Hormonal control of implantation in guinea pigs

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).     

Uterine estradiol and progesterone receptor concentration in relation to circulating hormone levels and histoarchitecture during high endometrial sensitivity and induced decidualization in guinea pigs

Alterations in nuclear and cytosolic estradiol (ER) and progesterone (PR) receptor concentration in the antimesometrial (AM) and mesometrial (M) segments of the uterus in relation to circulating hormone levels, histology and surface topography during the period of high endometrial sensitivity and development of trauma-induced decidualization in cyclic guinea pigs were investigated. The period of high endometrial sensitivity (i.e. day 5 of the estrous cycle) was characterized by elevated plasma estradiol and progesterone and their receptors in the nuclear and cytosolic fractions of the uterus. There was, however, no difference in the concentration of these receptors or the surface ultrastructure in the AM and M segments. Unilateral traumatization by scissor cut along the AM length of the uterus on day 5 of the estrous cycle induced decidual cell reaction resulting in a marked increase in weight of the decidualized (traumatized) uterine horn with advancing decidualization to reach maximum levels (926% of the contralateral nontraumatized uterine horn) 7 days after traumatization. This was associated with decidual transformation and a marked increase in nuclear and cytosolic ER and PR concentration in the AM segment of the traumatized uterine horn. An increase in receptor concentration in the M segment of the traumatized uterine horn or the AM segment of the nontraumatized uterine horn was transitory and of a low order. Receptor concentration in the M segment of the nontraumatized uterine horn remained low throughout days 8–12 of the cycle. Findings indicate a possible role of both estradiol and progesterone in induction of endometrial sensitivity and development and maintenance of decidua in the guinea pig.     

Ah receptor for 2,3,7,8- tetrachlorodibenzo-p-Dioxin: Ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P₁-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene–type compounds such as 3,4,3′4′-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (K_d for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene–type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

Blastocyst is the source of prostaglandins in the implantation site in the rabbit

The levels of prostaglandins (PGs) were measured by radioimmunoassay in the interimplantation and the implantation sites as well as in the implantation site without the blastocyst in the rabbit on day 7 of pregnancy (168h $\text{post coitum}$). The concentrations of PGs were also determined in the blastocyst (PGF:101.59_+?4.33 and PGE-A:29.74_+?3.11 ng/blastocyst, n=6) and the blastocel fluid (PGF:253.55_+?39.56 and PGE-A:83.29_+?6.60 ng/100 ul, n-4) on day 7. The levels of both PGF and PGE-A were significantly higher in the implantation site as compared to interimplantation site (PGF:73.63±6.68 vs. 0.59±0.21 and PGE-A:25.52±3.30 vs. 1.22±0.18 ng/100 mg wet weight n=8). The removal of the blastocyst from the implantation site drastically reduced the concentrations of PGs in this site (PGF:8.71±2.80 and PGE-A:1.64±0.12 ng/100 mg wet weight, n=8). The results provide evidence that the blastocyst is the major source of PGs which contribute to the hige concentration in the implantation site in the rabbit.     

Studies on sex-organ development. Ontogeny of cytoplasmic oestrogen receptor in chick Müllerian duct.

Oestradiol receptors were observed in the cytoplasm of the chick Müllerian duct at several embryonic stages. The sedimentation coefficients and the dissociation constants of the receptor protein remained unchanged throughout the various stages of development. Specific binding of cytoplasmic receptor to [3H]oestradiol assayed in vitro was shown to be saturable at concentration of 10nM or higher. The number of oeastradiol-binding sites on a per-cell basis increased linearly from day 8 to day 12 of incubation and then levelled off from day 12 to the fourth day after hatching. These results indicate that in the developing embryonic sex organ, the same receptor protein is present throughout prenatal development. The concentration of the oestradiol receptor increases and reaches a constant value, but the capacity for the receptor to interact with the hormone does not change.     

Angiotensin II induced increase in frequency of cytosolic and nuclear calcium waves of heart cells via activation of AT1 and AT2 receptors

The aim of this work is to verify if Angiotensin II (Ang II) affects the frequency of spontaneous cytosolic and nuclear Ca2+ waves in chick embryonic cardiomyocytes and if this effect is mediated via the activation of AT1 and/or AT2 receptors. Using the rapid scan technique of confocal microscopy, we observed that Ang II (10(-8)M) increases the frequency of cytosolic and nuclear Ca2+ waves. This effect was accompanied by a decrease in the amplitude of nuclear Ca2+ waves and an absence of effect on the amplitude of cytosolic Ca2+ waves. The effect of the octapeptide on both frequency and amplitude of the nuclear waves was prevented by the AT1 receptor antagonist L158809. However, blockade of the AT2 receptor using the antagonist PD123319 (10(-7)M) only prevented the effect of Ang II on the frequency of Ca2+ waves. Furthermore, the effect was prevented by both a PKC inhibitor (bisindolylmaleimide) and a PKC activator (phorbol 12,13-dibutyrate). In addition, the Ang II effect was not prevented by the blocker of the pacemaker current If. These results demonstrate that Ang II, via the activation of its receptors AT1 and AT2, affects the frequency of spontaneous Ca2+ waves and this effect seems to be mediated by the PKC pathway.     

Experssion of α-Bungarotoxin Receptro Subtypes in Chick Central Nervous System During Development

Abstract

Chick central nervous system (CNS) expresses a-bungarotoxin (aBgtx) receptors. We have recently reported the purification and characterization of two aBgtx receptor subtypes, a7 and a7-a8 from chick optic lobe (COL). In order to study whether other aBgtx receptor subtypes are present in other areas of the chick CNS, as well as their developmental expression, we used anti-a7 and anti-a8 subunitspecific antibodies to study aBgtx receptors at different developmental stages in COL, brain and retina. We found that only the a7 and a7-a8 subtypes are present at all developmental stages in chick COL and brain, where they represent 90% of all the aBgtx receptors at embryonic day 19 and 1 day post hatching (Dl). In chick retina, an a8 subtype representing 50% of all aBgtx receptors at D1 is present in addition to the a7 and a7-a8 subtypes, and the expression of this a8 subtype increases during neurodevelopment.     

Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.     

Receptors for estrogens and progesterone in the porcine cervix

In vitro binding and exchange methods were used to determine the levels of estradiol and progesterone receptors in cytosolic and nuclear fractions of cells obtained from the porcine cervix at different stages of the estrous cycle. The concentration of estradiol cytosolic receptors was about 4500 sites/cell during the luteal phase and increased to a maximum of approximately 7600 sites/cell on day 1 of the cycle, decreasing to a level of 2700 sites/cell on days 3-4. The estradiol nuclear receptor level increased between the end of the luteal phase and the onset of heat from 300 to 1200 sites/cell. No reduction in the number of nuclear sites was seen between day 1 and 3-4. The level of the progesterone cytosolic receptor and its cycle profile was very similar to that of the estradiol receptor. The nuclear receptor, however, reached its lowest level of 760 sites/cell on day 1 of the cycle, increased to a value of 4700 sites on days 3-4 and showed a steady level of about 1000 sites/cell during the luteal phase. The data obtained agree with present theories on the endocrine mechanisms regulating receptor levels in the uterus. Furthermore, these data support a concept in which the constriction of the cervix occurring in response to increased concentrations of circulating estradiol is mediated via steroid receptors.     

The oestrogen receptor in the rat uterus in relation to intra-uterine devices and the oestrous cycle.

We investigated the binding characteristics, content and intracellular distribution of nuclear and cytosolic oestrogen receptors in the uteri of rats bearing a unilateral intrauterine device, fitted 14--18 days earlier, at four phases of a 5-day oestrous cycle. The patterns of changes in wet weight and content of cytosolic and nuclear receptor that normally occur during the oestrous cycle were not altered by the presence of the device. At all stages of the cycle the intra-uterine-device-containing horn had a greater wet weight and a correspondingly higher content of cytosolic receptor than its contralateral control horn, the cellular concentration of cytosolic receptor being apparently maintained. However, the intra-uterine-device-containing horn had significantly lower cellular concentrations (i.e. per mg of DNA) of nuclear receptor, particularly at late dioestrus and pro-oestrus. Thus the treated horn showed a decreased translocation of receptor in response to increases in circulating oestrogens. Both horns contained equivalent amounts of an activating factor implicated in translocation and measured in vitro by binding of cytosol receptor to oligo(dT)-cellulose. The presence of an intra-uterine device neither altered the dissociation constants (Kd) of the nuclear and cytosolic oestrogen-receptor complexes nor the stability of the nuclear receptor complex in vitro. The decreased translocation cannot thus be directly attributed to changes in the physical properties of the receptor. This decrease may be responsible for the anti-fertility effect of the intra-uterine device (which affects only the treated horn of the bicornuate rat uterus), since implantation of the blastocyst requires correct concentrations of nuclear oestrogen receptor.     

