Gender- and compartment-specific bone loss in C57BL/6J mice: correlation to season?

Seasonal variation in bone mineral density (BMD) has been documented in humans, and has been attributed to changes in 25-hydroxyvitamin D [25(OH)D] synthesis. To test the hypothesis that seasonal changes in bone mass occur in laboratory mice, we measured body composition, femoral bone phenotypes, and serum bone markers in 16-wk-old male and female C57BL/6 (B6) mice during the summer (June-August) and winter (December-February) months at The Jackson Laboratory in Bar Harbor, Maine. Both male and female B6 mice had higher volumetric BMD in the summer than winter. Females showed reduced trabecular bone, whereas males showed changes in bone volume. Males, but not females, had higher insulin-like growth factor 1 in summer than in winter, and only males showed an increase in body weight during the winter. No seasonal differences in serum TRAP5b, osteocalcin, or 25(OH)D were noted for either sex. We conclude that seasonal variation in skeletal and body composition parameters in B6 mice is significant and must be considered when performing longitudinal phenotyping of the skeleton. Further studies are needed to determine the environmental factors that cue seasonal changes in body composition and the mechanisms that produce these changes.

Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice.

The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Furthermore, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F=16.49, P=0.0002), while no significant effect was found on total femur BMD (F=2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168-172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice.

Congenic mice provide in vivo evidence for a genetic locus that modulates serum insulin-like growth factor-I and bone acquisition.

We identified quantitative trait loci (QTL) that determined the genetic variance in serum IGF-I through genome-wide scanning of mice derived from C57BL/6J(B6) x C3H/HeJ(C3H) intercrosses. One QTL (Igf1s2), on mouse chromosome 10 (Chr10), produces a 15% increase in serum IGF-I in B6C3 F2 mice carrying c3 alleles at that position. We constructed a congenic mouse, B6.C3H-10 (10T), by backcrossing c3 alleles from this 57-Mb region into B6 for 10 generations. 10T mice have higher serum and skeletal IGF-I, greater trabecular bone volume fraction, more trabeculae, and a higher number of osteoclasts at 16 wk, compared with B6 (P < 0.05). Nested congenic sublines generated from further backcrossing of 10T allowed for recombination and produced four smaller sublines with significantly increased serum IGF-I at 16 wk (i.e. 10-4, 10-7, 10-10, and 10-13), compared with B6 (P < 0.0003), and three smaller sublines that showed no differences in IGF-I vs. age- and gender-matched B6 mice. Like 10T, the 10-4 nested sublines at 16 wk had higher femoral mineral (P < 0.0001) and greater trabecular connectivity density with significantly more trabeculae than B6 (P < 0.01). Thus, by comprehensive phenotyping, we were able to narrow the QTL to an 18.3-Mb region containing approximately 148 genes, including Igf1 and Elk-3(ETS domain protein). Allelic differences in the Igf1s2 QTL produce a phenotype characterized by increased serum IGF-I and greater peak bone density. Congenic mice establish proof of concept of shared genetic determinants for both circulating IGF-I and bone acquisition.

Congenic mice reveal sex-specific genetic regulation of femoral structure and strength.

Genetic linkage studies in C3H/HeJ (C3H) and C57BL/6J (B6) mice identified several chromosomal locations or quantitative trait loci (QTL) linked to femoral volumetric bone mineral density (vBMD). From QTL identified on chromosomes (chr) 1, 4, 6, 13, and 18, five congenic mouse strains were developed. In each of these mice, genomic DNA from the QTL region of the donor C3H strain was transferred into the recipient B6 strain. Here we report the effects of donated C3H QTL on femoral structure, cortical vBMD and bending strength. Femoral structure was quantified by the polar moment of inertia (Ip) at the mid-diaphysis, which reflects the bending or torsional rigidity of the femur. Although the C3H progenitor mice have a smaller Ip than B6 progenitor mice, the congenic mice carrying the C3H segment at Chr 4 had significantly increased Ip in both males and females, giving these mice stronger femora. In female mice from the congenic Chr 1 strain, Ip was increased whereas male mice from the Chr 1 strain had smaller femoral cross-sections and significantly reduced Ip. This sex-specific effect on femoral structure was seen to a lesser extent in Chr 18 congenic mice. In addition, cortical vBMD was measured using peripheral quantitative computed tomography. Cortical vBMD was similar among most congenic strains except in Chr 6 congenic mice, where cortical vBMD was significantly less in females, but not in males. We conclude that (1) chromosomal QTL from C3H mice, which are genetically linked to total femoral vBMD, also regulate femoral structure; (2) the QTL on Chr 4 improves femoral structure and strength; (3) QTL on Chr 1 and 18 impart sex-specific effects on femoral structure; and (4) the QTL on Chr 6 imparts a sex-specific effect on cortical vBMD and femoral strength.

Quantitative trait loci for femoral and lumbar vertebral bone mineral density in C57BL/6J and C3H/HeJ inbred strains of mice.

Significant differences in vertebral (9%) and femoral (50%) adult bone mineral density (BMD) between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice have been subjected to genetic analyses for quantitative trait loci (QTL). Nine hundred eighty-six B6C3F2 females were analyzed to gain insight into the number of genes that regulate peak BMD and their locations. Femurs and lumbar vertebrae were isolated from 4-month-old B6C3F2 females at skeletal maturity and then BMD was determined by peripheral quantitative computed tomography (pQCT). Estimates of BMD heritability were 83% for femurs and 72% for vertebrae. Genomic DNA from F2 progeny was screened for 107 polymerase chain reaction (PCR)-based markers discriminating B6 and C3H alleles on all 19 autosomes. The regression analyses of markers on BMD revealed ten chromosomes (1, 2, 4, 6, 11, 12, 13, 14, 16, and 18) carrying QTLs for femurs and seven chromosomes (1, 4, 7, 9, 11, 14, and 18) carrying QTLs for vertebrae, each with log10 of the odds ratio (LOD) scores of 2.8 or better. The QTLs on chromosomes (Chrs) 2, 6, 12, 13, and 16 were unique to femurs, whereas the QTLs on Chrs 7 and 9 were unique to vertebrae. When the two bone sites had a QTL on the same chromosome, the same marker had the highest, although different, LOD score. A pairwise comparison by analysis of variance (ANOVA) did not reveal significant gene x gene interactions between QTLs for either bone site. BMD variance accounted for by individual QTLs ranged from 1% to 10%. Collectively, the BMD QTLs for femurs accounted for 35.1% and for vertebrae accounted for 23.7 % of the F2 population variances in these bones. When mice were homozygous c3/c3 in the QTL region, 8 of the 10 QTLs increased, while the remaining two QTLs on Chrs 6 and 12 decreased, femoral BMD. Similarly, when mice were homozygous c3/c3 in the QTL region for the vertebrae, five of the seven QTLs increased, while two QTLs on Chrs 7 and 9 decreased, BMD. These findings show the genetic complexity of BMD with multiple genes participating in its regulation. Although 5 of the 12 QTLs are considered to be skeleton-wide loci and commonly affect both femurs and vertebrae, each of the bone sites also exhibited unique QTLs. Thus, the BMD phenotype can be partitioned into its genetic components and the effects of these loci on normal bone biology can be determined. Importantly, the BMD QTLs that we have identified are in regions of the mouse genome that have known human homology, and the QTLs will become useful experimental tools for mechanistic and therapeutic analyses of bone regulatory genes.

Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice.

