Paul Mak

Estrogen receptor (ER)-β isoforms: A key to understanding ER-β signaling

Estrogen receptor beta (ER-β) regulates diverse physiological functions in the human body. Current studies are confined to ER-β1, and the functional roles of isoforms 2, 4, and 5 remain unclear. Full-length ER-β4 and -β5 isoforms were obtained from a prostate cell line, and they exhibit differential expression in a wide variety of human tissues/cell lines. Through molecular modeling, we established that only ER-β1 has a full-length helix 11 and a helix 12 that assumes an agonist-directed position. In ER-β2, the shortened C terminus results in a disoriented helix 12 and marked shrinkage in the coactivator binding cleft. ER-β4 and -β5 completely lack helix 12. We further demonstrated that ER-β1 is the only fully functional isoform, whereas ER-β2, -β4, and -β5 do not form homodimers and have no innate activities of their own. However, the isoforms can heterodimerize with ER-β1 and enhance its transactivation in a ligand-dependent manner. ER-β1 tends to form heterodimers with other isoforms under the stimulation of estrogens but not phytoestrogens. Collectively, these data support the premise that (i) ER-β1 is the obligatory partner of an ER-β dimer, whereas the other isoforms function as variable dimer partners with enhancer activity, and (ii) a single functional helix 12 in a dimer is sufficient for gene transactivation. Thus, ER-β behaves like a noncanonical type-I receptor, and its action may depend on differential amounts of ER-β1 homo- and heterodimers formed upon stimulation by a specific ligand. Our findings have provided previously unrecognized directions for studying ER-β signaling and design of ER-β-based therapies.

Characterization of an estrogen receptor in the turtle testis

Using DNA-cellulose affinity chromatography, an estrogen-binding component having the properties of a classical estrogen receptor was characterized from testicular cytosol of the freshwater turtle, Chrysemys picta. This putative cytoplasmic receptor exhibited high affinity (Kd = 7.0 X 10(-10) M), low capacity (1-4 fmol/mg protein), and steroid binding that was specific for estrogens. It was not present in plasma, muscle, kidney, or lung. A temperature-dependent conversion of turtle testicular estrogen receptor from 4 to 5 S occurred on DNA-cellulose columns, and resembled that in mammalian testis and other target tissues. After a single injection of estrogen at 3 hr, cytoplasmic receptors were depleted with a concomitant increase of nuclear receptors. Identification in turtle testis of an estrogen-binding macromolecule having the physicochemical properties of mammalian estrogen receptors is further evidence that receptors have been widely conserved in many tissue types through vertebrate phylogeny and supports the idea that the testis is an important target of estrogen action.

The differentiation of Leydig cells, steroidogenesis, and the spermatogenetic wave in the testis of Necturus maculosus

The study of seminiferous tubule--Leydig cell interactions in relation to specific germ cell stages during the cycle of the seminiferous epithelium is extremely difficult in most mammalian species due to the continual presence of different spermatogenetic stages in the testis from the onset of puberty. The problem is also compounded by the uniform distribution of both seminiferous tubules and interstitial tissue throughout the entire testis. This difficulty can be circumvented, however, by studying certain species where there is a topographical distribution of germ cell stages within the testis. The urodele amphibian Necturus maculosus exhibits a breeding cycle during which a longitudinal wave of spermatogenesis occurs along the length of the testis, resulting in a spatial and temporal segregation of differentiating germ cells. Moreover, this topographical pattern of spermatogenesis is also reflected in the degree of development of adjacent Leydig cells. This anatomical arrangement allows distinct testicular regions to be obtained using a dissecting microscope. The isolated zones, containing germ cells and Leydig cells in various stages of development, were analyzed for 17 alpha-hydroxylase, C-17,20-lyase, and aromatase activities (key enzymes for the synthesis of androgens and estrogen), estrogen binding, and cytochrome P-450 content. Functional parameters were then correlated with the morphology of Leydig cells in the various zones observed by both light and electron microscopy. It was found that there existed a distinct correlation between the state of differentiation of the leydig cells, their steroidogenic potential, and the distribution of estrogen receptors. These results in Necturus indicate indicate in this species, at least, the steroidal microenvironment of different germ cell associations may be quite specific.

