The authors studied Witschi's theory of "corticomedullary inductors" with the; heterosexual grafts of the embryonal gonads of the rats in very close proximity or in remote distance each other for the effect of the inductor substance and the possibility of the substance acting as blood-borne agent when multiple embryonal testes and one or two ovaries were separately grafted in distant sites in the mammalian level. The heterosexual grafts of the embryonal gonads aging 16 days old were performed as the methods Macintyre (1956) used. Additionally the author grafted one or two embryonal ovaries of the same age in the subcapsular site and multiple embryonal testes of the same age in the similar site of the opposite kidney of the same host and allowed to develop at the sites for 3 weeks. The explants removed from the host, were fixed in Bouin's fluid, embedded in paraffin, sectioned serially at 6mu, and stained with hematoxylin and eosin. The embryonal transplanted ovary with testis in close contact, was inhibited and depressed to the one side probably due to the more rapid growth and differentiation of the testis as compared with the ovary and contained a tubular structure (seminiferous-like tubule) in which the degenerating oocytes were found. The author presumed the testicular effect upon the mutual ovary as the activity of the diffused inductor substance derived from the testis. In the group, which more than 15 embryonal testes (maximally 25 testes were grafted) were transplanted in the subcapsular site and one or two ovaries in the opposite site of the kidney of the same host, the ovarian grafts, which were at a distant site from the multiple testicular grafts, showed inhibited growth and differentiation by the similar appearance of the transplanted embryonal ovary with testis in mutual contact. By this observation the author considered the inhibited growth and differentiation of the effect of blood-borne inductor substance derived from the multiple testicular grafts of the opposite site of the host kidney.
Animals ; Castration ; Female ; Male ; Ovary/*embryology/*transplantation ; Rats ; Testis/*embryology/*transplantation

OBJECTIVETo assess the recovery of the reproductive endocrine function in rats following orthotopic transplantation of fetal ovarian allograft.

METHODSNinety female SD rats (50-60 days old) were randomized into graft recipient group (n=50), positive control group (n=20), and negative control group (n=20) to receive orthotopic transplantation of fetal (17-19 gestational days) ovaries following bilateral oophorectomy, sham abdominal surgery, and bilateral oophorectomy, respectively. At 45 days after the surgeries, serum estradiol and progesterone levels were measured and the ovaries were removed for evaluation of the ovarian volume and follicle development.

RESULTSOn day 45 after the operations, the estradiol or progesterone levels showed no significant difference between the recipient group and positive control group (P>0.05), but both were significantly lowered in the negative control group (P<0.05). The ovarian volume was comparable between the recipient group and positive control group (P>0.05), and optical microscopy showed follicles in different stages of development and formation of corpus luteum in the ovaries in both groups.

CONCLUSIONFetal rat ovary allografts can develop into functional ovaries capable of ovulation to restore the reproductive endocrine function of recipient female rats.

Animals ; Estradiol ; blood ; Female ; Fetus ; Ovariectomy ; Ovary ; physiology ; transplantation ; Ovulation ; physiology ; Pregnancy ; Progesterone ; blood ; Rats ; Rats, Sprague-Dawley ; Transplantation, Homologous

OBJECTIVE: To evaulate the factors affecting pubertal development after bone marrow transplantation (BMT) in girls. METHODS: We collected data from girls older than 14 on 2003, who had received bone marrow transplantation with or without total body irradiation. We checked their menstrual pattern, onset of menarche, growth rate before and after the transplantation using chart review. RESULTS: 41.6% of girls who took menarche before BMT mensurate regularly and 100% of girls who didn't take menarche before BMT suffered by amenorrhea. Time period from BMT to menarche is 0.6 years in the regular-menstruating group, and 2.14 years in the secndary amenorrhea group. 37.5% of girls who didn't take total body irradiation menstrate regularly, but only 15.3% of girls who took total body irradiation menstrate regularly. 100% of girls who showed decreasing growth rate after BMT diagnosed primary or secondary amenorrhea and 75% of girls showed increasing growth rate after BMT menstrated regularly. CONCLUSION: Ovary is more vulnerable before menarche. Time of menarche, time period between menarche and BMT, and radiation are the most important factors affecting ovarian function after bone marrow transplantation.
Amenorrhea ; Bone Marrow Transplantation* ; Bone Marrow* ; Female* ; Humans ; Menarche ; Ovary ; Whole-Body Irradiation

