A Male Sterile Pink-eyed Mutant Type in the Mouse. W. F. Hollander, Ph.D., J. H. D. Bryan, Ph.D., and John W. Gowen, Ph.D.

Transcription

1 A Male Sterile Pink-eyed Mutant Type in the Mouse W. F. Hollander, Ph.D., J. H. D. Bryan, Ph.D., and John W. Gowen, Ph.D. IN THE course of genetic analysis of mutations which have resulted from x irradiation of mature male mice, we found that one mutation is associated with male sterility. 12 The object of the present paper is to give a full account of this effect. The x-ray treatment must be considered here merely as a tool to induce genetic change, rather than as characteristically producing sterility mutations. Apparently no other radiation-induced mutants allelic to visible markers have hitherto been reported to show sterility in mice, although a number proved lethal Of course the direct effects of x rays on testis or ovary may be sterilizing to the treated individual by damage to gonial cells Also, irradiated spermatocytes, oocytes, and gametes may fail to yield viable progeny, but this "sterility" is in reality "dominant lethal" effects-chromosome aberration causing embryonic death Chromosome damage, particularly translocations, may get by for the first generation but give rise to sterility or "semisterility" in the next as a result of aneuploid segregation, 5 8 again generally embryonic death rather than true sterility. Such effects are in quite a different category from a mutation with regular Mendelian transmission, such as is discussed herein. MATERIALS AND METHODS The screening methods for detecting mutations at specific loci recoverable From the Genetics Department, Iowa State University, Ames, Iowa. This is Journal Paper J-3721 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project This work has received assistance from Contract AT (11-1) 107 from the U. S. Atomic Energy Commission. 316 r

2 Vol. 11, No. 3, 1960 STERILE MICE 317 from irradiated animals have been detailed elsewhere In the present study, the pelvic region of mature male mice of the highly inbred S and Z strains was exposed to a dosage of 500 r. The radiation source was a General Electric Maxitron, 250 pkv, 30 mamp., 0.25-mm. Cu and 1-mm. AI filters, dose rate about 170 r per minute. The treated males were immediately mated with females of the P strain, which are homozygous for several recessive marker genes, including p (pink-eyed dilute). Ordinarily the progeny are normal (wild type), but if one of the marker gene effects is visible a mutation is inferred to have been induced at the corresponding chromosome locus. The frequency of mutation per locus by this procedure is readily calculated, and in our data is on the order of 1/5000 of the functioning spermatozoa. This is a much higher rate than that observed for mutations from spermatogonia. Once obtained, such a mutation can be perpetuated by appropriate matings and isolated in a separate stock for further study. It is essential that the new mutant be removed from association with the original marker allele by which it was detected; where the 2 are very similar, additional marker genes for the chromosomes in question are required. Special tests of the mutant type will be detailed in a later section. THE PINK-EYED MUTANT TYPE A pink-eyed dilute F1 female from x-rayed S male was born March 9, 1955, with several normal sibs. Conception had occurred 3 or 4 days after the sire had been irradiated. From this F1 female a stock was developed, by outcrossing and appropriate tests, having only the new mutant, which we shall symbolize p", and the wild type allele. Segregation of homozygotes from matings of heterozygotes with one another was strictly Mendelian. Forty litters chosen at random from the data are composed of 437 progeny, 134 of which were p"p". The homozygous p"p" mice proved indistinguishable from ordinary pinkeyed dilute mice in color. Growth however was definitely inferior, averaging about two thirds of the normal. Their behavior was also distinctiverather nervous and slightly uncoordinated. At weaning and thenceforth they showed inability to chew food biscuits through ~4-inch mesh hardware-cloth hoppers and had to be given easier access to food; otherwise they starved. Even then, what appeared to be premature senility was also characteristic, most of these mice being unable to survive for a year. Difficulty in eating