A novel steroid-binding protein in the testis of the dogfish Squalus acanthias ☆

In an earlier study, we identified and characterized a testicular estrogen receptor in the spiny dogfish (Squalus acanthias); however, in this species estrogen receptors were located exclusively in nuclear subfractions. We describe here a nonreceptor, sex hormone-binding protein (SBP) present in Squalus testicular cytosol and distinguishable in its physicochemical characteristics from both the estrogen receptor and serum SBP of the same species. Cytosol (100,000g supernatant) was prepared by differential centrifugation of testicular homogenates and incubated with [3H]estradiol (E2) or [3H]testosterone (T) (10-15 nM +/- 100-fold excess radioinert competitor) to determine total and nonspecific bound radioactivity. The testicular SBP had a broad specificity: E2 = T greater than progesterone greater than 5 alpha-dihydrotestosterone greater than estrone, but diethylstilbestrol was not an effective competitor. It displayed a high affinity for both E2 and T (Kd = 2.2-2.5 X 10(-9) M), sedimented at 8-10 S in both low- and high-salt sucrose gradients, and migrated more slowly than BSA during polyacrylamide disc gel electrophoresis. The testicular SBP-E2 complex was relatively stable (t1/2 = 160 min) compared to the serum SBP-E2 complex (t1/2 = less than 30 min). The testicular SBP was not found in nuclear subfractions nor did it bind to DNA-cellulose affinity columns. Its intratesticular distribution was stage-dependent: Zone III (mature spermatids) greater than Zone II (spermatocytes) greater than Zone I (stem cells and spermatogonia). Moreover, increased binding activity corresponded exactly to the hypertrophy and differentiation of Sertoli cells in the same zones, pointing to this cell as the possible site of SBP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptors in the turtle brain

Estrogen binding activity in the CNS of the freshwater turtle, Chrysemys picta, was investigated using DNA-cellulose affinity chromatography. An estrogen binding component (EBC) with the characteristics of an estrogen receptor species was demonstrated in the brain cytosol extracts from both sexes. This EBC exhibited high affinity (K d = 10 -10 M), low capacity (n = 0.8 to 6.0 fmol/mg cytosol protein) and binding specificity. The bound estradiol-17 beta which adhered to DNA-cellulose was sensitive to excess synthetic and natural estrogens (DES, E2, E1 and E3) but not to progesterone and androgens (5 alpha- DHT and T). Specific estradiol-17 beta binding was not detected in plasma or non-target tissues such as lung, kidney and muscle. The topographic distribution of cytoplasmic EBC was similar in males and females, with binding highest in the hypothalamus-preoptic areas (HPOA) followed by the remaining forebrain (RFB). The mid/hindbrain (HB), consisting of the optic lobes, cerebellum and underlying brain stem had significantly lower concentrations of EBC. Monthly data (from May to October) suggest that variations in EBC concentrations occur during the year.

Estradiol binding activity in epididymal cytosol of the turtle, Chrysemys picta

Estrogen binding activity was investigated in the epididymis of the turtle, Chrysemys picta using DNA-cellulose affinity chromatography. A component binding estradiol-17 beta specifically with high affinity (Kd:8.0 X 10(-10) M) and limited capacity (20 fmol/mg protein) was demonstrated in the epididymal cytosol. In addition, binding of estradiol-17 beta was sensitive to excess (100-fold) diethylstilbestrol or natural estrogens (estradiol-17 beta, estrone, and estriol) but not to progesterone or androgens (testosterone and 5 alpha-dihydrotestosterone). The specific estrogen binding macromolecules eluted from DNA-cellulose columns sedimented at 4-5 S in linear 5-20% sucrose gradients. These characteristics suggest the presence of an estrogen receptor in this androgen target organ.

Self-starting mode locking in a Cr:forsterite laser by use of non-epitaxially-grown semiconductor-doped silica films

We demonstrate RF sputtered, non-epitaxially-grown semiconductor nanocrystallite-doped silica films for mode locking a Cr:forsterite laser. We controlled the size and the optical properties of the nanocrystallites by varying the ratio of InAs to SiO(2) during fabrication. Femtosecond pump-probe measurements were performed to characterize the nonlinear optical properties of these films, revealing their lower saturation fluences. Using the InAs-doped silica films as saturable absorbers permitted self-starting Kerr-lens mode locking (KLM), generating pulses of 25-fs duration with 91-nm spectral bandwidth at 1.3 microm . We also describe saturable-absorber mode-locked operation without KLM and investigate its dependence on intracavity dispersion.

Characterization of estrogen receptors in the hamster brain

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.

Ultrafast dynamics of non-epitaxially grown semiconductor-doped silica film saturable absorbers

Summary form only given. Semiconductor saturable absorbers are a widely used technology for generating femtosecond pulses in solid state lasers, providing advantages such as self-starting operation and the potential for compact laser cavities. The most common semiconductor saturable absorber devices are fabricated by molecular beam epitaxy (MBE) and have been used for both saturable absorber modelocking and initiation of Kerr lens modelocking (KLM) in many solid state laser systems. However, MBE has disadvantages including high complexity and cost as well as lattice matching constraints that limit material choice. Recently, we have developed non-epitaxially grown saturable absorber devices and applied them to self-starting KLM in a Ti:Al/sub 2/O/sub 3/ laser. The devices consist of InAs nanocrystallites doped into SiO/sub 2/ films and deposited on sapphire substrates using a non-magnetron radio frequency (RF) sputtering system. The current work focuses on methods for reducing the saturation fluence in these devices.