Reproduction in Domestic Animals

Endocrine Regulation of Porcine Spermatogenesis 283(a)(b)(c)(d)(e)(f)Fig. 2. Immunolocalization ofSertoli cell protein markers inpre-pubertal porcine testis tissue:(a) negative control (no primaryIgG), (b) GATA4, (c, f) cytokeratin18, (d) GATA1 and (e) androgenreceptor. Scale bars = 50 lmDegree of germ cell differentiation in porcine testis tissuegraftsThe extent of spermatogenesis in testis tissue fromneonatal boars of different ages was determined after22 weeks of grafting. Porcine testis tissue obtained from3-, 5-, 7- and 14-day-old neonatal boars were all capableof producing round and elongate spermatids aftergrafting (Fig. 4). Testis grafts from 14-day-old donorscontained a significantly greater (p < 0.05) percentageof seminiferous tubules with spermatids compared to allother donor ages (Fig. 4). In contrast, spermatogenesiswas established at similar rates in donor testis tissuefrom 3-, 5- and 7-day-old neonatal boars (Fig. 4). Theaverage percentage of tubules containing spermatids intestis tissue from 14-day-old donors was about eightfoldhigher (45.4%) when compared to other donor agesaveraging 5.4%. Photomicrograph cross-sections representingthe degree of germ cell differentiation withinporcine testis grafts can be found in Fig. 5.Effect of cryopreservation on porcine testis tissuedevelopment post-graftingWe evaluated the affect of pre-graft cryopreservationon the ability of porcine testis tissue grafts to grow,produce testosterone and support complete germ celldifferentiation by utilizing 5-day-old donor testis tissue(n = 3). Morphological evaluation prior to graftingrevealed that cryopreservation adversely affected seminiferouscord organization when compared to controls(Fig. 6a,b). Following the grafting period, no differenceswere observed between control and cryopreserved testistissue weight, seminiferous tubule number and diameteror testosterone production (data not shown). Establishmentof spermatogenesis in cryopreserved and controlporcine testis tissue grafts was similar as no differenceswere observed in the distribution of seminiferous tubulecross-sections containing spermatogonia, meiotic germcells and spermatids, respectively (Fig. 6c). Photomicrographcross-sections representing complete germ celldifferentiation in control and cryopreserved porcinetestis tissue grafts can be found in Fig. 6d,e.DiscussionIn the present study, porcine testis tissue grafts from alldonor ages initiated the first round of spermatogenesisassociated with pubertal age in a similar developmentaltimeline to the boar testis in situ, and were capable ofcomplete germ cell differentiation after 22 weeks ofgrafting. Yet, we observed a significant increase (eightfold)in the extent of germ cell differentiation in testisÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag