Role of steroidogenic factor 1 and aromatase in temperature-dependent sex determination in the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts.

This paper describes three distinct estrogen receptor (ER) subtypes: ERalpha, ERbeta, and a unique type, ERgamma, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERgamma arose through gene duplication from ERbeta early in the teleost lineage and indicates that ERgamma is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERgamma shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERgammas. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERbeta and ERgamma fusion proteins can each bind estradiol-17beta with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

Endocrine disruptors: present issues, future directions.

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.

Aromatase activity during embryogenesis in the brain and adrenal-kidney-gonad of the red-eared slider turtle, a species with temperature-dependent sex determination.

Gonadal sex in the red-eared slider turtle is determined by the incubation temperature that the embryo experiences during the mid-trimester of development. High temperatures result in female-biased sex ratios, and low temperatures produce male-biased sex ratios. The physiological equivalent of temperature appears to be a combination of the nature and abundance of steroidogenic enzymes and their products-including estradiol and its precursor, testosterone-and aromatase, the enzyme that converts testosterone to estradiol. Aromatase has been hypothesized to play a major role in the female developmental pathway in this species, and research in other species with temperature-dependent sex determination points to the brain as an organ that transduces the temperature signal into an aromatase response. In this study, we used a tritiated water assay to compare the pattern of estradiol biosynthesis at male- and female-producing temperatures in the brain and adrenal-kidney-gonad (AKG) through development. The pattern for both sexes in the AKG was one of increased activity after the temperature-sensitive period (TSP), but with no significant difference between sexes. In the brain, however, putative females exhibited a significantly higher level of aromatase activity than putative males at the beginning of the TSP, after which activity in both male and female brains decreased, dropping below detection in females before hatch. These results point to the brain as a site of aromatase response to temperature in this species, and they suggest that the product of aromatase activity, estradiol, may induce alterations in the neuroendocrine axis controlling gonadal sex steroid hormone production.

Developmental expression of steroidogenic factor 1 in a turtle with temperature-dependent sex determination.

A variety of reptiles possess temperature-dependent sex determination (TSD) in which the incubation temperature of a developing egg determines the gonadal sex. Current evidence suggests that temperature signals may be transduced into steroid hormone signals with estrogens directing ovarian differentiation. Steroidogenic factor 1 (SF-1) is one component of interest because it regulates the expression of steroidogenic enzymes in mammals and is differentially expressed during development of testis and ovary. Northern blot analysis of SF-1 in developing tissues of the red-eared slider turtle (Trachemys scripta), a TSD species, detected a single primary SF-1 transcript of approximately 5.8 kb across all stages of development examined. Analysis by in situ hybridization indicated nearly equivalent SF-1 expression in early, bipotential gonads at male (26 degrees C)- and female (31 degrees C)-producing incubation temperatures. In subsequent stages, as gonadal sex first becomes histologically distinguishable during the temperature-sensitive period, SF-1 expression increased in gonads at a male-producing temperature and decreased at a female-producing temperature, suggesting a role for SF-1 in the sex differentiation pathway. SF-1 message was also found in adrenal and in the periventricular region of the preoptic area and diencephalon, but there was no apparent sex bias in these tissues at any stage examined. The overall developmental pattern of SF-1 mRNA expression in T. scripta appears to parallel that found in mammals, indicating possible homologous functions.

Temperature-dependent sex determination in reptiles: proximate mechanisms, ultimate outcomes, and practical applications.

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an "all or none" process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes--rather than the organized/default system characteristic of vertebrates with genotypic sex determination--that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoding for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants.

Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain.

An isoform of the estrogen receptor messenger RNA (ER-mRNA) was identified in RNA from the brain of lizards and rats. Poly(A)+ RNA from brain and uteri was reverse transcribed using gene-specific primer for the ER. The resulting complementary DNA was amplified in a polymerase chain reaction followed by cloning and sequencing of the amplified products. This isoform lacks exon four and is designated delta 4 ER-mRNA. Although several isoforms of the ER have been described from cancerous cells, to our knowledge, none has been identified previously in the brain. Furthermore, the delta 4 isoform is the only isoform detected in normal tissue. The delta 4 isoform appeared most abundant in RNA from brain tissue, whereas uterine RNA contained only trace amounts of the isoform. Apparently, tissue-specific alternative splicing accounts for these differences in abundance. Because exon four encodes a part of the steroid-binding domain, we predict that the corresponding protein encoded by the isoform will not bind estradiol and may therefore belong to a growing subclass of the steroid/thyroid/vitamin superfamily known as orphan receptors. We predict that the putative delta 4 protein may function as a ligand-independent transcription factor that acts on the same DNA response elements as the conventional ER. The abundance of this isoform in the brain relative to the uterus raises fundamental questions regarding the regulation of estrogen-responsive genes in different tissues.

Counterproductive transcriptional and translational regulation of elongation factor 1-alpha synthesis during early development in sea urchins.

We have isolated a cDNA clone encoding elongation factor 1-alpha (EF1-alpha) and used probes prepared from this cDNA to measure levels of EF1-alpha transcripts during early development. We also determined the fraction of EF1-alpha transcripts in polysomes during this time period. Following the blastula stage there is a sharp increase in the amount of EF1-alpha mRNA. This pattern of accumulation is similar to other previously described sea urchin mRNAs. However, while the level of EF1-alpha mRNA is increasing, the fraction of EF1-alpha mRNA in polysomes decreases. Thus, there is an apparently counter-productive decrease in efficiency of recruitment into polysomes occurring concurrently with an increase in the overall amount of EF1-alpha mRNA.

Nurses and shiftwork: effects on physical health and mental depression.

An examination was made of relationships between the physical health and mental depression of nurse shiftworkers and their scores on relevant social and work-related variables. Nurses on the day, afternoon, night and rotating shifts from five hospitals (n = 463) were surveyed using a mail-back questionnaire. Two alternative models were examined in the study. The first model suggests that shift work influences the physical health and mental depression of nurses, which in turn affect social and work-related variables including: family relations; formal and informal social participation; solitary activities; job performance; and job-related stress. Shiftwork's disturbance of the body's circadian rhythm would exert a direct affect on nurses' physical health and mental depression, which in turn would then affect other aspects of the nurses' lives. The second model suggests instead that shift work primarily affects social and work-related variables, which then influence physical health and mental depression. Circadian rhythm desynchronization, while still being a consequence of shiftwork, might not directly affect physical health and mental depression. Contrary to either of the two models proposed for the study, shiftwork was not found to be significantly related to either the nurses' physical health or mental depression. However, certain factors, when considered in conjunction with the unique shift-related job characteristics of nursing, may help to explain the study's unexpected findings.

Nurses and shift work: effects on job performance and job-related stress.

The purpose of this research was to examine the influence of day, afternoon, night and rotating shift schedules on the job performance and job-related stress of nurses. Registered nurses from five hospitals (n = 463) were surveyed using a structured questionnaire which measured both job performance and job-related stress. Analysis of data indicated that both the nurses' job performance and their job-related stress were related to the type of shift they worked. Overall job performance was highest for the nurses on the day shift, followed by the night, afternoon, and rotating shifts. Rotating shift nurses experienced the most job-related stress, followed in turn by the afternoon, day, and night shift nurses. The findings are interpreted within a conceptual framework which examines the social organization of work in the hospital by shift and the effects of shift work on biological rhythm synchronization.

Identification of two forms of progesterone receptor from chick oviduct cytosol using non-denaturing gel electrophoresis.

Non-denaturing polyacrylamide gel electrophoresis and non-denaturing agarose gel electrophoresis have been used to resolve [3H]R5020-binding components from chick oviduct cytosol. From both gel systems 2 peaks of bound radioactivity are resolved which display these properties of authentic progesterone receptor: binding of R5020: steroid specificity, saturability, and restriction to target tissues. The two peaks are approximately equal in magnitude, and there is no evidence for interconversion of the 2 peaks. The presence or absence of 10-20 mM sodium molybdate during cytosol preparation had no effect on the magnitude or mobility of either peak. Neither peak contains salt-dissociable components which affect its electrophoretic properties, suggesting a possible alteration of native receptor forms during electrophoresis.

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form.

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.

Electrophoretic analysis of the estrogen receptor. Molybdate stabilization and identification of the classical estrogen receptor.

Conditions are defined which permit analysis of estrogen receptors from the mammalian uterus by polyacrylamide gel electrophoresis, thereby solving a longstanding problem encountered in previous attempts at such analysis, namely the failure of a large portion of the receptor population to enter such gels. A paramount requirement for entry of the estrogen-receptor complex into polyacrylamide gels is its maintenance in an untransformed state which does not form aggregates that are excluded from these gels. Of the multiple estrogen-binding proteins separated, only one (relative mobility of 0.5-0.6) possessed the definitive characteristics of the classical estrogen receptor. The inclusion of molybdate in extraction buffers selectively enhanced receptor recovery and facilitated its separation. Moreover, the estrogen-receptor complex so resolved is separated from other types of estrogen-binding proteins present in the uterine cytosol. These findings show that the molybdate-stabilized estrogen receptor exists in a single discrete form, but otherwise exhibits multiple forms that are probably artifactual. Electrophoresis in discontinuous buffers, but not in a continuous buffer system, promoted aggregate formation. This finding has implications concerning the subunit structure of the untransformed receptor.

