Perinatal exposure to low-dose DE-71 increases serum thyroid hormones and gonadal osteopontin gene expression.

Polybrominated diphenyl ethers (PBDEs) are flame retardants that have been widely used in manufacturing. They are major household and environmental contaminants that bioaccumulate. Humans are exposed primarily through dust inhalation and dietary ingestion of animal products. In animal studies, high doses of penta-brominated diphenyl ethers (penta-BDEs) in the mg/kg body weight (BW) range negatively impact brain development, behavior, memory, circulating thyroid hormone concentrations, the reproductive system and bone development. We investigated the effects of ingestion of a relatively low dose of the penta-BDE mixture DE-71 by pregnant and lactating rats on reproductive and thyroid parameters of the F1 offspring. F0 mothers received 60 µg/kg BW of DE-71 or vehicle daily by gavage from Day 1.5 of pregnancy through lactation (except the day of parturition). F1 pups were sacrificed at 21 d of age or outbred at approximately 80 d of age. Bred F1 females were sacrificed at Day 14.5 of pregnancy or at five months of age. Bred F1 males were sacrificed at five months of age. DE-71 treatment of the mothers affected the F1 females as evidenced by lower body weights at 80 d and five months of age, elevated serum T3 and T4 concentrations at Day 14.5 of pregnancy and increased thyroid gland weight and ovarian osteopontin mRNA at five months of age. Perinatal DE-71 exposure also increased testicular osteopontin mRNA in 21-day-old F1 males. Utilizing a granulosa cell in vitro model, we demonstrated that DE-71 activated the rat osteopontin gene promoter. Our results are the first to demonstrate that PBDEs increase rodent circulating T3 and T4 concentrations and gonadal osteopontin mRNA, and activate the osteopontin gene promoter. These changes may have clinical implications as others have shown associations between human exposure to PBDEs and subclinical hyperthyroidism, and overexpression of ovarian osteopontin has been associated with ovarian cancer.

Progress in pharmacogenomics and its promise for medicine.

Pharmacogenomics addresses the impacts of diverse and multiple genes in populations as determinants of responses of individual patients to drugs. The field has its roots in basic science, and is pivotal in drug development, elucidation of therapeutic efficacy, and constraining the risks of adverse drug reactions. Regulatory agencies are relying increasingly on pharmacogenomics for identification of patients who are particularly likely to benefit from treatment with specific agents and exclusion of those at risk of adverse drug reactions. Practical applications of pharmacogenomics already abound particularly in the use of drugs acting on the central nervous system and on the cardiovascular system. The Society for Experimental Biology and Medicine (SEBM) is proud and pleased to have devoted its 2008 symposium, presented at the annual Experimental Biology meeting in San Diego on April 6, 2008, to advances in pharmacogenomics with emphasis on drug development, regulatory agency considerations, and clinical applications.

Expression of sperm protein 22 (SP22) in the rat ovary during different reproductive states.

Sperm protein 22 (SP22) is correlated with fertility in rats. It has been identified in testis and implicated in sperm-egg interaction, protection against oxidative stress, and androgen receptor function. SP22 is widespread in rat and human tissues but has not yet been reported in the ovary. Using reverse transcription polymerase chain reaction, we identified the presence of SP22 transcripts in the rat ovary. We assessed the cellular distribution of the SP22 protein by collecting ovaries from rats in each of the following groups: 30, 60, and 90 days old; Days 9.5, 14.5, 16.5, 18.5, and 20.5 of pregnancy; and Days 1, 2, 8, and 19 of lactation. Tissue sections were stained immunohistochemically for SP22, and some serial sections were stained for relaxin or cytochrome P450 cholesterol side-chain cleavage enzyme (SCC). Weak staining for SP22 was evident in some corpora lutea (CL) and some interstitial gland cells in nonpregnant adult rats. At Day 9.5 of pregnancy, SP22 was detected in all CL, but staining intensity was weak. Staining intensity for SP22 in CL increased from Day 9.5 to 20.5 of pregnancy but was low on postpartum Day 1 and thereafter. A similar temporal pattern of staining intensity in CL was observed for relaxin. Strong immunoreactivity for SCC was present in the CL throughout pregnancy, and the spatial distribution of staining for SP22 in CL and in some areas of ovarian stroma was similar to that for SCC. There was weak staining of some theca cells in some antral follicles of pregnant and early postpartum rats when heat-induced antigen retrieval was used. There was inconsistent staining of oocytes for SP22, particularly in 30-day-old rats. In summary, the expression of SP22 was most prevalent in the CL and increased during pregnancy.

