete microma-

349

0012-1606190 $3.00 Copyright All rights

Q 1990 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

nipulation, zona pellucida drilling (Gordon and Talansky, 1986), was used to restore fertility to sterile ho males. Results are presented relevant to the potential of zona drilling for analysis of male infertility and enhancing the reproductive efficiency of valuable animal stocks.

VOLUME 137,199O

Library

Construction

and Screening

DNA was extracted from mutant mouse spleens, partially digested with MboI, size selected on agarose to isolate fragments of lo-20 kb and ligated to DNA of the bacteriophage EMBLS (Frischauf et al., 1983) which had been prepared by double digestion with BamHI and EcoRI. After in vitro packaging (Ish-Horowitz and MATERIALS AND METHODS Burke, 1981) and plating, 3.3 X lo5 colonies were DNA Extraction and Hybridization Analysis screened (Benton and Davis, 1977) with pFR400. DNA was extracted (Blin and Stafford, 1976) from Twenty-three clones were picked and rescreened with spleen. For Southern blots, 15 pg of DNA was digested pBR322, and 14 confirmed positives were isolated. Bewith the appropriate restriction enzyme (New England cause the founder transgenic animal carried 5-10 copies Biolabs), electrophoresed in 1% agarose, transferred to per cell of pFR400 (Gordon, 1986), a rapid test was denitrocellulose (Southern, 19’75), and hybridized to the vised to distinguish and exclude from analysis bacterioappropriate probe by the method of Wahl et al. (1979). phage carrying pFR400 only. The strategy relied upon Probes were labeled by random priming according to the fact that neither pFR400 nor EMBL3 (after prepaspecifications of a kit obtained from International Bio- ration for cloning) contains sites for the restriction entechnologies (“Prime Time”). Radiolabeled bacterio- zyme EcoRI (Simonsen and Levinson, 1983; Frischauf et phage h DNA (0.5 rig/ml) was also added to illuminate al, 1983). Bacteriophage minipreparations (Maniatis et al., molecular weight markers in Fig. 1A. For dot blot hybridizations, 3 pg of DNA was transferred to nitrocel- 1982) were evaluated for the presence of EcoRI sites, as lulose using a dot blotting device obtained from Be- recombinant phage with one or more EcoRI sites could only have acquired such sites from mouse DNA. Two thesda Research Laboratories.

FIG. 1. (A) MspI digested DNA from seven mutant animals (a-g), Materials and Methods). M, Molecular weight markers; pl, bands homologous to plasmid material. Note that all mutant animals have mutant (M), a hemizygous (H), and a normal (N) mouse hybridized mobility of Hind111 digested X DNA.

an obligate hemizygote (h), and a normal control (i) hybridized to 2.63 (see derived from plasmid material. Lower panel, longer exposure of bands more plasmid DNA than the carrier animal. (B) X&I digested DNA from a to the subcloned BP1 insert. Fragment sizes were calculated by measuring

GORDON ET AL.

Analysis

such clones were selected for further analysis. Recombinant bacteriophage sites were digested with a variety of restriction enzymes and tested for absence of hybridization to pFR400 and mouse repeated elements. Digestion of one bacteriophage, designated 2.6, with Sal1 produced one such 5-kb fragment, which was subcloned into the plasmid pGEM3 (Promega). The recombinant plasmid was called 2.63, and the mouse insert, S5. Restrictionmapping of bacteriophage 2.6 revealed the presence of numerous restriction-enzyme sites including MsipI between S5 and the pFR400 component of the phage. Because the presence of these sites made detection of restriction-fragment-length variants associated with pFR400 insertion difficult, S5 fragment instead was used as a reference hybridization probe when the relative amounts of pFR400 material were measured in mutant and carrier transgenic mice. A second 1-kb BamHI-PvuII fragment was subcloned from another phage, 2.1. This fragment, BPl, was closely approximated to the transgene component of the recombinant phage and was used to search for restriction-map alterations created by transgene insertion. For these studies BP1 was electroeluted from low-melt agarose and used directly for radiolabeling.

of the Hotfoot

351

(ho) Locus

1982). Motile cells were counted using a hemocytometer. Oocytes were inseminated at a final concentration of lo6 motile sperm/ml. After 6-8 hr of incubation, eggs were scored for fertilization with phase-contrast microscopy. Oocytes with two pronuclei and a second polar body were considered fertilized. When zona drilling was performed, healthy-appearing eggs with a visible first polar body were placed in a micromanipulation device equipped with two Leitz micromanipulators and fixed in position with a holding pipet. A microneedle loaded with acid Tyrode’s solution, pH 2.3, was placed tangentially against the zona pellucida, and the acid solution was expelled until localized dissolution of the zona was observed (Gordon and Talansky, 1986). After being drilled, oocytes were thoroughly washed, loaded into 30-ml microdrops for insemination, and inseminated in the same manner as the standard in vitro fertilizations. RESULTS

IdentiAcation and Characterization Mutation at the ho Locus

of an Insertional

The mutation was identified in 1 of 17 transgenic mouse lines established by microinjection of pFR400, a Sperm were expressed from caudae epididymides and 4.4-kb recombinant plasmid carrying an altered dihyvasa deferentia into 2 ml capacitation medium (Tha- drofolate reductase (dhfr) gene (Simonsen and Levindani, 1982) and incubated for 45 min to 1 hr. Cells were son, 1983). All 1’7 lines were screened for recessive incollected and gently pelleted. After being washed in sertional mutagenesis by inbreeding. In one line, ~447, phosphate-buffered saline (PBS) and repelleted, sperm crosses of transgenic hemizygotes resulted in the apwere fixed in 1.5% glutaraldehyde in sodium cacodylate pearance of offspring with a characteristic motor disbuffer, pH 7.2, for 1 hr. Sperm were then washed in order. Animals appeared normal until 12-15 days after cacodylate buffer containing 0.4 M sucrose, refixed in birth, when they developed a markedly ataxic gait. By 2% osmium tetroxide for 1 hr, dehydrated in ethanol, 25 days of age, mutants developed alternating stereoand embedded in Spurr resin. Thin sections were exam- typic, jerky flexions of the limbs, most pronounced in ined in a Phillips 300 electron microscope with an accel- the hind limbs, during locomotion. Histologic sections of brains, spinal cords, sciatic nerves, and dorsal root erating voltage of 60 or 80 kV. ganglia of these animals revealed no abnormalities. Affected males were sterile, as evidenced by the failure of In Vitro Fertilization and Zona Drilling normal females to become pregnant after several docuB6D2F1 females were superovulated with pregnant mented matings. To evaluate male sterility further, spermatozoa from mutant males were used for in vitro mares’ serum and human chorionic gonadotrophin (hCG) as previously described (Gordon and Talansky, fertilization. Results (Table 1) showed a marked reduc1986). The morning after hCG administration, females tion in the fertilization rate by mutant sperm relative were sacrificed and their oocytes were removed to in- to those of control males. The penetrance of the sterile semination medium (Thadani, 1982) containing 0.1% phenotype varied between males, such that some anihyaluronidase to remove cumulus cells. After thorough mals appeared fertile, while one was azoospermic washing, healthy eggs were loaded into 30-~1 micro- (Table 1). With the exception of the azoospermic anidrops of insemination medium under mineral oil (Mal- mal, sperm counts and the percentage of motile sperm linckrodt No. 6358). Sperm either from control B6D2F1 in mutant animals did not differ significantly from or transgenic males were expressed from caudae epidid- controls. These results were interpreted to indicate that ymides and vasa deferentia and capacitated by 45-60 while many animals are potentially fertile, the combination of reduced sperm fertilizing efficiency and neumin of incubation in capacitation medium (Thadani, Electron Microscopy

352

DEVELOPMENTALBIOLOGY TABLE

1

In Vitro FERTILIZATIONOF ZONA-INTACTOOCYTESBY ~44’7MUTANT SPERMAND NORMAL CONTROLS

Expt. no.

Mutant fert/insem

%

Wild-type fert/insem

%

25/40 14/18 21/40 13/20 27/40 31/40 19/19 -

63 78 53 65 68 78 100 -

150121’7

69

1

o/50

2 3 4 5 6 7 8”

11/13 o/43 o/20 5/43 l/41 5/23 -

0 85 0 0 12 2 22 -

Totals

22/223

9

Note. Fertilization rates are expressed as the ratio of eggs fertilized (fert) divided by the number inseminated (insem). a Mutant male was azoospermic, and fertilization test was aborted.

rologic impairment renders most males functionally sterile. Females, though subfertile, were able to produce litters. Progeny testing suggested that these neurologic and reproductive disorders resulted from recessive insertional mutagenesis. Breeding of two hemizygous transgenie mice resulted in the appearance of these anomalies in about 25% of progeny (Table 2). All mutant mice were transgenic. Offspring of mutant females mated to carrier males were affected approximately 50% of the time. Further, all of the progeny of affected females were transgenic, suggesting that mothers were homozygous for the foreign gene insert. No abnormal animals were born when transgenic mice were crossed to normal mice (Table 2). Breeding data suggesting the presence of a recessive insertional mutation were supported by Southern hybridization experiments which measured the amount of plasmid in mutant and carrier transgenic mice. Results of these transgene dosage studies are shown in Fig. 1A. When digested with Map1 and hybridized to the plasmid 2.63 (see Materials and Methods), a 5.2-kb band appeared both in normal and transgenic mouse DNA. This represented material homologous to the S5 genomic insert of 2.63. Two additional low molecular weight bands consisting of many small MspI fragments of pFR400, and homologous to the plasmid component of 2.63, were present only in transgenic samples. Thus, when transgenie mouse DNA was digested with M.spI and hybridized to 2.63, the intensity of low molecular weight fragments reflected the amount of pFR400 present, while the intensity of the 5.2-kb band was used as an indicator of the quantity of DNA assayed. Utilizing this strategy, the amounts of pFR400 in seven mutant animals derived from three separate

VOLUME137,199O

crosses were compared with that found in an obligate hemizygous transgenic mouse (Fig. lA, lower panel). In this figure, lanes a-g are DNAs from mutant animals; lane h contains DNA from the hemizygous mouse, and lane i is a normal control. In several lanes the quantities of DNA assayed were comparable between mutants and the carrier, and in one sample (lane c) less mutant DNA was used. Yet in all cases, the intensity of plasmid-speciiic bands in mutant animals was greater than that in the carrier mouse (Fig. lA, lower panel). Results thus supported progeny tests indicating that the mutant animals were homozygous for the transgene insert. To complement these experiments, BP1 was purified from agarose gels and used to probe transgenic animals for restriction fragment variants. Restriction patterns diagnostic of the transgene insertion were detected after digestion with XbaI, KpnI, PstI, and SacI. Figure 1B shows the results of restriction analysis using XbaI. As shown, the mutant animal is homozygous for a 4.0kb band homologous to BPl, while a normal animal has a single 7.1-kb band. Hemizygous transgenic mice show both bands, each with a relatively reduced hybridization intensity, as expected for carriers of one mutant and one wild-type allele. These results further strengthen the conclusion that mutant animals are homozygous for the foreign DNA insert. The Insertional with ho

Mutation

Shows Noncomplementation

These data did not formally rule out the possibility that expression of the exogenous dhfr gene rather than insertional mutagenesis was responsible for the abnormal phenotype. We had previously observed developmental anomalies in other transgenic lines carrying pFR400 (Gordon, 1986), though in no case did the defects resemble those seen in this line, and in all instances the anomalies were observed in transgenic carriers. Nonetheless, additive expression in homozygous mice of this line could, because of a pattern of transgene expression unique to this line, cause the observed disorders. TABLE

2

PROGENYTESTING OF TRANSGENIC MICE OF THE ~447 LINEAGE Cross F M W) x (HI W) X VU CM) x W

No.

No.

No.

No.

born

positive

abnormal

crosses

45 143 85

21 102 85

0 33 39

5 17 13

Note. M indicates a male parent, F a female parent. H, hemizygous transgenic mouse; W, wild-type mouse; M, mutant mouse.

GORDONET AL.

