Recipient Ovaries Research Articles

Ectopic Trophoblast Allografts in the Horse Resist Destruction by Secondary Immune Responses.

Invasive trophoblast from Day 34 horse conceptuses survives in extrauterine sites in allogeneic recipients that are immunologically naive to donor major histocompatibility complex class I antigens. The ectopic trophoblast retains its in utero characteristics, including similar lifespan, physiologic effect of its secreted product (equine chorionic gonadotropin) upon the recipient's ovaries, and induction of host immune responses. Immunologic memory has not been considered previously in this experimental system. We hypothesized that primary exposure to ectopic trophoblast would affect the recipient's immune status such that the survival time of subsequent transplants would be altered. Secondary transplant lifespans could be shortened by destructive memory responses, as has been observed in ectopic trophoblast studies in rodents, or lengthened, as occurs when male skin grafts follow multiple syngeneic pregnancies in mice. Eight mares received two closely spaced trophoblast transplants. Both grafts for each recipient were obtained from conceptuses sired by the same stallion to provide consistency in histocompatibility antigen exposure. Donor stallions were major histocompatibility complex class I homozygotes. Cytotoxic antibody production was tracked to monitor recipients' immune responses to the transplants. Detection of serum equine chorionic gonadotropin was used as a proxy for transplant lifespan. There was no significant difference between the distributions of primary and secondary transplant lifespans, despite evidence of immunologic memory. These data demonstrate that secondary ectopic trophoblast transplants in horses do not experience earlier destruction or prolonged survival following immune priming of recipients. Mechanisms responsible for the eventual demise of the transplants remain unperturbed by secondary immune responses or chronic antigenic exposure.

Skin-derived mesenchymal stem cells help restore function to ovaries in a premature ovarian failure mouse model.

Skin-derived mesenchymal stem cells (SMSCs) can differentiate into the three embryonic germ layers. For this reason, they are considered a powerful tool for therapeutic cloning and offer new possibilities for tissue therapy. Recent studies showed that skin-derived stem cells can differentiate into cells expressing germ-cell specific markers in vitro and form oocytes in vivo. The idea that SMSCs may be suitable for the treatment of intractable diseases or traumatic tissue damage has attracted attention. To determine the ability of SMSCs to reactivate injured ovaries, a mouse model with ovaries damaged by busulfan and cyclophosphamide was developed and is described here. Female skin-derived mesenchymal stem cells (F-SMSCs) and male skin-derived mesenchymal stem cells (M-SMSCs) from red fluorescence protein (RFP) transgenic adult mice were used to investigate the restorative effects of SMSCs on ovarian function. Significant increases in total body weight and the weight of reproductive organs were observed in the treated animals. Both F-SMSCs and M-SMSCs were shown to be capable of partially restoring fertility in chemotherapy-treated females. Immunostaining with RFP and anti-Müllerian hormone (AMH) antibodies demonstrated that the grafted SMSCs survived, migrated to the recipient ovaries. After SMSCs were administered to the treated mice, real-time PCR showed that the expression levels of pro-inflammatory cytokines TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFNγ were significantly lower in the ovaries than in the untreated controls. Consistent with this observation, expression of oogenesis marker genes Nobox, Nanos3, and Lhx8 increased in ovaries of SMSCs-treated mice. These findings suggest that SMSCs may play a role within the ovarian follicle microenvironment in restoring the function of damaged ovaries and could be useful in reproductive health.

Production of genetically diversified fish seeds using spermatogonial transplantation

Abstract Large-scale release of hatchery-produced seeds has been conducted in order to restore worldwide fishery production; however, concerns exist regarding the genetic effects of hatchery stock on wild fish populations, due to the reduced genetic variation often associated with hatchery-reared fish. Therefore, it is important that fish seeds used in stock enhancement possess sufficient genetic diversity to mitigate their genetic impact on wild fish populations. To promote genetic diversity of artificial seeds, seed production should be performed using a sufficiently large broodstock; however, maintenance of large broodstocks requires significant investments with respect to space, labor, and other associated costs. In order to circumvent the need for these investments, the present study proposed a means of producing gametes that possess a large amount of genetic diversity using only a small number of surrogate-broodstock through spermatogonial transplantation. We previously established spermatogonial transplantation methodology capable of producing functional eggs and sperm derived from donor-type A spermatogonia (ASGs). It was expected that if ASGs isolated from testes of different donor individuals were mixed prior to their transplantation into the peritoneal cavity of a single recipient, the resulting recipient would produce gametes derived from several donor individuals. To determine the feasibility of this scenario, mixed donor ASGs derived from vasa-GFP, vasa-DsRed and hsc-GFP transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into triploid recipients. At 20 days post-transplantation, approximately 80% of recipients possessed donor ASGs derived from all three strains within their gonads and mean numbers of incorporated ASGs derived from each donor strain were nearly equal. At 100 days post-transplantation, incorporated donor ASGs proliferated within the gonads of both male and female recipients, and some had differentiated into oocytes within recipient ovaries. Furthermore, in order to ascertain whether incorporated donor ASGs derived from the three strains could differentiate into functional gametes, gametes collected from mature male and female recipients were used in artificial insemination with gametes obtained from wild-type fish of the opposite sex. Phenotypic analysis of F1 offspring revealed that functional sperm and eggs derived from three donor strains were successfully produced in the gonads of individual recipients. These results suggested that individual recipients that received ASGs from multiple donors were capable of producing gametes with different genetic backgrounds. Therefore, it was concluded that the method of spermatogonial transplantation outlined in the present study could serve as a novel and efficient method of producing fish seeds with increased genetic diversity for use in stock enhancement.

Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries

Busulfan and cyclophosphamide (B/C)-treated mice exhibited a marked increase in apoptosis and a concomitant decrease in the ovarian weight. Histological and RT-PCR analysis indicate that the period of germ cell depletion in the B/C-treated ovaries coincides with decreased expression of genes Figla, Lhx8, Nobox, c-kit, and Sox3. However, depletion of the ovarian germ cells is mediated by autophagy-independent pathways that involve Fas/FasL-, TNF-, and/or p53-signalings. Treatment with B/C resulted in the cease of the reproductive function to produce their offspring during the 15th week post-treatment period in female mice. Furthermore, injection of the 3 × 106 GFP positive primordial follicles into the ovaries of the B/C treated mouse did not show apparent colonization of the transplanted follicles within the recipient ovaries. The present results suggest that B/C treatment is closely associated with an increased risk of premature ovarian failure.

Current progress and future direction in the biology of ovarian germ stem cells in mammals

Whether or not oogenesis continues after birth in mammalian ovaries remains controversial. Since the 1950's, it has been generally accepted that oogenesis takes place during embryogenesis in mammals and ceases at birth. At birth, germ cells in mammalian ovaries have progressed to the diplotene stage of meiotic prophase and have formed primordial follicles with surrounding somatic cells. These primordial follicles represent follicle reserves of the reproductive life. However, this view has been recently challenged by a growing body of evidence showing the isolation and propagation of germ stem cells from mouse and human ovaries. These ovarian germ stem cells are capable of regenerating functional oocytes when transplanted back into recipient ovaries. Despite the discovery of the potential germ stem cells in mammalian ovaries, it remains uncertain whether these cells exist and function in ovaries under physiological conditions. Herein we review the current progress and future direction in this infant area.

