GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function.

The growth hormone-releasing hormone receptor (GHRHR) is a member of the family of G protein-coupled receptors that is expressed on pituitary somatotrope cells and mediates the actions of GHRH in stimulating growth hormone (GH) synthesis and secretion. We report that the Ghrhr gene is located in the middle of mouse chromosome 6 in the same region as the little mutation. Mice homozygous for this mutation have reduced GH secretion and a dwarf phenotype. A missense mutation was identified in the extracellular domain of the little GHRHR that disrupts receptor function, suggesting that the growth deficit in these mice results from a defect in the GHRHR. Similar alterations in GHRHR might explain some isolated GH deficiencies in humans.

Two loci affecting B cell responses to B cell maturation factors.

B lymphocytes from DBA/2Ha mice have a genetic defect characterized by a failure to differentiate into antibody-secreting cells in response to a family of lymphokines termed B cell maturation factors (BMFs). By contrast, B cells from DBA/2Ha mice respond normally in PFC assays to the B cell mitogen LPS, and macrophages from these mice are activated by one of the three BMFs. Two loci are responsible for the B cell defect in DBA/2Ha mice. One locus (Bmfr-1) is constitutively expressed throughout life, and maps approximately 13 cM distal to the brown locus on chromosome 4. A second locus (Bmfr-2) becomes active only after sexual maturity and is closely linked to the dilute locus on chromosome 9. At both loci, alleles determining responsiveness to BMFs are dominant over nonresponder alleles. The effect of Bmfr-2 on B cell responsiveness may be related to levels of the steroid sex hormones. DBA/2Ha mice offer a tool for studying the genetic and hormonal regulation of the immune system.

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.

Receptor-associated resistance to growth hormone-releasing factor in dwarf "little" mice.

Anterior pituitaries from the dwarf mouse strain "little" did not release growth hormone or accumulate adenosine 3',5'-monophosphate (cyclic AMP) in response to human and rat growth hormone-releasing factor (GRF). Dibutyryl cyclic AMP, as well as the adenylate cyclase stimulators forskolin and cholera toxin, markedly stimulated growth hormone (GH) release. The basis of the GH deficiency in the little mouse may therefore be a defect in an early stage of GRF-stimulated GH release related either to receptor binding or to the function of the hormone-receptor complex.

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.

Gender-specific changes in bone turnover and skeletal architecture in igfbp-2-null mice.

IGF-binding protein-2 (IGFBP-2) is a 36-kDa protein that binds to the IGFs with high affinity. To determine its role in bone turnover, we compared Igfbp2(-/-) mice with Igfbp2(+/+) colony controls. Igfbp2(-/-) males had shorter femurs and were heavier than controls but were not insulin resistant. Serum IGF-I levels in Igfbp2(-/-) mice were 10% higher than Igfbp2(+/+) controls at 8 wk of age; in males, this was accompanied by a 3-fold increase in hepatic Igfbp3 and Igfbp5 mRNA transcripts compared with Igfbp2(+/+) controls. The skeletal phenotype of the Igfbp2(-/-) mice was gender and compartment specific; Igfbp2(-/-) females had increased cortical thickness with a greater periosteal circumference compared with controls, whereas male Igfbp2(-/-) males had reduced cortical bone area and a 20% reduction in the trabecular bone volume fraction due to thinner trabeculae than Igfbp2(+/+) controls. Serum osteocalcin levels were reduced by nearly 40% in Igfbp2(-/-) males, and in vitro, both CFU-ALP(+) preosteoblasts, and tartrate-resistant acid phosphatase-positive osteoclasts were significantly less abundant than in Igfbp2(+/+) male mice. Histomorphometry confirmed fewer osteoblasts and osteoclasts per bone perimeter and reduced bone formation in the Igfbp2(-/-) males. Lysates from both osteoblasts and osteoclasts in the Igfbp2(-/-) males had phosphatase and tensin homolog (PTEN) levels that were significantly higher than Igfbp2(+/+) controls and were suppressed by addition of exogenous IGFBP-2. In summary, there are gender- and compartment-specific changes in Igfbp2(-/-) mice. IGFBP-2 may regulate bone turnover in both an IGF-I-dependent and -independent manner.

Growth hormone enhances hepatic epidermal growth factor receptor concentration in mice.

The effect of growth hormone (GH) on binding of epidermal growth factor (EGF) to liver membrane preparations was investigated in hypophysectomized mice and partially GH-deficient, genetic mutant "little" (lit/lit) mice. The EGF binding of normal male mice and testosterone-treated females was higher than in normal females. Due to diminished receptor concentration, hepatic EGF binding was decreased in male and female lit/lit mice to a level that was unaffected by gender or androgen treatment. GH replacement therapy by intermittent injections and continuous infusion restored the EGF binding of hypophysectomized mice to normal male and female levels, respectively, suggesting a role for the more pulsatile GH secretion in normal males. In lit/lit mice, however, both continuous and intermittent GH resulted in EGF binding levels comparable to those in normal females. In normal males continuous GH suppressed EGF binding. In conclusion, endogenous GH secretion induces EGF receptors in mice and this effect may be modulated by sex differences in GH secretion.

Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency.

We have characterized a new mutant mouse that has virtually no beta-glucuronidase activity. This biochemical defect causes a murine lysosomal storage disease that has many interesting similarities to human mucopolysaccharidosis type VII (MPS VII; Sly syndrome; beta-glucuronidase deficiency). Genetic analysis showed that the mutation is inherited as an autosomal recessive that maps to the beta-glucuronidase gene complex, [Gus], on the distal end of chromosome 5. Although there is a greater than 200-fold reduction in the beta-glucuronidase mRNA concentration in mutant tissues, Southern blot analysis failed to detect any abnormalities in the structural gene, Gus-sb, or in 17 kb of 5' flanking and 4 kb of 3' flanking sequences. Surprisingly, a sensitive S1 nuclease assay indicated that the relative level of kidney gusmps mRNA responded normally to androgen induction by increasing approximately 11-fold. Analysis of this mutant mouse may offer valuable information on the pathogenesis of human MPS VII and provide a useful system in which to study bone marrow transplantation and gene transfer methods of therapy.

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.

Gonadotropin, steroid, and thyroid hormone milieu of young SWR mice bearing spontaneous granulosa cell tumors.

Young SWR mice possessing spontaneous ovarian granulosa cell (GC) tumors were examined for evidence of endocrine dysfunction associated with tumorigenesis. Tissue levels of hormones in tumor host and normal control females were measured by radioimmunoassays, ovarian luteinizing hormone (LH) receptors by uptake of 125I-labeled human chorionic gonadotropin (hCG) administered iv, and ovarian 3-beta-hydroxysteroid dehydrogenase (3-beta-OH) activity by histochemical techniques. When data from tumor host mice were compared with control data, hypothalamic gonadotropin releasing hormone content was not significantly different. Pituitary LH and follicle-stimulating hormone (FSH) contents were significantly decreased. Serum FSH, but not LH, levels were significantly reduced. No specific uptake of 125I-labeled hCG by tumor tissue was detected, whereas uptake by nontumorous contralateral ovaries was identified and found to be similar to that of control ovaries. With respect to serum steroids in tumor host mice, progesterone, dihydrotestosterone, and testosterone were significantly reduced, whereas androstenedione, dehydroepiandrosterone, corticosterone, estrone, and estradiol were normal. Frozen sections of tumor tissue failed to show any 3-beta-OH activity, whereas prominent activity was observed in non-tumorous contralateral and control ovaries. Serum thyroxine levels, evaluated because of the known depressive effects of hypothyroidism on reproductive function, were found to be significantly elevated in tumor host mice. The above results suggest that in SWR mice with spontaneous GC tumors, gonadotropins are moderately suppressed; the granulosa tumor cells do not have LH-hCG receptors; steroidogenesis by tumor tissue is reduced, whereas peripheral conversion of adrenal androgen precursors to estrogens is normal; and elevated serum thyroxine levels have a secondary role in established GC tumors.

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.

Spontaneous malignant granulosa cell tumors in ovaries of young SWR mice.

Granulosa cell tumors (GCT) of the ovary appear spontaneously at 4-6 weeks of age in SWR/J and in SWR/Bm inbred strain mice, with a maximum incidence reached by 10 weeks. Cancer was confirmed by metastasis to abdominal organs and by transplantability of primary tumors to histocompatible hosts. Results of genetic crosses showed that GCT appear in SWR X SJL F1 but not in SJL X SWR F1 nor in other F1 females derived from matings of SWR mice with A/HeJ, C57BL/6By, CBA/J, or DBA/2J mice. These findings suggest the maternal transmission of GCT susceptibility. Recombinant inbred strains SWXJ were produced from a progenitor mating of a SWR female to a SJL male. At F20, females in 3 of 14 SWXJ strains developed GCT, with one strain displaying a 5-fold increase in incidence. Embryo transfer studies with SWXJ-6 and -9 mice suggested that maternal transmission was most likely via the fertilized egg rather than through milk or placenta-uterine contact. Analysis of metaphase chromosomes indicated that the modal number in tumors and bone marrow was 40 (2n = 40) with 2 X chromosomes present. Gross chromosomal aberrations were not detected. A working hypothesis proposes that interaction of a unique SWR factor, perhaps cytoplasmic, with nuclear genomic material common to Swiss mouse stocks results in occurrence of GCT in young SWR and SWR-derived mice.

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.

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.

Reduced growth of human breast cancer xenografts in hosts homozygous for the lit mutation.

Insulin-like growth factor I (IGF-I) is a potent breast cancer mitogen. Growth hormone (GH) up-regulates hepatic IGF-I gene expression and circulating IGF-I level. Tissue IGF bioactivity is influenced not only by circulating IGF-I and IGF-II levels but also by autocrine and paracrine production of these growth factors and by IGF binding proteins. There is considerable person-to-person variability in GH-IGF-I physiology. Both laboratory and epidemiological data are consistent with the hypothesis that the host GH-IGF-I axis influences breast cancer behavior, but such an effect has not been directly demonstrated. To determine whether breast cancer growth in an in vivo model is influenced by the host GH-IGF-I axis, we compared the growth of human MCF-7 breast cancer cells in control mice to that in mice homozygous for lit, a missense mutation resulting in loss of function of the pituitary GH-releasing hormone receptor and secondary suppression of GH and IGF-I. Breast cancer growth was significantly reduced in lit/lit animals compared to control hosts [tumor size (mean +/- SD) on day 39,444 +/- 82 versus 845 +/- 444 mm3, respectively; P < 0.001, Mann-Whitney U test]. These data demonstrate that in our model, host GH-IGF-I axis physiology plays a role in determining breast cancer behavior. The results a) suggest that patient-to-patient variability in GH-IGF-I physiology may contribute to the large variability between patients regarding breast cancer behavior, and b) motivate clinical trials of novel hormonal treatment strategies that target the GH-IGF-I axis.

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.

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.

The effect of immunosuppression on streptozotocin-induced diabetes in C57BL/KsJ mice.

The objective of this study was to determine whether mature thymic-derived T-lymphocytes were required for streptozotocin (SZ)-induced insulitis. C57BL/KsJ male mice were immunocrippled by thymectomy at 3 wk of age followed 1 wk later by lethal irradiation (1000 R) and hematopoietic reconstitution with syngeneic bone marrow (pretreated with anti-Thy 1.2 antiserum and complement to eliminate mature T-lymphocytes). As a control for the systemic effects of lethal irradiation itself, thymus-intact males were also irradiated and reconstituted with anti-Thy-1.2-treated marrow cells. This latter treatment resulted in a reconstitution of functional T-lymphocytes. Independent of the presence or absence of functional T-lymphocytes, irradiation extensively damaged the testes and produced at least a 50% reduction in plasma testosterone levels. In such effeminized males, the hyperglycemic response following 6 daily injections of SZ (35 mg/kg) was reduced in comparison to unirradiated males. Pancreatic insulin content was reduced 50% in both thymus-intact and thymectomized groups receiving lethal irradiation and SZ treatment; this correlated with histologic findings of small, beta-cell-depleted islets. Focal leukocytic infiltrates of the exocrine pancreas were induced by the irradiation. Streptozotocin-induced insulitis was also observed regardless of the presence (in thymus-intact mice) or absence (in thymectomized mice) of phytohemagglutinin-responsive T-lymphocytes. Both groups exhibited intact B-lymphocyte function as measured by proliferative responsiveness to lipopolysaccharide. Severe immunosuppression of both T- and B-lymphocyte function was produced by subcutaneous injection of hydrocortisone into thymectomized mice 48 h prior to initiation of SZ treatments. This treatment prevented SZ-induced beta-cell necrosis and eliminated lymphocytic infiltrates in the endocrine and exocrine pancreas. We conclude that functional (mature) T-lymphocytes are not required to mediate the beta cytotoxicity of multiple low doses of SZ in inbred strains in which insulitis accompanies islet destruction. The ability of hydrocortisone to protect beta-cells from the direct cytotoxic action of SZ as well as to eliminate leukocytic infiltration in the pancreas would support the hypothesis that insulitis is a consequence of beta-cell destruction, in this model, rather than its cause. DIABETES 32:148-155, February 1983.

Atherogenic high-fat diet reduces bone mineralization in mice.

The epidemiological correlation between osteoporosis and cardiovascular disease is independent of age, but the basis for this correlation is unknown. We previously found that atherogenic oxidized lipids inhibit osteoblastic differentiation in vitro and ex vivo, suggesting that an atherogenic diet may contribute to both diseases. In this study, effects of an atherogenic high-fat diet versus control chow diet on bone were tested in two strains of mice with genetically different susceptibility to atherosclerosis and lipid oxidation. After 4 months and 7 months on the diets, mineral content and density were measured in excised femurs and lumbar vertebrae using peripheral quantitative computed tomographic (pQCT) scanning. In addition, expression of osteocalcin in marrow isolated from the mice after 4 months on the diets was examined. After 7 months, femoral mineral content in C57BL/6 atherosclerosis-susceptible mice on the high-fat diet was 43% lower (0.73 +/- 0.09 mg vs. 1.28 +/- 0.42 mg; p = 0.008), and mineral density was 15% lower compared with mice on the chow diet. Smaller deficits were observed after 4 months. Vertebral mineral content also was lower in the fat-fed C57BL/6 mice. These changes in the atherosclerosis-resistant, C3H/HeJ mice were smaller and mostly not significant. Osteocalcin expression was reduced in the marrow of high fat-fed C57BL/6 mice. These findings suggest that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblast progenitor cells.