Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10327-10331, November 1991 Genetics 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 oftransgenic mouse genetic locus affected by Lvs will illuminate a previously lines carrying the HCK protooncogene, we encountered one line undescribed and important function in spermatogenesis. in which males henmzygous for the transgene were sterile. The sterile males mated normally but failed to impregnate females. MATERIALS AND METHODS Light and electron microscopy revealed that spermatogenesis proceeds normally until nuclear condensation, which occurs Production of Transgenic Mice. A DNA fragment contain- but gives rise to a variety of abnormally shaped nuclei. ing an immunoglobulin enhancer, simian virus 40 (SV40) Expression of the transgene was not detectable. Thus, the promoter, a hematopoietic cell kinase (HCK) cDNA (9, 10), insertion itself probably caused the abnormal phenotype by and a SV40 poly(A) addition site and splicing signals (referred disrupting a gene (or genes) important in spermatogenesis. The to as plsh and shown in Fig. 1A) was generated by digestion mutation is genetically dominant, causing an abnormal phe- ofits parent plasmid by EcoRI and isolation on an agarose gel notype even though the sterile mice carry an ostensibly normal followed by electroelution (11) and CsCl gradient purification counterpart of the disrupted locus. The mutant phenotype is (12). The fragment was microinjected into the pronuclei of completely penetrant only in some genetic backgrounds, sug- fertilized (C57BL6/J x SJL/J)F2 embryos, which were used gesting a modifying influence from a second locus. Junctions to generate transgenic mice by standard protocols as de- between the inserted transgene and adjoining cellular DNA scribed (12). were cloned, allowing us to confirm the heterozygous nature of Identification of Transgenic Mice. High molecular weight the genetic disruption and to detect an associated deletion. We DNA from a small piece of mouse tail was isolated as have designated the mutation Lvs (lacking vigorous sperm) and described (12). DNA was digested with HindIII and then presume that it may define a previously undescribed locus analyzed by Southern blot analysis (13) using the SV40 early important in spermatogenesis. region as a probe. Hybridization was in 0.5 M Na2HPO4, pH 7.2/7% SDS/1 mM EDTA at 650C overnight, followed by two Transgenic mice offer a valuable means with which to eval- washes of20 min each at 650C in 0.5 M Na2HPO4, pH 7.2/1% uate the control and phenotypic effects ofgene expression (1, SDS/1 mM EDTA. 2). In addition, insertion of a transgene into the genome Genomic Cloning. Genomic DNA from a transgenic mouse occasionally disrupts a vital gene, providing a fortuitous was digested to completion with EcoRI and electrophoresed mutation that can reveal the physiological function of the on a 0.9o agarose gel. DNA ranging in size from 4.5 to 7 affected gene (3-5). Typically, an insertion mutation causes kilobases (kb) was electroeluted (11) and used to construct a loss offunction, is genetically recessive, and must be bred to partial genomic library in the phage vector AgtlO. The library homozygosity before its effects become evident. The virtue was screened using the SV40 early region as a probe. in such a mutation is that the presence of the transgene Subcloning and other standard molecular biological tech- provides a molecular "tag" that can facilitate cloning of the niques were performed as described (11). mutant gene and its normal counterpart. Microscopic Examination. For analysis by light micros- We have produced a series of 10 transgenic mouse lines copy, testes were dissected and then fixed in 4% formalin, with the protooncogene HCK. As the mice were bred, it dehydrated in a gradient ofethanol, cleared with toluene, and became evident that many males of one line were sterile and embedded in paraffin, followed by preparation of thin sec- that the sterility arose from a defect in spermatogenesis. tions and staining with hematoxylin and eosin. For analysis Since the sterility segregated with the hemizygous transgene by electron microscopy, mice were perfused through the and occurred in the absence of detectable expression of the heart with phosphate-buffered saline containing heparin (10 transgene, we concluded that the abnormal phenotype was units/ml) followed by perfusion with 1.5% glutaraldehyde in due to mutagenesis by insertion of the transgene. We desig- 0.1 M sodium cacodylate buffer. Testes were removed and nated the mutation Lvs for "lacking vigorous sperm." placed in additional fixative. The tissue was then further fixed Spermatogenesis has been well described morphologically, in 1% osmium tetroxide in acetate vernanal buffer for 1 hr at but little is known of the underlying molecular events (6-8). 