Serum insulin-like growth factor-1 (IGF-1) and femoral bone mineral density (BMD) differ between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), by approximately 30% and 50%, respectively. Similarly, skeletal IGF-1 content, bone formation, mineral apposition, and marrow stromal cell numbers are higher in C3H than in B6 mice. Because IGF-1 and several bone parameters cosegregate, we hypothesize that the serum IGF-1 phenotype has a strong heritable component and that genetic determinants for serum IGF-1 are involved in the regulation of bone mass. We intercrossed (B6 x C3H)F1 hybrids and analyzed 682 F2 female offspring at 4 months of age for serum IGF-1 by radioimmunoassay and femoral BMD by peripheral quantitative computerized tomography (pQCT). Genomic DNA was assayed by polymerase chain reaction (PCR) to determine alleles for 114 Mit markers inherited in F2 mice at average distances of 14 centimorgans (cM) along each chromosome (Chr). Serum IGF-1 levels in the F2 progeny were relatively normal in distribution, but showed a greater range than either progenitor, indicating that serum IGF-1 level is a polygenic trait with an estimated heritability of 52%. Serum IGF-1 correlated with femoral length (r = 0.266, p < 0.0001) and femoral BMD (r = 0.267, p < 0.0001). Whole genome scans for main effects associated with serum IGF-1 levels revealed three significant QTLs (in order of significance) on mouse Chrs 6, 15, and 10. The QTL on Chr 6 showed a significant reduction in IGF-1 associated with increasing C3H allele number, whereas the Chr 15 and Chr 10 loci showed additive effects with increasing C3H allele number. A genome-wide search for interacting marker pairs identified a significant interaction between the Chr 6 QTL and a locus on Chr 11. This interactive effect suggested that when the Chr 11 locus was homozygous for C3H, there was no effect of the Chr 6 locus on serum IGF-1; however, the combination of C3H alleles on Chr 6 with B6 alleles on Chr 11 was associated with reduced serum IGF-1 concentrations. To test this in vivo, we tested congenic mice carrying the Chr 6 QTL region from C3H on a B6 background (B6.C3H-6). Both serum IGF-1 and femoral BMD were significantly lower in female congenic than progenitor B6 mice. In summary, we identified three major QTLs on mouse Chrs 6, 10, and 15, and noted a major locus-locus interaction between Chrs 6 and 11. We named these QTLs IGF-1 serum levels (Igf1sl1 to Igf1sl4). Functional isolation of the Igf1sl1 QTL on Chr 6 for IGF-1 in B6.C3H-6 congenic mice demonstrated effects on both the IGF-1 and BMD phenotypes. The genetic determinants of these Igf1sl QTLs will provide much insight into the regulation of IGF-1 and the subsequent acquisition of peak bone mass.

Quantitative trait loci for bone density in C57BL/6J and CAST/EiJ inbred mice.

Genetic analyses for loci regulating bone mineral density have been conducted in a cohort of F(2) mice derived from intercross matings of (C57BL/6J x CAST/EiJ)F(1) parents. Femurs were isolated from 714 4-month-old females when peak adult bone density had been achieved. Bone mineral density (BMD) data were obtained by peripheral quantitative computed tomography (pQCT), and genotype data were obtained by Polymerase Chain Reaction (PCR) assays for polymorphic markers carried in genomic DNA of each mouse. Genome-wide scans for co-segregation of genetic marker data with high or low BMD revealed loci on eight different chromosomes, four of which (Chrs 1, 5, 13, and 15) achieved conservative statistical criteria for suggestive, significant, or highly significant linkage with BMD. These four quantitative trait loci (QTLs) were confirmed by a linear regression model developed to describe the main effects; none of the loci exhibited significant interaction effects by ANOVA. The four QTLs have been named Bmd1 (Chr 1), Bmd2 (Chr 5), Bmd3 (Chr 13), and Bmd4 (Chr 15). Additive effects were observed for Bmd1, recessive for Bmd3, and dominant effects for Bmd2 and Bmd4. The current large size of the QTL regions (6-->31 cM) renders premature any discussion of candidate genes at this time. Fine mapping of these QTLs is in progress to refine their genetic positions and to evaluate human homologies.

Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice.

