| First Author | Yu YE | Year | 1996 |
| Journal | Mouse Genome | Volume | 94 |
| Pages | 695-7 | Mgi Jnum | J:44593 |
| Mgi Id | MGI:1100477 | Citation | Yu YE, et al. (1996) Loss of spermatogenic function after puberty in transgenic males harboring an unbalanced product of the reciprocal translocation T(3;10). Mouse Genome 94:695-7 |
| abstractText | Full text of Mouse Genome contribution: LOSS OF SPERMATOGENIC FUNCTION AFTER PUBERTY IN TRANSGENIC MALES HARBORING AN UNBALANCED PRODUCT OF THE RECIPROCAL TRANSLOCATION T(3;10). Y. Eugene Yu(1), Margit Nemeth(2), Sen Pathak(2, 4), Marvin L. Meistrich(3), and Paul K.Y. Wong(1) 1-Science Park Research Division, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957; 2-Department of Cell Biology, 3-Department of Experimental Radiotherapy, and 4-Division of Laboratory Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030. To study the effect of the Moloney murine leukemia virus ts1 envelope protein (1) in activated T cells in vivo, we produced twelve transgenic founder mice by pronuclear injection of the transgene ts1-env(H) (Fig. 1). Five founders had an FVB/N genetic background; the other seven had a C57BL/6 x SJL/F2 genetic background. EY47 was the designation for one of the FVB/N founders. Transgenic progeny from the EY47 founder mouse exhibited two different profiles on Southern blot auto- radiograms: some mice had two hybridizing bands (3.9 kb and 8.0 kb, see lane 1 and 4 in Fig. 2), and others had only one 3.9 kb hybridizing band (see lane 2, 3, and 5 in Fig. 2). Hereinafter, these mice are referred to as EY47-2 and EY47-1 transgenic mice, respectively. The karyotypes of the embryonic fibroblasts, isolated from the EY47-2 transgenic fetuses (with two hybridizing bands) had evidence of a reciprocal translocation involving chromosomes 3 and 10 (Fig. 3). The karyotypes of the cells isolated from the EY47-1 transgenic fetuses (with a single 3.9 kb band) revealed that they were an unbalanced product of T(3;10) translocation, namely 39 chromosomes plus the 3-10 translocation chromosome (i.e. 3, 3-10, 10, 10). EY47-1 transgenic mice were detected in 18% of the transgenic progeny produced by EY47-2 transgenic females mated with nontransgenic males. Densitometric analysis of Southern blot autoradiograms suggested that EY47-1 and EY47-2 transgenic mice have approximately one and two copies of the transgene per cell, respectively (data not shown). The above results indicated that, like other translocations generated by insertion of the transgenes (3-5), the transgene ts1-env(H) was also apparently cosegregated with the translocation in EY47 line and suggest that the transgene represented by the 8.0-kb hybridizing band is located in translocation chromosome 10-3. Like the transgene in the translocation-associated line JCP-0#18 (5), the transgene ts1-env(H) probably was located on both translocation chromosomes. Thus, the transgene represented by the 3.9-kb hybridizing band may localize on the reciprocal translocation chromosome 3-10. To our knowledge, the present study is the first to report a transgene cosegregated with an unbalanced product of a reciprocal translocation. Fig. 1. (Legend). The ts1-env(H) transgene, 5ÕCSP-B, the 5Õ-flanking region of the human CGL-1/ granzyme B gene (2); ts1-env, ts1 virus envelope gene; ts1-LTR, ts1 virus long terminal repeat; H, Hpa I site; S, Sma I site; V, EcoR V site. Fig. 2. (Legend). Southern blot hybridization analysis of EcoR V-digested DNA from EY47 transgenic mice. Fig. 3. (Legend). Giemsa-banded karyotype of a male EY47-2 transgenic fetus showing a T(3;10) translocation. The genetic changes were then found to associate with the phenotypes of the impaired reproductive ability observed in EY47 mice. Similar to what has been reported in other mouse lines carrying reciprocal translocation, EY47-2 males had consistently small testes (36.7 mg +/- 2.9 [SD]/testis, based on the weights of 20 testes from mice of 2 months of age) and were sterile throughout their lifetimes. EY47-2 females have a shortened reproductive life span and a reduced litter size. Sterility was also observed in a fraction of EY47-1 females although the ovaries isolated from these infertile females appeared histologically normal and some of adult EY47-1 females died prematurely. The male-to-female ratio of EY47-1 mice at weaning was about 2.5:1 based on the data from 17 litters, which suggests that some of EY47-1 females may have died in utero. A more detailed characterization was focused on EY47-1 males who showed progressive impairment of spermatogenesis. The data from the mating experiments revealed that 21 of 34 EY47-1 males were fertile at 2 months old and produced litters of 3-12 mice. Ten of these 21 males lost their fertility around 4 months of age, while the other 11 did not do so until they were 5 months of age. Testes from three males 2-2.5 months old were examined histologically after the males had produced at least one litter, and although their testes weighed slightly less than normal, the overall testicular organization and approximate numbers of germ cells appeared normal (Fig. 4A). Testes from another three originally fertile 6-month-old transgenic males were examined by light microscopy and showed significant spermatogenic abnormalities (Fig. 4B-D). Unilateral orchidectomy was used to provide evidence for the progressive nature of defect. To do so, EY47-1 males were first mated at 5 weeks of age with normal females and then hemicastrated at 2 months of age when mated females were visibly pregnant and the presence of fetuses was confirmed by cesarean section. Removed testes were fixed in Bouins fixative and embedded in paraffin. Thin paraffin-embedded sections were then stained with hematoxylin and eosin. Quantitatively, the testis weights and condensed spermatid numbers of these transgenic males were 88% and 72% of those in normal controls (Tables 1-2), which suggests that these fertile 2 month-old EY47-1 males already showed some deficiency in spermatogenesis but no severe damage to spermatogenic function. However, by the second castration at 5.5 months of age, the weights of contralateral testes in these mice had decreased by 33% and their number of condensed spermatids by 76%, whereas wild-type FVB/N males showed a small increase in testis weights (Fig. 5) over a similar time period. These data, together with the temporal changes in testis weight summarized in Fig. 5, indicated that spermatogenesis was progressively impaired between 2 and 5.5 months of age in fertile EY47-1 males. A similar phenomenon has been observed in heterozygous weaver mice, who become sterile at 3.5 months of age (6). However, progressive spermatogenic degeneration seen in weaver mice is associated with neurohistopathological changes, and no such changes were detected in the CNS of EY47-1 or EY47-2 mice. No phenotypic-abnormalities were observed in mice from any other transgenic lines carrying the ts1-env(H) except for line EY832 (in that C57BL/6 x SJL/F2 lines, expression of the transgene was detected in thymus, and a fraction of the transgenic mice died prematurely with demyelination). Expression of the transgene in transgenic mice was examined using ribonuclease protection assay and the results revealed no expression of the transgene in the testis or other examined organs (cerebral cortex, brain stem, thymus, heart, liver, spleen or ovary) of EY47-1 or EY47-2 mice (data not shown). Therefore, the progressive impairment of spermatogenesis observed in EY47-1 males is probably not due to the expression of the transgene. Instead, the phenotype may have been caused by one of the following: (1) loss of part of chromosome 3 and/or gain of a small fragment of chromosome 10; (2) disruption of a structural gene by the inserted transgene or the breakpoint of the translocation; (3) gene fusion or activation induced by the translocation. In summary, EY47-1 male mice constitute a new transgenic line with a phenotype of postpubertal spermatogenic degeneration. These mice should be useful for further understanding the mechanism behind this kind of degeneration. Fig. 4. (Legend). Sections of tubules from testes of (A) a 2-month-old fertile EY47-1 transgenic male and (B-D) 6-month-old sterile EY47-1 transgenic males that were fertile at 2 months of age (X290). Table 1. Change in Body and Testis Weights of Transient Fertile EY47-1 Transgenic Mice Between 2 and 5.5 Months of Age. Mouse I.D. number: 1686; First castration Ð Body weight (g): 24.7; Left testis weight (mg): 82.9; Second castration Ð Body weight (g): 