Biomedical subjects

K E Mayo

Publications and source records attributed to K E Mayo.

At least 55 records · Page 3Linked to original sources

PubMed1992

Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.

Amino Acid Sequence↗

PubMed1992

Nonsteroidal signals originating in the gonads.

The discovery of the various peptide factors in the gonads followed different paths. A number of factors were specifically searched for because of physiological experiments that predicted that an activity from the gonads was necessary to explain phenomena. Such was the case for gonadal steroids and for such peptide factors as inhibin, MIS, OMI, FRP, seminal plasma inhibin, relaxin, PA factor and other proteases, and ABP. In the process other factors such as activin and follistatin were serendipitously discovered. A second group of factors was discovered because in vitro experiments of various combinations of gonadal cell types failed to replicate in vivo findings, suggesting missing signals. Such substances are the panoply of growth factors aiding in differentiation and growth promotion and inhibition: LS and LI, P-Mod-S, clusterin, and various components of the ECM. Finally, and most recently, another set of peptides has been identified because immunological or molecular probes have been used to search gonadal tissue for factors originally discovered elsewhere; these include POMC, GnRH-like peptide, oxytocin, AVP, angiotensin, ANF, CRF, neural peptides, and c-mos. Our understanding of the relationship of most of these peptides to the local signals necessary for gonadal function is still very elementary. Clearly some like relaxin and inhibin function as important hormones, and ABP, for example, probably functions importantly in transporting testosterone down the tubule. Most local paracrine or autocrine peptide signals appear to act in relationship to gonadotropin levels probably in local differentiation in the process of gamete maturation, but this is only conjecture at this point. No experimental verification that any of these factors is involved in follicle selection for recruitment or for atresia is yet available. For many of the factors local receptors have not yet been identified. The richness of the variety of peptides in the gonads suggests that microanalysis of cell-cell signaling would be rewarding, but at the time of this writing such investigations are not yet possible.

Animals↗

PubMed1992

Gonadotropin-releasing hormone, inhibin, and activin in human placenta: evidence for a common cellular localization.

The human placenta produces several hypophysiotropic factors that participate in the control of local hormone secretion. In particular, previous reports showed that inhibin and activin modulate both GnRH release and hormonal effect in cultured human placental cells. The present study evaluated the possible correlations among the synthesis and cellular distribution of inhibin, activin, and GnRH in placental villi at term. mRNA coding for inhibin alpha- and beta A-subunits and GnRH were localized in human placenta at term by in situ hybridization using the corresponding cDNA probes. Autoradiograms revealed that some placental cells express inhibin alpha- and beta A-subunit and GnRH mRNAs. A common localization of the three hormonal mRNAs has been found in the inner part of the villi. Using affinity-purified polyclonal antisera, immunocytochemical studies confirmed that in placental villi at term, immunoreactive inhibin alpha- and beta A-subunits and GnRH had a distribution that was superimposable in several areas. Both the outer layer and the inner trophoblasts contained immunoreactive hormonal products. The present data show that some placental cells at term can produce and release inhibin, activin, and/or GnRH. This complement of hypophysiotropic factors may, thus, represent a local paracrine/autocrine control mechanism within the placenta.

Activins↗

PubMed1992

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.

Adenoma↗

PubMed1991

Expression of ovarian inhibin during pregnancy in the rat.

We have examined the expression of the rat inhibin genes in the maternal ovary during pregnancy. RNA blot analysis indicates that the inhibin-alpha chain mRNA is expressed in the ovary throughout gestation at levels comparable to those observed in cycling rats. In situ hybridization shows that the inhibin-alpha and -beta A mRNAs are produced in the granulosa cells of developing antral follicles; little or no hybridization to functional corpora lutea is observed. Early in pregnancy, a large number of follicles hybridize to both alpha- and beta A-inhibin cDNA probes. Many of these follicles undergo atresia during the first half of pregnancy, and the number of inhibin-expressing follicles reaches a nadir on day 15. This is followed by an increase in inhibin-producing follicles, which peaks just before parturition. The increase in inhibin-expressing follicles observed in late pregnancy corresponds to a small rise in serum inhibin levels, as measured using an alpha chain-specific RIA. After the first postpartum ovulation, few hybridizing follicles are observed. Ovariectomy in either early (day 6) or mid (day 15) pregnancy results in a significant fall in serum inhibin levels and a robust increase in serum FSH levels 9 h after surgery. These results suggest that inhibin is produced by the maternal ovary during pregnancy, that its synthesis is modulated during late gestation, and that inhibin may play a role in regulating FSH secretion during pregnancy.

