Role of gonadotropin-releasing hormone-II and its receptor in swine reproduction.

The pig represents the only livestock mammal capable of producing a functional protein for the second mammalian form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). To examine the role of GnRH-II and its receptor in pig reproduction, we produced a unique swine line with ubiquitous knockdown of endogenous GnRHR-II levels (GnRHR-II knockdown [KD]), which is largely the focus of this review. In mature GnRHR-II KD males, circulating testosterone concentrations were 82% lower than littermate control boars, despite similar luteinizing hormone (LH) levels. In addition, nine other gonadal steroids were reduced in the serum of GnRHR-II KD boars, whereas adrenal steroids (except 11-deoxycortisol) did not differ between lines. Interestingly, testes from GnRHR-II KD males had fewer, hypertrophic Leydig cells and fewer, enlarged seminiferous tubules than control testes. As expected, downstream reproductive traits such as androgen-dependent organ weights and semen characteristics were also significantly reduced in GnRHR-II KD versus control boars. Next, we explored the importance of this novel ligand/receptor complex in female reproduction. Transgenic gilts had fewer, but heavier, corpora lutea with smaller luteal cells than littermate control females. Although the number of antral follicles were similar between lines, the diameter of antral follicles tended to be larger in GnRHR-II KD females. In regard to steroidogenesis, circulating concentrations of progesterone and 17ß-estradiol were lower in transgenic compared to control gilts, even though serum levels of follicle-stimulating hormone and LH were similar. Thus, GnRH-II and GnRHR-II represent a potential avenue to enhance fertility and promote the profitability of pork producers.

Role of gonadotropin-releasing hormone 2 and its receptor in human reproductive cancers.

Gonadotropin-releasing hormone (GnRH1) and its receptor (GnRHR1) drive reproduction by regulating gonadotropins. Another form, GnRH2, and its receptor (GnRHR2), also exist in mammals. In humans, GnRH2 and GnRHR2 genes are present, but coding errors in the GnRHR2 gene are predicted to hinder full-length protein production. Nonetheless, mounting evidence supports the presence of a functional GnRHR2 in humans. GnRH2 and its receptor have been identified throughout the body, including peripheral reproductive tissues like the ovary, uterus, breast, and prostate. In addition, GnRH2 and its receptor have been detected in a wide number of reproductive cancer cells in humans. Notably, GnRH2 analogues have potent anti-proliferative, pro-apoptotic, and/or anti-metastatic effects on various reproductive cancers, including endometrial, breast, placental, ovarian, and prostate. Thus, GnRH2 is an emerging target to treat human reproductive cancers.

The effect of varicocele on semen quality in boars exposed to heat stress.

Semen quality has a dramatic impact on reproductive efficiency in the swine industry, influencing both conception rate and litter size. The objective of this study was to assess whether the presence of varicocele hinders semen quality in both thermoneutral and heat stress (HS) conditions. At approximately 6 mo of age, ultrasonography was used to measure left and right pampiniform plexus area in order to detect varicocele in maternal line boars at the University of Nebraska-Lincoln. Between 10 and 12 mo of age, semen was collected from each boar (n = 28) twice weekly. Boars were collected under thermoneutral conditions, were then heat stressed for 7 d to exacerbate any semen quality issues, and semen was collected post-HS for 6 wk. Sperm characteristics were determined by computer-assisted semen analysis. The presence of varicocele had a significant effect on sperm concentration (P = 0.04) and trended toward significance for mean sperm head area (P = 0.06) throughout the duration of the study. An interaction existed between varicocele and collection time point at weeks 2-5 post-HS for distal droplet percentage, suggesting that boars with varicocele were possibly more susceptible to heat-stress-induced semen quality issues than boars without varicocele. Moreover, semen quality was reduced in boars with versus without varicocele under both thermoneutral and HS conditions. Therefore, detection of varicocele by ultrasound could represent a potential marker of fertility in young boars or as a component trait in selection indices for fertility.

A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues.

Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site. Tissue samples ( n=35) were collected from neonatal hemizygous ( n=3) and homozygous ( n=3) piglets and ZsGreen1 abundance was determined via immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar ( P>0.05). However, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within neural/endocrine tissues, ZsGreen1 abundance was highest in the anterior pituitary gland, intermediate in the cerebellum and lowest in the cerebrum, spinal cord and posterior pituitary ( P<0.05). In the digestive system, ZsGreen1 was more abundant in the salivary gland than esophagus, stomach, pancreas, duodenum, jejunum, ileum, spleen, colon, gallbladder and liver ( P<0.05). Interestingly, ZsGreen1 amounts also differed within an organ ( i.e., the right ventricle had 3-fold higher levels than the other heart chambers; P<0.05). These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig. Moreover, this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.

Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns.

Exosomes participate in cell-to-cell communication, facilitated by the transfer of RNAs, proteins and lipids from donor to recipient cells. Exosomes and their RNA cargos do not exclusively originate from endogenous synthesis but may also be obtained from dietary sources such as the inter-species transfer of exosomes and RNAs in bovine milk to humans. Here, we assessed the bioavailability and distribution of exosomes and their microRNA cargos from bovine, porcine and murine milk within and across species boundaries. Milk exosomes labeled with fluorophores or fluorescent fusion proteins accumulated in liver, spleen and brain following suckling, oral gavage and intravenous administration in mice and pigs. When synthetic, fluorophore-labeled microRNAs were transfected into bovine milk exosomes and administered to mice, distinct species of microRNAs demonstrated unique distribution profiles and accumulated in intestinal mucosa, spleen, liver, heart or brain. Administration of bovine milk exosomes failed to rescue Drosha homozygous knockout mice, presumably due to low bioavailability or lack of essential microRNAs.

RFamide-related peptide 3 and gonadotropin-releasing hormone-II are autocrine-paracrine regulators of testicular function in the boar.

Widespread use of artificial insemination in swine requires millions of doses of boar semen each year. Subfertility of boars remains a major constraint, which can impact the reproductive efficiency of thousands of sows, so a better understanding of testicular function is needed in order to develop methods to improve semen production. With this in mind, the effects of RFamide-related peptide 3 (RFRP3) and Gonadotropin-releasing hormone-II (GnRH-II) on gonadotropin secretion and testicular function of pigs are reviewed here. Receptors for RFRP3 are present in the pig hypothalamus, adenohypophysis, and testis. Evidence from in vitro studies indicates that RFRP3 could be a hypophysiotropic hormone in the pig by suppressing secretion of GnRH-I from the hypothalamus and luteinizing hormone (LH) from the pituitary gland; however, effects of RFRP3 on in vivo secretion of LH in pigs are minimal. Within the pig testis, RFRP3 suppresses testosterone secretion by inhibiting steroidogenic enzymes. GnRH-II and its receptor (GnRHR-II) are abundant in pig testes. Interstitial cells express GnRHR-II, and exogenous GnRH-II robustly stimulates secretion of testosterone in boars, despite minimal secretion of LH. Data illustrate that GnRH-II directly stimulates secretion of testosterone from the testes of mature boars. Thus, the primary function of RFRP3 and GnRH-II in the boar appears to be autocrine-paracrine inhibition and stimulation, respectively, of testosterone secretion within the testis. A better understanding of changes in the RFRP3 and GnRH-II systems within the testis during development will provide important clues about how to improve the testicular function of boars.

Relationship of neuropeptide FF receptors with pubertal maturation of gilts.

Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3 modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism.

Expression and Role of Gonadotropin-Releasing Hormone 2 and Its Receptor in Mammals.

Gonadotropin-releasing hormone 1 (GnRH1) and its receptor (GnRHR1) drive mammalian reproduction via regulation of the gonadotropins. Yet, a second form of GnRH (GnRH2) and its receptor (GnRHR2) also exist in mammals. GnRH2 has been completely conserved throughout 500 million years of evolution, signifying high selection pressure and a critical biological role. However, the GnRH2 gene is absent (e.g., rat) or inactivated (e.g., cow and sheep) in some species but retained in others (e.g., human, horse, and pig). Likewise, many species (e.g., human, chimpanzee, cow, and sheep) retain the GnRHR2 gene but lack the appropriate coding sequence to produce a full-length protein due to gene coding errors; although production of GnRHR2 in humans remains controversial. Certain mammals lack the GnRHR2 gene (e.g., mouse) or most exons entirely (e.g., rat). In contrast, old world monkeys, musk shrews, and pigs maintain the coding sequence required to produce a functional GnRHR2. Like GnRHR1, GnRHR2 is a 7-transmembrane, G protein-coupled receptor that interacts with Gαq/11 to mediate cell signaling. However, GnRHR2 retains a cytoplasmic tail and is only 40% homologous to GnRHR1. A role for GnRH2 and its receptor in mammals has been elusive, likely because common laboratory models lack both the ligand and receptor. Uniquely, both GnRH2 and GnRHR2 are ubiquitously expressed; transcript levels are abundant in peripheral tissues and scarcely found in regions of the brain associated with gonadotropin secretion, suggesting a divergent role from GnRH1/GnRHR1. Indeed, GnRH2 and its receptor are not physiological modulators of gonadotropin secretion in mammals. Instead, GnRH2 and GnRHR2 coordinate the interaction between nutritional status and sexual behavior in the female brain. Within peripheral tissues, GnRH2 and its receptor are novel regulators of reproductive organs. GnRH2 and GnRHR2 directly stimulate steroidogenesis within the porcine testis. In the female, GnRH2 and its receptor may help mediate placental function, implantation, and ovarian steroidogenesis. Furthermore, both the GnRH2 and GnRHR2 genes are expressed in human reproductive tumors and represent emerging targets for cancer treatment. Thus, GnRH2 and GnRHR2 have diverse functions in mammals which remain largely unexplored.

Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-B, specificity protein (SP)1-like and GATA-4 binding sites.

BACKGROUND: Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). METHODS: Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). RESULTS: Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-κB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-κB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-κB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05). CONCLUSIONS: Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).

Functional activity of the porcine Gnrhr2 gene promoter in testis-derived cells is partially conferred by nuclear factor-κB, specificity protein 1 and 3 (SP1/3) and overlapping early growth response 1/SP1/3 binding sites.

Unlike the classical gonadotropin-releasing hormone (GnRH1), the second mammalian isoform (GnRH2) is ubiquitously expressed, suggesting a divergent function. Indeed, we demonstrated that GnRH2 governs LH-independent testosterone secretion in porcine testes via interaction with its receptor (GnRHR2) on Leydig cells. Transient transfections with luciferase reporter vectors containing 3009bp of 5' flanking sequence for the porcine Gnrhr2 gene (-3009pGL3) revealed promoter activity in all 15 cell lines examined, including swine testis-derived (ST) cells. Therefore, ST cells were utilized to explore the molecular mechanisms underlying transcriptional regulation of the porcine Gnrhr2 gene in the testis. Reporter plasmids containing progressive 5' deletions of the Gnrhr2 promoter indicated that the -708/-490 region contained elements critical to promoter activity. Electrophoretic mobility shift assays (EMSAs) with radiolabeled oligonucleotides spanning the -708/-490bp region and ST nuclear extracts, identified specific binding complexes for the -513/-490, -591/-571 and -606/-581bp segments of promoter. Antibody addition to EMSAs indicated that the p65 and p52 subunits of nuclear factor-κB (NF-κB) comprised the specific complex bound to the oligonucleotide probe for the -513/-490bp promoter region, specificity protein (SP) 1 and 3 bound the -591/-571bp probe and early growth response 1 (EGR1), SP1 and SP3 bound the -606/-581 radiolabeled oligonucleotide. Transient transfections with vectors containing mutations of the NF-κB (-499/-493), SP1/3 (-582/-575) or overlapping EGR1/SP1/3 (-597/-587) binding sites reduced luciferase activity by 26%, 61% and 56%, respectively (P<0.05). Thus, NF-κB, SP1/3 and overlapping EGR1/SP1/3 binding sites are critical to expression of the porcine Gnrhr2 gene in ST cells.

LH-Independent Testosterone Secretion Is Mediated by the Interaction Between GNRH2 and Its Receptor Within Porcine Testes.

Unlike classic gonadotropin-releasing hormone 1 (GNRH1), the second mammalian isoform (GNRH2) is an ineffective stimulant of gonadotropin release. Species that produce GNRH2 may not maintain a functional GNRH2 receptor (GNRHR2) due to coding errors. A full-length GNRHR2 gene has been identified in swine, but its role in reproduction requires further elucidation. Our objective was to examine the role of GNRH2 and GNRHR2 in testicular function of boars. We discovered that GNRH2 levels were higher in the testis than in the anterior pituitary gland or hypothalamus, corresponding to greater GNRHR2 abundance in the testis versus the anterior pituitary gland. Moreover, GNRH2 immunostaining was most prevalent within seminiferous tubules, whereas GNRHR2 was detected in high abundance on Leydig cells. GNRH2 pretreatment of testis explant cultures elicited testosterone secretion similar to that of human chorionic gonadotropin stimulation. Treatment of mature boars with GNRH2 elevated testosterone levels similar to those of GNRH1-treated males, despite minimal GNRH2-induced release of luteinizing hormone (LH). When pretreated with a GNRHR1 antagonist (SB-75), subsequent GNRH2 treatment stimulated low levels of testosterone secretion despite a pattern of LH release similar to that in the previous trial, suggesting that SB-75 inhibited testicular GNRHR2s. Given that pigs lack testicular GNRHR1, these data may indicate that GNRH2 and its receptor are involved in autocrine or paracrine regulation of testosterone secretion. Notably, our data are the first to suggest a biological function of a novel GNRH2-GNRHR2 system in the testes of swine.

The role of RFamide-related peptide 3 (RFRP3) in regulation of the neuroendocrine reproductive and growth axes of the boar.

RFamide-related peptide 3 (RFRP3) has been implicated in regulating reproduction and growth. This regulation appears to be dependent upon sex, species, physiological status, and developmental stage. The objective of the present study was to evaluate the effects of RFRP3 on circulating concentrations of luteinizing hormone (LH) and growth hormone (GH) in mature boars. The hypothesis was RFRP3 would reduce circulating concentrations of LH and increase concentrations of GH. Meishan boars (716.6±2.8 days of age; 125.0±12.4kg BW) were randomly assigned to treatment: saline (n=4) or RFRP3 (8.5mg; n=5). Plasma was collected at 15-min intervals during 3 periods: pre-treatment, treatment, and post-treatment. During the treatment period, saline or RFRP3 were administered at 15-min intervals. Treatment was administered as a loading dose of 5mg RFRP3, followed by seven repeated injections of 0.5mg RFRP3. Pulsatile secretion of LH and GH were not affected by saline treatment. Mean concentrations of LH in RFRP3-treated boars were greater (P<0.01) in the pre-treatment period than in the treatment and post-treatment periods; however, the individual response to RFRP3 challenge was varied. RFRP3 suppressed (P<0.05) mean concentrations of GH during the treatment period. It is concluded that RFRP3 can act to suppress LH secretion in some boars, but the minimal and varied response between animals does not strongly support the idea that RFRP3 is a potent hypohysiotropic hormone in the pig. Results indicate that RFRP3 may function in regulating the growth axis of swine.

Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site.