3 HOLLANDER ET AL. Fertility & Sterility may have contributed to this reduced viability, as the incisor teeth generally developed unusual grooved wear patterns. Although the normal sibs of the homozygous p'ps mice were extremely prolific, it was found impossible to obtain young from p"p" X p"p". Crosses of p"p' females with normal males were more successful, often one or more litters being produced by a female. The young, however, were rarely nursed, and fostering them with normal females was necessary. Male p"p" homozygotes outcrossed to normal females proved practically sterile. About 80 males have been tested, usually with 4 normal females each, and only 2 of these sired any progeny. In 1 such case, 2 litters were sired ( 4 and 1 young, respectively); in the second case a single progeny was born. Thereafter these males proved sterile. Since these 3 litters were remarkably small, compared with the usual average of 8 or more young from such females, a paucity of spermatozoa might be suspected. To explore the reason for the sterility, semen from such males was examined and found to contain high proportions of very abnormal spermatozoa. Also, 12 males were mated with 4 normal females each, and these were examined daily for 2 weeks to determine the number of vaginal copulation plugs produced. Not a single plug was found. Evidently, then, the p"p" males have poor libido. ABNORMAL SPERMATOZOA In contrast to the semen from normal males, that from p"p" males shows little tendency to diffuse when expressed from a ductus deferens in physiologic saline. This fact suggests low sperm motility, and to some extent microscopic examination confirms the inference; but the failure to diffuse is more due to clumping, at least in part the result of tangling of filaments from the abnormal sperm heads. For study of motility and viability levels, the physiologic saline was made alkaline with 1% NaHC03 and colored with about 0.5% eosin Y. These modifications have no harmful effect on normal sperms, and the higher ph favors maximum motility. The eosin does not stain living sperms, whereas dead sperms rapidly take it up. This distinction is based on an ion exclusion principle which has long been used by others for identification of dead cells. Nigrosin, often used in semen study with eosin, has not been found advantageous in our observations. The method finally employed for semen study was to strip a little semen

4 V~l. 11, N~. 3,1960 STERILE MICE 319 Fig. I. Mouse spermatozoa under oil-immersion objective ( eosin-y stain). A, Normal, showing characteristic hook and rounded posterior part of head. B, C, D, E, Selected fields from semen smears of 4 males of the p p type, showing typical abnormalities of sperm heads and some of the granular debris.

5 320 HOLLANDER ET AL. Fertility & Sterility from the ductus of a freshly killed male into a few drops of the alkalineeosin saline on a slide. The semen was stirred with a toothpick, then covered with a glass cover slip and gently blotted. Percentages of abnormal sperms of various types were estimated by two observers for each sample. Semen from normal males showed very few abnormalities, except for varying percentages of flexed midpiece. Occasional sperms, generally not over 1 per cent, had small heads without hooks. By contrast, the semen from p"p" males had high frequencies of head abnormalities, ranging between per cent, with the majority over 75 per cent. Rather surprisingly, the sperms with abnormal heads were usually viable and motile, whereas the superficially normal sperms from p"p" males were commonly dead. For more detailed study of sperms, semen smears were prepared with distilled water instead of saline, then air dried and stained. However, many of the smears contained excessive plasma solids and debris so that the method was not continued. Instead, the semen was diluted with saline and centrifuged at 2700 rpm for 5 minutes. Next the sedimented sperms were resuspended in distilled water and centrifuged again. Two such washings often were required to give a clear field. The final suspension was simply smeared thinly on slides and air dried. Various staining procedures were used, with or without fixation, but eosin Y proved the simplest and was satisfactory. After staining and briefly washing in distilled water, the smears were blotted dry, air dried, dipped in xylene, and mounted in Euparal or Permount. With the Feulgen test normal sperm heads are deeply stained (positive reaction), while the very abnormal heads do not stain. Also, the granular debris so characteristic of p"p" semen is Feulgen-negative. The structural abnormalities of the sperm heads from p"p" males are so varied that strict classification is impossible. The following attempt to group them is rough but will assist in understanding. Nearly norrnal. These heads may be slightly larger than normal; the point is less hooked, and the posterior end tends to be truncated instead of rounded. These sperms resemble late spermatid stages 17 and are always Feulgenpositive. Triangular and oblate. These heads are near normal size and are Feulgenpositive as a rule; they show little or no hook and may have a lumpy shape. Spatulate. Heads resemble golf clubs; they are usually about half the normal diameter and always Feulgen-negative. f '

