Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels.

The nonsteroidal estrogenic compound bisphenol A (BPA) is a monomer used in the manufacture of polycarbonate plastics and resins. BPA may be ingested by humans as it reportedly leaches from the lining of tin cans into foods, from dental sealants into saliva, and from polycarbonate bottles into their contents. Because BPA is weakly estrogenic--approximately 10,000-fold less potent than 17beta-estradiol--current environmental exposure levels have been considered orders of magnitude below the dose required for adverse effects on health. Herein we demonstrate measurable effects on the offspring of Sprague-Dawley female rats that were exposed, via their drinking water, to approximately 0.1 mg BPA/kg body weight (bw)/day (low dose) or 1.2 mg BPA/kg bw/day (high dose) from day 6 of pregnancy through the period of lactation. Offspring exposed to BPA exhibited an increase in body weight that was apparent soon after birth and continued into adulthood. In addition, female offspring exposed perinatally to the high dose of BPA exhibited altered patterns of estrous cyclicity and decreased levels of plasma luteinizing hormone (LH) in adulthood. Administration of neither the doses of BPA that caused effects during perinatal exposure nor a 10-fold higher dose was able to evoke a uterotropic response in ovariectomized postpubertal females. These data indicate an increased sensitivity to BPA during the perinatal period and suggest the need for careful evaluation of the current levels of exposure to this compound.

Characterization and developmental expression patterns of testicular androgen-binding protein in the Djungarian hamster (Phodopus sungorus).

Studies were conducted to characterize Djungarian hamster androgen-binding protein and examine its expression during development. The cDNA encoding the full length testicular androgen-binding protein was cloned and, except for two suspected polymorphisms, shared a common primary sequence with hepatic sex hormone-binding globulin. A single androgen-binding protein/sex hormone-binding globulin gene was identified and a 1.7 kb mRNA encoding androgen-binding protein/sex hormone-binding globulin was present in both the testis and liver. Testicular homogenates contained specific 5 alpha-dihydrotestosterone-binding activity that was identified as androgen-binding protein. In the prepubertal testis, immunoreactive androgen-binding protein/sex hormone-binding globulin subunits ranged from 46 kDa to 60 kDa, with the majority of isoforms > 50 kDa. These subunits were distinct from the major 55 kDa and 51 kDa isoforms of sex hormone-binding globulin present in the serum of prepubertal hamsters. Deglycosylation studies demonstrated that size heterogeneities were the result of developmentally specific glycosylation patterns. Expression of androgen-binding protein by the testis was upregulated during puberty and coincided with a decline in serum sex hormone-binding globulin activity. The tissue- and age-dependent expression of specific androgen-binding protein and sex hormone-binding globulin variants suggests that these proteins play different roles in steroid-mediated sexual development.

Purification and characterization of the sex hormone-binding globulin in serum from Djungarian hamsters.

A high-affinity sex hormone-binding globulin (SHBG) was purified from the serum of prepubertal Djungarian hamsters (Phodopus sungorus). A purification of more than 2000-fold with an overall yield of 23% was achieved without the use of androgen affinity chromatography. Two predominant variants (51 and 55 kDa) were resolved by denaturing polyacrylamide gel electrophoresis. Both variants participated in the binding of dihydrotestosterone (DHT) and had identical amino-terminal sequences. The sequences obtained for Djungarian hamster SHBG (dhSHBG) showed a high degree of identity with those of other mammals. The affinity of purified dhSHBG for DHT (2.5 x 10(9) M(-1) was similar to that measured in unfractionated serum. This protein was isolated as a dimer with a single calcium-dependent steroid-binding site and a major pI of 4.7. The described purification procedure yielded active dhSHBG from small volumes of prepubertal serum. These studies also provide the first direct structural evidence that a SHBG-like protein, not of testicular origin, is expressed by a rodent during prepubertal development.

Hepatic expression of sex hormone-binding globulin associated with the postnatal surge of serum androgen-binding activity in the Djungarian hamster.

Serum androgen-binding capacity in Djungarian hamsters, as in many other mammals, increases within days after birth and remains elevated until puberty. This increased activity has been attributed to a hepatic glycoprotein, sex hormone-binding globulin (SHBG), but expression of SHBG by the postnatal liver has not been demonstrated. Therefore, a full-length SHBG cDNA was cloned from the liver of neonatal hamsters and the expression of SHBG during development was examined. Hepatic SHBG RNA levels, as measured by both competitive RT-PCR and Northern analysis, were very low in fetal animals but increased significantly within 24 h of birth. Maximal values were maintained for 1 week after parturition, and then declined to basal adult levels. The developmental pattern in hepatic SHBG immunoactivity, as determined by Western analysis, mirrored that of hepatic SHBG mRNA. However, changes in serum SHBG immunoactivity and steroid-binding activity occurred approximately 1 week later. There were no sex differences in the levels of hepatic SHBG mRNA or protein during development, but serum immunoactivity tended to be higher in females at puberty. Sex- and age-related differences in the relative abundance of SHBG isoforms were also noted. Results of these studies demonstrate that Djungarian hamsters express an authentic SHBG and indicate that the postnatal surge in serum androgen-binding activity is due to perinatal up-regulation of SHBG expression.

Seasonal influences on the control of plasma sex hormone-binding globulin by T4 in male little brown bats.

Male little brown bats showed a well-defined seasonal cycle of plasma thyroxine (T4). Total T4 concentrations increased during hibernation, reached peak levels in May at spring emergence, and subsequently declined to lowest levels in September. Thyroxine-binding globulin (TBG) activity increased only in summer, and seasonal changes in non-TBG-bound T4 generally paralleled those of total T4. During the fall and early hibernation period, bats studied under feral conditions or following photoperiod manipulation showed coincident changes in plasma T4 and sex hormone-binding globulin (SHBG) levels. In contrast, males studied during late hibernation or following spring arousal exhibited declining plasma T4 levels during periods of increased SHBG activity. Additionally, the prehibernation decline in SHBG did not depend on changing environmental conditions. Bats maintained under summerlike conditions during the fall showed normal seasonal reductions in SHBG and T4. These studies indicate that T4 is the likely physiological regulator of plasma SHBG activity during the fall and early hibernation, but that other factors modulate the timing of SHBG induction in the spring.

Sex hormone-binding globulin and male sexual development.

The masculinization of the brain, reproductive tract and many other structures is critically dependent on the testicular hormone, testosterone (T). In many species, T circulates bound with high affinity to sex hormone-binding globulin (SHBG). This protein has a wide phylogenetic distribution and SHBG or SHBG-like proteins are produced by the liver, testes, placenta, brain and other tissues. SHBG activity is detectable during gestation and its expression is both stage- and tissue-dependent. Although SHBG binds circulating androgens, it is argued that the trapping of steroids in the circulation is not the principal function of this protein. The specific binding and uptake of SHBG by various tissues has been observed and suggests that SHBG may directly affect the delivery of androgen signals to target tissues. Effects of SHBG on androgen metabolism, tissue retention, cellular targeting, and action are reviewed. Evidence to date indicates that SHBG is able to enhance or inhibit the uptake of androgens in a cell- and tissue-specific manner. Future work will be necessary to demonstrate whether such actions of SHBG are important for normal male reproductive development.

Variants of the human prostate LNCaP cell line as tools to study discrete components of the androgen-mediated proliferative response.

Androgens regulate the proliferation of their target cells through a sequential two-step mechanism. In the first step, androgens increase proliferation rates; following this proliferative response, a second step of proliferative shutoff ensues. Human prostate LNCaP cell variants were used to assess whether the proliferative and shutoff effects of androgens could be segregated selectively by manipulating the hormonal milieu. The LNCaP-FGC and LNCaP-LNO cell lines were derived from the same biopsy specimen but exhibited different proliferative responses. Cell proliferation was inhibited by treatment with 10% charcoal-dextran stripped human serum in the LNCaP-FGC variant but not in LNCaP-LNO cells. Physiological androgen concentrations induced a proliferative shutoff in LNCaP-LNO cells (G1 arrest), an effect also expressed by LNCaP-FGC cells. This G1 arrest was irreversible in the LNCaP-FGC variant but was reversed upon androgen withdrawal in LNCaP-LNO cells. A new variant (LNCaP-TJA) was selected from LNCaP-LNO cells treated with 30 nM methyltrienolone for 4 months; these cells proliferated maximally regardless of the presence of androgens. All three cell variants had functional androgen receptors, and androgens induced prostate-specific antigen secretion. The androgen-induced proliferative shutoff in LNCaP-FGC and LNO cells was partially antagonized by antiandrogens and was not mediated by autocrine factors. Finally, the variant LNCaP cell lines described herein are probably representative of phenotypes present in prostate cancer patients during the course of this disease and may arise from selective pressure imposed by therapeutic protocols aimed at modifying the hormonal milieu of the host.

Biological effects of sex hormone-binding globulin on androgen-induced proliferation and androgen metabolism in LNCaP prostate cells.

The effects of purified human sex hormone-binding globulin (SHBG) on androgen-sensitive cell proliferation were examined using a human prostatic cell line (LNCaP-FGC). Cells were grown for 5 days in medium supplemented with 10% charcoal-dextran-stripped human serum (10% CDHuS) and various concentrations of 5 alpha-dihydrotestosterone (DHT). In 10% CDHuS, without SHBG, the proliferative response of these cells to androgens was typically biphasic. At low androgen concentrations, cell yields were increased in a dose-dependent manner, reaching maximal levels at 0.3 nM DHT. However, at high androgen concentrations, cell proliferation was inhibited. Addition of purified human SHBG to the medium reduced the effectiveness of DHT on both phases of the proliferative response in a dose-dependent manner. These effects of SHBG appeared to be due primarily to the high affinity binding of DHT by SHBG. Proliferative responses induced by the synthetic androgen methyltrienolone (R1881), which binds poorly to SHBG, were not affected by added SHBG. Furthermore, analysis of the protein binding of DHT revealed that cell proliferation correlated best with the concentration of DHT not bound to SHBG. The presence of SHBG in the medium also altered the uptake and metabolism of DHT. LNCaP-FGC cells rapidly metabolized DHT to a polar glucuronidase-sensitive conjugate of DHT. In 10% CDHuS, LNCaP-FGC cells conjugated virtually all of the added DHT during the 5-day experiment. However, in medium containing SHBG, the SHBG-bound DHT remained unconjugated; more than 90% of the DHT initially bound to SHBG was present in the medium at the end of the experiment as unconjugated DHT. Uptake of radiolabeled DHT by cells was also inhibited by SHBG. In summary, these experiments provide evidence that 1) SHBG-bound DHT is not a signal for DHT-induced cell proliferation and 2) SHBG inhibits the uptake and metabolism of DHT by LNCaP-FGC cells.

Patterns of plasma sex hormone-binding globulin, thyroxine and thyroxine-binding globulin in relation to reproductive state and hibernation in female little brown bats.

A sex hormone-binding globulin (SHBG), which bound both oestradiol and dihydrotestosterone, was studied in the plasma of adult female little brown bats throughout the annual reproductive cycle. This protein was present at low baseline levels from September to May inclusive, months which correspond to the periods of hibernation, ovulation and early pregnancy. During the second half of pregnancy in June, SHBG levels increased 15- to 30-fold and remained increased throughout lactation and anoestrus/prooestrus (July-August). Although SHBG was increased during late pregnancy, the fact that levels were also high during and after lactation indicates that this protein is not specific to pregnancy. Plasma concentrations of thyroxine (T4) and the percentage binding of T4 to thyroxine-binding globulin (TBG) also showed marked seasonal variations, with T4 levels exhibiting a biphasic seasonal pattern. A major peak in plasma concentrations of T4 occurred around the time of spring arousal from hibernation and subsequent ovulation, while a second peak of lesser magnitude was measured in August, corresponding to the time of pro-oestrus and the onset of mating. The percentage binding of T4 by TBG was increased during the summer months in parallel with the increase in SHBG concentrations. Electrophoretic analysis of plasma T4 binding revealed a single peak of TBG activity throughout most of the year; however, during the early lactational period TBG was resolved as a double peak, suggesting the presence of a molecular variant during this reproductive stage.

Post-natal patterns of plasma androgen-binding activity in Djungarian (Phodopus sungorus) and golden (Mesocricetus auratus) hamsters.

The binding of sex steroids to plasma proteins was examined in post-natal Djungarian and golden hamsters. With dihydrotestosterone or testosterone as ligands, steady-state polyacrylamide gel electrophoresis revealed 2 androgen binding components in the plasma of young Djungarian hamsters of both sexes. The fast-moving component exhibited a low affinity and high capacity for androgens and corresponded to albumin in stained gels. In contrast, the slow moving component was a beta-globulin with high affinity (Ka = 10(9) M-1) and low capacity for androgens, and was identified as a specific sex steroid-binding protein (SBP; also SHBG). This SBP did not bind oestrogens or corticosteroids and was electrophoretically distinct from corticosteroid-binding globulin (CBG). In addition, this protein did not appear to be of testicular origin because it was present in immature females and in immature males that had been castrated for 8 days. Plasma concentrations of SBP in males as measured by a diethyl-aminoethyl-cellulose binding assay were low at birth, became significantly elevated shortly thereafter when plasma androgen values were also elevated, and subsequently fell to low levels during puberty. These changes follow the same general pattern that has been described for other mammals, including humans, during this period of reproductive development. Although the significance of elevated SBP concentrations during prepuberty has yet to be determined, it appears that the increased concentrations of high-affinity androgen binding in the plasma of Djungarian hamsters plays a role in the asynchronous pubertal development of the testes and accessory organs which occurs in this species. The post-natal SBP pattern in females was similar to that observed in males. Plasma SBP levels were low or undetectable in adults of both sexes. As previously described for adult golden hamsters, the plasma of post-natal male and female golden hamsters lacked a specific SBP: androgen binding in this species is apparently limited to albumin and CBG.

Testosterone regulation of proopiomelanocortin messenger ribonucleic acid in the arcuate nucleus of the male rat.

GnRH regulates the secretion of LH and FSH, which stimulate the secretion of testicular hormones. Acting in a reciprocal fashion, these hormones, including testosterone and inhibin, exert a negative feedback effect on GnRH and gonadotropin secretion. Endogenous opioid peptides (EOPs) have been implicated to play a role in steroid-mediated regulation of gonadotropin secretion. In this context, certain steroid hormones (e.g. testosterone) increase EOP activity and ultimately inhibit GnRH secretion; however, the cellular mechanism by which this occurs is unknown. beta-Endorphin is one of these EOPs, and it is derived from a larger precursor molecule, POMC. We tested the hypothesis that testicular hormones and testosterone, in particular, stimulate POMC gene expression in the arcuate nucleus of the male rat brain. First, we compared POMC mRNA levels between intact and castrated male rats. Adult male rats were killed 4 days (n = 4) and 21 days (n = 5) after castration. Intact animals (sham-operated; n = 6) were used as controls. Using in situ hybridization and a computerized image analysis system, we measured the POMC mRNA content in individual cells of the arcuate nucleus. POMC mRNA signal was significantly lower (P less than 0.0003) in both 4-day (126 +/- 2 grains/cell) and 21-day (117 +/- 5 grains/cell) castrates than in controls (142 +/- 2 grains/cell). In a second experiment we tested whether testosterone would reverse the castration-induced loss of POMC message. Again, we castrated animals and immediately implanted them with either empty (sham; n = 6) or testosterone-containing Silastic implants (n = 5) of a size that would deliver physiological levels of testosterone (3.6 +/- 1.5 ng/ml). We observed that testosterone-treated animals had significantly higher levels of POMC mRNA signal (121.8 +/- 3.8 grains/cell) than sham-treated castrates (111.4 +/- 3.6 grains/cell; P less than 0.03) and that the testosterone-treated castrates had POMC mRNA signal levels indistinguishable from those of intact controls (122.0 +/- 1.1 grains/cell). These observations lend credence to the theory that one mechanism by which testosterone may regulate GnRH secretion is by increasing the synthesis of POMC in the arcuate nucleus.

Effects of chronic infusions of sex steroid-binding protein on the testosterone-mediated inhibition of gonadotropin secretion and maintenance of sex accessory glands in male rats.