Spontaneous juvenile ovarian granulosa cell (GC) tumors that occur in young girls are similar to GC carcinomas that develop in SWR-derived inbred mice. We analyzed female offspring from a series of matings among SWR and SJL inbred mice for chromosomal loci underlying tumor susceptibility. Intercross F2 female mice were produced by reciprocal matings of (SWR x SJL)F1 and (SJL x SWR)F1 parents. Tumorigenesis in these F2 mice as well as in SWXJ recombinant inbred and congenic strains of mice derived from SWR and SJL showed significant (P < 0.001) association with Gct1, a dominant susceptibility locus on chromosome (CHR) 4 and with Gct2 on CHR 12. Suggestive (P < 0.01) association was found with Gct3 on CHR 15. A fourth susceptibility locus, Gct4 on CHR X, was demonstrated with a strong parent-of-origin effect associated with the paternal genotype. Imprinting and complex interactions among these four loci combine to establish the probability for GC tumorigenesis in this mouse model.

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 7 to X.

The SWXJ recombinant inbred (RI) set was developed for genetic analysis of heritable ovarian tumors. In this report we present data for 223 simple sequence length polymorphisms spanning Chromosomes (Chrs) 7-X to complete the genetic marking of this RI set. The strain distribution patterns (SDP) for these loci were combined with data from 19 other polymorphic genes, resulting in densely marked maps for Chrs 7-X. Combined with the 165 loci for Chr 1-6 reported previously (Svenson et al., Mamm. Genome 6, 867, 1995), the SWXJ RI set represents a powerful tool for mapping genes in neoplastic as well as other heritable disorders.

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 1 to 6.

We typed 147 simple sequence length polymorphisms in the SWXJ recombinant inbred (RI) strain set spanning Chromosomes (Chrs) 1-6. The strain distribution pattern for these loci was combined with data from 18 previously typed loci for SWXJ, resulting in new chromosome maps for this RI set, with an average density of 3.5 cM between loci. This is the first systematic effort to develop a more highly resolved genetic map for the SWXJ RI set and thereby improves the usefulness of this genetic tool for mapping genes underlying both simple and complex genetic disorders.

Improved engraftment of human spleen cells in NOD/LtSz-scid/scid mice as compared with C.B-17-scid/scid mice.

T and B lymphocyte-deficient mice homozygous for the severe combined immunodeficiency (SCID) mutation can be immunologically engrafted with human lymphocytes. However, low levels of human peripheral blood mononuclear cell engraftment are commonly observed, impeding full use of this model. We now demonstrate that strain background in mice homozygous for the scid mutation is a strong determinant of levels of human lymphocyte engraftment. NOD/LtSz-scid/scid mice support higher levels of engraftment of both human spleen and peripheral blood mononuclear cells than do C.B-17-scid/scid mice. We observed, using human spleen cell injected scid mice, 1), high levels of engraftment of the host peripheral lymphoid tissues with human CD45+ (leukocytes), CD3+ (T cells), CD4+ (helper/inducer), and CD8+ (suppressor/cytotoxic) lymphoid cells for up to 24 weeks in NOD/LtSz-scid/scid mice; 2), migration of high numbers of human lymphocytes to peripheral lymphoid and nonlymphoid organs in NOD/LtSz-scid/scid, but not in C.B-17-scid/scid mice; 3), higher levels of serum immunoglobulin of human origin in NOD/LtSz-scid/scid mice than in C.B-17-scid/scid mice; 4), histological lesions characteristic of human anti-mouse xenoreactivity in NOD/LtSz-scid/scid mice; and 5), human origin antibodies against filarial antigens after engraftment with native human spleen cells. The use of NOD/LtSz-scid/scid mice as recipients to achieve significantly enhanced human lymphopoietic cell engraftment will now enable human immunity to be more easily studied in animal models.

Increased B lymphopoiesis in genetically sex steroid-deficient hypogonadal (hpg) mice.