Andysis

This possibility was addressed by testing of the ~44’7 insertion for allelism with spontaneous neurologic mutations known to affect spermatogenesis. Transgenic mutants were noted to have a motor defect similar to that of mice homozygous for the spontaneous mutation ho, and the originally discovered alleles of ho were reported to cause male sterility (Dickie, 1966; Green, 1981). When ~447 hemizygotes were crossed with nontransgenic how/how animals, two of four offspring appeared with an abnormal neurologic phenotype indistinguishable from either ~447 or ho” homozygotes. In subsequent linkage tests (see below), allelism of the mutation associated with the transgene insert and hoW was confirmed with a large number of progeny. The finding that the ~44’7 insertion and ho fail to complement each other constitutes compelling evidence that disruption of the ho locus, not expression of pFR400, is the cause of the abnormal phenotype in ~447 homozygotes. hoJwg Maps to Mouse Chromosome 6

of th,e Hotfoot

353

(ho) Locus

TABLE 3 RESULTSFROMCROSSINGMP'~+/+ hdw to + hoU/+ ho” Mice Southern blot Phenotype No. pwe*y

Miwh

ho

21

+

ho

28

Miwh

+

+

+

9

Predicted genotype + hdwg ___ + h&’ MPh +

+ ho4J + + +

ho4J

Southern result expected Positive

No. positive/ No. tested ll/ll

Negative

O/10

Negative

O/8

Note. The “+” sign indicates the presence of the wild-type allele at each locus. Predicted genotypes are those expected from the phenotypit appearance of the animals.

zygotes. The 9 remaining animals carried neither ho nor Miwh and are presumed to result from crossing-over between the two loci. The failure to identify the other product of crossing-over-a carrier of both Miwh and the transgene-raised the possibility that chromosomal rearrangements associated with foreign gene integration might cause lethality when present in association with Miwh on the same chromosome. However, recent phenotypic analysis of 48 additional progeny has revealed at least one double mutant (data not shown). The observed recombination frequency between the neurologic trait and Miwh was 15.5 + 4.8% (9/58 progeny, Table 3), approximately equivalent to the 20-centimorgan map distance between the mi and the ho loci. Therefore, the neurologic trait associated with the pFR400 insertion site has been accepted as a new ho allele and has been given the designation hoJwg. The mouse strain has been named Jwg:p447. Further, the pFR400 insert followed precisely the same pattern of segregation as the new mutant allele: all 11 animals with the motor disorder characteristic of ho that had been tested by dot blot hybridization were also transgenic animals, while the 18 behaviorally normal animals were not transgenics (Table 3). No recombination has been observed between the plasmid insertion and the neurologic trait. The upper 95% confidence limit of the recombination frequency between the neurologic trait and the transgene is 5%, when calculated from data in Tables 2 and 3. The results thus establish that the foreign gene insert is tightly linked to hoJwg, is linked to Miwh, and therefore maps to chromosome 6.

Previous analyses of regions flanking transgenes inserted by microinjection have revealed a variety of host DNA rearrangements (Covarrubias et al., 1986, 1987; Palmiter and Brinster, 1986; Overbeek et al., 1986; Mahon et ab, 1988). In some instances the DNA linked to the donor material is not located at the site of foreign gene insertion (Covarrubias et al., 1987). These findings raised the possibility that the neurologic trait might be due to DNA rearrangements involving the ho locus, but that the transgene might not be located on chromosome 6. To address this issue, genetic studies were undertaken to map the chromosomal position of pFR400 and the neurologic trait in this line. White (Miwh) is a semidominant coat color mutation which maps to chromosome 6, approximately 20 centimorgans from the ho locus (Bunker and Snell, 1948; Miller et ab, 1971; Southard, 1981; Davisson et ab, 1988). To test for linkage of the transgene to chromosome 6, ~447 transgenic homozygotes were crossed to carriers of Miwh. Progeny carrying Miwh were then crossed to ho@ homozygotes. All 58 progeny of this triple backcross were scored for Miwh and the neurologic trait, and half were tested for inheritence of the transgene by Southern blotting. Transmission of Miwh was monitored by the appearance of a characteristic coat color dilution in progeny. Results of these studies, shown in Table 3, demonstrate nonrandom segregation of the neurologic trait and the transgene from Miwh. Of the 58 progeny born, 21 Evaluation and Rescue of the Male-Sterile Phenotype exhibited the motor abnormality characteristic of ho, Sterility in ho Jwghomozygous males provided the opand in no case did these animals also carry Miwh. Of 28 progeny carrying Miwh, none appeared to be ho homo- portunity to evaluate the role of the ho gene in male

354

GORDON ET AL.