77 SUCCESSFUL CRYOPRESERVATION OF SYRIAN HAMSTER OVARIES BY VITRIFICATION

In place of gametes, ovaries can be used as an alternative resource for cryopreservation. At the previous IETS meeting, we showed that in the Syrian hamster, pups could be successfully produced through the transplantation of fresh ovaries. In addition, the immunotolerance of interstrain ovarian transplantation was described. In this study, we attempted to produce pups via transplantation of cryopreserved ovaries to foster mothers. Our goal was to establish a preservation method for hamster strains based on ovarian cryopreservation in combination with ovarian transfer. Ovaries were cryopreserved by vitrification according to methods described for mouse ovaries (Migishima F et al. 2003 Biol. Reprod. 68, 881–887). In brief, ovaries were collected from 2- to 3-week-old HAW strain (white coat) females. Each ovary was cut into 4 sections, placed into 1 tube, and cryopreserved by vitrification using DAP213 solution (2 m dimethyl sulfoxide, 1 m acetamide, and 3 m propylene glycol; Nakagata N 1989 J. Reprod. Fertil. 87, 479–483). Thawed ovaries were transferred into the ovarian bursae (2 ovarian sections per ovarian bursa) of 3-week-old recipient females with agouti coat (Slc:Syrian) under anesthesia with isoflurane. Half of the recipients’ ovaries were removed immediately before transfer; i.e. both donor and recipient ovaries were present in each ovarian bursa. Successful ovarian transplantation was verified by examining the coat color (non-agouti color) of offspring produced in test matings between mature recipients and Slc:Syrian males. Out of 6 recipients tested, 2 became pregnant and delivered pups. According to the coat color of the pups, 1 of the 2 recipients delivered a pup produced from transplanted ovaries (1 out of 4 pups). Regardless of this low success rate, our results indicate that pups can be produced from cryopreserved ovaries via interstrain transplantation of ovaries to foster mothers in the Syrian hamster. Thus, ovarian cryopreservation by vitrification is a promising strategy for strain preservation of the Syrian hamster. This work was supported by a grant from the Ministry of Health, Labor, and Welfare, Japan.

Bone Marrow Transplantation Generates Immature Oocytes and Rescues Long-Term Fertility in a Preclinical Mouse Model of Chemotherapy-Induced Premature Ovarian Failure

Although early menopause frequently occurs in female cancer patients after chemotherapy (CTx), bone marrow (BM) transplantation (BMT) has been linked to an unexplained return of ovarian function and fertility in some survivors. Studies modeling this in mice have shown that BMT generates donor-derived oocytes in CTx-treated recipients. However, a subsequent report claimed that ovulated eggs are not derived from BM and that BM-derived oocytes reported previously are misidentified immune cells. This study was conducted to further clarify the impact of BMT on female reproductive function after CTx using a preclinical mouse model. Female mice were administered CTx followed by BMT using coat color-mismatched female donors. After housing with males, the number of pregnancies and offspring genotype were recorded. For cell tracking, BM from germline-specific green fluorescent protein-transgenic mice was transplanted into CTx-treated wild-type recipients. Immune cells were sorted from blood and analyzed for germline markers. BMT rescued long-term fertility in CTx-treated females, but all offspring were derived from the recipient germline. Cell tracking showed that donor-derived oocytes were generated in ovaries of recipients after BMT, and two lines of evidence dispelled the claim that these oocytes are misidentified immune cells. These data from a preclinical mouse model validate a testable clinical strategy for preserving or resurrecting ovarian function and fertility in female cancer patients after CTx, thus aligning with recommendations of the 2005 National Cancer Institute Breast Cancer Progress Review Group and President's Cancer Panel to prioritize research efforts aimed at improving the quality of life in cancer survivors.

Regeneration of Oocytes After Chemotherapy: Connecting the Evidence From Mouse to Human

Regeneration of Oocytes After Chemotherapy: Connecting the Evidence From Mouse to Human

