University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department 8-2017 Production of a gonadotropin-releasing hormone 2 receptor knockdown (GNRHR2 KD) swine line. Amy T. Desaulniers University of Nebraska-Lincoln, [email protected] Rebecca Middleton University of Nebraska-Lincoln, [email protected] Ginger A. Mills University of Nebraska-Lincoln, [email protected] Clay A. Lents USDA, ARS, USMARC, [email protected] Brett R. White University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/animalscifacpub Part of the Animal Sciences Commons, Genetics and Genomics Commons, and the Other Physiology Commons Desaulniers, Amy T.; Middleton, Rebecca; Mills, Ginger A.; Lents, Clay A.; and White, Brett R., "Production of a gonadotropin- releasing hormone 2 receptor knockdown (GNRHR2 KD) swine line." (2017). Faculty Papers and Publications in Animal Science. 976. http://digitalcommons.unl.edu/animalscifacpub/976 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Transgenic Res (2017) 26:567–575 DOI 10.1007/s11248-017-0023-4 BRIEF COMMUNICATION Production of a gonadotropin-releasing hormone 2 receptor knockdown (GNRHR2 KD) swine line A. T. Desaulniers . R. A. Cederberg . G. A. Mills . C. A. Lents . B. R. White Received: 30 August 2016 / Accepted: 11 May 2017 / Published online: 22 May 2017 Ó The Author(s) 2017. This article is an open access publication Abstract Swine are the only livestock species that GNRH2 and GNRHR2 mediate LH-independent produce both the second mammalian isoform of testosterone secretion from porcine testes. To further gonadotropin-releasing hormone (GNRH2) and its explore this ligand-receptor complex, a pig model with receptor (GNRHR2). Previously, we reported that reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentivi- Mention of trade names or commercial products in this ral particles were generated and microinjected into the publication is solely for the purpose of providing specific perivitelline space of zygotes, and embryos were information and does not imply recommendation or transferred into a recipient. One GNRHR2 knockdown endorsement by the U.S. Department of Agriculture. (KD) female was born that subsequently produced 80 piglets from 6 litters with 46 hemizygous progeny The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of (57% transgenic). Hemizygous GNRHR2 KD race, color, national origin, age, disability, and where applicable, (n = 10) and littermate control (n = 7) males were sex, marital status, familial status, parental status, religion, sexual monitored at 40, 100, 150, 190, 225 and 300 days of orientation, genetic information, political beliefs, reprisal, or age; body weight and testis size were measured and because all or part of an individual’s income is derived from any public assistance program (Not all prohibited bases apply to all serum was isolated and assayed for testosterone and programs). Persons with disabilities who require alternative luteinizing hormone (LH) concentrations. Body means for communication of program information (Braille, large weight of GNRHR2 KD boars was not different from print, audiotape, etc.) should contact USDA’s TARGET Center littermate controls (P = 0.14), but testes were smaller at (202) 720-2600 (voice and TDD). To file a complaint of 3 discrimination, write to USDA, Director, Office of Civil Rights, (P \ 0.05; 331.8 vs. 374.8 cm , respectively). Testos- 1400 Independence Avenue, S.W., Washington, D.C. terone concentrations tended (P = 0.06) to be reduced 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 in GNRHR2 KD (1.6 ng/ml) compared to littermate (TDD). USDA is an equal opportunity provider and employer. control (4.2 ng/ml) males, but LH levels were similar (P = 0.47). The abundance of GNRHR2 mRNA was A. T. Desaulniers and R. A. Cederberg contributed equally to this work. reduced (P \ 0.001) by 69% in testicular tissue from A. T. Desaulniers Á R. A. Cederberg Á C. A. Lents G. A. Mills Á B. R. White (&) USDA, Agricultural Research Service, U.S. Meat Animal Department of Animal Science, University of Nebraska- Research Center, Clay Center, NE 68933-0166, USA Lincoln, A224j Animal Science Building, 3940 Fair Street, Lincoln, NE 68583-0908, USA e-mail: [email protected] 123 568 Transgenic Res (2017) 26:567–575 mature GNRHR2 KD (n = 5) versus littermate con- production from COS cells overexpressing the porcine trol (n = 4) animals. These swine represent the first GNRHR2 (Neill et al. 2002b). genetically-engineered model to elucidate the function Studies in boars