Interleukin 7 (IL-7) responsive B lineage precursors were greatly expanded in genetically hypogonadal female (HPG/Bm-hpg/hpg) mice that have a secondary deficiency in gonadal steroidogenesis. Estrogen replacement in these mice resulted in a dose-dependent reduction in B cell precursors. More modest increases were documented in genetically normal mice that were surgically castrated. These findings complement other recent observations that B lymphopoiesis selectively declines in pregnant or estrogen-treated animals. Sex steroids have long been known to influence such disparate processes as bone physiology and tumor growth, in addition to their importance for reproductive function. We now show that these hormones are important negative regulators of B lymphopoiesis.

Influence of gonadal steroids on susceptibility to Brugia malayi in scid mice.

In the present study, we demonstrate that male scid/scid mice have higher adult worm burdens than do female scid/scid mice following equal challenge doses of infective Brugia malayi L3 larvae. Gonadectomy of four week old immature mice has no effect on worm burden in either sex, suggesting that the gender dichotomy between males and females does not depend on continued presence of endogenous gonadal steroids. The worm yield from female, but not male, mice can be increased by prepubertal oophorectomy combined with administration of either estradiol or testosterone in depot form. Our results raise the possibility that prepubertal steroid pulse(s) result(s) in physiological changes in male scid/scid mice that enhance worm growth. These studies confirm earlier reports of epidemiological data in humans suggesting a sexual dimorphism in susceptibility to filarial infection. Our data suggest that this gender difference is not due simply to the presence of adult gonadal steroids, but rather to ontogenic differentiative actions of sex steroids in the host.

Granulosa cell tumorigenesis in genetically hypogonadal-immunodeficient mice grafted with ovaries from tumor-susceptible donors.

The SWR and SWXJ recombinant inbred strains of mice develop heritable, pubertal onset ovarian granulosa cell (GC) tumors with characteristics similar to those observed for human juvenile GC tumors. We utilized this murine model to determine: (a) whether spontaneous tumorigenesis is an intrinsic property of the susceptible ovary; (b) whether pubertal developmental stage affects tumorigenesis; and (c) whether tumorigenesis depends on extraovarian regulation provided by an immune system or a hypothalamic-pituitary gonadotropin system. To test these questions, ovaries from tumor-susceptible donors were grafted beneath the kidney capsules of hosts with differing immunological and hormonal capabilities. Hosts for these ovarian grafts were: (a) immunologically intact, syngeneic mice; (b) immune-deficient, allogeneic mice homozygous for the severe combined immune deficiency (scid/scid) mutation; and (c) scid/scid mice segregating for the hypogonadal (hpg) mutation, yielding gonadotropin-deficient hpg/hpg scid/scid and gonadotropin replete +/? (hpg/+ or +/+) scid/scid littermates. Donors and hosts of differing ages were used to address questions of developmental effects on tumorigenesis. Grafts were examined 6 to 10 wk after implantation for ovarian morphology and tumor incidence. Results showed that ovary grafts from susceptible female mice formed spontaneous GC tumors equally well in both syngeneic and immune-deficient scid/scid hosts. In each type of host, the incidence of grafts exhibiting spontaneous tumor development declined significantly with increasing age of both donor and host. In addition, prepubertal ovary grafts formed spontaneous tumors in hormonally normal +/? scid/scid but not in hormonally deficient hpg/hpg scid/scid hosts. Finally, treatment of hpg/hpg scid/scid host mice with the androgenic steroid hormone precursor, dehydroepiandrosterone, resulted in GC tumor formation in the tumor-susceptible ovary grafts. We conclude that pubertal onset, spontaneous tumorigenesis in the susceptible ovaries is: (a) independent of an intact immune system; (b) terminated by completion of ovarian maturation as a cyclic organ; (c) not dependent on extraovarian factors unique to the genetically susceptible host; and (d) potentially initiated by androgenic steroids in the absence of an intact hypothalamic-pituitary gonadotropin axis. We hypothesize that ovarian androgens synthesized in response to normal gonadotropin stimulation initiate spontaneous tumorigenesis in the genetically susceptible ovary.