OBJECTIVE: To evaulate the factors affecting pubertal development after bone marrow transplantation (BMT) in girls. METHODS: We collected data from girls older than 14 on 2003, who had received bone marrow transplantation with or without total body irradiation. We checked their menstrual pattern, onset of menarche, growth rate before and after the transplantation using chart review. RESULTS: 41.6% of girls who took menarche before BMT mensurate regularly and 100% of girls who didn't take menarche before BMT suffered by amenorrhea. Time period from BMT to menarche is 0.6 years in the regular-menstruating group, and 2.14 years in the secndary amenorrhea group. 37.5% of girls who didn't take total body irradiation menstrate regularly, but only 15.3% of girls who took total body irradiation menstrate regularly. 100% of girls who showed decreasing growth rate after BMT diagnosed primary or secondary amenorrhea and 75% of girls showed increasing growth rate after BMT menstrated regularly. CONCLUSION: Ovary is more vulnerable before menarche. Time of menarche, time period between menarche and BMT, and radiation are the most important factors affecting ovarian function after bone marrow transplantation.
Amenorrhea ; Bone Marrow Transplantation* ; Bone Marrow* ; Female* ; Humans ; Menarche ; Ovary ; Whole-Body Irradiation

OBJECTIVETo evaluate the therapeutic effects of bone marrow transplantation (BMT) on ovarian injury induced by chemotherapy in mice.

METHODSForty-eight mice were randomized equally into normal control group (A), cyclophosphamide and BMT group (B), and cyclophosphamide group (C). The mice in groups B and C were treated with intraperitoneal injection of cyclophosphamide at the daily dose of 150 mg/kg for 3 consecutive days, and allogeneic bone marrow cell transplantation was performed in group B. The ovary coefficient and the amount of follicles were compared to evaluate the function of ovaries. For cell tracking, the bone marrow cells were labeled with Hoechst 33342 and detected through fluorescence microscope after transplantation.

RESULTSOn days 21 and 50 after cyclophosphamide treatment, the ovary coefficient and the amount of follicles were significantly lowered in groups B and C (P<0.05), but the reduction was obviously ameliorated in group B (P<0.05). Cell tracking showed the presence of the donor bone marrow cells in the ovaries of the recipients mice after BMT.

CONCLUSIONBMT can improve the ovarian function impaired by chemotherapy in mice.

Animals ; Bone Marrow Transplantation ; Cyclophosphamide ; adverse effects ; Female ; Mice ; Ovary ; pathology ; physiopathology

OBJECTIVE: Revascularization is critical for successful ovarian tissue transplantation. Vascular endothelial growth factor (VEGF) and angiopoietin-2 (angpt-2) are the principal mediators of neovascularization. This study was designed to assess VEGF and angpt-2 levels in cryopreserved ovarian tissue after heterotopic autotransplantation. METHODS: Ovarian tissues harvested from ICR mice at 5 to 6 weeks of age were stratified as follows: no cryopreservation (controls, group I); vitrification in VFS-40 (vitrification, group II); and gradual freezing in dimethyl sulfoxide (slow-freezing, group III). Frozen specimens were thawed at room temperature, assaying VEGF and angpt-2 levels 1 week after cryopreservation and 2 weeks after autotransplantation. RESULTS: VEGF and angpt-2 protein levels were significantly lower in cryopreserved ovaries of groups II and III than in controls (group I, P<0.05), whereas groups II and III did not differ significantly in this regard. After autotransplantation of cryopreserved ovarian tissue, VEGF and angpt-2 protein levels did not differ significantly by technique but tended to be lower than corresponding levels in controls. CONCLUSION: Expression of angiogenic factors in ovarian tissue is thought to vary by method of cryopreservation. Our findings indicate that levels of angiogenic factors expressed in cryopreserved ovarian tissue after autotransplantation do not differ appreciably from control levels, regardless of cryopreservation technique.
Angiogenesis Inducing Agents* ; Angiopoietin-2 ; Animals ; Autografts* ; Cryopreservation ; Dimethyl Sulfoxide ; Female ; Freezing ; Mice* ; Mice, Inbred ICR ; Ovary* ; Tissue Transplantation ; Transplantation ; Transplants ; Vascular Endothelial Growth Factor A ; Vitrification

