Skeletal effects of androgen withdrawal

Hypogonadism is considered to be one of the major risk factors for osteoporosis in men. Therefore, it is an important goal for skeletal research to improve our understanding of the skeletal effects of androgens. Androgen deficiency during growth is associated with a failure to acquire normal peak bone mass, and there is good evidence that the effects of androgens on skeletal growth and the development of a male skeletal phenotype are mediated through the androgen receptor. In adult men, acute withdrawal of androgens by surgical or chemical castration induces high turnover bone loss. Similarly, orchidectomy of aged, non-growing male rats is associated with a pronounced and sustained increase in bone turnover and with true loss of cancellous and cortical bone. Interestingly, the changes in bone turnover induced by orchidectomy are paralleled by a concomitant increase in B lymphopoiesis in bone marrow of rats and mice. Although there is firm evidence that male bone metabolism can be influenced by androgens and estrogen, a variety of clinical and animal experimental data have strongly suggested that, under physiological circumstances, the maintenance of cancellous bone mass in males involves the skeletal action of estrogen derived from aromatization of androgens. Aged male rats appear to closely mimic the conditions induced by androgen withdrawal in adult humans, and this animal model may be used 1) to elucidate further the role of muscle as a mediator of the actions of androgens on bone, 2) to explore the regulatory functions of androgens and estrogens in the male skeleton and the immune system, and 3) to find new treatment strategies for the prevention and treatment of osteoporosis in men.     

Testosterone alters duodenal calcium transport and longitudinal bone growth rate in parallel in the male rat.

Duodenal active calcium transport and longitudinal bone growth rate have been shown previously to be regulated in parallel by alteration of gonadal hormone status in sexually maturing female rats. The present study was designed to extend these observations to the sexually maturing male rat. Male rats were orchidectomized (ORX) and given Silastic implants containing either testosterone or estradiol at 6 weeks of age. At 9 weeks of age, duodenal active calcium transport was measured by the everted gut sac method and longitudinal bone growth rate was determined by tetracycline labeling. Decreases in body weight, longitudinal bone growth rate, duodenal calcium transport, and serum Ca and P were exhibited by ORX animals as compared with age-matched control animals. Testosterone administration to ORX animals resulted in an increase in body weight, longitudinal bone growth rate, duodenal calcium transport, and serum Ca and P as compared with ORX animals to a level not significantly different from that of age-matched control animals. Estradiol administration to ORX animals resulted in an additional decrease in body weight, although no significant effect on duodenal calcium transport, serum Ca, or P was noted as compared with ORX animals. There were no statistically significant alterations in the circulating levels of 1,25-dihydroxyvitamin D, parathyroid hormone, or osteocalcin in response to any of the experimental manipulations of gonadal status. These results indicate that, as in the female, gonadal hormone status affects intestinal calcium transport in sexually maturing male rats in parallel with changes in bone growth rate by mechanisms that are independent of circulating levels of 1,25-dihydroxyvitamin D.     

睾酮对去睾丸家兔骨密度及血清钙磷的影响

目的:观察去睾丸和睾酮补充对雄兔骨密度和血清钙、镁、磷的影响.方法:周龄相同的雄性新西兰白兔随机分成对照组、去睾丸组和睾酮补充组(去睾丸后肌注十一酸睾酮).同等条件下饲养20周后测量各组兔全身骨密度、腰椎骨密度、股骨颈骨密度,并检测血清总睾酮(TT)、雌二醇(E2),脱氢表雄酮(DHEA)水平以及血清钙(Ca2 )、游离钙([Ca2 ]i)镁(Mg2 )、磷(P)和碱性磷酸酶(AKP)浓度.结果:去睾丸组血清TT水平明显下降(P<0.01),睾酮补充组血清TT水平升高接近对照组(P>0.05).去睾丸组血清E2和E2/TT比明显高于对照组(P<0.01),睾酮补充组血清E2和E2/TT下降,接近对照组水平(P均>0.05).与对照组相比,去睾丸组血清Ca2 、[Ca2 ]i、Mg2 以及AKP浓度明显升高(P均<0.01),睾酮补充组血清Ca2 、[Ca2 ]i、Mg2 以及AKP浓度较去睾丸组低,接近对照组水平(P>0.05).股骨颈骨密度在去睾丸组明显低于对照组和睾酮补充组(P<0.01),而后两组无差别(P>0.05).结论:去睾丸后雄兔血清TT明显下降,E2和E2/TT比以及Ca2 、[Ca2 ]i、Mg2 和AKP浓度明显升高,骨密度显著下降,睾酮补充使上述异常明显改善.     

