A single genetic determinant that prevents sex reversal in C57BL-YPOS congenic mice.

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (YPOS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XYPOS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XYPOS normal males generated in these crosses were compared to XYPOS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XYPOS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the YPOS Sry gene.

The potential roles of estrogens in regulating Leydig cell development and function: a review.

It is generally agreed that estrogens, principally estradiol-17beta, are synthesized by and act in the testis of mammals, including humans. The site of estradiol synthesis in the testis is generally believed to begin in the Sertoli cell and switch to the Leydig cell during neonatal development where a gonadotropin-regulated aromatase is present. Numerous studies suggest that the primary target cell of estradiol in the testis at all ages is the Leydig cell. In fact, the Leydig cell is known to possess an estrogen receptor that binds estradiol in the classic manner. The mechanism of estradiol action and the role of its receptor in the testis, however, remain unresolved. In Leydig cells, estradiol appears to induce several alterations that are dependent in large part on the developmental stage of the Leydig cell. In the fetal and neonatal testes, estradiol appears to block the ontogenic development of Leydig cells from precursor cells. There is also evidence that estradiol similarly blocks the regeneration of Leydig cells in the testis of mature, ethane dimethylsulfonate-treated animals. Evidence indicates that the precursor cell possesses high levels of estrogen receptors relative to that of the Leydig cell. It is postulated that estradiol is a paracrine factor involved in regulating the interstitial population of Leydig cells. Evidence also indicates that estradiol acts directly in the mature testis to block androgen production. It appears to do so by inhibiting the activities of several steroidogenic enzymes involved in testosterone synthesis. Although the more conventional receptor-mediated mode of action is feasible, several studies have suggested that this action might entail direct competitive inhibition of key steroidogenic enzymes by estradiol. In summary, the net biologic effect of estradiol in the testis appears to be inhibition of androgen production, either by limiting development and growth of the Leydig cell population or through direct action in the Leydig cell.

Gene expression of luteinizing hormone receptor and steroidogenic enzymes during Leydig cell development.

Testicular Leydig cells (LC) are rapidly and selectively destroyed by an injection of ethane dimethane sulfonate (EDS). LC regeneration occurs in the testis of the EDS-treated rats from the differentiation of the precursor Leydig cells (PLC). This study was designed to investigate the patterns of change in the mRNAs for the luteinizing hormone receptor (LHR) and the steroidogenic enzymes, cholesterol side chain cleavage (P-450scc) and 17 alpha-hydroxylase (P-450(17 alpha)) during LC regeneration from PLCs. Mature (60 days of age) Sprague-Dawley male rats received a single intraperitoneal injection of EDS and were killed at different times between days 2 and 60 post-treatment. PLC- and LC-enriched fractions were isolated from the testes of the EDS-treated rats and age-matched control rats using a collagenase digestion-Percoll gradient method. Total RNA was extracted from these cell populations and subjected to Northern blot analysis. The LC fraction isolated from testes of control rats expressed four major transcripts of the LHR, sized 1.8, 2.5, 4.2 and 7.0 kb. The undifferentiated PLC fraction from controls expressed only a truncated form, the 1.8 kb transcript. This truncated LHR transcript was also the only LHR mRNA species detected in PLCs at day 2 post-EDS treatment. In contrast, all four transcripts of the LHR were detected in the PLC fraction at day 10 post-EDS treatment. The levels of the full length 7.0 kb transcript increased thereafter and reached a peak between days 24 and 36 post-EDS treatment in the PLC fraction. Concomitant with the increase in the 7.0 kb transcript, the truncated 1.8 kb transcript decreased in amount and reached a nadir between days 16 and 36 post-treatment. The changes observed in this cell fraction reflect the process of differentiation of PLCs into LCs. At day 45 post-EDS treatment, the level of the 7.0 kb transcript decreased while the 1.8 kb form increased in the PLC fraction, reflecting the completion of LC regeneration from this cell fraction. By day 60 post-EDS treatment, the levels of the 1.8 kb transcript rose to the value observed in undifferentiated control PLCs and the other transcripts were no longer detected in the PLC fraction, indicating that cells in the PLC fraction were again in an undifferentiated stage. Messenger RNAs for both the steroidogenic enzymes, P-450scc and P-450(17 alpha) were expressed in the control LC fraction. Neither of these two mRNAs were detected in the PLC fraction of the control rats. P-450scc and P-450(17 alpha) mRNAs were first expressed in the PLC fraction at day 10 post-EDS treatment. Thereafter, the levels of P-450scc and P-450(17 alpha) mRNAs increased in the PLC fraction and reached a peak between days 24 and 36 and days 24 and 45 post-EDS treatment respectively. P-450scc and P-450(17 alpha) mRNAs were no longer expressed in the PLC fraction at day 60 post-EDS treatment. These patterns also reflect the process of differentiation of PLCs into functional LCs. These results demonstrate for the first time that PLCs in the control testis are undifferentiated and do not express functional LHR and steroidogenic enzymes or their mRNAs. The PLCs are characterized, however, by the expression of a truncated 1.8 kb transcript of the LHR mRNA. Functional LHR and steroidogenic enzymes are expressed in PLCs only during their differentiation into LCs after EDS treatment. Subsequent to LC regeneration, the PLCs return to an undifferentiated stage.

