Dysregulation of angiogenesis-specific signalling in adult testis results in xenograft degeneration.

Ectopic xenografting of testis is a feasible option for preservation of male fertility and angiogenesis plays a pivotal role in xenograft survival and functionality. When compared to immature testis, the adult testis is unable to establish functional xenografts due to potentially lower efficiency to induce angiogenesis. The precise molecular mechanism, however, remains elusive. In the present study, we compared adult and immature testis xenografts for survival, maturation and germ cell differentiation. Further, we evaluated differential expression of angiogenesis signalling-specific proteins in adult and immature testis and their xenografts. Results showed that adult testis xenografts degenerated whereas immature testis xenografts survived and established spermatogenesis with the production of haploid germ cells. Protein expression analysis demonstrated that immature testis xenografts were able to establish angiogenesis either through eNOS activation via VEGF and PI3K/AKT or through EGFR-mediated STAT3 pathway. The role of ERK/MAPK pathway in xenograft angiogenesis was ruled out. The absence or reduced expression of angiogenesis-specific proteins in adult testis and its xenografts possibly resulted in poor angiogenesis and in their subsequent degeneration. This study provides insight into angiogenesis mechanism that can be utilized to augment testis xenografting efficiency.

Cryopreservation of adult cervid testes.

Several species of cervids are currently classified as threatened or endangered due to a rapid decline in their populations. Sperm cryopreservation, in association with assisted reproductive technologies, can find application for the conservation of endangered cervids. In cases of unsuccessful sperm retrieval through other means prior to the death of the animal, adult testis is the only source of sperm. Recovery of viable sperm from adult testes depends on the effective preservation of testicular tissues through optimization of cryopreservation protocols. The present study evaluated combinations of 10% dimethyl sulfoxide (DMSO) with 0% or 80% fetal bovine serum (FBS) and 20% DMSO with 0 or 20% FBS for the cryopreservation of testicular tissues of three adult cervids using uncontrolled slow freezing protocol. The cryopreserved testis was compared to chilled tissue without cryoprotectants. Results revealed that testicular tissues of barking deer cryopreserved in 20% DMSO (D20) had all the analyzed 7 parameters (number of TNP1-, PRM2 and acrosin-expressing cells/tubule and, the number of viable, morphologically normal, acrosome intact, Annexin V-negative sperm) comparable to the chilled testis. However, testicular tissues of sambhar and hog deer cryopreserved only in D20S20 had 5 of 7 parameters comparable to the chilled testis. In conclusion, D20 is acceptable for cryopreservation of barking deer and D20S20 for sambar and hog deer testes.

Replacement of serum with ocular fluid for cryopreservation of immature testes.

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species.

Comparative efficacies of six different media for cryopreservation of immature buffalo (Bubalus bubalis) calf testis.

Buffalo calves have a high mortality rate (~80%) in commercial dairies and testis cryopreservation can provide a feasible option for the preservation of germplasm from immature males that die before attaining sexual maturity. The aim of the present study was to evaluate combinations of 10 or 20% dimethylsulfoxide (DMSO) with 0, 20 or 80% fetal bovine serum (FBS) for cryopreservation of immature buffalo testicular tissues, subjected to uncontrolled slow freezing. Tissues cryopreserved in 20% DMSO with 20% FBS (D20S20) showed total, tubular and interstitial cell viability, number of early apoptotic and DNA-damaged cells, surviving germ and proliferating cells and expression of testicular cell-specific proteins (POU class 5 homeobox (POU5F1), vimentin (VIM) and actin α2 (ACTA2)) similar to that of fresh cultured control (FCC; P>0.05). Expression of cytochrome P450, family 11, subfamily A (CYP11A1) protein and testosterone assay showed that only tissues cryopreserved in D20S20 had Leydig cells and secretory functions identical to that of FCC (P>0.05). High expression of superoxide dismutase2 (SOD2), cold-inducible RNA-binding protein (CIRBP) and RNA-binding motif protein3 (RBM3) proteins in cryopreserved tissues indicated involvement of cell signalling pathways regulating cellular protective mechanisms. Similarity in expression of pro-apoptosis proteins transcription factor tumour protein P53 (TP53) and BCL2-associated X protein (BAX) in D20S20 cryopreserved tissues to that of FCC (P>0.05) suggested lower apoptosis and DNA damage as key reasons for superior cryopreservation.

Germ cell differentiation in cryopreserved, immature, Indian spotted mouse deer (Moschiola indica) testes xenografted onto mice.

Death of immature animals is one of the reasons for the loss of genetic diversity of rare and endangered species. Because sperm cannot be collected from immature males, cryobanking of testicular tissue combined with testis xenografting is a potential option for conservation. The objective of this study was to evaluate the establishment of spermatogenesis in cryopreserved immature testicular tissues from Indian spotted mouse deer (Moschiola indica) after ectopic xenografting onto immunodeficient nude mice. Results showed that testis tissues that were frozen in cryomedia containing either 10% DMSO with 80% fetal bovine serum (D10S80) or 20% DMSO with 20% fetal bovine serum (D20S20) had significantly more (P < 0.01) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled positive interstitial cells when compared with fresh testis tissues (46.3 ± 3.4 and 51.9 ± 4.0 vs. 22.8 ± 2.0). Xenografted testicular tissues showed degenerated seminiferous tubules 24 weeks after grafting in testes that had been cryopreserved in D20S20; alternatively, pachytene spermatocytes were the most advanced germ cells in testes that were cryopreserved in D10S80. Proliferating cell nuclear antigen staining confirmed the proliferative status of spermatocytes, and the increases in tubular and lumen diameters indicated testicular maturation in xenografts. However, persistent anti-Müllerian hormone staining in Sertoli cells of xenografts revealed incomplete testicular maturation. This study reports that cryopreserved testis tissue that had been xenografted from endangered animals onto mice resulted in the establishment of spermatogenesis with initiation of meiosis. These findings are encouraging for cryobanking of testicular tissues from immature endangered animals to conserve their germplasm.

Regeneration of Leydig cells in ectopically autografted adult mouse testes.

Ectopic autografting of testis tissue is a promising approach for studying testicular development, male germline preservation and restoration of male fertility. In this study, we examined the fate of various testicular cells in adult mouse testes following ectopic autografting at 1, 2, 4 and 8 weeks post grafting. Histological examination showed no evidence of re-establishment of spermatogenesis in autografts, and progressive degeneration of seminiferous tubules was detected. Expression of germ cell-specific proteins such as POU5F1, DAZL, TNP1, TNP2, PRM1 and PRM2 revealed that, although proliferating and differentiating spermatogenic germ cells such as spermatogonia, spermatocytes and spermatids could survive in autografts until 4 weeks, only terminally differentiated germ cells such as sperm persisted in autografts until 8 weeks. The presence of Sertoli and peritubular myoid cells, as indicated by expression of WT1 and ACTA2 proteins, respectively, was evident in the autografts until 8 weeks. Interestingly, seminal vesicle weight and serum testosterone level were restored in autografted mice by 8 weeks post grafting. The expression of Leydig cell-specific proteins such as CYP11A1, HSD3B2 and LHCGR showed revival of Leydig cell (LC) populations in autografts over time since grafting. Elevated expression of PDGFRA, LIF, DHH and NEFH in autografts indicated de novo regeneration of LC populations. Autografted adult testis can be used as a model for investigating Leydig cell regeneration, steroidogenesis and regulation of the intrinsic factors involved in Leydig cell development. The success of this rodent model can have therapeutic applications for adult human males undergoing sterilizing cancer therapy.