Chronic bovine besnoitiosis: intra-organ parasite distribution, parasite loads and parasite-associated lesions in subclinical cases.

Bovine besnoitiosis caused by Besnoitia besnoiti is a chronic and debilitating disease. The most characteristic clinical signs of chronic besnoitiosis are visible tissue cysts in the scleral conjunctiva and the vagina, thickened skin and a generally poor body condition. However, many seropositive animals remain subclinically infected, and the role that these animals may play in spreading the disease is not known. The aim of the present study was to assess the intra-organ parasite distribution, the parasite load and the parasite-associated lesions in seropositive but subclinically infected animals. These animals were seropositive at the time of several consecutive samplings, had visible tissue cysts in the past and, at time of slaughter, had detectable specific anti-Besnoitia spp. antibody levels, but they did not show evident clinical signs at culling. Thus, histopathological, immunohistochemical and molecular analyses of several samples from the respiratory tract, reproductive tract, other internal organs and skin from six cows were performed. The tissue cysts were located primarily in the upper respiratory tract, i.e., in the rhinarium and larynx/pharynx (four cows), followed by the distal genital tract (vulva/vagina) and the skin of the neck (three and two cows, respectively, out of the four cows with cysts in the respiratory tract). We were unable to detect any parasites in the two remaining cows. Cysts were associated with a significant non-purulent inflammatory infiltrate consisting predominantly of T lymphocytes and activated monocytes/macrophages in two cows. The parasite burden, estimated by quantitative real-time PCR, was very low. It is noteworthy that the only animal that showed a recent increase in the antibody titre had the highest parasite burden and the most conspicuous inflammatory reaction against the cysts. In conclusion, although these cows no longer displayed any visible signs of besnoitiosis, they remained infected. Therefore, cows without visible signs of disease may still be able to transmit the parasite.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

Exposure to stallion accelerates the onset of mares' cyclicity.

Horses (Equus caballus) belong to the group of seasonally polyestrous mammals. Estrous cycles typically start with increasing daylight length after winter, but mares can differ greatly in the timing of onset of regular estrus cycles. Here, we test whether spatial proximity to a stallion also plays a role. Twenty-two anestrous mares were either exposed to one of two stallions (without direct physical contact) or not exposed (controls) under experimental conditions during two consecutive springs (February to April). Ovarian activity was monitored via transrectal ultrasound and stallion's direct contact time with each mare was determined three times per week for one hour each. We found that mares exposed to a stallion ovulated earlier and more often during the observational period than mares that were not exposed to stallions. Neither stallion identity nor direct contact time, mare age, body condition, size of her largest follicle at the onset of the experiment, or parasite burden significantly affected the onset of cyclicity. In conclusion, the timing of estrous cycles and cycle frequency, i.e., crucial aspects of female reproductive strategy, strongly depend on how the mares perceive their social environment. Exposing mares to the proximity of a stallion can therefore be an alternative to, for example, light programs or elaborated hormonal therapies to start the breeding season earlier and increase the number of estrous cycles in horses.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

AN ASSESSMENT OF AFLATOXIN B1, AND OTHER SELECTED VARIABLES, IN A RURAL ENVIRONMENT AND ASSOCIATED CANCER RISK (TEXAS)

A study to assess possible exposure to carcinogenic metabolites (aflatoxins) from a mold Aspergillus flavus has been made in a rice producing area of Brazoria County, Texas. One hundred samples of unmilled rice were analyzed by thin-layer chromatography (TLC) for the amount of aflatoxin produced by the mold during rice growth and storage. Two well water samples and two rice elevator dust samples were also checked for possible aflatoxin content. The mortality rates from gastrointestinal and urinary tract cancers in the rice-growing part of the county were compared with mortality rates in the nonrice-producing areas of the same county.^ This study was an outgrowth of an earlier investigation by Cech and co-workers in Brazoria County which focused on environmental differences, specifically on the quality of drinking water in the former residences of decedents from primary liver cancer. It also compared subjects who died from other causes. The author of this dissertation participated in this phase of the overall investigation by performing some of the chemical analyses and by preparing synographic maps of water quality, and thus, part of those results from the early phase is also included in this manuscript.^ No aflatoxin was detected by TLC methods. However, when extracts of rice dust were checked for mutagenesis by the Ames Salmonella-microsome assay as a supplement to the TLC analysis, the result suggested that these dusts might have contained mutagenic material. The age-adjusted mortality rates in the rice-growing area were higher than those in the comparison area for both male and female gastrointestinal tract cancer and for male urinary tract cancer, but the differences were not statistically significant. ^