Podiatry: critical issues in the 1980s.

As part of a larger study on the place of podiatry within the American health care system, this report suggests four critical issues facing podiatry as an independent health profession in the 1980s. The four concerns are: low visibility and credibility, lack of a strong professional self-image, struggle for more liberal hospital privileges, and the threat of loss of federal financial support.

Podiatry: a medical care specialty in quest of full professional status and recognition.

Podiatry as a medical specialty has received little sociological attention. This omission leaves a sizeable gap in our understanding of the total health care system. Given the functional importance of this speciality and the growing need for the services of podiatrists this report represents an attempt to increase our knowledge in this area. The paper attempts to fill that gap by presenting a discussion of the development of podiatry as a health profession; documenting the educational and political development and attempting to explain the reasons which have inhibited the process of professionalization. Our findings suggest that podiatry as a profession has been unable to attain full autonomy over its anatomical area of expertise and this along with other factors has prevented podiatry from attaining full professional status and recognition.

Labor law and physicians' privileged position: an example of structural interest influence.

Health care delivery in the United States may be characterized as a dynamic system of conflicting interest groups. Since the reorganization of the medical profession in 1910, however, physicians have been able to maintain their position as a dominant structural interest group. A dominant structural interest in one which is served by the structure of social, economic, and political institutions. It does not have to reorganize continuously to protect its privileged position. Although several medical sociologists have noted the privileged position of physicians, few have attempted to explicate the process of status maintenance. This paper examines the development of labor law in health care as one example of structural interest influence. Labor law provides an excellent illustration of this influence in that its development and application are far removed from the physicians' sphere of direct influence. It is demonstrated that the ideology that physicians should hold a privileged position is so ingrained that their interests are protected even in their absence.

Modulation by estrogen of synthesis of specific uterine proteins.

The contemporary procedure for high resolution two dimensional gel electrophoresis was extended to include an initial nondenaturing dimension of electrophoresis. Use of the resulting three dimensional procedure revealed that the previously described single peak of estrogen-induced protein in the uterus of the rat contains at least three distinct proteins whose rates of synthesis are regulated by estrogen. These proteins were localized within partial protein maps, thereby providing definitive operational definitions for the detection and identification of each. It was unambiguously demonstrated that each of the three proteins is continuously synthesized in control uteri. These findings cast doubt on the simplistic hypothesis that estrogen induces a single key protein that triggers a "cascade" of sequential transcriptional events in the uterus. Our finding that the major uterine protein induced by estrogen is also synthesized in liver and muscle cells is significant in that it points to a more general cellular function for the protein, rather than a unique role within uterine cells. Finally, our procedure for three dimensional gel electrophoresis opens new avenues for the detection of minor proteins in heterogeneous protein mixtures, such as those from the tissues of higher animals.

Xenopus liver: ontogeny of estrogen responsiveness.

Estradiol-17 beta stimulates the synthesis of numerous proteins exported into the culture medium by Xenopus tadpole liver tissue obtained after stage 50 and throughout metamorphosis to stage 66. Although estrogen-induced vitellogenin can be detected as early as stage 54, it is a minor percentage of the exported proteins until after the completion of metamorphosis. In hepatic tissue obtained after metamorphosis, the hormone evokes the synthesis of vitellogenin specifically without affecting the labeling of other secreted proteins.

Evaluating inpatient costs: the staging mechanism.

Inpatient records at a short-term hospital over two years were analyzed according to the stage or degree of severity of their discharge diagnosis to examine their utilization of services. Patients with a more severe disease stage for surgical and medical conditions generated substantially higher total charges, ancillary charges, and had longer lengths of stay. At the 75th percentile (representing that value at which three-quarters of the cases fall below it in magnitude), increases in total charges from Stage I to II for ulcer of stomach, appendicitis, and diverticulitis were 103, 168, and 110 per cent, respectively. Ancillary charges for these diseases showed even greater increases, 167, 200, and 160 per cent, respectively. Components of ancillary charges revealed similar trends. The results suggest that a twofold review mechanism incorporating length of stay and charges, using the staging technique, would make the review procedure more discriminating in identifying cases appropriate for review.