Animals↗

PubMed1991

Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary.

The pituitary gonadotropins FSH and LH are key hormones for regulating gametogenesis and steroidogenesis in the ovary and testis. The cell surface receptors that mediate the biological activities of these hormones are thought to be expressed in a cell-specific fashion in the ovary and are regulated as animals progress through the reproductive cycle. Using cloned receptor cDNAs, we have examined the expression and hormonal regulation of the ovarian FSH and LH receptor mRNAs in the rat. A quantitative reverse transcription-polymerase chain reaction amplification scheme was used to measure relative levels of the FSH and LH receptor mRNAs, while in situ hybridization was used to localize FSH and LH receptor transcripts. In immature animals, low levels of FSH receptor mRNA are observed in the granulosa cells of small follicles, while low levels of LH receptor mRNA are found in the thecal cells of these same follicles. After stimulation with PMSG, levels of both mRNAs increase, and the LH receptor mRNA is localized in both the granulosa and thecal cells of large follicles. Further treatment of PMSG-primed animals with hCG results in down-regulation, particularly of the LH receptor mRNA in granulosa cells. In adult animals, LH receptor mRNA levels change dramatically during the estrous cycle, particularly after the preovulatory LH surge. FSH receptor mRNA levels show a similar pattern of change, but the FSH receptor mRNA is of lower abundance and is not as highly regulated as the LH receptor mRNA. FSH receptor mRNA is confined to the granulosa cells of healthy developing follicles, whereas LH receptor mRNA is localized predominantly to thecal cells of small follicles on estrous morning, then appears in the granulosa cells of growing follicles by diestrous morning. LH receptor mRNA is also found in interstitial tissues and corpora lutea throughout much of the estrous cycle. Our results indicate that the gonadotropin receptor genes are regulated in a complex fashion during the recruitment, maturation, and ovulation of the ovarian follicle.

Animals↗

PubMed1991

Regulation of the alpha inhibin gene by cyclic adenosine 3',5'-monophosphate after transfection into rat granulosa cells.

Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. To examine further the transcriptional regulation of the alpha inhibin gene by FSH and cAMP, we have isolated and characterized a genomic clone that contains the entire rat alpha inhibin gene. Sequence analysis of the alpha inhibin promoter region revealed several potential cAMP response elements (CREs) and transcription factor AP2-binding sites that might mediate cAMP regulation. To determine the functional importance of these sequences, fusion genes including the alpha inhibin 5' flanking region linked to a luciferase reporter gene were transiently transfected into primary granulosa cells isolated from immature rats. These fusion genes were both expressed and regulated by the adenylyl cyclase activator forskolin in transfected granulosa cells. Analysis of a series of 5' deletion mutants indicated that a construct containing as little as 170 basepairs up-stream of the alpha inhibin start site, which includes a single imperfect CRE and no AP2 sites, was regulated by forskolin. DNAse footprinting was used to demonstrate that bacterially expressed CRE-binding protein (CREB) binds to this CRE located 122 basepairs up-stream of the alpha inhibin gene transcriptional start site. To investigate further the role of this CRE in alpha inhibin gene expression, site-specific mutagenesis of the CRE was performed. The alpha inhibin promoter containing a mutated CRE was not regulated by forskolin in granulosa cells and did not bind the CREB protein. Interestingly, mutation of the CRE also substantially reduced basal expression of the alpha inhibin promoter. Lastly, a gel mobility shift assay was used to examine CRE-binding proteins from granulosa cell extracts. Granulosa cells contain a protein that specifically interacts with CRE-containing oligonucleotides or with the alpha inhibin promoter and that is recognized by antibodies against the CREB protein. Our results suggest that CREB or related transcription factors play an important role in both basal and cAMP-regulated expression of the alpha inhibin gene in ovarian granulosa cells.

Animals↗

PubMed1991

Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge.

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

Amino Acid Sequence↗

PubMed1990

Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus.

Photic information entrains a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus to environmental light/dark cycles. To determine whether light regulates c-fos gene expression in the SCN, we have measured c-fos mRNA levels in the SCN of the golden hamster. We report that, during the subjective night, light causes a rapid increase in levels of c-fos mRNA in the SCN. Light pulses of 5 min duration are sufficient to induce c-fos mRNA, and the highest mRNA levels occur 30 min following the onset of light. The minimum level of illumination required to induce an increase in c-fos mRNA is indistinguishable from the minimum irradiance that produces a phase shift in the hamster's circadian rhythm of activity. In addition, the induction of c-fos mRNA in the SCN by light is itself under circadian regulation. Light induction of c-fos mRNA occurs only during the subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, these data suggest that c-fos may be a molecular component of the photic pathway for entrainment of mammalian circadian rhythms.

Animals↗

PubMed1990

Regulation of inhibin synthesis in the rat ovary.

The lifespan of the female rodent is characterized by dynamic changes in the hormonal regulation of the reproductive axis. From the time ovarian follicular growth is initiated in peripubertal animals, through recruitment, ovulation and luteinization during the estrous cycle, to the quiescent follicular development of pregnancy, and ultimately to follicular exhaustion, tremendous changes in follicular architecture, hormonal responsiveness, steroid secretion, and inhibin and activin synthesis occur. Similar changes in both the biosynthetic functions and hormonal responsiveness of the pituitary are likely to occur. Some of the information on inhibin expression during the reproductive life-span of the rat reviewed here is summarized in a schematic fashion in Figure 3, which presents changes in serum FSH, ovarian inhibin production, and follicular development during different reproductive states. From recent observations regarding ovarian inhibin expression as a function of reproductive status, a partial picture of the complex interactions between steroid and peptide hormones necessary to maintain reproductive cyclicity in mammals is beginning to emerge. Study of the inhibin gene family and of other FSH-regulatory hormones is likely to further enhance our understanding of both normal reproductive processes and of reproductive disorders.

Aging↗

PubMed1990

The rat follistatin gene is highly expressed in decidual tissue.

Follistatin was originally described as an ovarian polypeptide hormone able to suppress pituitary follicle-stimulating hormone (FSH) secretion in in vitro systems. We have isolated rat ovarian follistatin cDNA clones and used these to investigate the possibility that decidual tissue expresses the follistatin gene. We found that the follistatin gene is highly expressed during early pregnancy in tissue derived from the site of blastocyst implantation. Follistatin mRNA was observed in tissue obtained from the implantation site at days 6, 8 and 10 of gestation, but was not found in the placenta at later stages of gestation. This same pattern of follistatin expression was observed in pseudopregnant animals in which decidualization had been artificially induced, indicating that decidual tissue is the primary source of follistatin mRNA. In situ hybridization was used to examine follistatin gene expression in decidua in greater detail. The mRNA is preferentially found in antimesometrial tissue, and is strictly confined to the endometrial-derived decidual cells. These results suggest that follistatin might function as an important endocrine hormone during implantation of the fetus, perhaps by acting to appropriately modulate maternal FSH secretion during early pregnancy.

Animals↗

PubMed1990

Gonadotropin-releasing hormone gene expression during the rat estrous cycle: effects of pentobarbital and ovarian steroids.