Chronic B cell deficiency from birth prevents age-related alterations in the B lineage

Aging is accompanied by a decline in B lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. The mechanisms underlying these changes are unclear. To explore whether aging in the B lineage is subjected to homeostatic regulation, we used mutant mice bearing chronic B cell deficiency from birth. We show that chronic B cell deficiency from birth, resulting from impaired maturation (CD19(-/-) and CD74(-/-)) or reduced survival (baff-r(-/-)), prevents age-related changes in the B lineage. Thus, frequencies of early and late hematopoietic stem cells, B lymphopoiesis, and the rate of B cell production do not substantially change with age in these mice, as opposed to wild-type mice where kinetic experiments indicate that the output from the bone marrow is impaired. Further, we found that long-lived B cells did not accumulate and peripheral repertoire was not altered with age in these mice. Collectively, our results suggest that aging in the B lineage is not autonomously progressing but subjected to homeostatic regulation.     

Cranberry juice improved antioxidant status without affecting bone quality in orchidectomized male rats

BACKGROUND: We reported that drinking citrus juice improves bone quality in orchidectomized senescent male rats. Because cranberry juice, like citrus, is rich in nutrients and phenolic compounds, beneficial effects of citrus juice might also be seen with cranberry juice. An experiment evaluated effect of drinking cranberry juice on bone quality in orchidectomized rats. METHODS: Thirty-two 1-year-old male rats were randomized to two groups: a sham-control group (n=8) and an orchidectomized group (n=24). The treatments for the 4 months duration of the study were SHAM, orchidectomy (ORX), ORX+drinking either 27% or 45% cranberry juice concentrate added to drinking water. At the termination of the study, the rats were euthanized, blood was collected for plasma antioxidant status and IGF-I. The femur, tibia and the 4th lumbar were evaluated for bone quality. Total calcium and magnesium concentration in the femurs were also evaluated. RESULTS: ORX did not affect red blood cell (RBC)-induced hemolysis despite lowering (p<0.05) plasma antioxidant capacity; reduced (p<0.05) plasma IGF-I, femoral density, femoral strength, time-induced femoral fracture, bone mineral content, bone mineral area; numerically (p=0.07) lowered 4th lumbar density; decreased (p<0.05) trabecular connectivity, trabecular number, femoral ash; increased (p<0.05) trabecular separation in comparison to the SHAM group. Drinking cranberry juice increased (p<0.05) plasma antioxidant status, protected RBC against hemolysis, but had no positive effect on bone quality or bone mineral status. CONCLUSIONS: Cranberry juice increases plasma antioxidant status without affecting bone quality.     

Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-alpha knockout mice.

To examine the role of the estrogen receptor-alpha (ERalpha) during male skeletal development, bone density and structure of aged ERalphaKO mice and wild-type (WT) littermates were analyzed and skeletal changes in response to sex steroid deficiency and replacement were also studied. In comparison to WT, ERalphaKO mice had smaller and thinner bones, arguing for a direct role of ERalpha to obtain full skeletal size in male mice. However, male ERalphaKO mice had significantly more trabecular bone as assessed both by pQCT and histomorphometry, indicating that ERalpha is not essential to maintain cancellous bone mass. Six weeks following orchidectomy (ORX), both WT and ERalphaKO mice showed high-turnover osteoporosis as revealed by increases in serum osteocalcin and decreases in trabecular (-38% and -58% in WT and ERalphaKO, respectively) and cortical bone density (-5% and -4% in WT and ERalphaKO, respectively). Administration of testosterone propionate (T, 5 mg/kg/day) completely prevented bone loss both in ERalphaKO and in WT mice. As expected, estradiol (E2, 60 microg/kg/day) replacement did not prevent cancellous bone loss in ORX ERalphaKO mice. However, E2 stimulated bone formation at the endocortical surface in ORX ERalphaKO, suggesting that osteoblasts may respond to nonERalpha-mediated estrogen action. In conclusion, although functional ERalpha may play a significant role during male skeletal development, this receptor does not seem essential for androgen-mediated skeletal maintenance in older male mice.     