In order to explore the feasibility of cryopreserving primordial follicles in attaining their developmental competence following freezing and thawing, ovaries from newborn mice were cryopreserved and the thawed ovaries were xenografted into kidney capsules of adult female mice. Ovaries were isolated from newborn B6C2F(1) female mice, infiltrated by Leibovitz 15 (L-15) medium containing 10% (V/V) fetal bovine serum (FBS) and 1.5 mol/L dimethylsulfoxide (DMSO), and then packed into 0.25 ml plastic straws. The ovaries contained in straws were frozen under nitrogen vapour at -40 degrees C in Cryocell 1200 programmable freezer, and stored in liquid nitrogen for periods ranging from 1 week to 6 months. Upon thawing, the straws were dipped into room temperature water for 10~20 s, after which the ovaries were collected and washed in L-15 buffer containing 10% (V/V) FBS without DMSO to remove cryoprotectant. The thawed ovaries were transplanted into kidney capsules of 8~12-week old adult B6C2F(1) female recipient mice by two protocols, with either 1 or 2 ovaries in each capsule. Upon withdrawal after at least 14 d of transplantation, only 45.00% (72/160) of the ovaries were recovered from 40 recipients transplanted with 2 ovaries in each capsule, compared to 82.50% (33/40) in 20 recipients with only 1 ovary in each capsule. The grafted ovaries exhibited similar follicular developmental progression to that of natural ovaries. There were antral follicles present in the transplanted ovaries on day 14, whose number increased more substantially on day 19 after transplantation. Following stimulation of the recipient mice with 10 IU PMSG on day 19 after xenografting, follicles further developed to preovulatory stage with appearance of cumulus oocytes and enlarged antrum. Oocytes from these fully grown antral follicles were collected and matured in vitro in modified essential medium-alpha (MEMalpha). After 16~17 h of culture, 40.90% of the oocytes exhibited germinal vesicle breakdown (GVBD) and among which 89.02% proceeded to the metaphase II (MII) stage as indicated by exclusion of the first polar body. The remaining oocytes were further cultured and 50.83% of which initiated GVBD by 20~21 h of culture, but only 21.40% of which proceeded to MII. The above results demonstrated that the primordial follicles in newborn mouse ovaries were capable of sustaining freezing and thawing, and reinitiating development following xenograft into kidney capsule in adult recipient female mice. Production of mature oocytes from such re-developed follicles following gonadotrophin priming and the subsequent oocyte in vitro maturation implied immense prospect of application of this method to preserve female germ cells, conserve endangered species, establish animal gene stock, and utilize oocytes in assisted reproductive techniques.
Animals ; Animals, Newborn ; Cryopreservation ; Female ; Mice ; Mice, Inbred BALB C ; Oocytes ; growth & development ; Oogenesis ; physiology ; Ovarian Follicle ; growth & development ; Ovary ; transplantation ; Transplantation, Heterologous

OBJECTIVE: To investigate the health safety of the offspring delivered following natural pregnancy after orthotopic fetal ovarian allotransplantation in rats. METHODS: Any symptoms of spontaneous abortion during pregnancy and of any possible still birth and death of infant rats within 3 days after the delivery were observed and compared in 19 pregnant rats (the study group) after the orthotopic fetal ovarian allotransplantation and in another 10 pregnant rats (the control group). Forty offspring rats from each group were selected randomly. The mean weight at day 35 after the birth of offsprings was measured and compared. By routine G-banding technique, the karyotype was analyzed and the chromosomal number and structure were observed. RESULTS: There was no spontaneous abortion, still birth, or death in the infants within 3 days after the birth in both groups. The body weight of offsprings at 35 days in both groups was (93.80 ± 4.93) g and (94.13 ± 4.53) g, respectively. There was no significant difference between the 2 groups (P> 0.05). The karyotype analysis indicated that the karyotype from both offspring groups was 42, XX or 42, XY. No chromosome abnormality (abnormal chromosomal number or abnormal chromosomal structure) was observed in both groups. CONCLUSION The health status of the offsprings of rats after orthotopic fetal ovarian allotransplantation is safe.
Animals ; Animals, Newborn ; Female ; Fetus ; Karyotype ; Male ; Ovary ; transplantation ; Ovulation ; physiology ; Pregnancy ; Pregnancy Outcome ; Rats ; Rats, Sprague-Dawley ; Transplantation, Homologous ; adverse effects

This study was conducted by consecutively transplanting spleens, which had gonads implanted previously. A total of 84 cases for infantile testicles and 106 cases for ovarian follicles were performed. In the case of ovarian implants, the results were determined by the total number of follicle implants. A modified spleen transplantation technique called double implantation of ovarian follicles was applied to increase the amount of the implants. In this technique, an extra spleen is implanted into the potential donor so that the ovarian follicles can be implanted to two different spleens, doubling the amount of implants. Through consecutive spleen transplantation, we observed the results beyond a typical rat's life span. In many of these cases, we found more aggressive forms of malignant tumor, seminoma and dysgerminoma. We present the results and discuss possible pathogenic mechanisms of tumor formation.
Animals ; Animals, Newborn ; Female ; Male ; Ovarian Neoplasms/*etiology ; Ovary/*transplantation ; Rats ; Rats, Inbred Lew ; Research Support, Non-U.S. Gov't ; Spleen/*surgery/*transplantation ; Testicular Neoplasms/*etiology/pathology ; Testis/*transplantation ; *Transplantation, Heterotopic

OBJECTIVETo investigate expression and clinical significance of VEGF and apoptosis in frozen-thawed mouse ovaries after transplantation.

METHODSOvaries from B6C2F1 (C57BL/6j x BALB/c) 4 week old mice were cryopreservation and the thawed ovaries were xenografted into kidney capsules of 8-12 week old adult male mice. The grafted were recovered 1 d, 2 d and 7 d after transplantation respectively, the grafts and frozen-thawed were removed for follicle counting and immunohistochemically, ultrastructure, and detection of the mRNA expression of vascular endothelial growth factor(VEGF).

RESULTSThe follicle numbers were decreased gradually after transplantation,the cell apoptosis increased especially in 48 h after transplantation. Transmission electron microscopy (TEM) showed the tissue damaged was severest 48 h after transplantation; the expression of VEGF increased after transplantation, peaked on day 7, the mRNA expression of VEGF120 and VEGF188 was more on 48 h after transplantation, decreased on day 7 (P < 0.05).

CONCLUSIONThe number of follicles was decreased after transplantation, the apoptosis index was increased especially in 48 h after transplantation; the expression of VEGF increased after transplantation, an increase in the VEGF188 and VEGF164 isoform might suggest the positive effect in the early stages of angiogenesis.

Animals ; Apoptosis ; physiology ; Cryopreservation ; Female ; Male ; Mice ; Organ Preservation ; adverse effects ; Ovarian Follicle ; pathology ; Ovary ; pathology ; transplantation ; Vascular Endothelial Growth Factor A ; metabolism