Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

Pretreatment of ovaries with collagenase before vitrification keeps the ovarian reserve by maintaining cell-cell adhesion integrity in ovarian follicles.

The mammalian ovarian follicle is comprised of the germ cell or oocyte surrounded by the somatic cells, the granulosa and theca cells. The ovarian stroma, including the collagen-rich matrix that supports the three-dimensional disk-like follicular structure, impacts the integrity of the ovarian follicle and is essential for follicular development. Maintaining follicular integrity during cryopreservation has remained a limiting factor in preserving ovarian tissues for transplantation because a significant proportion of developed follicles in the frozen-thawed ovaries undergo atresia after transplantation. In this study, we show for the first time that during vitrification of the mouse ovary, the attachment of the oocyte to the granulosa cells was impaired by the loss of the cadherin adhesion molecules. Importantly, exposure to a high osmotic solution greatly decreased the ratio of oocyte diameter to the diameter of its follicle but did not alter the collagen-rich matrix surrounding the follicles. By treating ovaries briefly with collagenase before exposure to the hyper-osmotic solution the ratio of oocyte diameter to follicle diameter was maintained, and cadherin adhesion junctions were preserved. When frozen-thawed ovaries were transplanted to the bursa of recipient hosts, pretreatment with collagenase significantly increased serum levels of AMH, the number of intact follicles and the total number of viable offspring compared to frozen-thawed ovaries without collagenase pretreatment, even 6 months after transplantation. Thus, the collagenase pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries in other species and clinically relevant situations.

Effect of intra-ovarian injection of mesenchymal stem cells in aged mares.

PURPOSE: This study aims to determine if intra-ovarian injection of bone marrow-derived mesenchymal stem cells (MSCs) improves or restores ovarian function in aged females. METHODS: Prospective randomized study of eight aged mares and six young mares receiving intra-ovarian injection of MSCs or vehicle. Main outcome measures were antral follicle count and serum anti-Müllerian hormone (AMH) (aged and young mares), and for aged mares, oocyte meiotic and developmental competence; gross and histological ovarian assessment; evaluation of presence of chimerism in recovered granulosa cells and in ovarian tissue samples; and gene expression in ovarian tissue as assessed by RNA sequencing. RESULTS: Injection of MSCs was not associated with significant changes in follicle number, oocyte recovery rate on follicle aspiration, oocyte maturation rate, or blastocyst rate after ICSI in aged mares, or in changes in follicle number in young mares. There were no significant changes in peripheral AMH concentrations, indicating a lack of effect on growing follicles. MSC donor DNA was not recovered in granulosa cells or in ovarian tissue, indicating lack of persistence of injected MSC. RNA sequencing revealed significant differences in gene expression between MSC- and vehicle-injected ovaries. CONCLUSIONS: Intra-ovarian injection of bone marrow-derived MSCs altered gene expression but did not improve ovarian function in aged mares.

HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway.

BACKGROUND/AIMS: Human adipose-derived stem cells (hADSCs) are a potential therapeutic option for clinical applications because of their ability to produce cytokines and their capacity for trilineage differentiation. To date, few researchers have investigated the effects of hADSCs on natural ovarian aging (NOA). METHODS: An NOA mouse model and human ovarian granule cells (hGCs) collected from individuals with NOA were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing NOA. Enzyme-linked immunosorbent assay was used to detect the serum levels of sex hormones and antioxidative enzymes. The proliferation rate and marker expression level of hGCs were measured by flow cytometry (FACS). Cytokines were measured by a protein antibody array methodology. Western blot assays were used to determine the protein expression levels of SIRT1 and FOXO1. RESULTS: Our results showed that hADSCs displayed therapeutic activity against ovarian function in an NOA mouse model, increasing the proliferation rate and marker expression level of hGCs. Furthermore, the yields of hADSC-secreted HGF and bFGF were higher than those of other growth factors. FACS showed that combination treatment with the growth factors HGF and bFGF more strongly promoted proliferation and inhibited apoptosis in hGCs than HGF or bFGF treatment alone. FACS and ELISA revealed that the combination treatment with both growth factors inhibited oxidative stress more forcefully than treatments with only one of these growth factors. In addition, protein assays demonstrated that combination treatment with both growth factors suppressed oxidative stress by up-regulating the expression of SIRT1 and FOXO1. CONCLUSION: These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which HGF and bFGF derived from hADSCs improved ovarian function during natural aging via reduction of oxidative stress by activating the SIRT1/FOXO1 signaling pathway.