Long-term effects of aging and orchidectomy on bone and body composition in rapidly growing male rats

The purpose of this study was to assess the long-term effects of aging and sex hormone deficiency on skeletal metabolism and body composition in rapidly growing male rats. Sprague-Dawley male rats were sham-operated (sham) or orchidectomized (ORX) at 3 months of age. Eight sham rats and eight ORX rats at each time point were serially sacrificed at 3, 4, 8, 12, 15, and 23 months of age. Bone mass in sham rats rapidly increased until 8 months of age, then slightly increased between 8 to 12 months of age; thereafter, an age-related decrease in bone mass was found between 12 to 23 months of age. In sham rats, bone formation parameters decreased between 3 and 8 months, and maintained at the lower level between 8 and 23 months of age, while bone resorption parameters decreased between 3 and 12 months, and thereafter, increased with age between 12 and 23 months of age. ORX significantly inhibited age-related gain in body weight, lean body mass, and cancellous and cortical bone mass and decreased peak bone mass (approximately 20% less versus sham). Further, we found that the lower bone and lean body mass in ORX rats was due to the lack of age-related gain rather than the net loss from basal controls. These data suggest that sex hormones are important factors for the accumulation of peak bone and lean body mass in male rats.     

Short-term androgen deprivation does not alter CaR and VDR mRNA expression in duodenal mucosa in male rats

Androgen deprivation is associated with decline in intestinal calcium absorption. The effect of androgen on CaR and VDR intestinal mucosa has not yet been studied. Calcium homeostasis, a real bone mineral density (aBMD, dual energy X-ray absorptiometry) and expression of CaR and VDR mRNA in duodenal mucosa of orchidectomized (ORX) and sham operated (Sham) adult Sprague Dawley rats at 4 week have been studied. There was no significant difference in serum calcium, alkaline phosphatase, calcidiol and calcitriol levels between both the groups. Serum testosterone (T) (ng/dl) and inorganic phosphorous (iP) (mg/dl) levels were significantly lower in ORX rats. As compared to sham rats, ORX rats had significant decline in in-vitro aBMD at proximal, middle and distal tibia, proximal, mid and distal femur and femoral neck (P < 0.05). Northern blot analysis revealed no significant alteration in the CaR and VDR mRNA expression in duodenal mucosa in ORX rats. CaR and VDR mRNA expression in duodenal mucosa is therefore, not affected by physiological concentrations of testosterone in rats.     

Sex differences and sex hormones in anxiety-like behavior of aging rats

Sex differences in the prevalence of affective disorders might be attributable to different sex hormone milieu. The effects of short-term sex hormone deficiency on behavior, especially on anxiety have been studied in numerous animal experiments, mainly on young adult rats and mice. However, sex differences in aged animals and the effects of long-term hypogonadism are understudied. The aim of our study was to analyze sex differences in anxiety-like behavior in aged rats and to prove whether they can be attributed to endogenous sex hormone production in males. A battery of tests was performed to assess anxiety-like behavior in aged female, male and gonadectomized male rats castrated before puberty. In addition, the aged gonadectomized male rats were treated with a single injection of estradiol or testosterone or supplemented with estradiol for two-weeks. Female rats displayed a less anxious behavior than male rats in most of the conducted behavioral tests except the light-dark box. Long-term androgen deficiency decreased the sex difference in anxiety either partially (open field, PhenoTyper cage) or completely (elevated plus maze). Neither single injection of sex hormones, nor two-week supplementation of estradiol in gonadectomized aged male rats significantly affected their anxiety-like behavior in the elevated plus maze. In conclusion, our results confirm sex differences in anxiety in aged rats likely mediated by endogenous testosterone production in males. Whether long-term supplementation with exogenous sex hormones could affect anxiety-like behavior in elderly individuals remains to be elucidated.     