The role of modern biology and medicine in drug development in academia and industry.

This symposium addresses careers in drug development in industry; the performance of translational research by academia, industry, and both; and numerous factors pertinent to alliances essential to drug discovery and development. Drug development is a complex process that regularly involves effective collaborations between academic and physician scientists and industry. There are specific occupational factors affecting recruitment of scientists and physicians in drug development programs in industry; ideal backgrounds for successful applicants for positions in industry in drug development; ethical and regulatory considerations particularly germane to the performance of scientists and physicians in drug development programs in industry and at universities; and particular gratifications available to scientists in industry working on drug development. Both similarities and differences characterize the performance of translational research in industry compared with academia. In industry, logistic, operational, and scientific oversight is complex, especially because it often involves relationships with clinical enterprises outside of the corporation. The process is long and arduous from formulation of a good idea in discovery to acceptance of a novel drug in the marketplace. Collaborations and partnerships by industry often involving academia and confrontation of multiple issues are pivotal.

The future of interdisciplinary research and training: how to conquer the silo guardians.

We have entered a new era in biomedical research in which large interdisciplinary teams are being established to answer important scientific questions. Scientists of multidisciplinary backgrounds within universities are combining forces and inter-institutional consortia that include alliances between academia and industry are springing up around the country to generate breakthrough advances. A number of driving forces are at work to establish these collaborative research approaches. By contrast, there also are barriers to be surmounted by institutions with silo mentalities for effective partnerships to be established. In order for this new era of research to reach maximal effectiveness, new approaches to education of the young and retraining of established administrators and scientists must take place. These issues were explored thoroughly at the 2006 annual meeting of the Association of Anatomy, Cell Biology and Neurobiology Chairpersons (AACBNC) that was held in Aruba from January 18 to 21. The theme of this historic meeting was the Future of Interdisciplinary Research and Training: Breaking Down the Barriers. In this introductory article, we discuss the formation of a trendsetting Institute of Biomedical Sciences and Technology, the concept of the AACBNC meeting, and the influence of the Institute on the content of the meeting. The proceedings of this meeting, including Nobel Laureate Papers and Nobel Round-Table Discussions on the future of interdisciplinary research and training, are contained in this special issue of Experimental Biology and Medicine, a journal dedicated to the publication of multidisciplinary and interdisciplinary research in the biomedical sciences.

Physiological proteomics: cells, organs, biological fluids, and biomarkers.

Proteomic research is accelerating rapidly because of marked advances in protein labeling techniques, mass spectrometry (MS), and bioinformatics. Two-dimensional difference gel electrophoresis (2D-DIGE) is being used effectively in conjunction with liquid chromatography tandem MS (LC-MS/MS) and/or matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-ToF MS) and database search software to quantify relative changes in the levels of proteins in two samples. It is now possible in a single study to identify and quantify large numbers of proteins and their posttranslational modifications in different biological samples. Comparisons can be made between groups of animals in different physiological states or in response to experimental treatment. Differences between normal individuals and those in disease states can form the foundation for elucidation of causative factors of disease and the identification of biomarkers for the diseased state. This symposium includes original research that compares the erythrocyte plasma membrane proteome in the normal and the sickle cell state, evaluates the anterior pituitary gland proteome in the ovariectomized rat in response to estrogen, and assesses proteomic methodology employed to identify potentially useful biomarkers in human cells and fluids for clinical medicine. It is directed not only to investigators working in these fields but also to a diverse group of scientists working in the biological and biomedical fields to stimulate cross-disciplinary awareness, interest, and collaboration.