4°C followed by 1 hr in 1% tannic acid at room temperature Therefore, we decided to pursue the nature of Lvs and the and then 1 hr at 37°C in Kellenburger buffer. The tissue was gene (or genes) that it presumably defines. Here we describe then dehydrated in a gradient of ethanol and embedded in the phenotype and genetic behavior of Lvs and the isolation Epox; ultrathin sections were then prepared. of DNA from the Lvs locus adjacent to the transgene. The Screening for Fertility. Males were tested for fertility by mutation appears to affect spermatogenesis at the time of mating with FVB/N females (mating was identified by the nuclear condensation and displays two unexpected proper- presence of a vaginal plug in the female after copulation) and ties: genetic dominance and a modifying effect of genetic then dissecting the females 7-10 days later to determine backgrounds. We anticipate that characterization of the Abbreviation: SV40, simian virus 40. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: G. W. Hooper payment. This article must therefore be hereby marked "advertisement" Foundation, Box 0552, University ofCalifornia, San Francisco, CA in accordance with 18 U.S.C. §1734 solely to indicate this fact. 94143-0552. 10327 Downloaded by guest on September 23, 2021 10328 Genetics: Magram and Bishop Proc. Natl. Acad. Sci. USA 88 (1991) whether the female was pregnant and, if so, the number of Table 1. Effect of genetic background on penetrance of sterility pups present. BALB/ RESULTS C57BL/6J cByJ SJL/J FVB/N Identification of Sterile Males Among Transgenic Mice. Ten F1 60% (6/10) 57% (4/7) 75% (3/4)* 100o (3/3) founder mice were generated by microinjection of fertilized F2 40% (2/5) 0%1 (0/6) 100lo (4/4) 10%to (4/4) eggs with the plsh construct (Fig. 1A). The founder female of F3 0% (0/3) 0%o (0/5) 100%o (4/4) 100%o (4/4) transgenic line pIsh 3 was mated to a (C57BL/6J x SJL/J)F1 Percentage of sterile transgenic male progeny derived from back- male (Fig. 1B). A transgenic female from the first cross was crossing two different transgenic females with males ofthe indicated subsequently mated to a different (C57BL/6J x BALB/ inbred strain. Data were pooled for preparation of the table. Num- bers in parentheses indicate sterile males of total number tested. F cByJ)Fl male due to the inability of the regular supplier to indicates the backcross generation number. provide the starting (C57BL/6J x SJL/J)F1 mice. Females *The one male that was not sterile was of very limited fertility; only from the resulting generation were also mated to (C57BL/6J one female of seven was pregnant and with only five pups. x BALB/cByJ)Fl males. Male progeny were tested for fertility. Because normal plugs were obtained with the ex- BALB/cByJ genetic backgrounds, transgenic males on pected frequency, it appeared that males exhibited appropri- SJL/J or FVB/N genetic background were sterile (Table 1). ate sexual behavior and copulated normally. Transgenic The differences in genetic background were apparent after a males fell into three different phenotypic classes. First, the single backcross and were completely established after three majority (2/3) were completely sterile: no pregnancy oc- backcrosses. These data suggest that the impenetrance ofthe curred in a minimum of five matings. The second class of Lvs mutation was due to the influence ofgenetic background. males had very limited fertility: no more than one female Testicular Hstology of Sterile Lvs Males. The testes of became pregnant in a minimum ofsix matings and that female nontransgenic and mutant adult males were fixed and pre- had no more than five pups (generally, one to three). The pared for light microscopy. Although overall organization third class of males had apparently normal fertility. In and numbers of germ cells within the tubules appeared contrast, all nontransgenic males displayed normal fertility, normal in sterile mice, mature spermatids appeared to be demonstrating cosegregation of the sterile phenotype with more darkly stained and misshapen (Fig. 2). Spermatids did the presence of the transgene. not appear to have the normal elongated shape but rather Effect of Genetic Background on Penetrance of the Pheno- were short and wide. The morphologies of both Sertoli and type. One possible explanation for the appearance of various Leydig cells were indistinguishable between sterile trans- phenotypic classes was differences in genetic background. genic testes and nontransgenic testes. To test this hypothesis, transgenic females were backcrossed Electron Microscopy of Spermatids from Sterile Lvs Males. to males of four different inbred strains and resultant female Further characterization by electron microscopy revealed progeny were used to continue backcrossing onto the same the absence of normally shaped mature spermatids in the inbred strain.