RESUMO
Fertility preservation is not only a concern for humans with compromised fertility after cancer treatment. The preservation of genetic material from endangered animal species or animals with important genetic traits will also greatly benefit from the development of alternative fertility preservation strategies. In humans, embryo cryopreservation and mature-oocyte cryopreservation are currently the only approved methods for fertility preservation. Ovarian tissue cryopreservation is specifically indicated for prepubertal girls and women whose cancer treatment cannot be postponed. The cryopreservation of pre-antral follicles (PAFs) is a safer alternative for cancer patients who are at risk of the reintroduction of malignant cells. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation, or research purposes. Vitrification is being used more and more as it seems to yield better results compared to slow freezing, although protocols still need to be optimized for each specific cell type and species. Several methods can be used to assess follicle quality, ranging from simple viability stains to more complex xenografting procedures. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. This review will focus on recent developments in (human) fertility preservation strategies, which are often accomplished by the use of in vitro animal models due to ethical considerations and the scarcity of human research material.
Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Folículo Ovariano/citologia , Ovário , Vitrificação , Animais , Criopreservação/veterinária , Feminino , Preservação da Fertilidade/veterinária , HumanosRESUMO
Maternal metabolic pressure due to a cow's negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of ß-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocyte's micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.
Assuntos
Líquido Folicular , beta Caroteno/metabolismo , Animais , Bovinos , Metabolismo Energético , Ácidos Graxos não Esterificados/sangue , Feminino , Lactação/metabolismo , Folículo Ovariano/metabolismoRESUMO
Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (> 13 mm; LF group; high fertility phenotype) or smaller (< 12 mm; SF group) follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4) and 7 (D7) days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF). Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that regulate endometrial tissue receptivity. Data was deposited in the SRA database from NCBI (SRA Experiment SRP051330) and are associated with the Bio-Project (PRJNA270391). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65450. Further assessment of the data in combination with other data sets exploring the transcriptional profile of the endometrial tissue during early diestrus may potentially identify novel molecular mechanisms and/or markers of the uterine receptivity.
RESUMO
The increasing number of cancer survivors the past decades, has sparked the need for fertility preservation strategies. Due to predominantly ethical constraints, human research material is scarce. A bovine in vitro model is a valuable alternative. Therefore, the following objectives were defined: 1) to xeno-graft bovine ovarian cortex tissue in immune deficient mice as a study-model for female fertility preservation strategies; 2) to stereologically quantify vascularization in Vascular Endothelial Growth Factor (VEGF)-treated and non-treated tissue; 3) to study preantral follicular survival in situ, after xenotransplantation. Bovine ovarian tissue strips were incubated with or without VEGF prior to grafting into female, neutered BALB/c-nu mice (n=16). Non-transplanted cortical tissue was used as a control. At time zero (control), two (2 weeks) and four (4 weeks) weeks after transplantation, grafts were retrieved and assessed by von Willebrand Factor and caspase-3 immunostaining. Data were analyzed using a linear mixed model. In the VEGF+ grafts, 31% of the follicles were considered 'alive' 2 weeks after transplantation, compared to only 17% in the VEGF- grafts (P<0.05). However, no difference could be detected 4 weeks after transplantation (P=0.76) with less follicles being considered 'alive' after transplantation (22%), compared to the control (47.5%) (P<0.05). Finally, the vascular surface density was significantly less in the grafts, irrespective of the transplantation period or the use of VEGF. Although the transplantation process overall negatively influenced the number of viable follicles and vascular density, VEGF exposure prior to transplantation can favor follicle survival during a 2 weeks transplantation period.
Assuntos
Folículo Ovariano/transplante , Ovário/transplante , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Caspase 3/metabolismo , Bovinos , Feminino , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Transplante Heterólogo/métodos , Fator de von Willebrand/metabolismoRESUMO
This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.
Assuntos
Diestro/fisiologia , Endométrio/metabolismo , Transcriptoma/genética , Animais , Apoptose , Caspase 3/fisiologia , Bovinos , Proliferação de Células , Cloprostenol/farmacologia , Biologia Computacional , Endométrio/crescimento & desenvolvimento , Ativação Enzimática/fisiologia , Matriz Extracelular/metabolismo , Feminino , Luteolíticos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , GravidezRESUMO
To provide new insights in the molecular mechanism controlling preantral follicular development and to unravel the needs to support in vitro follicular development of early-stage preantral follicles (PAFs), there is a need for alternative in vitro bovine follicle culture methods. In this study, we aimed to characterize follicular dynamics using an IVC system of isolated and individually cultured bovine early PAFs during 10 days to generate individual follicle follow-up data. Preantral follicles (<50 µm) were isolated from slaughterhouse ovaries and cultured individually for 10 days. Individual follicle morphology, growth, survival, quality, and cell proliferation were evaluated in time by combining noninvasive and invasive assessment methods. The PAFs were light microscopically evaluated during culture to assess follicular dynamics, stained with neutral red to determine follicle viability, stained with 4',6-diamidino-2-phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling to evaluate cell proliferation and follicle quality, and processed for histologic evaluation to assess follicle morphology. On the basis of their morphology, follicles were subdivided in three categories, with category 1 follicles showing the best morphologic features. On Day 0, only category 1 follicles were selected, but follicle categories were reassigned on evaluation Days 1, 2, 4, 7, or 10. Although 67% of the follicles survived 10 days of IVC, the number of follicles exhibiting a normal morphology decreased significantly from Day 7 onward and the apoptotic index increased significantly from Day 10. Both category 1 and 2 follicles showed a significant increase in follicular diameter (Day 10: 21.80 ± 0.86 and 11.82 ± 0.80, respectively). This increase in follicular diameter showed to be correlated with an increase in the total cell number. In conclusion, this culture system showed to support follicular development until Day 10, although the proportion of follicles showing normal morphologic features and the follicular quality decreased after 10 days of IVC. Follicles maintaining their category 1 morphologic features over time seem to be of a better quality and show a higher developmental competence as compared to category 2 and 3 follicles.