Analysis

reproduction. Homozygous males were found to be capable, despite their motor disorder, of mating with females. When used for in vitro fertilization, sperm from hoJw/hoJwg were found (with a single exception) to be present in normal numbers, but deficient in egg penetration (Table 1). In addition, sperm appeared to move abnormally, when examined by phase-contrast microscopy, with increased lateral head amplitude and decreased forward progression. Sperm from sterile hoJwg/hoJwg males were then evaluated ultrastructurally. Figure 2 shows transmission electron micrographs of sperm from one such male. Two obvious irregularities were seen. First, many cells exhibited a bizarre reduplication of the flagellar structure (Fig. 2A). In the example shown, 11 sections of flagella are enclosed within a single plasma membrane. Additional microtubules are scattered outside the flagella (Fig. 2A). Another frequently observed abnormality was juxtaposition of distal portions of flagella to the head (Fig. 2B). Variable numbers of normal-appearing spermatozoa were seen in all transgenic samples. To study further the basis of the sterile phenotype, a new micromanipulation technique, zona drilling (Gordon and Talansky, 1986), was employed. Zona drilling entails making a hole in the zona pellucida to allow sperm direct access to the oolemma. Fertilization in the mouse is normally the culmination of an orderly series of interactions between sperm and oocyte. The ability of ho mutant sperm to fertilize only after zona drilling would indicate that the normal sequence of interactive events could not be carried out, but that circumvention of these normal prerequisites for sperm penetration could lead to gamete fusion. To assess this possibility, sperm were recovered from hoJwg homozygous males and tested for their ability to fertilize in vitro with and without zona drilling. Table 4 summarizes data from seven such tests. After creating a gap in the zona, 71% of eggs were fertilized, whereas insemination of zonaintact oocytes by sperm from mutant males in previous experiments gave an overall fertilization rate of 9% (Table 1). Thus, ho affects the ability of spermatozoa to penetrate the oocyte investments, but not to fertilize once the egg is reached. To test the developmental potential of zona-drilled oocytes fertilized by these defective spermatozoa, zygotes were implanted into pseudopregnant females. Impaired development would be expected to manifest as a reduced birth rate and/or the appearance of pheno-

of the Hotfoot

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(ho) Locus

typically abnormal offspring. Table 4 shows that 35% of embryos implanted gave rise to normal young, a percentage not statistically different from previous experiments (Gordon and Talansky, 1986) in which eggs subjected to zona drilling and fertilized by normal sperm were transferred. The gross appearance of all newborn animals was normal. All 29 progeny have been raised to adulthood, and 15 have been crossed to either wild-type mice or each other. All animals are fully fertile. Nine of the animals are now more than a year old. Because sterility originally arose from a recessive mutation caused by foreign gene insertion, we were able to confirm by Southern blotting that sperm from sterile animals gave rise to normal young. Since homozygous transgenics were those previously found to be infertile, all offspring of mutants rescued by zona drilling would be expected to be transgenics. All 29 progeny were subsequently found to be transgenic by Southern or dot blot analysis (Table 4). Results from 12 of the offspring tested by dot hybridization are shown in Fig. 3. DISCUSSION

The data presented demonstrate that microinjection of a plasmid into mouse embryos has resulted in a recessive mutation which causes a motor disorder and male infertility. The mutation, which is probably insertional in origin, exhibits allelism with the previously recognized mutation hog which maps to mouse chromosome 6. Independent linkage studies localize the plasmid DNA in these transgenic animals to the same chromosome. The genetic distance of the transgene from another marker on chromosome 6, Miwh, is the same as that of ho< Males homozygous for this new allele hoJW have severely reduced fertility, a characteristfc preiiously seen in several of the spontaneous ho mutant alleles. The present studies demonstrate that infertility in ho animals is due to a disordered sperm morphology associated with failure to penetrate the zona pellucida. Zona pellucida drilling leads to fertilization by mutant sperm and development of normal offspring. An interesting aspect of these findings is that another of the original 1’7 lines produced by microinjection of pFR400 (Gordon, 1986) also proved to carry an insertional mutation causing neurologic impairment and male infertility (Krulewski et aZ., 1989). This mutation, in line 432, was allelic with Purkinje cell degeneration, a

FIG. 2. Transmission electron micrographs of sperm from sterile p447 homozygous males. (A) Eleven cross sections through the principal piece of the flagellum are enclosed within a single plasma membrane. Microtubules are located outside the flagella (arrows). X64,000. (B) An acrosome-reacted spermatozoon from a sterile transgenic male. The head appears normal; however, a distal portion of the flagellum is located immediately behind the head (arrow). X57,000.