6 Vol. 11, No. 3, 1960 STERILE MICE 321 Fig. 2. Camera Iucida drawings of sperm heads usually with a portion of midpiece. All stained with eosin-y and drawn to same scale. Left column, Normal sperms from 10 normal males. Second column, Nearly normal sperms from p p males. Third column, Triangular and oblate class of sperms from p p males. The remaining drawings are of representative spatulate, elongated, and filamentous or unclassifiable types of abnormalities from p p males. These are shown with lighter stippling to indicate poor stainability as well as Feulgen-negative character.

7 322 HOLLANDER ET AL. Fertility & Sterility Elongated. Heads are very long and thin, often constricted in the middle; no hook; always Feulgen-negative. Filamentous. Heads are diffuse or small knobs, attached to midpiece by one to several fine threads. The filaments are often so twisted and entangled with those from other sperms as to be inseparable. These are always Feulgennegative. There was an apparent relation between frequency of abnormal sperm heads and the vigor of the p"p" males; that is, the smaller and older males tended to have the most abnormalities. To search for the origin of the abnormalities, smears from the epididymis at different levels also were examined. The same abnormal types were found even in the upper end (caput); testis smears also showed them. Testis sections showed normal meiotic stages. Evidently, therefore, spermiogenesis was somehow faulty. Spermiogenesis was next studied by means of squashes of individual testis tubules in saline, examined with a phase-contrast microscope. Much variation in acrosome development was found; in males with 5 per cent or fewer normal sperm heads acrosomes were rare. Spermatids with well-developed tails and no acrosome were common, a condition never observed in the normal testis. \ DISCUSSION It seems reasonable to deduce that in males of the p"p" genotype spermiogenesis usually fails to go to completion because of faulty acrosome development. Even the "nearly normal" class of sperms rarely appears beyond Oakberg's ( 1956) Stage 11 or 12. Even a slight anomaly of the acrosome apparently can result in sterility, according to HancocP 0 and Bishop and Austin, 2 so that sterility of the p"p" males is to be expected. Rather, it is surprising that some sperms in the conglomeration have apparently been morphologically and functionally normal. If the faulty acrosome development is responsible for the abnormalities of head formation, as seems most probable, the genetic control of acrosome development must be imprecise. This is the same conclusion reached by Rajasekarasetty 18 in his study of another genetic type of mouse showing sperm anomalies. His abnormal types were mostly different from those reported here, except that hook formation was interfered with in both. That type of mouse also was charac-

8 Vol. 11, No. 3, 1960 STERILE MICE terized by almost complete sterility of the males, 7 a condition termed "quasisterility."9 18 As noted by Rajasekarasetty, it was shown by Schrader and Leuchtenberger2 that the acrosomal material originates from the dictyosomes ( Golgi body), and that the latter are produced in the gonial, diploid cells. If the acrosome governs the morphology of the spermatid nucleus, rather than vice versa, the genotype of the primary spermatocyte should be more important in determining the general course of spermiogenesis than is the genetic constitution of the spermatid itself. This appears to be the case; heterozygotes for p" show no increase in abnormalities although half the spermatids do not have the normal allele. The perfect Mendelian segregation from heterozygotes has been cited in an earlier section. The suggestion by Beatty1 that the sperm's morphology may be a function of its own genetic constitution, therefore, has not been supported in these studies. The rather tenuous nature of genotypic control over sperm morphology is again indicated in recent work by Hughes et al. Deuterium oxide ( 30 per cent in drinking water) for mice caused male sterility associated with defective sperm head structure. The Feulgen-negative reaction of the more abnormal heads in the present study is most interesting. Since most of these showed motility, they can hardly be considered "dead," but perhaps they are dead from the neck forward. Tails separated from normal heads are often active, so that a functional head is obviously not necessary for motility. Since the Feulgennegative heads generally are much smaller than the normal, one might inquire whether any nucleus is present in them or whether more or less nuclear material has been expelled. The filamentous appearance of many heads and the abundance of granular debris in the semen of these males support this interpretation. The failure of these heads and the granules to give a Feulgenpositive reaction characteristic of deoxyribonucleic acid can be attributed to degradation of the chromatin. 323 SUMMARY A recessive mutation, symbolized p", obtained from an x-rayed male, has been produced in homozygous condition. Male homozygotes show almost total sterility, partly because of failure to copulate and partly because of high proportions of abnormal sperm heads. The abnormalities are described and figured, and their origin traced to defective acrosome development. A

