Colin M. Clay

Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback.

Kisspeptins are encoded by the gene KiSS‐1 and regulate gonadotrophin‐releasing hormone (GnRH) and gonadotrophin secretion in various species, including humans. Here, we quantify gene expression of KiSS‐1 in the arcuate nucleus (ARC) across the ovine oestrous cycle and demonstrate an increase in the caudal division of the ARC during the preovulatory period. These data strongly suggest that kisspeptins are involved in the generation of the preovulatory GnRH and luteinising hormone surge.

Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of proximal 5' flanking region.

Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by the hypothalamus. Upon binding to specific high-affinity receptors on gonadotrope cells of the anterior pituitary gland, GnRH stimulates the synthesis and secretion of LH. In light of the critical role of GnRH in reproduction much effort has been directed toward understanding the regulation of this hormone and its cognate receptor. The recent availability of genomic clones for the GnRH receptor has facilitated research to address the molecular mechanisms underlying regulation of GnRH receptor gene expression. We have expanded the analysis of the promoter for the mouse GnRH receptor gene and report that in addition to transcriptional start sites located within 100 bp of the translation start codon there is a more distal transcriptional start site approximately 200 bp 5′ of the initiation codon. The initiation of transcription from this more distal site was sufficient to confer cell-specific expression on luciferase. Further, transient expression assays of constructs containing progressive 5′ deletions in the GnRH receptor gene promoter reveal the presence of one or morecis-acting elements located between −500 and −400 (relative to ATG) necessary for transcriptional activity in the gonadotrope-derived αT3 cell line. Finally, αT3 but not COS-7 cell nuclear extract contained protein(s) that bind to at least two separate motifs contained within the −500 to −400 region. We suggest that activation of GnRH receptor gene expression in the αT3 cell line requires the binding of at least two transcriptional regulatory proteins to basal enhancer elements located within a 100 bp region between −500 to −400 relative to the translation start codon in the mouse GnRH receptor gene.

Expression of Growth and Differentiation Factor-9 in the Ovaries of Fetal Sheep Homozygous or Heterozygous for the Inverdale Prolificacy Gene (FecXI)

Abstract Abnormal follicular and oocyte growth in ovaries of sheep homozygous (II) for the Inverdale gene, FecXI, suggest that this gene may influence a fundamental event in initiation of folliculogenesis, with two copies of the gene inhibiting growth at the primordial/primary stage. In addition, striking similarities in ovarian morphology between mice deficient in growth and differentiation factor-9 (GDF-9) and II sheep suggest a relationship between the FecXI gene and GDF-9 function in the ovary. Therefore, it was hypothesized that GDF-9 mRNA expression would be inhibited in ovaries of II fetal sheep. To test this hypothesis, in situ hybridization was used to characterize GDF-9 mRNA expression in ovaries of homozygous (II), heterozygous (I+), and control (++) fetal sheep at Day 135 of gestation. GDF-9 mRNA expression was localized exclusively to oocytes from the type 1 follicle stage onward in all genotypes and is the first demonstration of GDF-9 mRNA expression in ovaries of fetal sheep. In addition, GDF-9 mRNA expression was detected in oocytes of abnormal type 2 follicles in the ovaries of II sheep. Thus, it does not appear that inhibition of GDF-9 gene expression is the mechanism of action whereby the FecXI gene exerts its influence. However, the possibility of translation at specific stages of follicular development cannot presently be ruled out. In addition, the FecXI gene may be involved, either directly or indirectly, in regulating expression of receptors for GDF-9. At present, however, neither the FecXI gene product nor the GDF-9 receptor has been isolated or characterized.

Cellular Mechanisms by Which Oxytocin Mediates Ovine Endometrial Prostaglandin F2α Synthesis: Role of Gi Proteins and Mitogen-Activated Protein Kinases

Abstract Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2α synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2α synthesis. The objective of experiment 1 was to determine whether Gi proteins mediate oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of Gi proteins, had no effect on the ability of oxytocin to induce PGF2α synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF2α synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF2α synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to Gi proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2α synthesis in ovine endometrium.

Atrazine inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity to a gonadotropin-releasing hormone receptor agonist in female Wistar rats.

Abstract Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-tri-azine] is one of the most commonly used herbicides in the United States. Atrazine has been shown to suppress luteinizing hormone (LH) release and can lead to a prolongation of the estrous cycle in the rat. The objectives of this study were to examine the effects of atrazine on normal tonic release of LH and to elucidate the site of action of atrazine in the hypothalamic-pituitary-gonadal axis. Episodic release of gonadotropin-releasing hormone (GnRH) and the corresponding release of LH from the anterior pituitary gland are required for normal reproductive function. To determine if atrazine affects pulsatile LH release, ovariectomized adult female Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle control for 4 days. On the final day of atrazine treatment, blood samples were obtained using an indwelling right atrial cannula. In the group receiving 200 mg/kg, there was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude. To determine if the effects of atrazine on LH release were due to changes at the level of the pituitary, animals were passively immunized against endogenous GnRH, treated with atrazine, and challenged with a GnRH receptor agonist. Atrazine failed to alter pituitary sensitivity to the GnRH receptor agonist at any dose used. Taken together, these findings demonstrate that high doses of atrazine affect the GnRH pulse generator in the brain and not at the level of gonadotrophs in the pituitary.

Biological and Anatomical Evidence for Kisspeptin Regulation of the Hypothalamic-Pituitary-Gonadal Axis of Estrous Horse Mares

The purpose of the present study was to evaluate the effects of kisspeptin (KiSS) on LH and FSH secretion in the seasonally estrous mare and to examine the distribution and connectivity of GnRH and KiSS neurons in the equine preoptic area (POA) and hypothalamus. The diestrous mare has a threshold serum gonadotropin response to iv rodent KiSS decapeptide (rKP-10) administration between 1.0 and 500 microg. Administration of 500 microg and 1.0 mg rKP-10 elicited peak, mean, and area under the curve LH and FSH responses indistinguishable to that of 25 microg GnRH iv, although a single iv injection of 1.0 mg rKP-10 was insufficient to induce ovulation in the estrous mare. GnRH and KiSS-immunoreactive (ir) cells were identified in the POA and hypothalamus of the diestrous mare. In addition, KiSS-ir fibers were identified in close association with 33.7% of GnRH-ir soma, suggesting a direct action of KiSS on GnRH neurons in the mare. In conclusion, we are the first to reveal a physiological role for KiSS in the diestrous mare with direct anatomic evidence by demonstrating a threshold-like gonadotropin response to KiSS administration and characterizing KiSS and GnRH-ir in the POA and hypothalamus of the diestrous horse mare.

Developmental Profile and Sexually Dimorphic Expression of Kiss1 and Kiss1r in the Fetal Mouse Brain

The hypothalamic-pituitary-gonadal axis (HPG) is a complex neuroendocrine circuit involving multiple levels of regulation. Kisspeptin neurons play essential roles in controlling the HPG axis from the perspectives of puberty onset, oscillations of gonadotropin releasing hormone (GnRH) neuron activity, and the pre-ovulatory LH surge. The current studies focus on the expression of kisspeptin during murine fetal development using in situ hybridization (ISH), quantitative reverse transcription real-time PCR (QPCR), and immunocytochemistry. Expression of mRNA coding for kisspeptin (KISS1) and its receptor KISS1R was observed at embryonic (E) day 13 by ISH. At E13 and other later ages examined, Kiss1 signal in individual cells within the arcuate nucleus (ARC) appeared stronger in females than males. ISH examination of agonadal steroidogenic factor-1 (Sf1) knockout mice revealed that E17 XY knockouts (KO) resembled wild-type (WT) XX females. These findings raise the possibility that gonadal hormones modulate the expression of Kiss1 in the ARC prior to birth. The sex and genotype differences were tested quantitatively by QPCR experiments in dissected hypothalami from mice at E17 and adulthood. Females had significantly more Kiss1 than males at both ages, even though the number of cells detected by ISH was similar. In addition, QPCR revealed a significant difference in the amount of Kiss1 mRNA in Sf1 mice with WT XY mice expressing less than XY KO and XX mice of both genotypes. The detection of immunoreactive KISS1 in perikarya of the ARC at E17 indicates that early mRNA is translated to peptide. The functional significance of this early expression of Kiss1 awaits elucidation.

Microarray analysis of Foxl2 mediated gene regulation in the mouse ovary derived KK1 granulosa cell line: Over-expression of Foxl2 leads to activation of the gonadotropin releasing hormone receptor gene promoter.