Although hypothalamic GnRH release is known to be modulated by neural and hormonal factors, the relationship between altered GnRH secretion and GnRH synthesis remains unclear. In an attempt to address this question, we examined GnRH gene expression in the rat hypothalamus using in situ hybridization histochemistry. An 25S-labeled antisense RNA probe was used to identify neurons expressing GnRH mRNA in an area that included the diagonal band of Broca, the organum vasculosum of the lamina terminalis, and the preoptic area. The number of GnRH mRNA-expressing cells was determined at various times during the rat estrous cycle. During proestrus, the number of GnRH mRNA-expressing cells decreased somewhat at 1400-1600 h, increased significantly at 1800 h (the time of the LH surge), then gradually returned to basal levels at 2200 h. Expression did not change substantially at other times during the estrous cycle. To understand this close temporal relationship between the LH surge and increased GnRH mRNA levels, we examined GnRH gene expression in proestrous animals in which the LH surge was blocked with pentobarbital. Pentobarbital treatment blocked the increase in the number of GnRH mRNA-expressing cells normally observed at 1800 h in saline-treated controls, suggesting that the increase in GnRH gene expression is closely coupled to secretion of GnRH from the hypothalamus. Finally, we addressed the question of whether ovarian steroids have direct effects on GnRH gene expression by examining GnRH mRNA levels in ovariectomized steroid-treated rats at a time before (1100 h) and a time after (1800 h) hypothalamic GnRH hypersecretion. At 1100 h, no significant changes were observed, but at 1800 h, estrogen-treated rats showed a significant increase in both the number of GnRH mRNA-expressing cells and serum LH levels. This suggests that estrogen influences GnRH gene expression indirectly, perhaps by altering hypothalamic GnRH release. Our results in each of these models suggest that GnRH mRNA levels increase in response to GnRH hypersecretion at the time of the LH surge.

Animals↗

PubMed1989

Production of transgenic sheep with growth-regulating genes.

Pronuclei of fertilized sheep ova were injected with fusion genes consisting of the mouse metallothionein-I promotor/regulator ligated to either the structural gene for bovine growth hormone (mMTbGH) or to a minigene for human growth hormone-releasing factor (mMThGRF). From a total of 842 sheep ova injected with mMTbGH and transferred into recipient ewes, 47 lambs were born. Two of the lambs were transgenic with mMTbGH, and both had bGH mRNA present in liver, kidney, and gut. In one lamb, plasma growth hormone was as high as 700 ng/ml. From a total of 435 sheep ova injected with mMThGRF and transferred to recipients, 54 lambs were born and 9 fetuses were collected. Nine of the 63 had integrated the mMThGRF gene. One of the nine had high concentrations of immunoassayable hGRF in its plasma and high variable plasma concentrations of ovine growth hormone. The lamb that expressed the hGRF gene did not release GH in response to an hGRF challenge. Four of five fetal offspring of a nonexpressing mMThGRF transgenic ram also contained the mMThGRF gene and, like the sire, failed to express the gene as determined by either liver hGRF mRNA or by plasma hGRF. Growth of the single transgenic lamb expressing hGRF was similar to control lambs. These studies demonstrate efficient introduction of genes into the sheep genome and indicate that transgenes are expressed and heritable.

Animals↗

PubMed1989

Unilateral ovariectomy increases inhibin messenger ribonucleic acid levels in newly recruited follicles.

We have used the unilaterally ovariectomized (ULO) rat as an experimental model to assess transient relationships between follicular recruitment and inhibin mRNA levels in the ovary. Animals were divided into three experimental groups (ULO, sham-operated, and intact) and killed 0, 9, or 24 h postsurgery. Inhibin alpha- and beta A mRNA were measured within individual ovarian follicles by in situ hybridization. Serum FSH concentrations in ULO animals increased significantly at 9 h and returned to presurgery levels by 24 h. There was no effect of surgery on serum LH concentrations. In the ULO animals, compensatory follicular recruitment was observed in the remaining ovary at 24 h as a significant increase in the number of follicles with mean diameter greater than 350 micron. Two distinct populations of healthy maturing follicles, based on diameter and hybridization intensity, were observed in ovaries from 24 h post-ULO rats. The first class, NRF-1 (newly recruited follicle-1), were those recruited 48 h previously by the secondary FSH surge of the preceding cycle. These large follicles (greater than 500 micron mean diameter) hybridized to both inhibin probes. The second class of follicles (NRF-2), which represent those newly recruited by the ULO-induced increase in serum FSH, were smaller in size than NRF-1 (350-500 micron mean diameter). The intensity of hybridization of both inhibin probes to NRF-1 was higher than that to NRF-2 in the majority of follicles. Each NRF-1 contained higher levels of both alpha- and beta A-inhibin mRNA than nonrecruited follicles (150-350 micron diameter). These data suggest that subtle changes in serum FSH concentrations, which are sufficient to induce follicular recruitment into a size class capable of ovulating within a given cycle, play the additional role of enhancing inhibin mRNA levels in developing follicles.