Proteomics of the anterior pituitary gland as a model for studying the physiology of a heterogeneous organ.

The anterior pituitary gland (AP) secretes six established hormones that collectively control hundreds of biological and behavioral functions. Because of advances in mass spectrometry (MS), protein labeling, and bioinformatics, it is now possible to characterize, compare, and quantify the AP hormones together with large numbers of nonhormonal AP proteins. For example, by using high-performance liquid chromatography in line with tandem MS we characterized 145 proteins in sub-cellular fractions of the AP of young adult male Golden Syrian hamsters and 115 proteins in subcellular fractions of the AP of young adult male mice. These included hormones, proteins involved in hormone synthesis and release, and housekeeping proteins. We also used difference gel electrophoresis in conjunction with MS and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Ovariectomized rats were administered 50 microg of estradiol valerate subcutaneously and studied 48 hrs later, before the onset of the anticipated surges of gonadotropins in blood. Following DeCyder image analysis, we identified by MS and peptide mass fingerprinting 26 protein spots that were upregulated and 19 protein spots that were downregulated. Estrogen increased levels of acidic isoforms of growth hormone and prolactin, several proteins involved in protein synthesis, folding and secretion, and several metabolic enzymes. Most of the downregulated proteins are involved in RNA or DNA interactions. We followed up on the results with RT-PCR and immunohistochemical techniques to demonstrate that one protein identified by MS in hamster AP, fertility protein SP22, is synthesized in the AP and localized primarily in somatotropes and thyrotropes. These experiments demonstrate the efficacy of our proteomics approach to characterize AP proteins and quantify changes in them. The approaches used to study the AP could serve as a model to investigate other heterogeneous organs.

Estrogen regulation of the rat anterior pituitary gland proteome.

Estrogen is known to affect the regulation of all six of the established anterior pituitary gland (AP) hormones, but little is known of the specifics of its regulation of the AP hormones, their isoforms, and nonhormonal AP proteins. We used difference gel electrophoresis in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Two-month-old rats were ovariectomized and used at 6 months of age. They were injected subcutaneously with sesame oil vehicle or 50 mug estradiol valerate in vehicle and studied 48 hrs later, approximately 3 hrs before the time of the anticipated onset of the estrogen-induced surges of gonadotropins in blood. The APs were pooled, and the soluble protein fraction was examined in replicate analyses. After DeCyder software analysis, we identified 26 protein spots that had a 1.5-fold or greater average increase in the experimental group relative to the controls. Nineteen showed a 1.5-fold or greater decrease. Estrogen increased levels of the more acidic isoforms of growth hormone and prolactin and of proteins involved in protein synthesis, folding, and secretion (e.g., eukaryotic translation elongation factor 2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit, and secretogranin II) and of some metabolic enzymes (e.g., arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase, and aldolase A). The majority of the downregulated proteins were involved in RNA or DNA interactions (e.g., five heterogeneous nuclear ribonucleoproteins, DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein), but isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III were also reduced. Our results indicate that estrogen acts in vivo within 48 hrs to modulate levels of a significant number of AP proteins.

Localization of fertility factor SP22 to specific cell types within the anterior pituitary gland.

