scholarly journals In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

2020 ◽  
Vol 103 (3) ◽  
pp. 455-470
Author(s):  
Leah E Simon ◽  
T Rajendra Kumar ◽  
Francesca E Duncan
Keyword(s):  
Wildlife Conservation ◽  
Culture Media ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Ovarian Follicles ◽  
Culture Methods ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Culture Techniques

Abstract Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Three-dimensional in vitro ovarian follicle culture

2011 ◽  
pp. 167-176
Author(s):  
Eugene Galdones ◽  
Lonnie D. Shea ◽  
Teresa K. Woodruff
Keyword(s):  
Ovarian Follicle ◽  
Three Dimensional ◽  
Follicle Culture

Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes

2020 ◽  
Author(s):  
Jongwon Kim ◽  
Jung Kyu Choi
Keyword(s):  
Culture System ◽  
Ovarian Follicle ◽  
Formation Rate ◽  
Germinal Vesicle ◽  
Ovarian Follicles ◽  
Germinal Vesicle Breakdown ◽  
Follicle Culture ◽  
Nuclear Maturation ◽  
Follicle Diameter

This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes 325–500 mm and treated them with ovine follicle stimulating hormone OFSH at different concentrations 0–400 mIU. Follicle diameter, antrum formation and cumulus oocyte complex COC recovery rate were significantly higher p andlt; 0.05 under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations 100, 200 and 400 mIU. Additionally, follicles cultured for 3 and 4 d differed significantly p andlt; 0.05 in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250–300, 301–400 and 401–500 mm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250–300, 301–400 and 401–500 mm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250–300 mm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.

scholarly journals Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea Jones ◽  
Beatriz Peñalver Bernabé ◽  
Vasantha Padmanabhan ◽  
Jun Li ◽  
Ariella Shikanov
Keyword(s):  
Childhood Cancer ◽  
Time Course ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Transcriptome Profiling ◽  
Culture Methods ◽  
Time Course Experiment ◽  
Underlying Mechanisms ◽  
Survivors Of Childhood Cancer

AbstractIn vitro ovarian follicle culture is an active area of research towards providing fertility options for survivors of childhood cancer. Late-stage murine follicles (multilayer secondary and onwards) can be cultured successfully to maturity to obtain a meiotically competent oocyte for fertilization, but primordial and primary follicles usually die in culture because many key components of early follicle development are still unknown and difficult to mimic in vitro. To engineer a biomimetic three-dimensional culture system with high efficacy and reproducibility for the clinic, detailed mechanisms of early folliculogenesis must be uncovered. Previous studies have shown that primary murine follicles co-cultured in groups, in contrast to single follicles cultured in isolation, can reach preovulatory size and produce competent oocytes, but the factors accounting for the synergy of follicle co-culture are still unknown. To probe the underlying mechanisms of successful follicle co-culture, we conducted a time-course experiment for murine follicles encapsulated in 0.3% alginate hydrogels and compared between two conditions: groups of 5 (5X) versus groups of 10 (10X). For every 2 days during the course of 12 days, follicles were dissociated and somatic cells were isolated for microarray-based gene expression analysis (n = 380 follicles for 5X and n = 430 follicles for 10X). Gene activities in follicles co-cultured in larger groups (10X) had a distinct transcriptomic profile of key genes and pathways such as prolactin signaling and angiogenesis-related genes when compared to cells from follicles co-cultured in the smaller cohort (5X). To benchmark the results for follicles grown in culture, we compared our microarray data to data from murine follicles freshly isolated from the ovary at comparable stages of development previously published by Bernabé et al. Comparison of these datasets identified similarities and differences between folliculogenesis in the native microenvironment and the engineered in vitro system. A more detailed understanding of follicle growth in vitro will not only allow for better culture methods but also advance the field towards providing improved fertility options for survivors of childhood cancer.

scholarly journals In Vitro Mouse Ovarian Follicle Growth and Maturation in Alginate Hydrogel: Current State of the Art

Acta Naturae ◽  
2015 ◽  
Vol 7 (2) ◽  
pp. 48-56 ◽  
Author(s):  
M. A. Filatov ◽  
Yu. V. Khramova ◽  
M. L. Semenova
Keyword(s):  
Culture Media ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Alginate Hydrogel ◽  
Factors Affecting ◽  
Current State ◽  
Substrate Rigidity ◽  
Vitro Development

