Reproduction in Domestic Animals

Endocrine Regulation of Porcine Spermatogenesis 281recipient mouse serum follicle-stimulating hormone(FSH) would exist between the donor ages. Completionof these objectives will provide a better understanding ofthe mechanisms regulating neonatal germ and somaticcell development associated with establishing the firstwave of spermatogenesis and future spermatogeniccapacity in the boar testis.Materials and MethodsChemicals and reagents were all purchased from Sigma(http://www.sigmaaldrich.com) unless otherwise stated.Donor animals and tissue collectionThe Washington State University Animal Care and UseCommittee approved all animal procedures. Neonataltestis was obtained from 3-, 5-, 7- and 14-day-old whiteline composite boars at the Washington State UniversitySwine Facility using standard castration protocols. Aminimum of three boars were randomly chosen torepresent each donor age. To assess age-related differencesin testicular characteristics of porcine donormaterial prior to grafting, individual testis weights,Sertoli cell number and markers of Sertoli cell maturitystatus were measured (n = 6 neonatal boars per age).We also evaluated these traits in 21-day-old boars.Ectopic testis xenograftingTesticular parenchyma tissue was cut into 5 mg piecesand ectopically xenografted onto castrated, recipientimmunodeficient NCr nude mice (Taconic, http://www.taconic.com; CrTac:NCR-Fox1 ) or placedin Bouin’s fixative for 4 h at 4°C followed by dehydrationand storage in 70% ethanol for eventual morphologicalevaluation. Briefly, mice were anesthetized withketamine (0.1 mg ⁄ kg body weight, BW) and xylazine(0.5 mg ⁄ kg BW) castrated using surgical procedures,and managed as previously described (Oatley et al.2004).Analysis of donor grafts and recipient miceRecipient mice were sacrificed 22 weeks after grafting.Testis grafts were removed, weighed and processed forhistological analysis of establishment of spermatogenesisas previously described (Schmidt et al. 2006). Atsacrifice, mouse vesicular gland weights were recordedas a bioassay for androgen biosynthesis, and bloodwas also collected from recipient mice by cardiacpuncture and serum evaluated for FSH and testosteroneby radioimmunoassay (RIA). The extent of germcell differentiation in testis tissue grafts was visuallyevaluated by comparing the average number ofseminiferous tubules cross-sections containing spermatogonia,meiotic germ cells, differentiating spermatidsor only Sertoli cells within the largest centre section ofeach tissue sample. Digital images were captured usinga Leica DFC 280 camera and a Leica DMEcompound microscope (Leica Microsystems ImagingSolutions Ltd, http://www.leica-microsystems.com) at400 · magnification.ImmunohistochemistryAll antisera were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA). Tissues were fixed inBouin’s solution and embedded in paraffin accordingto standard procedures. The tissues were sectioned(thickness, 5 lm), deparaffinized, rehydrated and microwavedon high for 15 min while submerged in0.01 M of sodium citrate (pH 6.0) to retrieve antigens.Tissues were submerged for 20 min in 3% hydrogenperoxide (in methanol) to quench endogenous peroxidaseactivity. Sections were then blocked with 10%normal serum for 30 min at room temperature, andincubated overnight at 4°C with an affinity-purified,rabbit anti-mouse GATA4 polyclonal antiserum(C-20), mouse cytokeratin 18 monoclonal antiserum(C-04), rabbit anti-human GATA1 polyclonal antiserum(H-200), each diluted 1 : 200; or rabbit antihumanandrogen receptor (AR) polyclonal antiserum(1 : 100). As a negative control, serial sections wereprocessed without any primary antibody. At roomtemperature, sections were then washed in phosphatebufferedsaline (PBS) (2 · 5 min) and incubated withbiotinylated secondary antibody diluted 1 : 300 for 1 h,rinsed in PBS (2 · 5 min) and exposed to streptavidin⁄ horseradish peroxidase (HRP) for 30 min. Immunoreactivitywas detected following a 5-min incubationin diaminobenzidine (DAB) and sections were counterstainedlightly with haematoxylin. Slides were storedat room temperature until morphological analysis.Photomicrograph cross-sections representing immunolocalizationof GATA-4, cytokeratin 18, GATA-1 andAR can be found in Fig. 3.CryopreservationOn the day of collection, four pieces of testis tissue from5-day-old boars (n = 3) were ectopically grafted ontocastrated NCr nude mice as a pre-cryopreservationcontrol. The remainder of the samples that was notgrafted immediately was frozen in a dimethyl sulfoxide(DMSO)-based cryopreservation medium using a programmableembryo freezer. Briefly, freezing media wasprepared by mixing 0.68 g sucrose (0.1 M) and 0.2 gbovine serum albumin (1%) into Dulbecco’s modifiedeagle medium (DMEM) to a final volume of 17.87 ml.Freezing media was then filtered with a 0.4 mm syringefilter, and 2.13 ml of DMSO (1.5 M) was added. About4–8 pieces of testis tissue were added to 1 ml of freezingmedia in cryovials. The cryovials were placed in aCrylogic (Model CL856) freezer at 20°C. The vials werecooled at 2°C ⁄ min to )7°C and seeded with liquidnitrogen cooled tongs. The freezing procedure continuedat 0.3°C ⁄ min to )30°C and then at 0.1°C ⁄ min to )35°C.Once the tissue reached )30°C, it was transferred to an)80°C freezer for 1 h and then submersed in liquidnitrogen. Tissue was stored for 1 week in liquid nitrogen,removed and subsequently thawed. After removalfrom liquid nitrogen, the cryovials were placed at roomtemperature for 1 min and then incubated for 1 min in a37°C water bath. Samples were washed in mediacontaining decreasing concentrations of DMSO (1.0and 0.5 M) in DMEM for 3 min, each followed byÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag