RESUMO
Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.
Assuntos
Macrófagos/fisiologia , Folículo Ovariano/fisiologia , Comunicação Parácrina , Células Estromais/fisiologia , Células Tecais/fisiologia , Análise de Variância , Androgênios/metabolismo , Androstenodiona/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/metabolismo , Citocinas/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Macrófagos/metabolismo , Camundongos , Folículo Ovariano/metabolismo , Fenótipo , Células Estromais/metabolismo , Células Tecais/metabolismo , Fatores de TempoRESUMO
Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.
Assuntos
Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Animais , Criopreservação , Técnicas de Cultura Embrionária , Feminino , Hormônio Foliculoestimulante/farmacologia , Humanos , Fase Luteal , Meiose , Modelos Animais , Oócitos/efeitos dos fármacos , Folículo Ovariano/citologia , Papio , Injeções de Esperma Intracitoplásmicas , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Young female adult and adolescent cancer patients facing life-preserving but fertility-threatening chemo- or radiation-therapy are increasingly seeking options to protect their reproductive potential. Ovarian tissue cryopreservation with transplantation is a promising technique to safeguard fertility in cancer patients. However, this method may risk re-introduction of the original cancer to the survivor of the disease. Thus, developing a method for in vitro growth of immature follicles may provide a method for fertility restoration in the future. METHODS: Human secondary follicles were isolated from ovarian tissues obtained from cancer patients and grown in vitro within a bio-engineered culture system for 30 days. RESULTS: Human ovarian follicles became steroidogenically active, and developed from the early secondary to antral stage in vitro. The follicles contained healthy, growing oocytes that were connected by transzonal projections between the somatic cells and oocyte. CONCLUSIONS: Our data support the notion that human follicle development can be achieved in vitro in a bio-engineered culture system. More studies are required to investigate whether the fully sized oocytes obtained from in vitro grown follicle are competent to resume meiosis and be fertilized.
Assuntos
Neoplasias , Oócitos/crescimento & desenvolvimento , Oogênese , Folículo Ovariano/crescimento & desenvolvimento , Coleta de Tecidos e Órgãos , Adolescente , Adulto , Técnicas de Cultura de Células , Criopreservação , Feminino , Hormônios Esteroides Gonadais/metabolismo , Humanos , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Técnicas de Cultura de TecidosRESUMO
More than half of the primordial follicles that are formed by Day 6 of postnatal life in the mouse will be eliminated from the ovary by the time of puberty. Apoptosis, a form of programmed cell death, is one mechanism by which these follicles could be actively lost. To investigate whether apoptosis is responsible for the loss of primordial follicles, follicular atresia was examined during the prepubertal period, when follicles die and are cleared from the ovary at an extremely high rate. Four hallmarks of classical apoptosis were measured in follicles present in prepubertal ovaries. The primordial follicle cohort was not positively associated with nuclear condensation or cell shrinkage, activation of caspase 3, cleavage of poly(ADP ribose) polymerase 1 (PARP1), or fragmentation of DNA. These data are consistent with a nonapoptotic pathway that is responsible for small follicle death.
Assuntos
Apoptose/fisiologia , Folículo Ovariano/fisiologia , Maturidade Sexual/fisiologia , Animais , Caspases/metabolismo , Contagem de Células/métodos , Morte Celular , Feminino , Camundongos , Modelos Biológicos , Modelos Teóricos , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors alpha1 and beta1, which are commonly referred to as TRalpha1 and TRbeta1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TRalpha1 and TRbeta1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TRalphaKO), Thrb knockout (TRbetaKO), and wild-type (WT) mice. Triiodothyronine (T3) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRbetaKO mice versus that in their untreated controls, whereas T3 had a diminished effect on TRalphaKO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T3-treated WT and TRbetaKO testes. In contrast, T3 had no effect on these parameters in TRalphaKO mice. In untreated adult TRalphaKO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRbetaKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRbetaKO mice showed comparable increases following hypothyroidism. Our results show that TRalphaKO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRbetaKO mice, but not TRalphaKO mice, have normal Sertoli cell responsiveness to T3. Thus, effects of exogenous manipulation of T3 on neonatal Sertoli cell development are predominately mediated through TRalpha1.
Assuntos
Células de Sertoli/fisiologia , Receptores alfa dos Hormônios Tireóideos/fisiologia , Tri-Iodotironina/fisiologia , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27 , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Receptores alfa dos Hormônios Tireóideos/genética , Tri-Iodotironina/farmacologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.