Sperm protein 22 (SP22) was recently identified in the anterior pituitary gland (AP) of male Golden Syrian hamsters using ion trap mass spectrometry. SP22 has been implicated in apoptosis, androgen receptor function, fertility, and ontogeny of early-onset Parkinson's disease. However, the role of SP22 in the pituitary has not been investigated. We cloned the cDNA for full-length SP22 from AP and posterior lobe (posterior pituitary and intermediate lobe) of the pituitary gland in adult male rats and Golden Syrian hamsters, confirming the presence of SP22 mRNA in the AP and posterior lobe. Because gonadal steroids are important regulators of AP function, and SP22 is associated with androgen receptor function, we used Western blots to compare SP22 in the AP of intact and orchidectomized male rats given placebo or a low or high dose of testosterone. SP22 did not differ with treatment, indicating that AP SP22 concentration was not regulated by testosterone. To localize SP22 to specific cells of the AP, mirror-image paraffin sections were labeled against SP22 and either luteinizing hormone (LH)beta, thyroid-stimulating hormone (TSH)beta, prolactin, adrenocorticotropic hormone (ACTH), or growth hormone (GH) using peroxidase-conjugated secondary antibody. Additional sections were colabeled with SP22 and one of the AP hormones using fluorescent secondary antibodies. SP22 colocalized in somatotropes and thyrotropes in rat and hamster. We identified SP22 in a small percentage of corticotropes, gonadotropes, and lactotropes. This is the first report that SP22 mRNA is present specifically in the AP, and SP22 is localized primarily in somatotropes and thyrotropes. SP22 may help regulate AP function and be particularly important for the control of GH and TSH secretion.

Mouse anterior pituitary gland: analysis by ion trap mass spectrometry.

We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively. A total of 115 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, and luteinizing hormone-beta. Groups of other identified proteins included hormone-processing, secretion granule-associated, non-hormonal endoplasmic reticulum-associated, calcium-binding, protein kinase C-associated, histones, non-histone chromosomal, other RNA-binding, heterogeneous nuclear ribonucleoproteins, splicing factors, helicases, lamins, ribosomal, microtubule-associated, microfilament-associated, adenosine triphosphate- and guanosine triphosphate-associated, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, enzymes in glycolysis and the tricarboxylic and urea cycles and the pentose phosphate path, heat-shock, glutathione-associated, peroxidases, ubiquitin-associated, catabolic, protease inhibitors, other, and blood proteins. The 115 proteins reported in this study and the 145 proteins reported in a previous study on the AP of the adult male Golden Syrian hamster are compared and form a foundation for defining the proteome in normal adult male AP.

Fetal alcohol syndrome: an assessment of the field.

Fetal exposure to alcohol is the major known cause of mental retardation in the Western world. For more than half of the 20th century, the placenta was widely believed to be an effective barrier against environmental agents. The discovery that offspring of pregnant women who were exposed to German measles or administered thalidomide were often malformed raised awareness that teratogens could be any environmental agent, including viruses and drugs, that caused abnormal development. Alcohol was not identified as a teratogen until the 1970s. Fetal exposure to alcohol can cause fetal alcohol syndrome (FAS), which is characterized by specific physical traits and central nervous system dysfunctions. The development of animal model systems has facilitated our study of the effects of fetal alcohol exposure and the elucidation of the mechanisms involved in alcohol-induced abnormal development. Despite our current understanding of the effects of fetal alcohol exposure, the occurrence of FAS and associated fetal alcohol spectrum disorders is still widespread and the associated health-care costs are staggering. This symposium provides an up-to-date analysis of fetal exposure to alcohol and FAS. It is directed not only to investigators working in the field but to a diverse group of scientists working in the biological and biomedical fields to stimulate cross-disciplinary awareness, interest, and collaboration.

Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy.

Immunohistochemical studies were undertaken to determine the distribution of GATA-4 and GATA-6 in rat fetal gonad and the postnatal ovary during development and pregnancy. In the undifferentiated gonad, GATA-4 was expressed in the somatic cells of both sexes. After differentiation of the ovary and testis, GATA-4 expression continued in both ovarian and testicular somatic cells; whereas, GATA-6 was expressed in both somatic and germ cells. In the ovary of postnatal rats, granulosa and thecal cells of healthy follicles expressed both GATA factors. In the adult rat, GATA-4 expression was lower in corpora lutea as compared to follicles; whereas, GATA-6 was strongly expressed in both structures. GATA-4 expression was greater in functional corpora lutea than regressing corpora lutea. GATA-6 was expressed in both functional and regressing corpora lutea. In all postnatal ovaries, the expression of P450scc localized with tissue expressing GATA-4 and/or GATA-6, but GATA expression also occurred in P450scc negative cells.