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DEVELOPMENTALBIOLOGY

TABLE 4 DEVELOPMENTOF EMBRYOSFERTILIZEDBY ~447 MUTANT SPERM AFTER ZONADRILLING AND in vitro FERTILIZATION

VOLUME 137.1990

intact zona. In addition, the phenotypic variability seen between males, including one animal that lacked sperm entirely (Table l), is consistent with the notion that severe cases of disruption of spermiogenesis lead to No. transg/ No. born/ No. fert/ azoospermia. The variability seen between animals is No. transf No. born No. insem Expt. no. (%) (WI presently attributed to effects of modifying alleles 17 o/o 11’6 1 which, because the transgenic mice were produced on an 100 o/o 6/6 2 outbred background (Gordon, 1986), segregate in combi6/6 48 6/20 30 20142 3 nations which variably augment the effect of ho on 3/3 100 3/3 100 313 4 37 O/7 0 7/19 5 spermatogenesis. It is unclear why the infertile pheno6/6 so 6/15 40 36/40 6 type is relatively variable and the motor disorder 95 14/38 37 14114 38/40 7 stable, both within the ~447 line and between indepen29/29 71 29183 35 Totals 1111156 dently discovered ho alleles. Many of the remaining questions regarding the acNote. For each experiment, the number of eggs fertilized of those tion of the ho gene should be answered when portions of inseminated (No. fert/No. insem) is shown, and the percentage calculated. The number and percentage of animals born of the embryos the coding sequence are cloned from hoJwg animals. For example, it is unclear why some alleles affect male fertransferred (No. born/No. transf) is also indicated. Hyphens indicate that embryos were not implanted because foster females were not tility while others do not. It is possible that two closely available. The number of transgenic animals among those born (No. linked genes are involved in generating the phenotype transg/No. born) also contains hyphens where no animals were born of ho, one of which affects motor function and the other for analysis. spermatogenesis. Efforts have been initiated to recover coding sequences from the region flanking pFR400 in these animals. Preliminary studies with the BP1 mouse long-recognized recessive mutation that maps to chro- fragment indicate that mRNA homologous to this mamosome 13. Our best explanation is that these two un- terial is present in brain and testis, but not in liver and linked insertional mutations that cause similar pheno- heart (data not shown). Experiments are currently in typic abnormalities occurred by chance. The mutations progress to determine if hybridization is altered in hoW arose in separate lines and mapped to different chro- or hoJwg homozygotes. While DNA rearrangements mosomes. When the lines were interbred to produce known to be associated with microinjection of genes can compound hemizygous transgenic animals, no abnor- complicate cloning, and have not been ruled out in this malities appeared. line, the demonstration that the transgene is present at Our inability to identify histologic changes in the the ho locus indicates that cloning of the ho gene is brains of ~44’7mutants is consistent with allelism to ho. feasible. Because mutations of several genes affect both Previous searches for histopathology in the ho/ho ner- neurologic tissues and male reproductive development, vous system have likewise been negative. However, the it is possible that these genes are members of an imcreation of the hoJwg allele has provided information portant family whose members share sequence homolregarding the role of this locus in male reproductive ogy. If so, cloning of one gene may make possible the development. Mutant alleles causing male sterility do isolation of some or all of the others. not interrupt meiosis, nor do they block fertilization The production of these mutant animals has allowed and development of embryos after the spermatozoa gain access to the oocyte surface. However, mutant sperm exhibit gross morphological abnormalities (Fig. 2) and are unable to penetrate zona-intact eggs. These results indicate that spermatozoa of hoJwg/hoJwQ males are specifically deficient in their inability to penetrate investments surrounding the oocyte. Whether this deficiency relates to abnormal motility or to the absence of a surface molecule(s) required for interaction with the zona is not yet known. A reasonable hypothesis from the disordered flagellar morphology is that spermiogenesis is disrupted and that the morphologically abnormal sperm lack motility characteristics required FIG. 3. Dot blot hybridization analysis of offspring from mutant for penetration. This explanation is favored by the find- transgenic males. Al-6, Bl-6, offspring born after zona drilling; B7, ing that sperm are rarely capable of penetrating the normal mouse DNA.