148 PRODUCTION OF PUPS BY OVARIAN TRANSFER IN THE SYRIAN HAMSTER

Instead of gametes, ovaries could be used as alternative resources for cryopreservation. In this study, we attempted to produce pups by transplantation of fresh ovaries to foster mothers as a first step to establish a preservation method of hamster strains on the basis of the cryopreservation of ovaries in combination with ovarian transfers. We also checked the possibility of inter-strain transfers of ovaries, since immunological tolerance among hamster strains is suggested by the fact that most Syrian hamsters were derived from only a small number of animals. Fresh ovaries collected from 3-weekold females of HAW (white coat) and APA (albino) strains were transferred into ovarian bursae of 3-week-old recipient females with agouti-coat (Slc:Syrian) under anesthesia with xylazine and ketamine. Halves of ovaries of recipients were removed immediately before the transfers, i.e., both donor and recipient ovaries were present in each ovarian bursa. Successful transplantation of ovaries was checked by the coat color (non-agouti color) of offspring produced by test matings of recipients with Slc:Syrian males after the recipients became mature. Five and six recipients received HAW and APA ovaries, respectively. All 11 recipients tested became pregnant and delivered pups. According to the coat color of the pups, three of five recipients which received HAW ovaries and one of six recipients which received APA ovaries delivered pups derived from transplanted ovaries (two out of eight pups, one out of seven pups, and three out of 11 pups for HAW; one out of five pups for APA). Our results indicate that transplantation of fresh ovaries to foster mothers could be used for production of pups from grafted ovaries in the Syrian hamster. In particular, immunological tolerance for ovarian transfers among at least three strains suggests that recipient strains might not need to be the same as donor strains for practical ovarian transfers in the Syrian hamster. Our results would facilitate the development of a strain preservation system for the Syrian hamster based on ovarian cryopreservation. This work was supported by a grant from the Ministry of Health, Labor, and Welfare of Japan.

Ovarian Transplantation In A Series Of Identical Twins Discordant For Ovarian Failure

To investigate the prospects for restoring spontaneous menstrual cycles and fertility by ovarian isotransplantation between monozygotic twin sisters. A series of 4 otherwise healthy monozygotic twins, where 1 had idiopathic premature menopause & the other had normal ovarian function. 4 pairs of monozygotic twins (aged 24, 25 and 38[n=2] years of age) requested ovarian transplantation from the fertile sister to their sibling who had premature ovarian failure (POF). 3 of the donor sisters had normal menstrual cycles and had completed their target family sizes of 2 or 3 children; a 25 y.o. twin donor had no children and was on oral contraception. All 4 prospective recipients had FSH levels > 90 mIU/ml, no menses for ≥10 years, and complete absence of ovarian follicles in ovarian biopsies. They were menopausal from ages 14, 15, 16, and 22 years. 2 of the twins underwent transplantation at age 24 and 38, and 2 are awaiting. One ovary was removed from the donor and cortical tissue was dissected ex vivo in Leibovitz-L15 medium at 4°C. Donor ovarian cortex was then sutured microsurgically into the recipient’s medulla via a mini-laparotomy. This procedure was outpatient, with prompt & uneventful recovery. Surplus tissue from the youngest twin-pair was cryopreserved for future use with a slow-freezing protocol using 1,2-propanediol & sucrose as cryoprotective agents. Ovarian tissues & peripheral blood from both women was cultured for analysis by karyotyping and analyzed by FISH for sex chromosome mosaicism. All twin-pairs were genetically fingerprinted, and DNA from the sterile sisters was tested for fragile X mutation. All the twin-pairs were confirmed by genetic fingerprinting to be homozygous, implying that immunosuppression was unnecessary for transplant survival. Histology confirmed that the recipient ovaries were sterile, whereas the donor ovaries contained numerous primordial and small growing follicles, consistent with the antral follicle counts obtained preoperatively by transvaginal ultrasound. Karyotypes were normal 46,XX and there was no expansion of CCG repeats in the FMR-1 gene. The first of the ovary transplant recipients menstruated lightly at 12 weeks postoperatively and conceived naturally with an intrauterine pregnancy at the next cycle (∼20 weeks postoperatively). At 22 weeks of pregnancy, ultrasound revealed a normal girl and, at the time of writing, the pregnancy is proceeding uneventfully at the 33rd week. The 2nd twin-pair has also undergone transplantation with similar genetic and histologic findings, and 2 additional pairs are awaiting the same procedure. Evidence we present elsewhere indicates that POF within discordant twin-pairs is less rare than expected. These first cases of ovarian tissue transplantation between women are made possible by virtue of a genetically identical twin willing to be a donor. There is a latent period of several months before ovulation is restored & the endocrine profile is normalized, but in our first case, conception occurred promptly thereafter & pregnancy was normal. If this success can be repeated, ovarian transplantation may provide an alternative to conventional egg donation for comparable patients, and it confirms the feasibility of natural conception after transplanting thawed ovarian tissue in cancer patients. Finally, ongoing molecular study of such twin-pairs may eventually shed light on the causes of premature ovarian failure.%3:45 p.m.

