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.

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.

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.

Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we reported that GNRH2 and GNRHR2 mediate LH-independent testosterone secretion from porcine testes. To further explore this ligand-receptor complex, a pig model with reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentiviral particles were generated and microinjected into the perivitelline space of zygotes, and embryos were transferred into a recipient. One GNRHR2 knockdown (KD) female was born that subsequently produced 80 piglets from 6 litters with 46 hemizygous progeny (57% transgenic). Hemizygous GNRHR2 KD (n = 10) and littermate control (n = 7) males were monitored at 40, 100, 150, 190, 225 and 300 days of age; body weight and testis size were measured and serum was isolated and assayed for testosterone and luteinizing hormone (LH) concentrations. Body weight of GNRHR2 KD boars was not different from littermate controls (P = 0.14), but testes were smaller (P < 0.05; 331.8 vs. 374.8 cm3, respectively). Testosterone concentrations tended (P = 0.06) to be reduced in GNRHR2 KD (1.6 ng/ml) compared to littermate control (4.2 ng/ml) males, but LH levels were similar (P = 0.47). The abundance of GNRHR2 mRNA was reduced (P < 0.001) by 69% in testicular tissue from mature GNRHR2 KD (n = 5) versus littermate control (n = 4) animals. These swine represent the first genetically-engineered model to elucidate the function of GNRH2 and its receptor in mammals.

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.