BACKGROUND: Regulation of gonadotropin-releasing hormone (GnRH) receptor (GnRHR) numbers on gonadotropes within the anterior pituitary gland represents a critical point for control of reproductive function. Binding of GnRH to its receptor regulates follicle stimulating hormone (FSH) and luteinizing hormone (LH) release and levels of this G-protein coupled receptor on the surface of gonadotropes determines their sensitivity to GnRH pulses. While transcriptional regulation of this gene has been studied in mice, rats, humans and sheep, little is known about its regulation in the pig, an important agricultural species and human research model. METHODS: We isolated 5118 bp of 5' flanking sequence for the porcine GnRHR gene and generated luciferase reporter vectors. Deletion and mutation constructs were evaluated in gonadotrope-derived alphaT3-1 cells to determine regions important for gene transcription. Additionally, electrophoretic mobility shift assays (EMSAs) were performed to identify transcription factors binding to the GnRHR promoter. RESULTS: Transient transfections revealed that the GnRHR promoter was functional in alphaT3-1 cells but not in cells of non-gonadotrope origin. Mutation of the highly conserved gonadotrope specific element (GSE) located at -179/-171 of proximal promoter completely ablated luciferase activity, whereas mutation of another GSE at -315/-310 reduced activity by 34%. Consistent with this, EMSAs using alphaT3-1 nuclear extracts and a steroidogenic factor (SF)1 antibody confirmed SF1 binding to both GSEs. EMSAs also demonstrated that a retinoid X receptor (RXR) binding site at -279/-274 binds RXRalpha and RXRbeta and mutation of this site eliminated promoter activity. Transient transfection of alphaT3-1 cells with reporter vectors containing selective removal of 5' flanking region for the porcine GnRHR gene indicated that the -1915/-1431 segment was important for promoter activity. Definition of this region via transfection assays and EMSAs revealed an upstream enhancing region located at -1779/-1667 that increases porcine GnRHR gene expression in alphaT3-1 cells and includes a SF1 binding site at -1760/-1753. CONCLUSIONS: Porcine GnRHR promoter activity in alphaT3-1 cells is partially conferred by a distal GSE, two proximal GSEs and a RXR binding site. Basal gonadotrope expression of the porcine GnRHR gene uniquely involves three GSEs and RXR is newly identified as a regulator of GnRHR promoter activity.

Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells.

BACKGROUND: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development. AIM OF THE STUDY: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells. METHODS: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified. RESULTS: Biotin concentrations in culture media correlated negatively with expression of the biotin transporter SMVT, as judged by cellular transport rates of biotin, abundance of SMVT protein, and transcriptional activity of SMVT reporter-gene constructs. Notwithstanding this homeostatic mechanism, biotin concentrations in media correlated positively with activities of biotin-dependent propionyl-CoA carboxylase, abundance of biotinylated carboxylases, and with biotinylation of histones. Biotin deficiency was associated with decreased rates of thymidine uptake into JAr cells [pmol thymidine/( 10(6) cells x 24 h)]: 1.6 +/- 0.1 (25 pmol/L biotin) versus 2.3 +/- 0.2 (250 pmol/L biotin) versus 3.7 +/- 0.4 (10,000 pmol/L biotin), suggesting that cell proliferation depends on biotin. Secretion of progesterone was reduced in biotin-deficient cells; this effect was caused by reduced generation of new cells in deficient media rather than by an immediate effect of biotin on progesterone secretion at the singlecell-level. CONCLUSIONS: This study provides evidence that choriocarcinoma cells cannot maintain normal activities of biotin-dependent metabolic pathways if biotin concentrations in culture media are low. It is uncertain whether activities of biotin-dependent pathways in placenta affect fetal development in vivo.

Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells.

Here the hypothesis was tested that monocarboxylate transporters (MCT) mediate biotin transport in human lymphoid cells. Uptake of [(3)H]biotin was measured in human lymphoid cells [peripheral blood mononuclear cells (PBMC) and Jurkat cells] under conditions known to affect MCT-mediated transport. When biotin uptake into PBMC was measured in the presence of excess concentrations of competitors (MCT substrates) and MCT inhibitors, transport rates decreased significantly to <75 and <67%, respectively, of controls. Biotin uptake correlated with the concentration of protons in culture media, consistent with cotransport of protons and the carboxylate biotin by MCT. Efflux of biotin from PBMC was stimulated by extracellular lactate (a known substrate for MCT), consistent with countertransport of the two substrates by the same transporter. PBMC responded to proliferation with parallel increases of transport rates for both biotin and lactate, providing circumstantial evidence that the same transporter mediates uptake of the two substrates in PBMC. Transfection of Jurkat cells with an expression vector encoding MCT1 caused a 50% increase in biotin uptake; in contrast, overexpression of MCT1 did not affect biotin uptake in various nonlymphoid cell lines. These findings are consistent with the hypothesis that MCT mediate biotin uptake in human lymphoid cells.

c-Jun N-terminal kinase activation of activator protein-1 underlies homologous regulation of the gonadotropin-releasing hormone receptor gene in alpha T3-1 cells.

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in alpha T3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH(3)). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in alpha T3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.