Use of a rat cDNA probe specific for the Y chromosome to detect male-derived cells.

A cDNA probe that exhibits specificity for the rat Y chromosome was generated by using a set of primers specific to the murine Sry gene, the sex-determining region of the Y chromosome. A 459-base pair (bp) DNA fragment was obtained by polymerase chain reaction (PCR) amplification from male, but not female, rat genomic DNA (EMBL Nucleotide Sequence Database accession number X89730). This DNA fragment was purified, cloned using a vector, and digested with EcoR1 to yield a 270-bp DNA fragment. This 270-bp cDNA fragment, when used as a probe in Southern blot analysis of rat DNA, was observed to bind to three separate bands of approximately 2.3, 5.0, and 7.0 kb in size. The binding was demonstrated with male, but not female, genomic DNA. Another set of primers was generated to sequences within the 270-bp fragment that produced a PCR product of 104 bp. This DNA fragment, when used as a probe in Southern blot analysis, enabled PCR detection of at least 0.1% male cells in a mixed population of female cells. These cDNA probes should prove useful in studies designed to track cell populations (e.g., tumor metastasis and hemopoietic cells after bone marrow transplantation) in syngeneic male/female pairs. In addition, a cDNA probe that is specific for the rat Sry gene might be valuable in studies of fetal male sexual development or the study of spermiogenesis.

Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells.

PURPOSE: Bovine corneal epithelial cells (BCEC) were cultured to determine whether endothelin (ET) receptor subtype stimulation affects ET isoform expression (ET-1, ET-2, and ET-3) through capacitative Ca2+ influx. To probe in the isolated bovine corneal epithelium (BCE) for ET isoform and ET (i.e., ETA and ETB) receptor gene expression. METHODS: [Ca2+]i transients were characterized with microfluorometry. Endothelin isoform and ET receptor gene expression were probed with RNase protection analysis. Enzyme-linked immunosorbent assay was used to measure levels of ET-1-like immunoreactivity (ET-1-LI) in conditioned medium. RESULTS: ET-1 (10(-6) M) increased [Ca2+]i more than twofold. After treatment with 10(-7) M alltrans retinoic acid (an inducer of differentiation), 10(-6) M sarafotoxin (S-6-c) (a selective ETB agonist), had a similar effect. Preincubation with either 5 microM U73122 (an inhibitor of IP8 formation) or 10 microM cyclopiazonic acid, which depletes intracellular Ca2+ store content, eliminated ET agonist-mediated [Ca2+]i increases. With a nominally Ca(2+)-free solution containing 10 microM cyclopiazonic acid, simultaneous 10(-6) M ET-1 and extracellular Ca2+ additions transiently increased [Ca2+]i twofold, whereas 10(-6) M S-6-c increased it by only 20%. This augmentation was eliminated by preexposure to either BQ123 (10 microM), selective ETA receptor antagonist, U73122 (5 microM), or SKF 96365 (3 x 10(-5) M), an inhibitor of stores-operated channels. ET-1, ET-2 isoforms, and ET receptor mRNAs were identified. S-6-c (10(-6) M) increased the level of ET-1-LI after 12 hours by approximately ninefold. CONCLUSIONS: In BCEC, capacitative calcium influx is involved in mediating a positive feedback relationship between ETB receptor stimulation and ET protein expression. Identification of ET-1 and ET-2 gene expression in BCE strengthens the notion that this regulation could be autocrine mediated.

Estrogen receptor messenger ribonucleic acid changes during Leydig cell development.

Mature (60-65 days old) male Sprague-Dawley rats received a single i.p. injection of ethane dimethane sulfonate (EDS, 100 mg/kg BW) and were killed at different times from Days 2 to 60 posttreatment. Bands of cells enriched in precursor Leydig cells (PLCs) and Leydig cells (LCs) were isolated from the testis of EDS-treated rats and age-matched controls using a collagenase digestion-Percoll gradient method. Total RNA extracted from the PLC and LC fractions was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) to detect estrogen receptor (ER) mRNA. The RT-PCR results demonstrated that ER mRNA was present in both LC and PLC fractions. Quantitative RT-PCR analysis, using rabbit beta-globin mRNA as the internal standard, showed that ER mRNA in the PLC fraction was 20-fold higher than in the LC fraction in control testis. After EDS treatment, ER mRNA levels in the PLC fraction decreased and reached a nadir at Day 16 posttreatment. Thereafter, ER mRNA in the PLC fraction gradually increased and returned to control PLC levels. In contrast, ER mRNA levels in the LC fraction in controls and at Days 16-45 posttreatment remained constant. To correlate the changes in ER mRNA levels with LC differentiation, in vitro testosterone (T) production by PLC- and LC-enriched fractions in the presence or absence of 50 mIU hCG was measured by RIA. T production in the control PLC fraction was low (1/10th that in the control LC fraction), and hCG addition resulted in only a 1.5-fold stimulation (relative to a 7.5-fold stimulation in LCs). In the PLC fraction, T production was not detectable at Days 2 and 10 after EDS treatments, began to respond to hCG stimulation with increased T production at Day 16, and reached a maximum between 4 and 6 wk after EDS treatment. By Day 60 posttreatment, T production in the PLC fraction decreased and returned to control PLC levels. Testosterone production in the LC fraction was not detectable at Days 2 and 10 posttreatment. From Days 16 to 60 posttreatment, LC basal and hCG-responsive T production increased gradually and returned to control LC levels. It is concluded that functional LCs are regenerated from the PLCs and that both these cell types possess ER mRNA. It is interesting to note that PLCs exhibit higher levels of ER mRNA than do LCs. A decrease in ER mRNA in PLCs appears to coincide with the early differentiation process to yield LCs. Thus, estradiol-17 beta produced locally in the testis by the LCs might act via its receptor as a paracrine substance to impede PLC development into LCs. It is therefore possible that either a decrease in E2 production or a decrease in ER and its mRNA in PLCs would then release the PLCs to begin the regeneration process.

ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells.

PURPOSE: To determine if there is a heterogeneous pattern of endothelin (ET) receptor subtype (i.e., ETA and ETB) gene expression in the bovine corneal epithelium (BCE). To determine if ET receptor subtype stimulation increases the effectiveness of epidermal growth factor (EGF) to accelerate wound closure in a primary culture of bovine corneal epithelial cells (BCEC). METHODS: In situ hybridization histochemistry was used to characterize ETA and ETB gene expression in the BCE. A wound closure assay evaluated wound healing rates in BCEC after 4 to 7 days in culture. [3H] thymidine incorporation and MTT assay measured proliferation. RESULTS: ETA gene expression was appreciably higher in the basal cells than in the suprabasal cells, whereas the pattern for ETB was reversed. Epidermal growth factor (5 ng/ml) maximally increased wound closure by 145% above the control. With 5 ng/ml EGF, either 10(-9) M ET-1 or 10(-8) M sarafotoxin-6-c (s-6-c) increased wound closure by an additional 39% (P < 0.001) above that measured with 5 ng/ml EGF alone. BQ123 (10(-7) M) did not alter any of these effects of ET-1 or s-6-c. Epidermal growth factor stimulated wound closure through a selective increase in proliferation. Neither ET-1 nor s-6-c alone had any effect on proliferation or migration. CONCLUSIONS: Both ETA and ETB genes are expressed in BCE. However, in BCEC only, ETB stimulation increases the effectiveness of EGF to stimulate wound closure. This response was caused by an increase in cell migration rather than proliferation because, after treatment with mitomycin C, neither ET-1 nor EGF stimulated wound closure.

Biosynthesis and metabolism of carnitine.

This review article presents the biosynthesis, metabolism, sources, levels, and general functions of carnitine. Emphasis is placed on the expression of carnitine deficiency and insufficiency as well as the causes of these conditions. The various functions of carnitine are discussed as they may relate to disease treatment.

Cell specific distribution of LH/hCG receptor messenger ribonucleic acid in rat testicular Leydig cells.

By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.

17 beta-estradiol inhibition of Leydig cell regeneration in the ethane dimethylsulfonate-treated mature rat.

This study was designed to determine the effects of 17 beta-estradiol (E2) on Leydig cell development in the rat. Mature (60 to 65 days old) male rats received a single intraperitoneal injection of ethane dimethylsulfonate (EDS, 100 mg/kg body weight); untreated rats served as controls. In one series of experiments, groups of EDS-treated rats also received daily injections of either E2 (25 micrograms/100 g body weight), human chorionic gonadotropin (hCG, 20 IU/day), a combination of the two, or vehicle only (EDS controls). Animals were killed on days 2, 4, 10, 16, 24, 30, and 36 after EDS treatment. In another series of experiments, groups of EDS-treated rats received daily injections of hCG and E2 during days 0 through 5, 5 through 30, or 16 through 30 after EDS treatment, and were killed on day 30. In both series of experiments, the steroidogenic capacity and hCG binding capacity of the Leydig cells were examined in short-term in vitro incubations using collagenase-dispersed interstitial cells. Testes were also prepared and examined histologically by light and electron microscopy. E2 treatment of animals during the initial 5 days after EDS administration had no effect on the regeneration of interstitial cells and Leydig cells. Treatment with E2 during days 5 to 30 post-EDS blocked the regeneration of Leydig cells and thereby significantly reduced the increase in interstitial cell numbers. Finally, when E2 treatment was delayed until 16 days post-EDS, there was no significant reduction in the regeneration of interstitial or Leydig cells. These data suggest that an important developmental process that is necessary for Leydig cell regeneration occurs between days 5 and 16 post-EDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Interstitial cell proliferation in the testis of the ethylene dimethane sulfonate-treated rat.

This study was conducted to examine interstitial cell proliferation in the testis of the ethylene dimethane sulfonate (EDS)-treated rat. Initial autoradiographic studies demonstrated a peak of [3H]thymidine incorporation by interstitial cells at 2 and 4 days post-EDS treatment. Subsequent studies were designed using in vivo pulse labeling regimens in an attempt to identify interstitial cell proliferation associated with Leydig cell regeneration. Rats were injected with [3H]thymidine at days 2 and 4 post-EDS and were killed 6 hours later or at 30 days post-EDS. Although cells labeled at 2 and 4 days post-EDS appeared to undergo subsequent division, the Leydig cells visible at 30 days post-EDS were not labeled. In a second study, rats were injected with [3H]thymidine at days 10 and 20 post-EDS and were killed either 6 hours later or at 24 days post-EDS. In the 10-day post-EDS group, interstitial cells were labeled at both the 6-hour and 24-day time points; however, Leydig cells present at 24 days were not labeled. In contrast, the testes of rats that were killed at 20 days post-EDS (6 hours labeling period) contained Leydig cells that displayed grains over the nucleus, thus suggesting that Leydig cell proliferation had occurred. In addition, a high number of the Leydig cells observed at 24 days post-EDS were labeled, suggesting that they arose from divisions occurring during the 20- to 24-day post-EDS period. These studies demonstrate that interstitial cell proliferation occurs in several stages following EDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Testosterone and androstanediol production by regenerating Leydig cells in the ethylene dimethane sulphonate-treated mature rat.

Mature (60-65 day old) male Sprague-Dawley rats received a single intraperitoneal injection of ethylene dimethane sulphonate (EDS; 100 mg/kg) and were subsequently killed at various times from day 2 to day 40 post-treatment. Testes were removed from these animals and age-matched controls and utilized either for light and electron microscopical analyses or for in-vitro assessment of Leydig cell function. Interstitial cells were prepared by collagenase digestion and used to measure 125I-labelled human chorionic gonadotrophin (hCG) binding capacity and androgen production in the presence or absence of hCG or dibutyryl cyclic AMP (dbcAMP). At day 2 after EDS treatment, 125I-labelled hCG binding capacity was reduced to 10% of control values, while the production of testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (adiol) were non-detectable. Histological observations confirmed the lack of identifiable Leydig cells at day 2-16 after EDS treatment. Between days 24 and 40 post-treatment, Leydig cell regeneration occurred, as indicated by a rise in 125I-labelled hCG binding capacity, increased androgen production and the presence of histologically identifiable Leydig cells. A pattern of adiol production similar to that seen in the immature rat during Leydig cell development was observed with peak synthesis occurring at day 30 post-treatment. Adiol production fell to barely detectable levels by day 36 and remained low at day 40. It is concluded that the steroidogenic pattern of regenerating Leydig cells in the EDS-treated animal is similar to that of developing Leydig cells in the immature animal.

