Rebecca A. Cederberg

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

ABSTRACT 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.

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.

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

The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. ZsGreen1 was detected in all tissues examined; however, quantification revealed that the level of ZsGreen1 expression was tissue-specific. For example, ZsGreen1 was most abundantly produced in the salivary gland, moderately produced in the esophagus and lowly expressed in the stomach. Interestingly, expression of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. The expression patterns of ZsGreen1 were similar between homozygous and hemizygous piglets. Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig.Background The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. Results ZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets. Conclusions Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

A transgenic pig model expressing differential levels of ZsGreen1 reporter across an extensive array of tissues

The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. ZsGreen1 was detected in all tissues examined; however, quantification revealed that the level of ZsGreen1 expression was tissue-specific. For example, ZsGreen1 was most abundantly produced in the salivary gland, moderately produced in the esophagus and lowly expressed in the stomach. Interestingly, expression of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. The expression patterns of ZsGreen1 were similar between homozygous and hemizygous piglets. Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig.Background The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. Results ZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets. Conclusions Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

Differential regulation of the ZsGreen1 reporter gene by the human cytomegalovirus promoter in 35 tissues of neonatal transgenic swine.

The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. ZsGreen1 was detected in all tissues examined; however, quantification revealed that the level of ZsGreen1 expression was tissue-specific. For example, ZsGreen1 was most abundantly produced in the salivary gland, moderately produced in the esophagus and lowly expressed in the stomach. Interestingly, expression of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. The expression patterns of ZsGreen1 were similar between homozygous and hemizygous piglets. Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig.Background The advent of genetically engineered pig production has revealed a wide array of opportunities to enhance both biomedical and agricultural industries. One powerful method to develop these models is transgenesis; however, selection of a suitable promoter to drive transgene expression is critical. The cytomegalovirus (CMV) promoter is the most commonly used viral promoter as it robustly drives transgene expression in a ubiquitous nature. However, recent reports suggest that the level of CMV promoter activity is tissue-dependent in the pig. Therefore, the objective of this study was to quantify the activity of the CMV promoter in a wide range of porcine tissues. Swine harboring a CMV-ZsGreen1 transgene with a single integration site were utilized for this study. Thirty five tissue samples were collected from neonatal hemizygous (n = 3) and homozygous (n = 3) transgenic piglets and analyzed for ZsGreen1 abundance via immunoblot. Results ZsGreen1 was detected in all tissues examined; however, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within organs of the digestive system, for example, ZsGreen1 was most abundant in the salivary gland, moderately produced in the esophagus and levels were lowest in the stomach. Interestingly, abundance of ZsGreen1 also differed within organ. For instance, levels were highest in the right ventricle compared with other chambers of the heart. There was no effect of transgene dose as ZsGreen1 expression patterns were similar between homozygous and hemizygous piglets. Conclusions Ultimately, these results elucidate the tissue-specific activity of the CMV promoter in the neonatal pig. Moreover, this model can serve as a useful tool for research applications requiring reporter gene activity in mammalian organs.

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.

5 TESTICULAR GnRH-II RECEPTOR KNOCKDOWN IMPAIRS DIURNAL TESTOSTERONE SECRETION IN THE BOAR

The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are ubiquitously expressed, with elevated levels in the testis. Gene coding errors prevent their production in many species, but both genes are functional in swine. We demonstrated that GnRHR-II localizes to porcine Leydig cells, and exogenous GnRH-II robustly stimulates testosterone production in vivo, despite minimal luteinizing hormone (LH) secretion. These data suggest that GnRH-II directly effects steroidogenesis in the boar testis. To explore this hypothesis, we produced a GnRHR-II knockdown (KD) swine line. Upon characterisation of this line, serum testosterone concentrations were reduced in GnRHR-II KD compared with littermate control males during pubertal development. However, concentrations of LH were unaffected, indicating that GnRHR-II KD impairs steroidogenesis directly at the testis rather than inhibiting gonadotropin secretion from the anterior pituitary gland. Based on these results, the objective of this study was to compare diurnal secretory patterns of testosterone in mature GnRHR-II KD (n=5) and littermate control (n=5) males. Boars were fit with indwelling jugular cannulae and blood was collected every 15min for 8h. Serum was assayed for testosterone concentration via radioimmunoassay. Next, GnRHR-II KD (n=5) and littermate control (n=4) boars were killed, testis weight was recorded, and testicular tissue was collected for RNA isolation. To confirm KD in these animals, digital droplet PCR was performed to quantify GnRHR-II mRNA abundance (normalized to β-actin). Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with line (transgenic or control) as the fixed effect and litter as a random effect. For hormone data, time and line×time interaction were included as fixed effects, with time as a repeated measure. Although there was no effect of time or line×time interaction (P>0.05) on serum testosterone concentrations, we observed a line effect (P<0.05). Differences between lines were dramatic; testosterone was reduced by 82% in GnRHR-II KD (0.75±0.05ngmL-1) compared with littermate control (4.09±0.29ngmL-1; P<0.05) males. Despite divergent testosterone levels, testis weights were similar between lines (P>0.05) indicative of altered Leydig cell function as opposed to hypertrophy/hyperplasia. Given that testicular GnRHR-II mRNA levels were reduced by 69% in transgenic animals (P<0.001), these data demonstrate that GnRH-II and its receptor play a critical role in testosterone biosynthesis within porcine Leydig cells. Thus, this report reveals novel mediators of testicular function in the boar and challenges the central dogma of testosterone regulation. Because testosterone dictates male reproductive success, GnRH-II and its receptor represent unique targets to improve fertility in swine.

Erratum to Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-kB, specificity protein (SP)1-like and GATA-4 binding sites. [Reproductive Biology and Endocrinology (2016) 14, 36]. DOI: 10.1186/s12958-016-0170-0

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Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene Is Conferred by an Element(s) Located Between -290/-270 bp of Proximal Promoter.

Our results indicated that glucocorticoid responsiveness of the porcine GnRHR gene promoter is conferred by element(s) located within 287 to 307 bp upstream of the translational start site. Consistent with this, EMSA analysis indicated enhanced binding at this site with nuclear extracts from glucocorticoid-treated α T3-1 cells compared to those treated with vehicle. Finally, signaling pathways other than protein kinase A and C are stimulated following treatment of αT3-1 cells with dexamethasone. References