In vivo transplantation of 3D encapsulated ovarian constructs in rats corrects abnormalities of ovarian failure.

Safe clinical hormone replacement (HR) will likely become increasingly important in the growing populations of aged women and cancer patients undergoing treatments that ablate the ovaries. Cell-based HRT (cHRT) is an alternative approach that may allow certain physiological outcomes to be achieved with lower circulating hormone levels than pharmacological means due to participation of cells in the hypothalamus-pituitary-ovary feedback control loop. Here we describe the in vivo performance of 3D bioengineered ovarian constructs that recapitulate native cell-cell interactions between ovarian granulosa and theca cells as an approach to cHRT. The constructs are fabricated using either Ca++ or Sr++ to crosslink alginate. Following implantation in ovariectomized (ovx) rats, the Sr++-cross-linked constructs achieve stable secretion of hormones during 90 days of study. Further, we show these constructs with isogeneic cells to be effective in ameliorating adverse effects of hormone deficiency, including bone health, uterine health, and body composition in this rat model.

A novel three-dimensional culture system allows prolonged culture of functional human granulosa cells and mimics the ovarian environment.

The development of techniques allowing the growth of primordial follicles to mature follicles in vitro has much potential for both reproductive medicine and developmental research. However, human primordial and preantral follicles fail to grow after isolation from the surrounding ovarian stroma. Granulosa cells, which normally undergo apoptosis after ovulation, contain a subpopulation of ovarian follicular cells remaining viable in vitro over prolonged periods when cultured in the presence of leukemia-inhibiting factor. However, when cultured as monolayers, they progressively lose all their characteristics, such as follicle-stimulating hormone receptor and cytochrome P450-aromatase. Here, we describe a three-dimensional culture system containing type I collagen, which, together with leukemia-inhibiting factor, allowed the survival and growth of a subpopulation of granulosa cells isolated from mature ovarian follicles and supported them to proliferate into spherical structures exhibiting steroidogenic capacity, as demonstrated by P450-aromatase and 3beta-hydroxysteroid dehydrogenase. After transplantation into the ovaries of immunodeficient mice, these cells became localized preferentially within antral follicles and the prolonged expression of follicle-stimulating hormone receptor was confirmed as well. With this optimization of the culture conditions, an environment was created, which acts as a niche closely mimicking the development of early ovarian follicles in vitro.

Xenotransplantation by injection of a suspension of isolated preantral ovarian follicles and stroma cells under the kidney capsule of nude mice.

OBJECTIVE: To develop and test a novel approach to xenotransplantation of isolated preantral follicles underneath the kidney capsule of immunodeficient mice. DESIGN: Prospective experimental animal study. SETTING: Academic research unit. ANIMAL(S): Healthy adult nude mice. INTERVENTION(S): Bovine ovaries from fetuses (n = 3) and calves (n = 3) were enzymatically disaggregated and subsequently filtered. Isolated preantral follicles were suspended in phosphate buffered saline, and granulosa and stroma cells originating from the ovarian digest served as embedding matrix. The suspension was injected under the kidney capsule of adult nude mice. MAIN OUTCOME MEASURE(S): Fourteen days after transplantation, follicular survival and proliferation in grafts was assessed by histology and proliferating cell nuclear antigen (PCNA) immunostaining, and was compared with ungrafted control tissue. RESULT(S): Primordial follicles decreased from 58.2% in control tissue to 17.1% in transplants in the fetal group, and from 76.0% to 17.2% in the calf group. Concomitantly, primary follicles increased from 13.4% to 62.2% in the fetal group, and from 5.4% to 63.5% in the calf group. Follicular proliferation measured by PCNA immunolabeling exhibited an increase from 40.6% growing follicles to 81.9% in the fetal group, and from 21.0% to 80.7% in the calf group. CONCLUSION(S): The massive follicular activation following transplantation indicates that isolated preantral follicles are able to survive and grow 14 days after renal subcapsular xenotransplantation.