A novel method to 'exercise' rats: making rats rise to erect bipedal stance for feeding - raised cage model

We employed a novel method to exercise rats: making them rise to bipedal stance for feeding using raised cages. We studied its effects on the skeletons of 6 and 10-month-old intact or orchidectomized (ORX) rats. Body and hindlimb muscle weights, tibial BMC and periosteal cortical bone formation increased after housing in raised cages, but more so in 6-month-old animals than in 10-month-old ones. In 6-month-old orchidectomized rats, raised cages partially prevented ORX-induced bone loss by stimulating periosteal cortical bone (TX) formation and decreased bone resorption next to marrow. In 10-month-old male orchidectomized rats, raised cages also decreased the endosteal and trabecular bone resorption, but not enough to prevent completely ORX-induced net bone losses. Because the osteogenic effects of raised cages alone were only partial, we also studied the interaction between raised cage and prostaglandin E(2) (PGE(2)) in 10-month-old retired female breeders. When treated with combined raised cage and PGE(2), both cortical (TX) and trabecular bone mass of the proximal tibial metaphysis and lumbar vertebral body increased over either raised cages or PGE(2) treatment alone, that was accompanied by dramatic increased bone formation at periosteal and endosteal surfaces. Thus making rats rise to erect bipedal stance for feeding helps to prevent bone loss after orchidectomy; it amplifies the anabolic effects of PGE(2), and it provides an inexpensive, non-invasive and reliable way to increase mechanical loading of certain bones of the rat skeleton.     

Inhibition of 5alpha-reductase blocks prostate effects of testosterone without blocking anabolic effects

We studied the effect of the 5alpha-reductase inhibitor MK-434 on responses to testosterone (T) in orchiectomized (ORX) male Brown Norway (BN) rats aged 13 mo. At 4 wk after ORX or sham surgery, a second surgery was performed to implant pellets delivering 1 mg T/day or placebo pellets. During the second 4 wk of the study, rats received injections of MK-434 (0.75 mg/day) or vehicle injections. Treatment with T elevated serum T to 75% above that for sham animals (P = 0.002) and did not affect serum dihydrotestosterone (DHT) or serum estradiol. T treatment also caused an elevation of prostate T and a marked elevation of prostate DHT. During the second half of the study, ORX rats lost an average of 18.86 +/- 4.62 g body wt. T completely prevented weight loss, and the effect was not inhibited by MK-434 (P < 0.001). ORX produced a nonsignificant trend toward a small (5%) decrease in the mass of the gastrocnemius muscle (P = 0.0819). This trend was also reversed by T, and the effect of T was not blocked by MK-434. T caused a significant 16% decrease in subcutaneous fat that was not blocked by MK-434 (P < 0.05). Finally, T caused a 65% decrease in urine excretion of deoxypyridinoline, a marker of bone resorption, and again the effect was not blocked by MK-434 (P < 0.0001). In contrast, T caused a greater than fivefold increase in prostate mass, and the effect was almost completely blocked by MK-434 (P < 0.0001). This study demonstrates that 5alpha-reductase inhibitors may block the undesirable effects of T on the prostate, without blocking the desirable anabolic effects of T on muscle, bone, and fat.     

Brain aromatase is neuroprotective

The expression of aromatase, the enzyme that catalyzes the biosynthesis of estrogens from precursor androgens, is increased in the brain after injury, suggesting that aromatase may be involved in neuroprotection. In the present study, the effect of inactivating aromatase has been assessed in a model of neurodegeneration induced by the systemic administration of neurotoxins. Domoic acid, at a dose that is not neurotoxic in intact male mice, induced significant neuronal loss in the hilus of the hippocampal formation of mice with reduced levels of aromatase substrates as a result of gonadectomy. Furthermore, the aromatase substrate testosterone, as well as its metabolite estradiol, the product of aromatase, were able to protect hilar neurons from domoic acid. In contrast, dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone and a nonaromatizable androgen, was not. These findings suggest that aromatization of testosterone to estradiol may be involved in the neuroprotective action of testosterone in this experimental model. In addition, aromatase knock-out mice showed significant neuronal loss after injection of a low dose of domoic acid, while control littermates did not, indicating that aromatase deficiency increases the vulnerability of hilar neurons to neurotoxic degeneration. The effect of aromatase on neuroprotection was also tested in male rats treated systemically with the specific aromatase inhibitor fadrozole and injected with kainic acid, a well characterized neurotoxin for hilar neurons in the rat. Fadrozole enhanced the neurodegenerative effect of kainic acid in intact male rats and this effect was counterbalanced by the administration of estradiol. Furthermore, the neuroprotective effect of testosterone against kainic acid in castrated male rats was blocked by fadrozole. These findings suggest that neuroprotection by aromatase is due to the formation of estradiol from its precursor testosterone. Finally, a role for local cerebral aromatase in neuroprotection is indicated by the fact that intracerebral administration of fadrozole enhanced kainic acid induced neurodegeneration in the hippocampus of intact male rats. These findings indicate that aromatase deficiency decreases the threshold for neurodegeneration and that local cerebral aromatase is neuroprotective. Brain aromatase may therefore represent a new target for therapeutic approaches to neurodegenerative diseases.     

Effects of triiodothyronine and estrogen administration on bone mass, mineral content and bone strength in male rats.

Experimental hyperthyroidism had a negative effect on bone mineral density, but did not significantly alter mechanical properties of femur and femoral bone thickness. Estradiol at a dose used in humans for the treatment of osteoporosis decreased seminal vesicle weight and concentration of testosterone but increased bone density in male rats compared to intact animals. In these rats, the mechanical analysis revealed an increased mechanical femur strength higher than the increase in bone density and femoral cortical thickness. When hyperthyroid male rats with low bone density were treated with estradiol in spite of a low plasma testosterone, the changes in bone density resulting from hyperthyroidism were entirely prevented. Estrogens protect the male skeleton against resorbing action of T (3). Treatment with estradiol in male rats with hyperthyroidism did not increase mechanical bone strength or femoral cortical thickness as it did with estradiol administration alone. Our results suggest that exogenously administered estrogens may have therapeutic value in preventing bone loss accompanying triiodothyronine administration, even in male rats with a low testosterone levels. At the concentration studied, estradiol increased in spite of low plasma testosterone, bone mineral density, mechanical strength of femur, and femoral cortical thickness.     

Novel, non-steroidal, selective androgen receptor modulators (SARMs) with anabolic activity in bone and muscle and improved safety profile

A novel approach to the treatment of osteoporosis in men, and possibly women, is the development of selective androgen receptor modulators (SARMs) that can stimulate formation of new bone with substantially diminished proliferative activity in the prostate, as well as reduced virilizing activity in women. Over the last several years, we have developed a program to discover and develop novel, non-steroidal, orally-active selective androgen receptor modulators (SARMs) that provide improved therapeutic benefits and reduce risk and side effects. In recent studies, we have used a skeletally mature orchiectomized (ORX) male rat as an animal model of male hypogonadism for assessing the efficacy of LGD2226, a nonsteroidal, non-aromatizable, and non-5alpha-reducible SARM. We assessed the activity of LGD2226 on bone turnover, bone mass and bone strength, and also evaluated the effects exerted on classic androgen-dependent targets, such as prostate, seminal vesicles and muscle. A substantial loss of bone density was observed in ORX animals, and this loss was prevented by SARMs, as well as standard androgens. Biochemical markers of bone turnover revealed an early increase of bone resorption in androgen-deficient rats that was repressed in ORX animals treated with the oral SARM, LGD2226, during a 4-month treatment period. Differences in architectural properties and bone strength were detected by histomorphometric and mechanical analyses, demonstrating beneficial effects of LGD2226 on bone quality in androgen-deficient rats. Histomorphometric analysis of cortical bone revealed distinct anabolic activity of LGD2226 in periosteal bone. LGD2226 was able to prevent bone loss and maintain bone quality in ORX rats by stimulating bone formation, while also inhibiting bone turnover. LGD2226 also exerted anabolic activity on the levator ani muscle. Taken together, these results suggest that orally-active, non-steroidal SARMs may be useful therapeutics for both muscle and bone in elderly hypogonadal men through their anabolic activities. Since SARMs both prevent bone loss, and also stimulate formation of new bone, they may have significant advantages relative to currently used anti-resorptive therapies. Coupled with their activity in muscle and their ability to maintain or restore libido, they offer new therapeutic approaches for male and female hormone replacement.     