indirectly suggested that a testic- of GNRH2 and its receptor in mammals. ular GnRH isoform mediates steroidogenesis within the porcine testis (Bowen et al. 2006; Wise et al. 2000; Keywords GNRH2 Á GNRHR2 Á Porcine Á Zanella et al. 2000). However, GNRHR is absent in Transgenic Á Testosterone Á Testis Á Gene knockdown Á swine testes (Zanella et al. 2000); therefore, we shRNA hypothesized that the results of the aforementioned studies could be ascribed to GNRHR2. In support of this hypothesis, the GNRHR2 gene promoter is active Introduction in a swine testis-derived (ST) cell line (Brauer et al. 2016) and both GNRH2 and GNRHR2 protein are Gonadotropin-releasing hormone (GNRH1; pGlu- abundantly produced within the boar testis (De- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly) is well-re- saulniers et al. 2015). We also demonstrated that garded as the master regulator of reproduction in GNRHR2 immunolocalized to porcine Leydig cells mammals. A second GnRH isoform (GNRH2; pGlu- and exogenous GNRH2 treatment stimulated LH- His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly) has also independent testosterone secretion from testes of boars been identified in mammals (White et al. 1998). (Desaulniers et al. 2015). These data suggest that Expression of GNRH2 is ubiquitous (Neill 2002) and swine maintain a unique testicular GNRH2-GNRHR2 is most abundant in tissues outside of the brain (White system capable of regulating testosterone secretion in et al. 1998), suggesting a divergent role from GNRH1. a paracrine manner that is independent from the Indeed, GNRH2 is a poor stimulator of gonadotropin classical androgen stimulator, LH. However, the release; GNRH2-induced secretion of luteinizing function and cellular mechanisms of GNRH2-medi- hormone (LH) and follicle-stimulating hormone ated testosterone secretion remain unknown. Thus, we (FSH) is about 10% that of GNRH1 (Millar et al. developed a GNRHR2 knockdown (KD) swine line to 1986). further examine the role of GNRH2 and its receptor in A 7-transmembrane, G-protein-coupled receptor the boar testis. specific for GNRH2 (GNRHR2) has also been iden- tified in mammals (Millar et al. 2001; Neill et al. 2001). Interestingly, GNRH1 receptor (GNRHR) and Materials and methods GNRHR2 are quite different; their homology is only about 40% and GNRHR2 has an intracytoplasmic tail Animals which GNRHR lacks (Millar 2003). Like its ligand, the GNRHR2 gene is also expressed ubiquitously, White crossbred pigs from the University of Nebraska- including extra-pituitary reproductive tissues (Millar Lincoln (UNL) Agricultural Research and Develop- et al. 2001; Neill et al. 2001). Interestingly, GNRHR2 ment Center (ARDC) swine unit (Mead, NE) were mRNA levels were highest in the marmoset testis used to generate the founder animal. Yorkshire boar compared to 31 other organs (Millar et al. 2001). semen, purchased from Swine Genetics International However, most mammalian species are unable to (Cambridge, IA), was used to produce progeny from produce a functional GNRHR2 due to gene deletions the founder female. Farrowing and subsequent hous- or gene coding errors (Gault et al. 2004; Millar 2003; ing occurred within the UNL Animal Science Building Morgan et al. 2003; Neill et al. 2002a; Pawson et al. (Lincoln, NE). Pre-pubertal pigs were group housed 2003). In fact, the pig is the only livestock species to with ad libitum access to feed and water. Adult pigs maintain the appropriate sequence to encode a full- were housed individually with ad libitum access to length GNRHR2 (Stewart et al. 2009). Furthermore, water and fed approximately 2.5 kg of feed daily. All this receptor is considered functional since exogenous diets were formulated to meet National Research GNRH2 treatment stimulated inositol triphosphate Council guidelines for each stage of production. 123 Transgenic Res (2017) 26:567–575 569 Vector preparation, production of lentiviral Quantitative PCR particles and ST cell transduction Ribonucleic acid from ST cells was DNase treated with Utilizing oligomer design software (Clontech, Moun- RQ1 (Promega) and quantitated using a Nanodrop tain View, CA), two potential target small hairpin spectrophotometer (NanoDrop Technologies, Inc., (shRNA) sequences, shRNA1 (TGGCTACT- Wilmington, DE). The RNA was reverse transcribed CAGTTTCCTTC) and shRNA2 (CTGTCATGAC- into cDNA with M-MLV (Promega) and random CATCTGCTA), specific to the porcine GNRHR2 hexamers (Promega). The PCR amplification of (NCBI accession number NM_001001639.2) were