Cryopreservation and xenotransplantation of human ovarian tissue: an ultrastructural study.

OBJECTIVE: To analyze the ultrastructure of human ovarian follicles after cryopreservation and short-term xenografting. DESIGN: Prospective experimental study. SETTING: Academic gynecology and anatomy research units. PATIENT(S): Ovarian cortical biopsy specimens were obtained from 13 patients. INTERVENTION(S): Each ovarian biopsy specimen was dissected into pieces of 1 mm(3) and divided into three groups: [1] fresh tissue, [2] frozen-thawed tissue, and [3] frozen-thawed tissue xenografted onto the peritoneum of nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Follicular ultrastructure was assessed by light and transmission electron microscopy in [1] fresh, [2] frozen, and [3] frozen-grafted tissue. RESULT(S): Thirty-five ovarian follicles were analyzed by light and transmission electron microscopy. Twenty-five primordial and primary ovarian follicles were found. Most of them exhibited ultrastructurally well preserved features (fresh [N = 8/10], frozen [N = 7/10], and frozen-grafted [N = 4/5] tissue). Ten secondary follicles were present in xenografts. By transmission electron microscopy, all the healthy-looking secondary follicles (70%) were shown to contain intact oocytes, with features typical of earlier developmental stages, surrounded by several layers of follicular cells. CONCLUSION(S): The present study demonstrates, for the first time, that cryopreservation and xenotransplantation do not appear to greatly affect human primordial/primary follicle ultrastructure. Interestingly, in frozen-thawed xenografts, secondary human ovarian follicles presented a well preserved ultrastructure, but asynchrony between oocyte and granulosa cell development was detected. The possible causes for this asynchrony are discussed.

Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells.

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.

Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes.

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum-fed donor cells. There were no significant differences (P > 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10,11, and 13 (7, 11.5, and 14%, respectively, P < 0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.

A method for transplantation of luteinizing granulosa cells: evidence for progesterone secretion.

A method is described to enable the investigation of luteal tissue that is formed in the absence of ovarian theca lutein cells. Immature rats were given a s.c. injection of pregnant mare's serum gonadotropin (PMSG) followed 48 h later by an i.v. injection of human chorionic gonadotropin (hCG). Both ovaries were removed 8 to 10 h after the injection of hCG, the preovulatory follicles punctured, and the granulosa cells expressed into medium using gentle pressure with a dissecting scalpel. The cells were centrifuged at low speed (50 X g) for 5 min, resuspended and recentrifuged. The cells were aspirated into PE 90 polyethylene tubing, the tubing sealed at one end and then placed into a microcapillary hematocrit tube. The cells were packed by centrifugation in a microhematocrit centrifuge for 2 min, after which they were autotransplanted by injecting them beneath the kidney capsule. Progesterone, determined in blood samples obtained 4 through 12 days after transplantation, was elevated on most days compared to ovariectomized controls without cell transplants. In a second experiment, serum progesterone in ovariectomized-adrenalectomized rats with autotransplants was also elevated (4 to 6 ng/ml) compared to controls without cell transplants in which progesterone was about 0.1 ng/ml. Morphology of transplants revealed luteal cells with considerable vascularity and with the typical appearance of cells observed in in situ corpora lutea. This procedure, with modifications to include homotransplants in inbred strains, holds promise for investigation of the endocrine characteristics of granulosa lutein cells devoid of cells of thecal origin.