GORDONET AL.

Analysis

us to evaluate the efficacy of zona drilling in compensating for disorders of spermatogenesis which affect motility and morphology. We find that zona drilling is capable of rescuing sterile animals whose sperm are ordinarily unable to fertilize eggs and that the offspring produced are phenotypically normal. In addition to providing important clues as to the effect of the ho mutation on spermatogenesis, the data indicate that zona drilling may have wide applicability in the management of reproductive problems in animals with valuable phenotypic traits and in humans as well. In instances where transgenic animals or valuable livestock are unable to reproduce, zona drilling could be used in conjunction with in vitro fertilization to enhance the reproductive capacities of the animals. In cases of severe male infertility in humans, we have already demonstrated a marked improvement in the fertilization rate of oocytes exposed to abnormal sperm after zona drilling (Gordon et aZ.,1988), and Malter and Cohen (1989) have established three pregnancies after zona drilling. Thus, this new micromanipulative procedure has use not only for delineating the cause of infertility, but also for overcoming at least some reproductive disorders. We acknowledge Drs. Philip Leder and Lydia Villa-Komaroff for their critical review of this manuscript. This work was supported by NIH Grants CA42103 and HD20484 and March of Dimes Grant l-1026 to J.W.G. and by an NIH Grant NS20820 to Dr. Richard Sidman. This is manuscript No. 16 of the Brookdale Center for Molecular Biology, Mt. Sinai Medical Center. REFERENCES BENTON,W. D., and DAVIS, R. W. (1977). Screening Xgt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182. BLIN, N., and STAFFORD,D. E. (1976). A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3,2302-2308. BREINDL, M., HARBERS, K., and JAENISCH, R. (1984). Retrovirus-induced lethal mutation in collagen I gene of mice is associated with an altered chromatin structure. CeU38,9-16. BUNKER, H., and SNELL, G. D. (1948). Linkage of white and waived-l. J. Hered

39.28.

CATTANACH, B. M., and MOSLEY, H. J. (1974). Mouse News L&t. 50, 41-42. COVARRUBIAS,L., NISHIDA, Y., and MINTZ, B. (1986). Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain. Proc. Natl. Acad Sci. USA 83,6020-6024. COVARRUBIAS,L., NISHIDA, Y., TERAO, M., D’EUSTACHIO, P., and MINTZ, B. (1987). Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryo. Mol. Cell Biol. 7,2243-2247. DAVISSON,M. T., RODERICK,T. H., HILLYARD, A. L., and DOOLITIZE, D. P. (1988). Mouse News Lett. 81,12-19. DICKIE, M. M. (1966). Mouse News L&t. 34,30.

of thx Hotfoot

(ho) Locus

357

FRISCHAUF, A.-M., LEHRACH, H., POUSTKA, A., and MURRAY, N. (1983). Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170,82?-842. GORDON,J. W. (1986). A foreign dihydrofolate reductase gene in transgenic mice acts as a dominant mutation. Mol. Cell. Biol. 6, 2158-2167. GORDON,J. W., GRUNFELD,L., GARRISI, G. J., TALANSKY, B. E., RICHARDS,C., and LAUFER, N. (1988). Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil. Steril. 50,68-73.

GORDON,J. W., SCANGOS,G. A., PLOTKIN, D. J., BARBOSA,J. A., and RUDDLE,F. H. (1980). Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380-7384. GORDON,J. W., and TALANSKY, B. E. (1986). Assisted fertilization by zona drilling: A mouse model for correction of oligospermia. J. Exp. ZOOL 239,34?-354.

GREEN,M. C. (1981). Catalog of mutant genes and polymorphic loci. In “Genetic Variants and Strains of the Laboratory Mouse” (M. C. Green, Ed.), pp. 8-278. Gustav Fischer Verlag, Stuttgart. HARTUNG, S., JAENISCH, R., and BREINDL, M. (1986). Retrovirus insertion inactivates mouse al(I) collagen gene by blocking initiation of transcription. Nature (London) 320,365-367. ISH-HOROWICZ,D., and BURKE,J. F. (1981). Rapid and efficient cosmid cloning. Nucleic Acids Res. 9,2989-2998. JAENISCH,R. (1976). Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Nat1 Acad Sci. USA 73,1260-1264.