Sex steroid-binding protein (SBP) is a plasma glycoprotein which, in man and many other mammals, binds significant amounts of circulating testosterone (T) with high affinity. To evaluate the effects of T binding by SBP on the control of reproductive function in males, SBP was purified from human plasma and administered by chronic infusion to castrated T-treated rats, animals that lack endogenous SBP. Plasma T concentrations in males implanted with 20-mm T capsules and infused with vehicle for 48 h were maintained at 1.37 +/- 0.14 ng/ml, and plasma LH and FSH concentrations remained at intact levels. Castrates implanted with identical T capsules but infused with human (h) SBP at a rate that produced a steady state plasma hSBP concentration of 75 nM, had markedly higher plasma T concentrations (3.44 +/- 0.32 ng/ml); however, circulating LH and FSH concentrations were not significantly different from those of the vehicle-infused controls. Similarly, the pituitary LH response to a bolus injection of LHRH was not affected by hSBP infusions. Measurements of tissue concentrations of T, dihydrotestosterone, and 3 alpha-androstanediol in the ventral prostate and seminal vesicles, as well as the weights of these organs, failed to reveal any effects of hSBP infusions on the uptake, metabolism, or action of T. Analysis of T distribution in plasma showed that the hSBP infusions produced marked increases in total plasma T levels but resulted in only marginal reduction in the concentration of T not bound to hSBP. These results are consistent with the hypothesis that T not bound to SBP is the major feedback-effective moiety and that under conditions of constant release of T into the circulation, an increase in plasma T binding by SBP increases the total plasma T concentration but does not appear to significantly alter the feedback-effective levels of T in the circulation. Therefore, it would appear that moderate changes in the amount of high affinity binding of T in the circulation would have little, if any, effect on androgen balance in adult males.

Changes in populations of LHRH-immunopositive cell bodies following gonadectomy.

One day after castration of male rats, plasma LH rose and the number of LHRH immunopositive neuronal perikarya decreased. As plasma LH continued to rise six days and three weeks post-castration, the number of LHRH immunopositive neurons also increased. The largest population of LHRH immunopositive neurons was detected three weeks post-castration and the cell group that showed the greatest increase was in the rostral preoptic area. In females, the largest population of LHRH immunopositive neurons was observed one day post-ovariectomy; at this time plasma LH levels were not significantly elevated above diestrous levels. Six days post-ovariectomy, LH levels were elevated and the number of LHRH immunopositive cells decreased. As LH levels continued to rise three weeks post-ovariectomy, the population increased in size. In males, primarily LHRH cells of the rostral preoptic area increased in in number; in females, the cell groups that increased were scattered over the diagonal band of Broca, preoptic and anterior hypothalamic areas. Although LHRH neurons demonstrated these variations following gonadectomy, there was no evidence of alteration(s) in molecular processing of precursor hormone.

Plasma sex steroid binding in Chiroptera.

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

Morphological evidence that luteinizing hormone-releasing hormone neurons participate in the suppression by estradiol of pituitary luteinizing hormone secretion in ovariectomized rats.

Morphological characteristics of LHRH neurons identified by immunocytochemistry were studied using light and electron microscopy in female rats in which estradiol was replaced at the time of ovariectomy ('pseudo-intact' rats) or 3 weeks after ovariectomy (long-term ovariectomized, estradiol-treated). While estradiol levels were equivalent in these two groups, the rise in LH after ovariectomy was prevented by the immediate administration in the pseudo-intact rats, while the augmented plasma LH levels present three weeks following ovariectomy were only reduced by 50% as a result of delayed estradiol treatment. The LHRH content of the medial basal hypothalamus (MBH) including the median eminence (ME) was greater in pseudo-intact females than in untreated long-term ovariectomized control females or long-term ovariectomized, estradiol-treated females, both 1 and 14 days after estradiol exposure. Immunocytochemistry revealed fewer LHRH-immunopositive neuronal processes coursing throughout the MBH and terminating in the ME of long-term ovariectomized, estradiol-treated rats compared to those in pseudo-intact rats. However, within individual neurovascular terminals in the ME, image analysis revealed that the area of reaction product was greater in long-term ovariectomized, estradiol-treated animals. Equivalent amounts of LHRH were assayed in the MBH within each group of animals by several LHRH antisera regardless of their different binding requirements (R42, IJ29 and A-R743), suggesting that the predominant moiety present in neuronal terminals is the fully mature decapeptide. In contrast, in the preoptic area-anterior hypothalamus (POA-AH) these antisera assayed amounts of LHRH that varied as a function of binding characteristics, although the quantities did not vary with the estradiol treatment schedule. Immunocytochemical results paralleled these assay data; antisera requiring an interior sequence of amino acids (A-R743 and A-R419) detected approximately 3 times as many immunoreactive perikarya in the POA-AH as did an antiserum requiring the free amidated C terminal (IJ29). The estradiol treatment schedules had no effect on the total number of LHRH-immunopositive neurons detected by each antiserum or the distribution of LHRH-immunopositive neuronal perikarya. These data support the hypothesis that the predominant moieties present in neuronal cell bodies are precursor forms. The fine-structural characteristics of LHRH-immunopositive neuronal cell bodies are consistent with greater secretory and biosynthetic activity in LHRH neurons of long-term ovariectomized, estradiol-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of sex steroid-binding protein (SBP) in the male little brown bat: relationship of plasma thyroxine levels to the induction of plasma SBP in immature males.