Study on cellular events in postthymectomy autoimmune oophoritis in mice. I. Requirement of Lyt-1 effector cells for oocytes damage after adoptive transfer.

Neonatal thymectomy during the critical period, 2-4 d after birth, can induce various organ-specific autoimmune diseases including oophoritis in A/J mice. The oophoritis thus induced was passively transferred into neonatal mice by injection of spleen cells obtained from syngeneic donors with the disease. Recipient ovaries were rapidly damaged with remarkable mononuclear cell infiltration and destruction of follicular structures. The phenotype of effector cells responsible for successful adoptive transfer was found to be Thy-1+, Lyt-1+,23-, Ia-, Qa-1-, and was sensitive to antithymocyte serum treatment but resistant to cyclophosphamide treatment or in vitro X-ray irradiation. The compatibility between donor and recipient at the major histocompatibility complex was not required for the effector phase of transfer. The oophoritis induced in BALB/c (nu/+ or +/+) was also shown to be transferred into athymic BALB/c nude mice with resulting ovarian lesion and circulating autoantibodies against oocytes. In this transfer system, the effector cells were also demonstrated to be T cells with the Lyt-1+,23- phenotype. Adoptive transfer experiments in both systems revealed that the destruction of ovaries in postthymectomy autoimmune oophoritis was mediated by Lyt-1 T cells. Whether these T cells can be distinguished from other Lyt-1 cells, such as T helper cells and effector T cells in delayed-type hypersensitivity (DTH), is not clear at present, but the results suggest that the effector mechanisms may be closely related to a DTH reaction.

ACTION OF ESTROGEN ON RELEASE OF HYPOPHYSEAL LUTEINIZING HORMONE

SummaryThe pituitaries of adult female rats, under normal estrogen influence, stimulated moderate luteinization in normal and hypophysectomized recipient immature female rats. The pituitaries of adult female rats freed of estrogen stimulation through oophorectomy, invariably caused extensive luteinization of the recipient ovaries. This indicated that liberation of the hypophyseal luteinizing factor was minimal in the absence of estrogen; the factor remaining stored within the pituitary gland.Release of the luteinizing factor by estrogen was suggested also by the type of ovarian response produced by pituitaries from oophorectomized adult female rats which had been injected with estradiol benzoate daily for 30 to 45 days. These pituitaries produced follicular development but no corpora lutea, due to removal of the luteinizing factor by the estrogen treatment.

Action of Estrogen on Release of Hypophyseal Luteinizing Hormone

SummaryThe pituitaries of adult female rats, under normal estrogen influence, stimulated moderate luteinization in normal and hypophysectomized recipient immature female rats. The pituitaries of adult female rats freed of estrogen stimulation through oophorectomy, invariably caused extensive luteinization of the recipient ovaries. This indicated that liberation of the hypophyseal luteinizing factor was minimal in the absence of estrogen; the factor remaining stored within the pituitary gland.Release of the luteinizing factor by estrogen was suggested also by the type of ovarian response produced by pituitaries from oophorectomized adult female rats which had been injected with estradiol benzoate daily for 30 to 45 days. These pituitaries produced follicular development but no corpora lutea, due to removal of the luteinizing factor by the estrogen treatment.