Effects of 4-tert-octylphenol given in drinking water for 4 months on the male reproductive system of Fischer 344 rats.

The environmental pollutant 4-tert-octylphenol (OP) is both toxic and estrogenic to mammalian cells, and injection of OP into adult male rats has devastating effects on their reproductive system. We now report the effects of OP in drinking water ( 1 x 10(-5), 1 x 10(-7) or 1 x 10 (-9) M) on the male reproductive system. Exposure of adult male rats for 4 months to any tested dose of OP had no significant effect on water or food consumption; body weight gain; hematocrit; reproductive organ weights; mean serum LH, FSH or testosterone concentrations; germ cell yield or relative numbers of different classes of testicular cells; or testicular sperm number. In contrast, all doses of OP caused an increase in epididymal sperm with tail abnormalities that would interfere with sperm motility, and the highest dose decreased epididymal sperm number. Our findings raise the possibility that consumption of OP in drinking water may adversely influence male reproductive fertility.

Analysis of the golden Syrian hamster anterior pituitary gland proteome by ion trap mass spectrometry.

We utilized mass spectrometry (MS) and bioinformatics to investigate the proteome of the anterior pituitary gland (AP). Subcellular fractions of APs from 2-month-old male Golden Syrian hamsters were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography MS/MS and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 x g and cytosolic fractions we identified 76, 52 and 52 different proteins, respectively. A total of 145 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, luteinizing hormone-beta and follicle-stimulating hormone-beta. Groups of other identified proteins included hormone processing, secretion granule associated, non-hormonal endoplasmic reticulum associated, calcium binding, protein kinase C associated histone and non-histone chromosomal material, other RNA-binding, splicing factors, heterogeneous nuclear ribonucleoproteins, helicases, lamins, microfilament associated, microtubule associated, adenosine triphosphate and guanosine diphosphate associated, keratins, lysosomal, ribosomal, enzymes in glycolysis and the tricarboxylic and pentose phosphate paths, glutathione associated, transmethylation, catabolic and unknown protein products as well as blood hemoglobins. Proteins previously not reported in the AP, such as fertility protein SP22, were identified. The proteins identified in the present study form a foundation for defining the proteome in normal adult male AP.

Teaching medical histology at the University of South Carolina School of Medicine: Transition to virtual slides and virtual microscopes.

We describe how the histology course we teach to first-year medical students changed successfully from using glass slides and microscopes to using virtual slides and virtual microscopes. In 1988, we taught a classic medical histology course. Subsequently, students were loaned static labeled images on projection slides to introduce them to their microscope glass slides, and we made laser disks of histological images available in the teaching lab. In 2000, we placed the static labeled images and laboratory manual on the Web. We abandoned the Web-based approach in 2001. Faculty selected specific areas on microscope glass slides in student collections for scanning at a total magnification of 40, 100, 200, or 400. Christopher M. Prince of Petro Image, LLC, scanned the glass slides; digitized, encoded, and compressed (95%) the images; and placed them on CD-ROMs. The scanned images were viewed up to a magnification of 400 using the MrSID viewer (LizardTech software) and the computer as a virtual microscope. This viewer has many useful features, including effective microscope and telescope functions that provide greater versatility for sample study and speed in localizing structures than was possible with the actual microscope. Image detail is indistinguishable from that viewed under the light microscope at equivalent magnifications. Static labeled images were also placed on CD-ROMs to introduce students to the virtual slides. Students could view all the images on their CD-ROMs at any time and in any place with their laptop computers without going online. Students no longer rented light microscopes in 2002. Both students and faculty have shown strong support for using this approach to teaching histology during the past 2 years.