JAENISCH,R., HARBERS,K., SCHNIEKE,A., LOHLER,J., CHUMAKOV,I., JAHNER, D., GROTKOPP,D., and HOFFMAN,E. (1983). Germline integration of the Moloney murine leukemia virus at the Mov 13 locus leads to recessive lethal mutation and early embryonic death. Cell 32,209-216. KOTHARY, R., CLAPOFF,S., BROWN,A., CAMPBELL,R., PETERSON,A., and ROSSANT,J. (1988). A transgene containing ZacZ inserted into the dystonia locus is expressed in neural tube. Nature (London) 335, 435-437. KRUWLEWSKI,T. F., NEUMANN, P. E., and GORDON,J. W. (1989). Insertional mutation in a transgenic mouse allelic with Purkinje cell degeneration. Proc. Natl. Acad. Sci. USA, in press. MAHON, K. A., OVERBEEK,P. A., and WESTPHAL,H. (1988). Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation. Proc. Natl Acud S& USA 85,1165-1168. MALTER, H. E., and COHEN,J. (1989). Partial zona dissection of the human oocyte: A nontraumatic method using micromanipulation to assist zona pellucida penetration. Fe&l. Steril. 51,139-148. MANIATIS, T., FRITSCH, E. F., and SAMBROOK,J. (1982). “Molecular Cloning.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MCNEISH, J. D., SCOTT,W. J., JR., and POWER, S. S. (1988). Legless, a novel mutation found in PHT-1 transgenic mice. Science 241. 837-839. MILLER, 0. J., MILLER, D. A., KOURI, R. E., ALLDERDICE,P. W., DEV, V. G., GREWAL,M. S., and HUTTON,J. J. (1971). Identification of the mouse karyotype by quinacrine fluorescence, and tentative assignment of 7 linkage groups. Proc. Natl. Acad. Sci USA 68,1530-1534. MULLEN, R. J., EICHER, E. M., and SIDMAN, R. L. (1976). Purkinje cell degeneration, a new neurological mutation in the mouse. Proc. Natl. Acad Sci. USA 73,208-212.

MULLEN, R. J., and LAVAIL, M. M. (1975). Two new types of retinal degeneration in cerebellar mutant mice. Nature (London) 258, 528-530. OVERBEEK,P. A., LAI, S.-P., VAN QUILL, K. R., and WESTPHAL, H. (1986). Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences. Science 231,15?4-1577.

358

DEVELOPMENTALBIOLOGY

PALMITER, R. D., and BRINSTER,R. L. (1986). Germ-line transformation of mice. Annu. Rev. Genet. 20,465-499. PALMITER, R. D., WILKIE, T. M., CHEN, H. Y., and BRINSTER,R. L. (1983). Transmission distortion mosaicism in an unusual transgenic mouse pedigree. Cell 36,869~877. SCHNIEKE, A., HARBERS, K., and JAENISCH, R. (1983). Embryonic lethal mutation in mice induced by retrovirus insertion into the al(I) collagen gene. Nature (Lonclon) 304,315-320. SIDMAN, R. L., DICKIE, M. M., and APPEL, S. H. (1964). Mutant mice (quaking and jimpy) with deficient myelination in the central nervous system. SIMONSEN,C. C., and LEVINSON,A. D. (1983). Isolation and expression of an altered dihydrofolate reductase cDNA. Proc. Nat1 Acad. Sci. USA 80,2495-2499.

VOLUME13’7,199O

SOUTHARD,J. L. (1981). Mouse News Lett. 64,60. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. THADANI, V. M. (1982). Mice produced from eggs fertilized in vitro at a very low sperm:egg ratio. J. Exp. Zool 219,2’77-283. WAHL, G. M., STERN,M., and STARK,G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization using dextran sulfate. Proc. NatL Acad. Sci. USA 76,3683-3687.

WOYCHIK, R. P., STEWART,T. A., DAVIS, L. G., D’EUSTACHIO,P. D., and LEDER, P. (1985). An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature (London) 318, 36-40.