BackgroundThe Foxl2 transcription factor is required for ovarian function during follicular development. The mechanism of Foxl2 regulation of this process has not been elucidated. Our approach to begin to understand Foxl2 function is through the identification of Foxl2 regulated genes in the ovary.MethodsTransiently transfected KK1 mouse granulosa cells were used to identify genes that are potentially regulated by Foxl2. KK1 cells were transfected in three groups (mock, activated, and repressed) and twenty-four hours later RNA was isolated and submitted for Affymetrix microarray analysis. Genesifter software was used to carry out analysis of microarray data. One identified target, the gonadotropin releasing hormone receptor (GnRHR) gene, was chosen for further study and validation of Foxl2 responsiveness. Transient transfection analyses were carried out to study the effect of Foxl2 over-expression on GnRHR gene promoter-luciferase fusion activity. Data generated was analyzed with GraphPad Prism software.ResultsMicroarray analysis identified 996 genes of known function that are potentially regulated by Foxl2 in mouse KK1 granulosa cells. The steroidogenic acute regulatory protein (StAR) gene that has been identified as Foxl2 responsive by others was identified in this study also, thereby supporting the effectiveness of our strategy. The GnRHR gene was chosen for further study because it is known to be expressed in the ovary and the results of previous work has indicated that Foxl2 may regulate GnRHR gene expression. Cellular levels of Foxl2 were increased via transient co-transfection of KK1 cells using a Foxl2 expression vector and a GnRHR promoter-luciferase fusion reporter vector. The results of these analyses indicate that over-expression of Foxl2 resulted in a significant increase in GnRHR promoter activity. Therefore, these transfection data validate the microarray data which suggest that Foxl2 regulates GnRHR and demonstrate that Foxl2 acts as an activator of the GnRHR gene.ConclusionsPotential Foxl2 regulated ovarian genes have been identified through microarray analysis and comparison of these data to other microarray studies. The Foxl2 responsiveness of the GnRHR gene has been validated and provided evidence of Foxl2 transcriptional activation of the GnRHR gene promoter in the mouse ovary derived KK1 granulosa cell line.

Internalization Rates of Murine and Ovine Gonadotropin-Releasing Hormone Receptors1

Abstract Rates of internalization of the murine GnRH receptor fused via its C-terminus to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamster ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (LβT2 cells). The resulting rates of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinated [d-Ala6]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, then cells were warmed to 37°C to permit internalization. Surface-bound radioligand began to decrease as soon as the cells were warmed and had decreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LβT2 cells (38% and 41%, respectively, of initial value; P < 0.05). Internalization of radioligand began immediately after warming the cells to 37°C, and a significant proportion of surface ligand had been internalized by 20 min. A steady-state maximum of internalization was reached after 60 min in both CHO cells and LβT2 cells (29% and 28%, respectively, of total cell-associated ligand; P < 0.05). Changes in surface-bound radioligand and internalized radioligand in sheep pituitary cells were similar to those in CHO cells and LβT2 cells, but the amount of radioligand internalized after 60 min (40% of total cell-associated ligand) was 1.4 times higher than in CHO cells and LβT2 cells (P < 0.05). In a separate experiment, the effect of estradiol on the rate of internalization of GnRH-R in ovine pituitary cells was examined. Although treatment of ovine pituitary cells with estradiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show that rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in LβT2 cells and in sheep pituitary cells, respectively, are similar, but amounts of ovine GnRH-R internalized are greater than those for murine GnRH-R. Further, the rate of internalization of occupied receptor is similar in gonadotroph and nongonadotroph cells, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.

Differential Impact of Intracellular Carboxyl Terminal Domains on Lipid Raft Localization of the Murine Gonadotropin-Releasing Hormone Receptor

Abstract The mammalian type I GNRH receptor (GNRHR) is unique among G protein-coupled receptors (GPCRs) because of the absence of an intracellular C-terminus. Previously, we have found that the murine GNRHR is constitutively localized to low-density membrane microdomains termed lipid rafts. As such, association of the GNRHR with lipid rafts may reflect both a loss (C-terminus) and a gain (raft association address) of structural characteristics. To address this, we fused either the full-length C-terminus from the nonraft-associated LH receptor (LHCGR; GNRHR-LF) or a truncated (t631) LHCGR C-terminus to the GNRHR. These chimeric receptors are trafficked to the plasma membrane, bind ligand, and display increased agonist-induced receptor internalization, but they do not partition into lipid rafts. Thus, a heterologous C-terminus from a nonraft-associated GPCR redirects localization of the GNRHR to nonraft domains. In contrast to the murine GNRHR, the catfish GNRHR (cfGNRHR) possesses an intracellular C-terminus. We found that the cfGNRHR was localized to lipid rafts and that the cfGNRHR C-terminus did not alter raft localization of the mammalian receptor. Consistent with placement in different lipid microenvironments within the plasma membrane, fluorescence recovery after photobleaching revealed different lateral diffusion phenotypes of the raft-associated GNRHR and cfGNRHR versus the nonraft-associated GNRHR-LF fusion protein. We conclude that whereas an intracellular C-terminus is capable of redirecting the GNRHR to nonraft compartments, this is not a generalized feature of GPCR C-terminal tails. Thus, constitutive raft localization of the GNRHR is not simply a result of the loss of an intracellular C-terminus.