9 324 HOLLANDER ET AL. Fertility & Sterility high percentage of the abnormal sperms, although motile, give negative Feulgen reaction, probably because of ruptured nucleus. REFERENCES 1. BEATTY, R. A. Genetics of mammalian spermatozoa. Proc. X Int. Congress Genetics (Montreal) 2:16, BISHOP, M. W. H., and AusTIN, C. R. Mammalian spermatozoa. Endeavour 16: 137, BRYAN, J. H. D., and GowEN, J. W. The effects of 2560 r of x-rays on spermatogenesis in the mouse. Biol. Bull. 114:271, BRYAN, J. H. D., and GoWEN, J. W. The regenerative response of the mouse testis to two different levels of x-irradiation ( 320 r and 2560 r). Proc. X Int. Congress Genetics (Montreal) 2:37, CARTER, T. C. Genetic implications of irradiation in man. Advances in Radiobiology, Proc. V Int. Conference Radiobiology, Stockholm, Edinburgh, Oliver and Boyd, 1957, p CARTER, T. C. Radiation-induced gene mutation in adult female and foetal male mice. Brit.]. Radial. 31:407, DuNN, L. C. Genetically determined variations in male fertility in the house mouse. Studies on Testis and Ovary, Eggs and Sperm, Proc. Conference Committee on Human Reproduction, Nat. Res. Council, E. T. Engle, ed. Springfield, Ill., Thomas, GRIFFEN, A. B. Occurrence of chromosomal aberrations in pre-spermatocytic cells of irradiated male mice. Proc. Nat. Acad. Sc. 44:691, GRUNEBERG, H. The genetics of the mouse. Bibliographia Genetica 15:1, HANCOCK, J. L. The spermatozoa of sterile bulls. Brit. ]. Exp. Biol. 80:50, HANKS, J. H., and WALLACE, J. H. Determination of cell viability. Proc. Soc. Exp. Biol. Med. 98:188, HoLLANDER, W. F. Sperm abnormality of a mutant type involving the p-locus in the mouse. Proc. X Int. Congress Genetics (Montreal) 2:12.'3, HoLLANDER, W. F., and GowEN, J. W. Estimation of the induction rate for dominant lethal effects in mouse sperms by x-rays (abstract). Genetics 40:575, HoLLANDER, W. F., and GowEN, J. W. An extreme non-agouti mutant in the mouse. ]. Heredity 47:221, HuGHES, A. M., BENNETT, E. L., and CALVIN, M. Production of sterility in mice by deuterium oxide. Proc. Nat. Acad. Sc. 45:581, AKBERG, E. F. Degeneration of spermatogonia of the mouse following exposure to x-rays, and stages in the mitotic cycle at which cell death occurs. ]. Morph. 97:39, AKBERG, E. F. A description of spermiogenesis in the mouse. Am. ]. Anat. 99:391, RAJASEKARASETTY, M. R. Studies on a new type of genetically determined quasisterility in the house mouse. Fertil. & Steril. 5:68, RussELL, L. B., and RussELL, W. L. Genetic effects of radiation in the female mouse. Proc. X Int. Congress Genetics (Montreal) 2:245, RussELL, W. L., and RussELL, L. B. The genetic and phenotypic characteristics of radiation-induced mutations in mice. Radiation Res. Suppl. 1:296, ScHRADER, F., and LEucHTENBERGER, C. The cytology and chemical nature of some constituents of the developing sperm. Chromosoma 4:404, ( -.