Genetic susceptibility for C19 androgen induction of ovarian granulosa cell tumorigenesis in SWXJ strains of mice.

Susceptibility to pubertal onset, malignant granulosa cell (GC) tumors of the ovary is inherited in SWR/Bm and certain SWR-related SWXJ recombinant inbred strains of mice. In some SWXJ strains, GC tumors occur spontaneously (spontaneous strains), and in others GC tumors can only be induced by treatment with dehydroepiandrosterone (DHEA-dependent strains). A gene controlling susceptibility to both spontaneous and DHEA-induced GC tumorigenesis, Gct, has been assigned to Chromosome 4. Additional research on the role of steroids in GC tumorigenesis has revealed a second gene controlling response to C19 androgenic steroids. Spontaneous strains showed increased tumor frequency after treatment with testosterone (T), whereas DHEA-dependent strains showed no GC tumors following T treatment. Within treatment groups, serum steroid data from DHEA, T, and control treated mice showed no consistent differences between spontaneous and DHEA-dependent strains with respect to progesterone, DHEA, androstenedione, dihydrotestosterone, T, estrone, or estradiol. Thus, observed differences in GC tumor responsiveness to exogenous steroids were not due to different patterns of steroid metabolism among spontaneous and DHEA-dependent strains. Further studies on the range of effective C19 steroids were conducted using one spontaneous and one DHEA-dependent strain. The spontaneous strain showed increased GC tumor frequency in response to dihydrotestosterone and androsterone treatment, whereas the DHEA-dependent strain showed no response. This result suggests that spontaneous strains may be sensitive to a broad range of C19 steroids. To determine whether genetic differences in endogenous steroid levels have a role in spontaneous GC tumorigenesis, serum steroid levels were measured in SWR/Bm and SJL/Bm progenitor strains during the developmental period of risk between 22 and 38 days of age. With the exception of transiently increased DHEA at 22 days, there were no consistent differences in steroid levels analyzed. Thus, serum steroid profiles were not reliably prognostic for GC tumorigenesis. In conclusion, GC tumor induction in response to T treatment has co-segregated with susceptibility to spontaneous GC tumors in the SWXJ recombinant inbred strains. Thus, the second gene in our ovarian granulosa cell tumor model regulates responsiveness to T. We propose to name this gene spontaneous ovarian tumorigenesis (Sot), with alleles for susceptibility (s) carried by spontaneous strains and resistance (r) carried by DHEA-dependent strains.

Ovarian granulosa cell tumorigenesis in SWR-derived F1 hybrid mice: preneoplastic follicular abnormality and malignant disease progression.

A high incidence (27.5%; 174 of 633) of spontaneous, malignant ovarian granulosa cell tumors develop in (SWR x SWXJ-9)F1 hybrid females between 3 and 6 weeks. Granulosa cell tumors developed in predictable stages, starting as preneoplastic lesions appearing as hyperemic follicles on the ovarian surface. These follicles were characterized by hypertrophied theca, degenerating oocytes, and large fluid- or erythrocyte-filled antra lined by irregular masses of granulosa cells. Rapidly proliferating granulosa cells filled the antra and the theca/interstitial cells became more dysplastic as granulosa cell tumors developed. Thus the morphology of the preneoplastic lesion suggests that disturbed mechanisms for normal follicular development underlie granulosa cell tumor initiation. Estradiol treatment before but not after preneoplastic lesions appeared inhibited granulosa cell tumor formation. By 6 to 9 months 42% of these mice show metastases in major abdominal and thoracic organs. Thus this model can be experimentally analyzed both for mechanisms of granulosa cell tumor initiation and subsequent malignant progression.

Gene for ovarian granulosa cell tumor susceptibility, Gct, in SWXJ recombinant inbred strains of mice revealed by dehydroepiandrosterone.