Thyroid hormone receptors of developing chick brain are predominantly in the neurons

The relative concentration of the triiodothyronine (T₃) receptors in the neuronal and glial nuclei of developing chick brain have been studied. Scatchard analysis indicate that the number of T₃ binding sites in the neuronal nuclei increases from 400 to 1600 sites/nucleus between 7–11 day of embryonic development without any concomitant change in the level of glial nuclear receptors (130 – 200 sites/nucleus). Both sites are of high affinity (K_a = 1–3 × 10⁹ M^?1) at all ages examined. The abundance of the T₃-receptors in the neuronal nuclei and the close coincidence of the period of rise in the level of these receptors in these nuclei (7–11 day) with that of maximal neuronal growth and synaptogenesis (7–13 day) suggest that the neurons are the primary site of action of T₃ in the developing brain.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica)

The timing of development was examined in 496 embryos from female Monodelphis domestica, collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96–100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.© 1994 Wiley-Liss, Inc.     

The nuclear-bound form of the progesterone receptor is generated through a hormone-dependent phosphorylation

The solubilized ("cytosolic") receptor present in the rabbit uterus in the absence of hormone and the chromatin-bound ("nuclear") receptor obtained after injection of a progestin were compared. Crude cellular extracts were analyzed by immunoblotting and receptors were purified by immunoaffinity chromatography. With both methods it was observed that the electrophoretic mobility of the "nuclear" receptor was slower than that of the "cytosolic" receptor. This difference in mobility appeared to be due to the existence of variably phosphorylated forms of receptor. The phosphorylation reaction was examined in uterine slices. In the absence of hormone the cytosolic receptor was phosphorylated. When hormone was added the phosphorylation of receptor was markedly enhanced and the electrophoretic mobility of the "nuclear" receptor was decreased. These experiments thus show that the receptor in its "cytosolic" form is a phosphoprotein. Under the effect of the hormone the receptor is further phosphorylated on some supplementary site(s). This polyphosphoprotein is the chromatin-bound, putatively active, form of the receptor. In this respect the intracellular progesterone receptor is similar to various membrane receptors for hormones and growth factors which are phosphorylated upon binding of their ligand.     

The ontogeny of cytosol and nuclear progestin receptors in male rat brain and its male-female differences

The fetal and postnatal development of the progestin receptor systems in the intact male rat brain was investigated by means of the in vitro cytosol binding and the nuclear exchange assay using [³H]R5020 ([17α-methyl-³H]17α,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione). The cortical cytosol receptors, first detectable at day 0, rapidly increased at day 7, reaching a maximum at day 10, then gradually declined thereafter. The receptors in the HPOA appeared clearly at day 1, increased during the first 10 days, then remained constant at days 14–21. The postnatal developmental patterns of cytosol brain progestin receptors in males were essentially similar to those in females, but there were some differences between both sexes. The male HPOA at days 10–14 contained more receptors than the female one. Nuclear progestin binding was low in the neonatal male brain at days 1–3. Despite the low level of serum progesterone, the cortical nuclear binding suddenly increased at days 7–10, then remained high at days 14–21. A similar, though less pronounced, pattern was seen in the HPOA. The male pattern of nuclear binding, thus, essentially resembled the female one. However, lower binding in the cortex and, possibly, HPOA was found in males than in females at days 10–21. After progesterone injection postnatal male rats accumulated a lower concentration of progestin receptors in the cortex and, possibly, HPOA than similarly-treated females.It is concluded from these results that progestin receptors in male rat brain appear immediately after birth and develop differentially in the cortex and HPOA. The sudden onset of increased nuclear translocation of endogenous progestin receptor complexes may occur in the brain at around days 7–10. There is a marked sex difference in the nuclear progestin receptor system in the postnatal brain, particularly the cortex. Moreover, the postnatal male brain has lower capacities of nuclear receptor translocation than does the female one. The progestin receptor system in the cortex and, possibly, HPOA of rats in the early postnatal life might be involved with some processes in the mechanism of sexual differentiation of the brain.     

Ontogeny, regional distribution and properties of thyroid-hormone receptors in the developing chick brain