The effects of reduced O2 and antioxidants on steroidogenic capacity of cultured rat Leydig cells.

We have examined the effects of reduced O2 tension and the antioxidant dimethylsulfoxide (DMSO) to determine if O2-derived free radicals are the cause of decreased steroidogenic capacity (testosterone and progesterone production) of cultured rat Leydig cells. Rat Leydig cells were initially cultured under standard conditions of 5% CO2, 95% air (19% O2) with or without DMSO. Addition of DMSO resulted in increased basal testosterone production on days 2, 3 and 4 of culture. hCG (10 mIU)-stimulated testosterone secretion was 2-3 times greater on days 2 and 3 in the presence of DMSO. Lowering the O2 concentration to 5% in the presence of DMSO resulted in even greater hCG-stimulated testosterone production on days 1 to 3. However, the effect of DMSO or low O2 and DMSO were not seen after 5 days. The reduced O2 concentration resulted in an increase in hCG (10 mIU)-stimulated progesterone synthesis throughout the culture, particularly on days 4 to 8. Also, when total steroid (progesterone and testosterone) was determined, cells cultured under reduced O2 conditions responded with increased steroid production on days 1 to 8 in comparison to controls (19% O2). These results demonstrate that lowered O2 concentration and DMSO provide a protective effect resulting in the maintenance of testosterone production and an increase in progesterone synthesis. These findings suggest that free radical-mediated damage of enzymes may result in decreased steroidogenic capacity of cultured Leydig cells.

Fibrocystic breast disease: the significance of beta-human chorionic gonadotropin and other polypeptides in breast cyst fluid.

Breast cyst fluids from 118 women, aged 29 to 69 years, were analyzed by radioimmunoassays for beta-human chorionic gonadotropin (beta-hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and thyroid-stimulating hormone (TSH). Blood was drawn at the same time in many cases to compare hormonal levels in serum with those in the breast cyst fluids (BCF). The levels of beta-hCG in BCF were relatively high, with a mean (+/- standard error of the mean [SEM]) of 58.9 +/- 16.8 mIU/ml; serum levels of beta-hCG were negligible. LH and TSH also were elevated in BCF compared with serum levels, exhibiting mean values (+/- SEM) of 26.7 +/- 4.3 mIU/ml and 6.4 +/- 0.44 muIU/ml, respectively. The levels of FSH and PRL in BCF were equivalent to the levels in the serum. The presence of biologically active hCG was suggested in several BCF samples using the rat ovarian hyperemia test. Samples of BCF were assessed for the capacity to stimulate Leydig cell testosterone production in vitro in the presence or absence of an anti-hLH antiserum. Testosterone production was significantly (P less than 0.05) enhanced, even in the presence of the antiserum. These data suggest that BCF contains biologically active hCG.

Localization of gamma-butyrobetaine hydroxylase in the rat testis.

The activity of gamma-butyrobetaine hydroxylase in testes is comparable on a protein basis to the amount of activity found in liver and accounts for a significant amount of the total activity found in the rat. The enzyme is localized to the seminiferous tubules of the testes. In attempting to further localize this enzyme, activity remained the same when reported on a protein basis in cryptorchid testes as compared to controls, and activity was found in bovine sperm cells obtained from the caput epididymis. These results are consistent with the localization of this enzyme in both the Sertoli cells and the germ cells.

Depletion-replenishment of the testicular estrogen receptor: sensitivity to cycloheximide and actinomycin D.

Male rats (30-35 days old) were utilized to examine the process of depletion and replenishment of the testicular cytosolic estrogen receptor and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose dependence and temporal nature of receptor depletion and replenishment were investigated. Maximum depletion (greater than 90%) occurred by 1 h after in vivo administration of either 5 or 10 micrograms estradiol-17 beta. Depletion of the cytosolic receptor at 1 h occurred concomitant to a marked increase in nuclear receptor thus indicating translocation. Receptor replenishment to control levels was observed by 6 h post treatment. To determine the requirements for transcriptional and translational events in the replenishment process, actinomycin D and cycloheximide were administered in vivo. Simultaneous treatment with cycloheximide and estradiol resulted in a significant inhibition of replenishment at 6 and 12 h post treatment of 46 and 60% below control levels, respectively. Cycloheximide treatment alone had no effect on receptor levels. A significant inhibition of replenishment at 6 h was also shown when cycloheximide was given 3 h after estradiol treatment. Cycloheximide administration at 6 h after estradiol significantly suppressed receptor levels at 12 h suggesting that replenished receptor levels at 6-12 h are in a state of rapid turnover. Receptor replenishment exhibited a different response to actinomycin D treatment in that significant inhibition was observed only when the drug was administered at 3 h after estradiol treatment. These results demonstrate that testicular cytosolic receptor depletion is dose and time dependent. The results further demonstrate that receptor replenishment involves protein synthesis and suggests that synthesis of new RNA might also be required.

