Development Of Oocytes And Follicles Of Ovarian Allografts In Male Recipient Mice

Ovarian transplantation is an emerging technology with potential value applied to preservation and utilization of female gametes. Ovaries grafted can survive and develop in female recipients, and mature ovaries grafted can survive and develop in male recipients.To investigate further the development of oocytes and follicles of ovarian allografts in male recipient mice,ovaries from one-day-old mice were allografted underneath the kidney capsule of male recipients. The recipients were treated with or without castration and/or gonadotrophins, and same age female ovaries as control of ovarian grafts.Ovarian grafts were recovered 21-49 days after transplantation.Development of the oocytes and follicles of ovarian grafts and expression of genes related to follicular development were evaluated with morphology, histology,in vitro maturation, in vitro fertilization and semiquantitative RT-PCR. The results are as follows.Two hundred and sixty-four male recipient mice were used and 588 ovaries from one-day-old mice grafted. Ovarian grafts were successfully recovered from 84.5 % of the male recipients in total, and the ovary recovery rate was 73.3% in total. Ovarian grafts grew and increased in size significantly (P<0.01) 21, 28, 35, 42 and 49 days after transplantation, and the mean diameter of ovarian grafts increased significantly(P <0.05) after stimulated with exogenous gonadotrophins. Ovarian grafts displayed a pattern of growth similar to that observed in in situ mouse ovaries.Primordial follicle formation, recruitment and growth can be initiated in ovarian grafts in male recipient mice. Ovarian grafts contained follicles at all stages of folliculogenesis and the follicles can develop to maturation stage 21-49 days after transplantation, but didn't find corpus luteum in ovarian grafts. Ovarian grafts transplanted under the kidney capsule of castrated male mice displayed a pattern of development of follicles similar to that observed in intact male mice, but with corpus luteum formation in ovarian graft 49 days after transplantation. The follicles could develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus, with expanded cumulus-oocyte complex, mucified cumulus cell and corpus luteum.The majority of oocytes collected from the grafts in intact male recipients were at the GV stage. Mature metaphase II oocytes as well as immature GV oocytes were retrieved from the 28 d grafts. The yield of oocytes per ovarian graft in the 21 d, 28 d and 35 d groups was 40.5±29, 28.4±21.1 and 11.1±7.6, respectively. Ovarian grafts in the 35 d group had a significantly lower oocyte yield than that in the 21 d group(P<0.05). The mean diameter of oocytes from ovarian graft in the 21 d, 28 d and 35 d groups was 73.6±2.5, 74.6±3.4 and 74.7±5.3μm, respectively, with significant difference in the mean diameter of oocytes between the 21 d group and 35 d group (P<0.05). It seems that the yield of oocytes per ovarian graft decreases and the mean diameter of oocytes from ovarian graft increases with the graft period extended during test period.Mature metaphase II oocytes as well as immature GV oocytes were retrieved from the 21 d grafts in castrated male recipients. The yield of oocytes per ovarian graft was 32.8±14.8, and the mean diameter of oocytes was 73.6±3.6μm, without significant difference compared with that in intact male recipients(P>0.05).The yield of oocytes per ovarian graft in intact male recipients in the 21 d, and 28 d gonadotrophin-treated groups was 31.5±20.3 and 28.9±21.2, respectively, without significant difference compared with the non-treated groups (P>0.05). The yield of mature metaphase II oocytes was 9.0±7.9 and 14.6±13.4, respectively, with significant difference compared with the non-treated groups(P <0.01). Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation.The two-cell cleavage rate of in vitro matured GV oocytes collected from non-stimulated 21 d grafts was 16.0%.Some of the two-cell embryos cleaved to morula but not to blastocyst. GV oocytes from 21 d grafts of gonadotrophin treated recipients had a higher two-cell cleavage rate and blastocyst rate after IVM and IVF (32.8% and 1.3%, respectively) than the oocytes from non-stimulated 21 d grafts(P <0.01). There was significant difference in the cleavage rate (37.6%)( P<0.05) and blastocyst rate (12.8%)( P<0.07)between GV and MII eggs from from 21 d grafts of gonadotrophin treated recipients. Oocytes collected from ovarian grafts in intact male recipients can be fertilized in vitro, and stimulation with exogenous gonadotrophins can improve oocyte development.GDF-9, AMH, FSHR, LHR, ERβ, P450-arom, P450-scc, IGFBP 4, Bax and Bcl-2 mRNA expression were detected in ovarian grafts and normal ovaries. Expression of target genes related to follicular development, differentiation and follicle atresia was not influenced by transplantation, sex and glands of recipients. Exogenous gonadotrophins stimulation upregulated the expression of LHR. In conclusion, the pattern of follicular development, differentiation and follicle atresia in ovarian grafts may be similar to the ovaries of normal female mice. The follicles in ovarian grafts from intact male recipients can grow and develop to maturation stage.