Inflammatory biomarker, neopterin, suppresses B lymphopoiesis for possible facilitation of granulocyte responses, which is severely altered in age-related stromal-cell-impaired mice, SCI/SAM

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre-B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-alpha and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.     

Anabolic effects of testosterone are preserved during inhibition of 5alpha-reductase

At replacement doses, testosterone produces only modest increases in muscle strength and bone mineral density in older hypogonadal men. Although higher doses of testosterone are more anabolic, there is concern over increased adverse effects, notably prostate enlargement. We tested a novel strategy for obtaining robust anabolic effects without prostate enlargement. Orchiectomized (ORX) male rats were treated for 56 days with 1.0 mg testosterone/day, with and without 0.75 mg/day of the 5alpha-reductase inhibitor MK-434. Testosterone administration elevated the prostate dihydrotestosterone concentration and caused prostate enlargement. Both effects were inhibited by MK-434. ORX produced a catabolic state manifested in reduced food intake, blunted weight gain, reduced hemoglobin concentration, decreased kidney mass, and increased bone resorption, and in the proximal tibia there was both decreased cancellous bone volume and a decreased number of trabeculae. In soleus and extensor digitorum longus muscles, ORX reduced both the percentage of type I muscle fibers and the cross-sectional area of type 1 and 2 fibers. Testosterone administration caused a number of anabolic effects, including increases in food intake, hemoglobin concentration, and grip strength, and reversed the catabolic effects of ORX on bone. Testosterone administration also partially reversed ORX-induced changes in muscle fibers. In contrast to the prostate effects of testosterone, the effects on muscle, bone, and hemoglobin concentration were not blocked by MK-434. Our study demonstrates that the effects of testosterone on muscle and bone can be separated from the prostate effects and provides a testable strategy for combating sarcopenia and osteopenia in older hypogonadal men.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.     

Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of New Zealand black mice

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate resulting in a deficiency of B lineage precursors in adult (greater than 15 wk old) animals. To study the characteristics of B lineage cells in young (4 wk) and old (6 mo) NZB mice, bone marrow from these animals was used to initiate long term lymphoid bone marrow cultures (LBMC) that permit the long term maintenance of B cells and their precursors. Age-matched cultures from BALB/c mice and NZB.xid marrow were established in parallel. Primary LBMC were readily established from these strains and showed similar patterns of growth for the 3-mo observation period. No significant differences in numbers of 14.8 positive cells were observed. However, NZB mice at both ages had a higher percentage of membrane IgM (mIgM)-expressing cells. Significant levels of supernatant IgM were found only in cultures of 6-mo NZB and BALB/c mice; levels were highest in NZB culture supernatants and were often more than 500 ng/ml; significant, although much lower, levels of IgG were likewise detected. Lymphoid cells from NZB.xid mice were unable to generate significant levels of IgM in supernatant fluids indicating the effects of the xid gene were displayed in vitro. Autoantibodies were not detected in any of the culture supernatants. Additional evidence for NZB hyperactivity in primary B lymphopoiesis was observed upon initiation of primary myeloid bone marrow cultures (MBMC) from these strains of mice and subsequently transferring them to LBMC conditions. This results in the cessation of myelopoiesis at the initiation of B lymphopoiesis. At the time of converting MBMC to LBMC, cultures of NZB and BALB/c mice morphologically resembled myeloid cultures and had neither B cell colony-forming units nor cells that expressed 14.8 or mIgM. However, following the switch, NZB mice had a 5-fold higher number of B cell colony-forming units. Further, MBMC established from NZB bone marrow cells had a reduced capacity to form colonies in the granulocyte-macrophage colony-forming unit assay. These studies indicate that defects of NZB hemopoietic cells are manifest in vitro and suggest the use of in vitro long term cultures as a valuable technique to further dissect the hematopoietic abnormalities of NZB mice and possible underlying microenvironmental defects.