(Table 1) Lipid content and organohalogen contaminant concentration in plasma of incubating glaucous gulls (Larus hyperboreus) from Bjørnøya, Norwegian Arctic in 2006

The factors influencing prolactin (PRL) variation in birds and in wildlife in general have rarely been investigated with respect to the physiological impacts of exposure to environmental contaminants. We investigated the associations between circulating baseline PRL levels and concentrations of eight persistent organohalogen contaminant (OHC) classes (i.e., major organochlorines and brominated flame retardants, and associated metabolic products) in blood (plasma) of free-ranging glaucous gulls (Larus hyperboreus), a top predator in the Norwegian Arctic, engaged in the process of incubation. We further examined whether plasma OHC concentrations were associated with the variation of PRL in glaucous gulls exposed to a standardized capture/restraint protocol. Plasma OHC concentrations in male glaucous gulls were 2-to 3-fold higher relative to females. Baseline PRL levels tended to be higher in females compared to males, although not significantly (p = 0.20). In both males and females, the 30-min capture/restraint protocol led on average to a 26% decrease in PRL levels, which resulted in a rate of PRL decrease of 0.76 ng/mL/min. The baseline PRL levels and the rate of decrease in PRL levels tended to vary negatively with plasma OHC concentrations in males, but not in females, although several of these associations did not adhere with the criterion of significance (alpha = 0.05). Present results suggest that in highly OHC-exposed male glaucous gulls, the control of PRL release may be affected by the direct or indirect modulating actions of OHCs and/or their metabolically derived products. We conclude that potentially OHC-mediated impact on PRL secretion in glaucous gulls (males) may be a contributing factor to the adverse effects observed on the reproductive behavior, development and population size of glaucous gulls breeding in the Norwegian Arctic.

Principales efectos de la aplicación de una metodología destinada a inducir la muda en ponedoras comerciales sobre su producción cuantitativa y cualitativa durante el segundo ciclo de puesta

En la presente Tesis Doctoral se ha estudiado el efecto de una metodología para inducir la muda en dos estirpes de gallinas ponedoras comerciales, una ligera y otra semipesada, mediante el suministro de tres alimentos: salvado de trigo, cebada en grano y un pienso comercial de ponedoras aportado en cantidad restringida. Se realizaron dos pruebas en dos lotes diferentes de gallinas. En la primera de ellas se utilizaron 472 animales (236 de cada estirpe) alojados en jaulas con cuatro o seis gallinas por jaula, con una estructura factorial 2 x 3 x 2 (2 estirpes, 3 alimentos, 2 densidades) y una duración total de 32 semanas (4 de muda y 28 de producción posmuda). En la segunda prueba fueron 432 animales los utilizados (216 de cada estirpe), alojados en grupos de cuatro aves por jaula, con una estructura factorial 2 x 3 (2 estirpes, 3 alimentos) y una duración de 27 semanas (4 de muda y 23 de producción postmuda). En los dos experimentos realizados se estudió el efecto del uso de los alimentos citados para inducir de la muda sobre los resultados cuantitativos: pérdida de peso vivo durante la muda e intensidad de puesta, peso medio del huevo y masa de huevo diaria, durante y después de la muda, así como la distribución de la puesta en clases comerciales durante el segundo ciclo de puesta. Así como sobre los resultados cualitativos después de la muda: color de la cáscara de los huevos morenos, espesor de la cáscara, peso específico del huevo, altura del albumen, unidades Haugh y color de la yema. En la primera prueba se estudió, además, el efecto que la inducción de la muda mediante los tres alimentos considerados tuvo sobre la regresión del aparato reproductor de las gallinas durante este período de muda. En el primer experimento se observaron diferencias entre estirpes. Las gallinas ligeras tuvieron una más rápida regresión del aparato reproductor (ovario+oviducto) (P=0,003) que las semipesadas, aunque la regresión total no presentó diferencias significativas. Las gallinas semipesadas tuvieron mejores resultados después de la muda en intensidad de puesta (P<0,0001), en peso medio del huevo (P<0,0001) y en masa de huevo diaria (P=0,0002). También hubo diferencias significativas para las variables cualitativas espesor de la cáscara (mayor en huevos de gallinas semipesadas) mientras que los huevos procedentes de gallinas ligeras presentaron mejores valores de altura del albumen, de unidades Haugh y de color de yema; todas estas variables tuvieron un nivel de significación P<0,0001. El suministro restringido de pienso dio lugar a un mayor porcentaje de pérdida de peso vivo (P<0,0001) aunque la regresión de los órganos del aparato reproductor fue la más baja (P<0,003), no habiéndose encontrado diferencias entre los otros dos alimentos utilizados. Con este alimento también fue más lenta (P<0,0001) la disminución de la puesta durante la muda, aunque fue mayor la producción durante el segundo ciclo (P<0,0001). La única variable cualitativa afectada fue el espesor de cáscara (P<0,0001), con valores más altos en los huevos producidos por las gallinas mudadas con cebada. Los grupos de seis gallinas por jaula produjeron más huevos durante la muda (P<0,0001) aunque después de ésta la densidad de animales no tuvo efecto significativo, como tampoco lo hubo sobre los parámetros de calidad del huevo. En la segunda prueba las gallinas semipesadas experimentaron un menor porcentaje de pérdida de peso corporal (P<0,01) pero tuvieron mayores índices de puesta (P<0,001) y de huevos clasificables (P<0,001) durante la muda. En cambio, durante el segundo ciclo de producción las gallinas ligeras produjeron más huevos (P=0,0041), de menor peso (P<0,02) y con menor consumo de pienso (P<0,001). Los huevos puestos por las gallinas semipesadas tuvieron mayor espesor de cáscara y mayor color de yema, pero peor calidad de albumen (P<0,0001). La mayor pérdida de peso la experimentaron las gallinas mudadas con salvado (P<0,02). La producción durante la muda fue mayor (P<0,001) en las gallinas que consumieron pienso en cantidad restringida y también fueron las que tuvieron menor intensidad de puesta (P<0,006) y masa de huevo diaria (P<0,042) durante el segundo ciclo. El tratamiento de muda no tuvo efecto significativo sobre la calidad del huevo en esta segunda prueba. La principal conclusión que merece destacarse es que es posible inducir la muda a las gallinas ponedoras utilizando alimentos bajos en energía o en proteína, o altos en fibra, con un porcentaje de pérdida de peso vivo no tan alta como las recomendaciones tradicionales, y alcanzar buenos resultados productivos, tanto cuantitativos como cualitativos, durante el segundo ciclo de puesta. ABSTRACT Present Doctoral Thesis has studied the effect of a methodology to induce molting in two strains of commercial laying hens, one light and another semi-heavy one, through the provision of three feed: wheat bran, barley grain and a commercial laying hens feed provided in limited quantity. Two tests were performed in two different lots of layers. In the first 472 animals were used (236 of each strain) housed in cages with four or six hens per cage, with a structure 2 x 3 x 2 factorial (2 strains, 3 meals, 2 densities) and a total duration of 32 weeks (4 of molt and 28 of postmolting). In the second test 432 animals were used (216 each strain), housed in groups of four birds per cage, with a structure factorial 2 x 3 (2 strain, 3 meals) along 27 weeks (4 of molt and 23 of postmolting). In both experiments, we studied the effect of the use of above mentioned foods to induce molting on the quantitative results: body weight lost during the molt and laying index, average egg weight and egg mass daily during and after the molt, as well as on grading in commercial classes during the second laying cycle. As well as on qualitative outcomes after the molt: colour of Brown eggsshell, shell thickness, specific density, albumen height, Haugh units and yolk colour. In the first test was studied, in addition, the effect of induction of molting through the three feed considered on the regression of the reproductive tract of hens during molting period. In the first experiment, differences between strains were observed. Light hens had a faster regression of the reproductive tract (ovary+oviduct) (P=0,003) than semi-heavy hens, although the total regression did not present significant differences. Semi-heavy hens had better outcomes after the molt in laying index (P<0.0001), in average egg weight (P<0.0001) and daily egg mass (P=0, 0002). There were also significant differences for the qualitative variables (higher in semi-heavy hen eggs) as shell thickness while light chicken eggs showed better values of albumen height, Haugh units and yolk color; all these variables had the same level of significance (P<0.0001). Restricted supply of layer feed resulted in a greater percentage of live weight loss (P<0.0001) although the regression of the reproductive organs was the lowest (P<0.003), having not found differences between the other two feed used. With this food decreasing of laying during molting period was also slower (P<0.0001), although production was higher during the second cycle (P < 0.0001). The only qualitative variable affected was shell thickness (P<0.0001), with higher values in the eggs produced by hens molted with barley. Groups of six hens per cage produced more eggs during the moult (P<0.0001) but after this animal density had no significant effect, as neither had it on egg quality parameters. In the second trial hens semi-heavy experienced a lower percentage of body weight loss (P<0.01) but had higher rates of egg production (P<0.001) and of grading eggs (P<0.001) during the moult. On the other hand, during the second production cycle light hens produced more eggs (P=0,0041), of lower weight (P < 0.02) and with less feed intake (P<0.001). Eggs from semi-heavy hens had thicker shell and greater color yolk, but poorer quality of albumen (P<0.0001). The greater weight loss was experimented by the hens molted with wheat bran (P=0.02). Production during molting period was greater (P<0.001) in hens which consumed feed in restricted quantities and were also less the laying index (P=0.006) and daily egg mass (P=0.042) during the second cycle. The molting treatment had no significative effect on the quality of the egg in this second test. The main conclusion that deserves to stand out is that it is possible to induce molting hens using foods low in energy or in protein or high in fiber, with a percentage of live weight loss not as high as the traditional recommendations, and achieve good productive, both quantitative and qualitative results during the second implementation cycle.

Can seed production and restricted dispersal limit recruitment in Pinus pinaster Aiton from the Spanish Northern Plateau?

Natural regeneration faces increasing difficulties in dry forests from the Mediterranean basin, including for normally well-regenerating species such as maritime pine (Pinus pinaster Aiton). In this paper, we studied female fertility, seed dispersal and spread rates in P. pinaster from the Spanish Northern Plateau, where natural regeneration failure is a main concern for forest managers. For this purpose we periodically collected data from seed traps and trees located at two core locations across several years. We found significant variation in interannual cone production, with the best seed trees being the same across years. In addition, we found highly skewed distributions of female reproductive effort and large fertility differences across stands located few kilometres away. Annual seed dispersal kernels fitted lognormal or 2Dt models depending on the stand analysed, with median dispersal distances between 14 and 25 m. Kernels fitted for maximum dispersal periods showed an outstanding intraseasonal variation of median dispersal distances, from 10 to 54 m, in association to variable patterns of rainfall and maximum wind speed. The amount of seed produced appeared to be enough to guarantee the natural regeneration of the stands during the typical 20-year regeneration period. Colonisation simulations concluded that Mediterranean maritime pine has a notable dispersion capacity, which is strongly influenced by levels of fecundity and, especially, by the number and frequency of long-distance dispersal events. The latter play a key role in tree dispersion processes through enlarging the occupied area and fostering the invasion of abandoned crop land.

Apoptosis regulator Bcl-w is essential for spermatogenesis but appears otherwise redundant

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w−/− mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-α (ER-α). By using ER-α “knock-out” (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17β (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-α. A primary estrogen, estradiol-17β (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-α and the recently cloned ER-β. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

How flowering plants discriminate between self and non-self pollen to prevent inbreeding.

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.

Thyroid hormone and estrogen interact to regulate behavior.

Environmental perturbations that increase plasma thyroid hormone (T3) concentrations also profoundly affect female reproductive behavior and physiology. We explored whether these effects were mediated by interactions between T3 receptor (TR) and estrogen receptor (ER). This hypothesis was of interest because the half-site of a consensus T3 response element DNA sequence is identical to an ER response element (ERE), and TRs bind to a consensus ERE. Molecular data presented in the accompanying paper [Zhu, Y.-S., Yen, P.M., Chin, W.W.& Pfaff, D.W. (1996) Proc. Natl. Acad. Sci. USA 93, 12587-12592] demonstrate that TRs and ERs are both present in rat hypothalamic nuclear extracts and that both can bind to the promoter the hypothalamic gene preproenkephalin and that interations between liganded TRs and ERs affect preproenkephalin transcription. In this paper, we show that molecular interactions between TRs and ERs are sufficient to mediate environmental effects on estrogen-controlled reproductive behavior. Ovariectomized (OVX) rats treated with high doses of T3 showed significantly lower levels of lordosis behavior in response to estradiol benzoate (EB) compared with OVX females treated with EB alone. Conversely, thyroidectomized/OVX females treated with EB showed significantly greater levels of lordosis behavior compared with OVX females treated with EB, showing the effect of endogenous T3. Thyroid hormone interference with EB-induced behavior could not be explained by a reduction in plasma E2 concentrations or by a general reduction in responsiveness of EB-sensitive tissues. Moreover, numbers of hypothalamic ER-immunoreactive cells increased dramatically following T3 treatment. These data suggest that T3 may reduce EB-dependent sexual behavior through interactions between TR and ER in the nuclei of behaviorally relevant hypothalamic neurons, envisioning for the first time a functional consequence of interactions between two nuclear hormone receptors in brain. These results also open up the possibility of molecular interactions on DNA encoding environmental signals, a new field for the study of neuronal integration.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.