29.2; Right testis weight (mg): 52.6 Mouse I.D. number: 1830; First castration Ð Body weight (g): 23.8; Left testis weight (mg): 82.1; Second castration - Body weight (g): 30.0; Right testis weight (mg): 50.0 Mouse I.D. number: 1833; First castration Ð Body weight (g): 21.8; Left testis weight (mg): 79.6; Second castration Ð Body weight (g): 29.5; Right testis weight (mg): 57.9 Mouse I.D. number: 1834; First castration Ð Body weight (g): 23.4; Right testis weight (mg): 82.8; Second castration Ð Body weight (g): 31.4; Left testis weight (mg): 52.9 Mouse I.D. number: 1843; First castration Ð Body weight (g): 21.3; Right testis weight (mg): 80.4; Second castration Ð Body weight (g): 28.1; Left testis weight (mg): 46.4 Mouse I.D. number: 1889; First castration Ð Body weight (g): 27.4; Right testis weight (mg): 79.9; Second castration Ð Body weight (g): 32.7; Left testis weight (mg): 64.5. Table 2. Number of Germ Cells per Sertoli Cell and Testis Weights in Transient Fertile EY47-1 Transgenic Versus Nontransgenic Males. (+/- SD). Germ cell stage: Spermatogonia; First castration of nontransgenic males (2 months old) (n=6)*: 1.6 +/- 0.4; First castration of transgenic males (2 months old) (n=6): 1.5 +/- 0.5; Second castration of transgenic males** (5.5 months old) (n=6): 1.3 +/- 0.5. Germ cell stage: Pachytene: First castration of nontransgenic males (2 months old) (n=6)*: 5.9 +/- 1.8; First castration of transgenic males (2 months old) (n=6): 5.4 +/- 1.4; Second castration of transgenic males** (5.5 months old) (n=6): 4.0 +/- 2.0(+). Germ cell stage: Round spermatids: First castration of nontransgenic males (2 months old) (n=6)*: 18.5 +/- 9.0; First castration of transgenic males (2 months old) (n=6): 17.2 +/- 5.2; Second castration of transgenic males** (5.5 months old) (n=6): 6.7 +/- 4.7(++). Germ cell stage: Condensed spermatids: First castration of nontransgenic males (2 months old) (n=6)*: 10.3 +/- 6.8(+); First castration of transgenic males (2 months old) (n=6): 7.4 +/- 6.8; Second castration of transgenic males** (5.5 months old) (n=6): 1.8 +/- 1.6(++). Testis weight (mg): First castration of nontransgenic males (2 months old) _n-6)*: 92.8 +/- 3.9(++); First castration of transgenic males (2 months old) (n=6): 81.3 +/- 1.5; Second castration of transgenic males** (5.5 months old) (n=6): 54.1 +/- 6.4(++). * No. of animals per group shown in parentheses ** In 5.5-month-old EY47 transgenic males, the exact stage could not be determined reliably because development of spermatids past step 8 was markedly reduced. Therefore, average numbers of different germ cells counted in stages I to VIII were used for statistical analysis in all mice (7). Tubules in stage I to VIII were identified by the existence of round spermatids and absence of two generations of spermatocytes. One tubular cross-section from every 30th section was randomly sampled, and the first five tubular cross-sections per testis were analyzed under a 50x objective. (+) Different from transgenic males at 2 months of age, P < 0.05. (++) Different from transgenic males at 2 months of age, P < 0.001. Fig. 5. (Legend). Temporal change in testis weights in EY47-1 transgenic versus nontransgenic males. 0, EY47-1 transgenic males (changes in testis weights from 2 to 5.5 months old are based on results shown in Table 1); °, nontransgenic males. Number of testes per group is shown in parentheses. Bars, SD. Acknowledgments: P. Wong is the corresponding author. The authors thank Debra Hollowell and Vicki Hopwood for technical assistance. This work was supported by Public Health Service Grants CA 45124 and AI 28283 to P.W. Microinjections to generate C57BL/6 x SJL/F2 transgenic mice were done at the National Transgenic Development Facility (supported by National Institute of Child Health and Human Development Contract N01-HD-0-2911) at DNX, Inc. in Princeton, NJ. References: 1. Wong, PKY, et al. Proc Natl Acad Sci USA 1991; 88:8991-8995. 2. Hanson RD, et al. J Biol Chem 1991; 266:24433-24438. 3. Mahon KA, et al. Proc Natl Acad Sci USA 1988; 85:1165-1168. 4. Gordon JW, et al. Somatic Cell Mol Genet 1989; 15:569-578. 5. Francke U, et al. Mamm Genome 1992; 3:209-216. 6. Verina T, et al. J Neurogenet 1995; 94:251-265. 7. Kangasniemi M, et al. Endocrinology 1996; 137:949-955. |