Animals↗

PubMed1989

Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges.

We have examined the role of the primary gonadotropin surges in regulating inhibin alpha- and beta A-subunit mRNA levels in rat ovarian follicles. Inhibin subunit mRNA levels decline dramatically on the evening of proestrus in follicles of the ovulatory pool. Because this decline is temporally associated with primary gonadotropin surges, we investigated the contribution of LH and FSH to this process. The primary gonadotropin surges were blocked by injection of a GnRH antagonist (WY45760) at 1200 h on proestrus. This resulted in sustained elevation of inhibin mRNA levels through 0700 h of the subsequent day, a time when inhibin mRNA levels would normally be very low. Replacement of either exogenous LH or FSH in ovulatory doses to an antagonist-treated animal at 1530 h on proestrus resulted in a decrease in inhibin mRNA levels by 4-5 h postreplacement. We conclude that LH and FSH act via a common mechanism to repress inhibin mRNA levels in stimulated preovulatory follicles.

Animals↗

PubMed1989

Mouse growth-hormone-releasing hormone: precursor structure and expression in brain and placenta.

To pursue questions concerning the regulation of somatic growth in a species amenable to both genetics and germ-line manipulation, we have isolated and characterized a full-length cDNA clone encoding mouse GH-releasing hormone (mGHRH). A GHRH cDNA clone isolated from a mouse placental library contains an open-reading frame of 309 basepairs that predicts a 103 amino acid mouse GHRH precursor protein. The mature mouse GHRH is predicted to be 42 amino acids with a free carboxyl-terminus. Although the mGHRH precursor sequence is clearly related to those determined for rat and human, the mature mGHRH peptide differs at seven of its 42 positions from all previously characterized GHRH peptides. RNA blot analysis of mouse tissues indicates that the mature 750 nucleotide mGHRH mRNA is found in hypothalamus and placenta, while testis contains a larger GHRH-related transcript. In situ hybridization analysis of GHRH gene expression in the mouse brain indicates that GHRH mRNA is localized predominantly to the arcuate nucleus of the hypothalamus. In the placenta, GHRH mRNA levels are developmentally regulated and peak on days 16-17 of gestation. GHRH mRNA is localized predominantly to trophoblast giant cells and to cytotrophoblasts of the placental labyrinth.

Amino Acid Sequence↗

PubMed1988

Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle.

The alterations in morphology and function of the ovarian follicle as it matures, ovulates, and becomes a corpus luteum are dramatic. A variety of steroid and polypeptide hormones influence these processes, and the ovary in turn produces specific hormonal signals for endocrine regulation. One such signal is inhibin, a heterodimeric protein that suppresses the secretion of follicle-stimulating hormone from pituitary gonadotrophs. Rat inhibin complementary DNA probes have been used to examine the levels and distribution of inhibin alpha-and beta A-subunit messenger RNAs in the ovaries of cycling animals. Striking, dynamic changes have been found in inhibin messenger RNA accumulation during the developmental maturation of the ovarian follicle.

Animals↗

PubMed1988

Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene.

A transgenic animal model system was used to analyze the mitogenic effects of GRF on its target cell, the pituitary somatotroph. We have previously established a strain of mice that express a mouse metallothionein-I/human GRF (hGRF) fusion gene, and that grow to be abnormally large due to GH hypersecretion. We show here that chronic GRF production in these mice leads to the development of enormous pituitary glands. The increase in pituitary size appears to be largely the result of a selective proliferation (hyperplasia) of somatotrophs, the GH-producing cells. This observation provides direct evidence that a neuropeptide may act as a specific trophic factor for its target cell. In addition to this effect on pituitary development, we find that the pituitary is a major site of expression of mouse metallothionein-I/hGRF mRNA, and of hGRF peptide. This tissue specificity was unexpected in that neither component of the fusion gene is highly expressed in the normal pituitary. It suggests that pituitary somatotrophs might produce and respond to GRF in an essentially autocrine fashion in these transgenic animals.

Animals↗