Studies on the thyroid-hormone receptors in the nuclei of developing chick brain revealed a single class of binding sites for tri-iodothyronine (T3) and thyroxine (T4) at all embryonic and adult ages. High-affinity [Ka = (1.85-3.3) X 10(9)M-1 and (0.3-0.6 X 10(9)M-1 for T3 and T4 respectively] receptors were detected in the brain as early as day 7 of embryonic development; their level increased progressively rapidly until day 13, and thereafter the value remained essentially constant during development. Occupancy of the receptor site with endogenous hormone was 75-90% at 7-11 days, 50-60% during the late phase of embryogenesis (13-17 days), and 80% after hatching. Comparison of the binding properties of the receptors with T3 and T4 indicates that, although the binding capacities per nucleus are almost identical, T4 has four to five times less binding affinity than T3. The half-lives of dissociation of solubilized T3- receptor complexes were 20-30h between 0 degrees and 7 degrees C, about 4h at 20 degrees C and less than 15 min at 37 degrees C. Studies of the regional distribution of receptors in the brain indicate that cerebrum has the highest concentration of T3 receptors (4000-7000 sites per nucleus); this concentration is 2-4-fold higher than that in the cerebellum, optic lobe or medulla oblongata. The overall results indicate that between 7 and 13 days of embryonic development the thyroid-hormone receptors in the embryonic chick brain, particularly in the cerebrum, assume a very high level and appear to be mostly saturated with endogenous hormone. This, and the temporal correspondence of the phenomenon with the period of neuronal growth and synaptogenesis, strongly indicate the influence of the hormone in the maturation of the developing brain.     

Effects of aromatic cationic molecules on bovine viral diarrhea virus and embryonic development

Bovine viral diarrhea virus (BVDV) has been shown to replicate in embryo culture systems and remain associated with bovine embryos developing in vitro. In this study, novel antiviral agents were evaluated for capability to inhibit replication of BVDV without affecting embryonic development. Serial concentrations of 2-[5(6)-{2-imidazolinyl}-2-benzimidazolyl]-5-(4-aminophenyl)furan (DB456) or 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) were prepared in IVC medium. Then, bovine uterine tubal epithelial cells (UTC) were placed in IVC media with varying concentrations of DB456 or DB606. Within 1h, a genotype I or II strain of BVDV was added to the cultures. Cultures were maintained for 7 days. Infectious virus was quantitated in IVC media collected on days 3 and 7 and in UTC lysates harvested on day 7. The effective antiviral concentrations of DB606 were much lower than effective antiviral concentrations of DB456. In subsequent experiments, IVF presumptive zygotes were cultured in IVC medium with or without DB456 or DB606 at multiple concentrations for 7 days to evaluate effect of the compound on conceptus development. On day 7, stage of embryonic development was observed, and blastocysts were harvested and stained using Hoechst 33342 to enumerate embryonic cells. While DB456 inhibited blastocyst development, DB606 at 20 times the effective antiviral concentration did not hinder blastocyst development or reduce the mean number of cells per blastocyst. These preliminary results indicated that bovine embryo cultures might be safely supplemented with effective concentrations of an antiviral agent.     

Decreased nuclear binding of estrogen receptors in the pituitary of mice: correlation with biological effects throughout aging

We have determined the biological impact of our previously reported decreased activation of pituitary E2 receptors (PER) in mice. Young (4-8 months), middle-aged (10-14 months) and aged (15-18 months) female mice were studied either intact (metestrous) or gonadectomized (ovx) for 2 weeks prior to decapitation. Recombination studies using heat-treated cytosolic PER and enriched nuclear fractions of various age groups showed a markedly reduced ability for nuclear binding with advancing age, despite unchanged affinity of activated PER for nuclear acceptor sites. Cross incubation studies of heat-activated cytosolic PER with nuclei from mice of various age groups suggested that the activation defect followed the onset of anestrous whereas a reduced nuclear ability to support receptor binding preceded this manifestation. In parallel with these endocrine changes we observed a decrease in baseline cytosolic progesterone receptor (RcP) concentration and in the activity of the enzyme G6PDH in intact aging mice. Ovx induced a further decrease of both markers in young and middle-aged, but not in old mice, while E2 administration induced a decreased pituitary response in RcP but not in G6PDH in aged mice. Our results suggest functional changes in cytosolic nuclear interactions in the pituitary of aging female mice.     

Nuclear binding sites for juvenile hormone III in the male accessory reproductive glands of Melanoplus sanguinipes

Summary

We have identified a potential nuclear juvenile hormone (JH) receptor in the long hyaline tubules (LHT), part of the male accessory reproductive gland (MARG) of M. sanguinipes. The MARG was incubated in vitro with [³H]JH III, and the distribution of the [³H]JH III among the cellular fractions of the LHT was determined. Some 37±4% of the radioactivity was associated with the crude nuclear pellet, while the cytosolic, microsomal and mitochondrial fractions contained 30±3%, 23±2% and 10±1%, respectively. The bound JH III was measured in nuclear extracts of LHT from males up to 15 days post-eclosion. These results revealed that JH binding increased in an age-dependent manner up to day 7, then levelled off to day 12, to increase again on day 14. The nuclear-binding component in the LHT had a very strong affinity for JH III, with a K_D value of 0.8 nM. Our observations are considered in relation to the potential site and mode of action of JH.