The seasonally reproductive male little brown bat (Myotis lucifugus lucifugus) exhibits marked increases in plasma concentrations of sex steroid-binding protein (SBP) in the spring following arousal from hibernation. In this species an increase in SBP levels is induced prematurely in male bats aroused during the first half of hibernation and housed under long photoperiods; however, this rise is inhibited in bats housed under short photoperiods. In order to investigate the physiological role of the thyroid gland in the regulation of plasma SBP activity, plasma total thyroxine (T4) and SBP concentrations were determined in immature male little brown bats prematurely aroused from the first half of hibernation and maintained on either a short or long photoperiod. For this purpose a radioimmunoassay for the measurement of total T4 in bat plasma was established and validated. The results showed that immature male little brown bats aroused prematurely from hibernation and housed under a long spring-like photoperiod exhibited marked increases in plasma T4 and SBP concentrations, while animals housed under a short photoperiod showed only marginal increases in SBP, and plasma T4 remained undetectable. These results suggest that the thyroid gland, through the action of T4, may normally play an important role in the control of the post-arousal rise in plasma SBP concentrations in the little brown bat.

Annual variations in plasma sex steroid-binding protein and testosterone concentrations in the adult male little brown bat: relation to the asynchronous recrudescence of the testis and accessory reproductive organs.

The annual reproductive cycle of the male little brown bat, in contrast to seasonal reproductive patterns of other mammals, is differentiated by an asynchronous recrudescence of the testis and the accessory reproductive glands. Spermatogenesis occurs during the summer, whereas fully stimulated accessory organs, stored epididymal spermatozoa, and sexual behavior are expressed later during a mating period that extends, albeit interrupted by hibernation, from late summer until early spring. To investigate whether changes in high affinity androgen-binding activity in the circulation are related to the delayed renewal of the accessory organs, plasma sex steroid-binding protein (SBP) and total testosterone (T) levels were measured throughout the year. From these data and determinations of association constants for T binding to SBP and albumin at both hibernating (4 degrees C) and active (40 degrees C) temperatures, estimates of the unbound ("free") and albumin-bound T fractions were made and correlated with changes in the accessory reproductive organs. Plasma SBP concentrations (mean +/- SEM) exhibited wide seasonal fluctuations: they were baseline in May (10 +/- 2 nM) following spring arousal, increased dramatically in June (184 +/- 24 nM), and reached peak levels in early July (262 +/- 29 nM), where they remained until August. In late August they began to fall (104 +/- 23 nM) and then returned to baseline during the hibernation period (October-April). Although total T levels were also elevated in June, it appeared that the unbound ("free") and the unbound plus albumin-bound T fractions did not increase until late July. Since the accessory gland weights did not begin to increase until late July as well, it was concluded that increases in the unbound and albumin-bound T fractions may be an important factor in the recrudescence of the accessories and that increased SBP activity in early summer may play a role in the regression and delayed renewal of these organs. However, what factor(s) maintain the accessory glands, epididymal spermatozoa, and sexual behavior during the breeding and hibernation periods when all T fractions were low are, as yet, undetermined.

Control of plasma sex steroid-binding protein (SBP) in the little brown bat: effects of thyroidectomy and treatment with L- and D-thyroxine on the induction of SBP in adult males.