Spontaneous, malignant ovarian granulosa cell (GC) tumors occur in pubertal SWR and specific SWXJ recombinant inbred strains of mice. Treatment of these mice with dehydroepiandrosterone (DHEA), an adrenal secretory steroid with anticancer actions against spontaneous and carcinogen-induced tumors of different tissues, gave unexpected results. Diet supplemented with 0.4% DHEA (a) induced significantly more GC tumors in spontaneous tumor-susceptible strains (SWR and SWXJ-1, -4, and -9), (b) induced the first GC tumors observed in five previously tumor-free strains (SWXJ-6, -7, -8, -10, and -12), and (c) failed to induce GC tumors in SJL and in the remaining six SWXJ strains (SWXJ-2, -3, -5, -11, -13, and -14). The strain distribution pattern of DHEA-induced GC tumor susceptibility versus resistance was compared with strain distribution patterns for 35 different loci known to distinguish SWR and SJL progenitor strains. A complete match of DHEA-induced GC tumors with pancreas-2 (Pan-2) on mouse chromosome 4 was found. We have named this new locus GC tumor susceptibility (Gct), with the Gcts (susceptible) allele found in SWR and the Gctr (resistant) allele found in SJL mice. The Gct locus is closely linked to pancreas-2, Pan-2, but the order of genes is not yet confirmed. In addition, data from F1 progeny of matings between SWR and selected inbred strains provide suggestive evidence for a second gene controlling GC tumor incidence that we hypothesize involves steroid metabolism. Differences in GC tumor incidence data from reciprocal F1 progeny of matings between SWR and SJL mice reveal a strong maternal effect that may represent yet a third gene. These data support a heritable basis for GC tumorigenesis in the SWR model involving a small number of genes.

Induction of ovarian granulosa cell tumors in SWXJ-9 mice with dehydroepiandrosterone.

Spontaneous ovarian granulosa cell (GC) tumors develop in SWXJ-9 inbred mice at approximately the time of puberty. The effect of dehydroepiandrosterone (DHEA), a steroid secreted by the adrenals and reported to have antitumor actions, was examined in this ovarian tumor model. In contrast with expectations, administration of diet supplemented with 0.4% DHEA or Silastic capsules containing 10 mg DHEA resulted in a significant multifold increase in GC tumor incidence. Similar studies with metabolites of DHEA, i.e., testosterone (TESTO), dihydrotestosterone (DHT), and 17 beta-estradiol (E2), revealed that TESTO was as effective as DHEA in increasing GC tumor incidence. DHT was without effect, and E2 suppressed GC tumor incidence. Serum steroid levels and steroid target tissue responses were assessed to determine if a correlation between a change in level or response to specific steroids and GC tumorigenesis existed. In both tumor-free and GC tumor host mice, dietary or capsular treatment with DHEA, TESTO, or DHT resulted in substantial alteration in one or more of serum steroids, DHEA, androstenedione, TESTO, and DHT, in addition to the administered steroid. No consistent correlation was observed between changes in a single steroid or pattern of steroids and GC tumorigenesis. Although significant increases in serum estrogens could be detected in GC tumor hosts treated with DHEA but not TESTO, estrogens did not induce these tumors. Treatment with E2 increased only serum E2 levels. In tumor-free mice, DHEA and E2 treatments were associated with vaginal cytological evidence of estrogen action, whereas the androgens induced a leukocytic pattern. Eighty-eight % of GC tumor host mice, regardless of steroid treatment, showed a vaginal cytology pattern that included cornified cells. The evidence presented in this report leads us to hypothesize that (a) spontaneous and steroid-induced GC tumorigenesis in these mice have the same mechanism, and (b) subtle increases in DHEA or a closely related metabolite during the peripubertal period may initiate GC tumors in these genetically susceptible mice. The mechanism whereby these steroids initiate GC tumorigenesis remains to be determined.

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice.

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.