Assuntos
Bovinos , Folículo Ovariano/anatomia & histologia , Animais , Apoptose , Proliferação de Células , Feminino , Corantes Fluorescentes , Marcação In Situ das Extremidades Cortadas , Indóis , Técnicas de Cultura de Órgãos/métodos , Técnicas de Cultura de Órgãos/veterinária , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Coloração e Rotulagem , Técnicas de Cultura de Tecidos/veterináriaRESUMO
Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 µm for primordial follicles, 47 ± 6.3 µm for primary follicles and 67.1 ± 13.1 µm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.
Assuntos
Bovinos/fisiologia , Cabras/fisiologia , Recuperação de Oócitos/veterinária , Folículo Ovariano/fisiologia , Ovinos/fisiologia , Animais , Sobrevivência Celular , Feminino , Recuperação de Oócitos/métodos , Folículo Ovariano/citologiaRESUMO
PURPOSE: Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles. METHODS: Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham's F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5% CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h. RESULTS: Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 µg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture. CONCLUSIONS: In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 µg/ml for 30 min, being perfectly compatible with PAF vitrification.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/administração & dosagem , Vermelho Neutro/administração & dosagem , Folículo Ovariano/crescimento & desenvolvimento , Animais , Bovinos , Criopreservação , Meios de Cultura/química , Feminino , Humanos , Folículo Ovariano/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Vitrificação/efeitos dos fármacosRESUMO
The metabolic state of pregnant mammals influences the offspring's development and risk of metabolic disease in postnatal life. The metabolic state in a lactating dairy cow differs immensely from that in a non-lactating heifer around the time of conception, but consequences for their calves are poorly understood. The hypothesis of this study was that differences in metabolic state between non-lactating heifers and lactating cows during early pregnancy would affect insulin-dependent glucose metabolism and development in their neonatal calves. Using a mixed linear model, concentrations of glucose, IGF-I and non-esterified fatty acids (NEFAs) were compared between 13 non-lactating heifers and 16 high-yielding dairy cows in repeated blood samples obtained during the 1st month after successful insemination. Calves born from these dams were weighed and measured at birth, and subjected to intravenous glucose and insulin challenges between 7 and 14 days of age. Eight estimators of insulin-dependent glucose metabolism were determined: glucose and insulin peak concentration, area under the curve and elimination rate after glucose challenge, glucose reduction rate after insulin challenge, and quantitative insulin sensitivity check index. Effects of dam parity and calf sex on the metabolic and developmental traits were analysed in a two-way ANOVA. Compared with heifers, cows displayed lower glucose and IGF-I and higher NEFA concentrations during the 1st month after conception. However, these differences did not affect developmental traits and glucose homeostasis in their calves: birth weight, withers height, heart girth, and responses to glucose and insulin challenges in the calves were unaffected by their dam's parity. In conclusion, differences in the metabolic state of heifers and cows during early gestation under field conditions could not be related to their offspring's development and glucose homeostasis.
Assuntos
Animais Recém-Nascidos/metabolismo , Bovinos/fisiologia , Glucose/metabolismo , Criação de Animais Domésticos , Animais , Peso ao Nascer , Bovinos/sangue , Ácidos Graxos não Esterificados/sangue , Feminino , Insulina/administração & dosagem , Fator de Crescimento Insulin-Like I/análise , Lactação , Masculino , Paridade , GravidezRESUMO
Producing bovine in vitro embryos individually is a challenge as it generally leads to impaired embryo development. Earlier research optimised a single embryo in vitro production (IVP) protocol using serum, cumulus cells and oil during culture. As some of these factors are undesirable in certain circumstances, the present study investigated their necessity and possible interactions, and defined their role during single-embryo culture. Although the cumulus cell monolayer produced progesterone, it appeared not to be a key factor in supporting single-embryo development. Because in vitro culture in large medium volumes was shown to impair single-embryo development, two new oil-free culture protocols were tested. Using a 30-µL droplet of medium in 96-well plates with a small surface area resulted in comparable blastocyst rates to those obtained under oil. When serum was used, co-culture with cumulus cells seems necessary, leading to consistently high blastocyst rates. Finally, a serum-free, oil-free culture system using insulin, transferrin, selenium and BSA resulted in embryos with similar total cell numbers and apoptotic cell ratios, but blastocyst rates did not equal those obtained with serum and co-culture. This research additionally stresses the fact that specific interaction mechanisms between somatic cells and a developing in vitro embryo are far from unravelled.
Assuntos
Bovinos/embriologia , Técnicas de Cocultura/veterinária , Células do Cúmulo/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Animais , Blastocisto/fisiologia , Meios de Cultura , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Técnicas de Cultura Embrionária/métodos , Fertilização in vitro/veterinária , Progesterona/biossíntese , Zigoto/crescimento & desenvolvimentoRESUMO
Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.