The male little brown bat is a seasonally reproductive mammal that exhibits dramatic increases in plasma concentrations of sex steroid-binding protein (SBP) in the spring, following arousal from hibernation. Adult male bats, aroused prematurely from hibernation, were found to exhibit increases in plasma SBP titers that were comparable to those observed during normal spring arousal. To evaluate the role of the thyroid gland in the control of SBP in this species, plasma SBP concentrations were determined at weekly intervals in adult male bats that were either thyroparathyroidectomized (TRX) or sham operated (SHAM) after arousal from hibernation. Plasma SBP titers in SHAM males increased markedly within the first week after arousal and by 3 wk had reached levels 20-fold higher than those measured in hibernating controls. In contrast, plasma SBP values in the TRX animals did not increase significantly following arousal but were maintained at low basal levels throughout the experiment. The postarousal rise in SBP, which was blocked by TRX, was completely restored by implantation of either L- or D-thyroxine pellets. In male bats, TRX also hindered the normal postarousal atrophy of the sex accessory glands and resulted in attenuation of the postarousal increases in plasma testosterone concentrations. These effects of TRX were also prevented by treatment with thyroxine. Thus, the thyroid appears to play a significant role in the control of the postarousal rise of SBP in the little brown bat and may be an important factor in the regulation of reproductive function in this species.

Relationship of food intake to the induction of plasma sex steroid-binding protein and testicular activity in immature male little brown bats (Myotis lucifugus lucifugus).

Immature male little brown bats were aroused prematurely from their first hibernation and fed ad libitum (Group AL) or given a restricted diet (Group FR). All animals were weighed daily and the food intake of Group FR males was restricted to maintain their body weights at or near initial post-arousal values (5.3-6.8 g). The average body weights of Group AL males increased during the first week after arousal and then stabilized at a level which was 20% higher than those of Group FR males. The post-arousal induction of plasma SBP was similar in Groups FR and AL: plasma SBP activity was significantly increased 1 week after arousal and by 3 weeks had reached levels which were more than 10-fold higher than those of immature hibernating males which served as controls. Although food restriction had no effect on plasma SBP levels, it did inhibit reproductive development. Arousal-induced increases in testicular and epididymal (head/body) weights in Group FR males were less than 50% of those in Group AL males. However, histological examination of the testes revealed similar degrees of spermatogenic activation in both groups. Plasma testosterone concentrations were increased markedly in Groups FR and AL; values were generally lower in Group FR but wide individual variations were observed. Despite these elevated peripheral testosterone values, the accessory sex glands in both groups remained unstimulated.

Perinatal and postnatal patterns of plasma sex steroid-binding protein and testosterone in relation to puberty in the male little brown bat.

The little brown bat (Myotis lucifugus lucifugus) possesses a specific plasma sex steroid-binding protein (SBP) which exhibits high affinity for both testosterone (T) and estradiol. To examine the peri- and postnatal patterns of plasma androgen binding in relation to puberty, concentrations of SBP and total T levels were measured in males at various intervals throughout the first year of life. In addition, reproductive organs were examined histologically over this period. SBP levels were low (less than 7 nM) in near-term fetuses (mid-June) but increased more than 30-fold by 1 week of age (range, 245-602 nM). Elevated titers of this protein were present until 3 weeks of age. By 4 weeks of age (onset of puberty, initiation of the first spermatogenesis), SBP concentrations had fallen significantly, and by 12 weeks, just before the first winter hibernation period, plasma SBP levels had returned to low prenatal-like values (3-12 nM). These levels were maintained throughout winter dormancy. The total plasma T concentrations were also elevated during the early postnatal period and were comparable to or even exceeded those previously measured in spermatogenically active adults. Plasma T levels then declined in parallel to those of SBP and reached a nadir (less than 0.6 ng/ml) by 12 weeks of age. As with SBP, T also remained low throughout hibernation. Although a complete spermatogenic cycle was found to occur in postnatal bats during the summer of their birth, this cycle was greatly reduced compared to that which occurs in sexually mature adults during the same period. Maximal testicular weights from the postnatal males were only one fifth of those previously measured in adult males. In addition, the epididymides in the young bats remained virtually devoid of sperm, and the accessory glands showed little evidence of stimulation. Thus, this first spermatogenic cycle in Myotis does not result in the attainment of sexual maturity and, therefore, appears to be "silent." Since sexual maturity is reached during the following summer when the bats are approximately 1 yr of age, the pubertal process in Myotis is prolonged and appears to be biphasic. Nevertheless, the existence of a perinatal rise in SBP levels (which is repeated in this species when the pubertal process is reinitiated during the second summer) as well as a decline in SBP levels during the onset of puberty resembles that previously described for other mammals.