Gonadotropin regulation of Leydig cell DNA synthesis.

Adult male rats were injected s.c. with either saline, 100 IU hCG, 100 micrograms FSH, 50 micrograms LH, 100 micrograms PRL, 50 micrograms estradiol-17 beta, 500 micrograms or 10 mg testosterone; 50 micrograms estradiol-17 beta; animals were sacrificed at 12-120 h post-injection. Collagenase-dispersed interstitial cells (150-200 X 10(6) cells/2 ml) were incubated in vitro with 10 microCi [3H-methyl]thymidine for 1 h at 32 degrees C. Centrifugation of the cells on discontinuous 11-27% metrizamide gradients revealed thymidine incorporation in the regions of population I and II Leydig cells. A significant increase in thymidine incorporation into DNA after treatment with either hCG or LH was first detectable at 48 h, was equivalent to control values at 72 h and was again significantly increased at 96 h in population I and at 120 h in population II cells. [3H]Thymidine incorporation at 48 h, expressed as dpm/10(6) cells, was 2205 +/- 432 and 4119 +/- 929 vs. 16473 +/- 3795 and 11648 +/- 3427 for control and hCG-treated population I and II cells, respectively. Addition of 20 mM hydroxyurea suppressed [3H]thymidine incorporation, 97% and 96% in hCG-treated population I and II cells, respectively. Autoradiographic analyses revealed that nuclei from control and 48 h hCG-treated population I and II cells exhibited 1.2% and 2.3% vs. 7% and 6.8% silver grains, respectively. PRL had no influence on LH/hCG-enhanced DNA synthesis; however, estradiol-17 beta administration for 48 h dramatically suppressed thymidine incorporation. Population I Leydig cells exhibited a higher level of LH/hCG-stimulated DNA synthesis compared to population II cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal influence on experimental infections by a toxic shock strain of Staphylococcus aureus.

Subcutaneous infection chambers in rabbits were infected with a strain of Staphylococcus aureus isolated from a patient with toxic shock syndrome. Estrogens (mestranol and 17-beta-estradiol) protected male rabbits and prolonged survival. Neither androgens (testosterone and dihydrotestosterone) nor progesterone affected the susceptibility of intact or ovarihysterectomized female rabbits.

Temporal effects of diethylstilbestrol administration in vivo on testosterone production in Leydig cells.

The temporal nature of estrogenic suppression of Leydig cell testosterone production was investigated. Adult rats were injected SC with 50 micrograms/100 g BW of DES or vehicle. Animals were sacrificed at 4, 8, or 12 h following a single injection or at 12 h following the latter of two daily injections for 1 or 2 days. Collagenase-dispersed interstitial cells were obtained, and population I and II Leydig cells were subsequently isolated on metrizamide gradients. Population I and II Leydig cells produced in vitro testosterone levels of 7.19 +/- 0.86 and 12.84 +/- 1.86 ng/10(6) cells/3 h, respectively. These levels were increased to 10- to 13-fold in the presence of hCG of dbcAMP. No significant difference was noted in the responsiveness of these two populations to the in vitro additions. DES administration in vivo for 8-48 h resulted in dramatic and significant decreases in basal and stimulated testosterone production in vitro in both populations. However, DES treatment for 4 h was relatively ineffective in blocking testosterone production in vitro. The inhibitory patterns exhibited by the two populations differed considerably. Population I displayed a uniform degree of inhibition throughout the treatment, whereas population II exhibited a more transient suppression by estrogen. Thus, population II appeared to be less sensitive to the estrogenic effects than population I at 48, 24, and 12 h of treatment. These data indicate that both population I and population II Leydig cells become sensitive to the inhibitory effects of estrogens between 4 and 8 h of in vivo treatment and suggest that certain differences exist between the two populations with respect to the temporal action of estrogens.