Reproduction in Domestic Animals

282 KC Caires, JA Schmidt, AP Oliver, J de Avila and DJ McLeanwashing in Hank’s balanced salt solutions (HBSS)(0.0 M DMSO). After the final wash, donor tissues(n = 3) were grafted onto nine mice (three mice perdonor) in the same manner as the controls. To determinethe immediate affects of cryopreservation on tissuemorphology, samples of control and cryopreserved pregrafttestis tissue were also processed for evaluation bylight microscopy.Statistical analysisGerm cell proportions were averaged per mouse toprevent unequal weighting due to variation in thesetraits. All statistical comparisons were performed usingthe Proc GLM function of Statistical Analysis System(SAS) software, version 9.13 (SAS Institute, Cary, NC,USA), and differences were considered significant at thep £ 0.05 level. Pair-wise comparisons were evaluatedbetween donor ages using the Duncan’s multiple rangetest for significance. Data are presented as the mean ± -SEM and differences between donor ages were consideredsignificant at the p £ 0.05 level.ResultsNeonatal donor testicular characteristicsTesticular growth and Sertoli cell numberTestis weight increased with advancing age(p < 0.001). The average increase in testis weight after3 days post-partum (dpp) in neonatal boars was about1.5-, 2-, 6- and 12-fold at ages 5, 7, 14 and 21 dpp,respectively (Fig. 1). The number of Gata4-positiveSertoli cells per seminiferous cord cross-section also(a)(b)Fig. 1. Porcine testis tissue weight and Sertoli cell number from prepubertalboars at 3, 5, 7, 14 and 21 days post-partum (dpp). (a)Testicular growth and (b) number of Gata4-positive Sertoli cell nucleiper seminiferous tubule (cross-section). Bars with different lettersindicate significant differences between means (p < 0.05)increased with advancing age (p < 0.05, Fig. 1b).Sertoli cell nuclei reacted strongly with the Gata4antibody, but a low level of expression was alsolocalized to Leydig cell nuclei in the interstitial compartment(Fig. 2b), as previously described (McCoardet al. 2003).Protein markers of Sertoli cell maturationTo determine the maturation status of Sertoli cells theexpression of cytokeratin 18 and Gata1, proteinmarkers for immaturity and differentiation wereimmunolocalized. Strong cytoplasmic staining forcytokeratin 18, a foetal Sertoli cell marker, wasvisualized in Sertoli cells within 3, 5, 7, 14 and 21dpp boar testis tissues (Fig. 2c,f). Immunolocalizationof GATA1, a lineage specific differentiation marker,revealed weak but specific cytoplasmic staining nearthe apical region of Sertoli cell nuclei of all neonatalages evaluated (Fig. 2d) as is consistent with theexpression pattern of precursor Sertoli cells in the prenataland early neonatal mouse testis (Yomogida et al.1994). Nuclear immunoreactivity for AR was visualizedin Sertoli, peritubular myoid, Leydig cells andgonocytes within 3, 5, 7, 14 and 21 dpp boar testistissues (Fig. 2e).Porcine testis tissue graft growth, androgen biosynthesisand recipient mouse serum follicle-stimulating hormoneconcentrationTo assess age-related differences in growth potential ofporcine donor tissue at removal, testis graft weightswere measured and the numbers of seminiferoustubule cross-sections were determined. Analysis ofgrowth potential indicated donor-age-related variationin testis tissue graft weight and tubule number. Bothparameters for graft growth were significantly higher(p < 0.05) in testis grafts from 3-day donor tissue,when compared to all other ages (Fig. 3). In contrast,donor testis tissue from 5-, 7- and 14-day-old neonatalboars had similar growth rates as no differences weredetected in graft weight or seminiferous tubule number.To assess the ability for testis tissue grafts tosynthesize biologically active androgen, recipient mouseserum testosterone levels and vesicular gland weightswere compared between donor age groups. Despitevariation in donor age, RIA for serum testosterone andbiological assay of vesicular gland weights indicated nodifferences in the ability of testis tissue grafts to producetestosterone (Table 1). We also investigated the abilityof porcine testis tissue xenografts to produce factors thatexert negative feedback on pituitary FSH synthesiswhen compared to normal, intact mice of similar ageand strain by RIA. Follicle-stimulating hormone concentrationsin recipient mice supporting testis graftsfrom 5-, 7- and 14-day-old donor tissue did not differand were similar to normal physiological levels in agematched,intact nude mice (Table 1). Yet, serum FSHlevels were significantly lower (p < 0.05) than normal inrecipient mice supporting testis grafts from 3-day-olddonor tissue.Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag