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.

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.

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.

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.