This review describes the main factors affecting the in vitro development of mouse ovarian follicles under conditions of three-dimensional alginate hydrogel system. The factors discussed include concentration of alginate hydrogel, presence of additives (collagen, fibrin) influencing substrate rigidity; culture conditions; composition of culture media; substances that act like antioxidants (salts of ascorbic acid, glutathione) and contribute to the improvement of lipid metabolism (L-carnitine), hormones and growth factors. The methods for follicle group cultivation in alginate hydrogel and cocultivation of different cell populations with follicles encapsulated in alginate hydrogel are covered in the present article.

Three-dimensional systems for in vitro follicular culture: overview of alginate-based matrices

2014 ◽  
Vol 26 (7) ◽  
pp. 915 ◽  
Author(s):  
Ivina R. Brito ◽  
Isadora M. T. Lima ◽  
Min Xu ◽  
Lonnie D. Shea ◽  
Teresa K. Woodruff ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Conditions ◽  
Physical Interaction ◽  
Oocyte Growth ◽  
Follicle Culture ◽  
Culture Systems ◽  
Extracellular Matrix Components

The in vitro culture of ovarian follicles has provided critical insight into the biology of the follicle and its enclosed oocyte and the physical interaction and communication between the theca and granulosa cells and the oocyte that is necessary to produce meiotically competent oocytes. Various two-dimensional (2D) and three-dimensional (3D) culture systems have been developed to evaluate the effect of growth factors, hormones, extracellular matrix components and culture conditions on follicle development and oocyte growth and maturation. Among these culture systems, 3D systems make it possible to maintain follicle structure and support communication between the various cell compartments within the follicle. In this review article, we will discuss the three main approaches to ovarian follicle culture: 2D attachment systems, 3D floating systems and 3D encapsulated systems. We will specifically emphasise the development of and advances in alginate-based encapsulated systems for in vitro follicle culture.

scholarly journals A New Microfluidic Concept for Successful in Vitro Culture of Mouse Embryos

2021 ◽  
Author(s):  
Vanessa Mancini ◽  
Paul McKeegan ◽  
Alexandra C. Schrimpe-Rutledge ◽  
Simona Gabriela Codreanu ◽  
Stacy D. Sherrod ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Media ◽  
Mouse Embryos ◽  
Developmental Competence ◽  
Mass Spectrometry Data ◽  
Culture Methods ◽  
Preimplantation Embryo Development ◽  
Culture Techniques

Abstract Innovative techniques for gene editing have enabled accurate animal models of human diseases to be established. In order for these methods to be successfully adopted in the scientific community, the optimization of procedures used for breeding genetically altered mice is required. Among these, the in vitro fertilization (IVF) procedure is still suboptimal and the culture methods do not guarantee the development of competent embryos. Critical aspects in traditional in vitro embryo culture protocols include the use of mineral oil and the stress induced by repetitive handling of the embryos. A novel microfluidic system was designed and fabricated in poly dimethyl siloxane (PDMS) to allow for efficient in vitro production of mouse embryos. Culture experiments conducted by completing the industry gold standard Mouse Embryo Assay excluded any harmful fluidic stress and plastic toxicity. The developmental competence of the embryos developed in the device was consistently confirmed by high blastocyst rate (>80%), hatching and outgrowth rate, and matched with analysis of energy substrate metabolism and expression of genes related to implantation potential. Metabolomics analyses of spent culture media allowed for biologically important metabolite changes to be observed throughout embryo development, and for identification of specific overrepresented metabolic pathways affected by the microfluidic environment. Moreover, mass spectrometry data identified plastic-related compounds released in medium, and confirmed leaching of low molecular weight species into the culture medium that might be associated to un-crosslinked PDMS. Finally, these data show the potential for the system to study preimplantation embryo development and to improve the embryo culture techniques used for human assisted conception.

scholarly journals Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shauna Kehoe ◽  
Katarina Jewgenow ◽  
Paul R. Johnston ◽  
Susan Mbedi ◽  
Beate C. Braun
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Ovarian Follicle ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Ovarian Follicles ◽  
Domestic Cat ◽  
Transcriptional Analysis ◽  
Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

scholarly journals STUDI BACILLUS FIRMUS SEBAGAI KANDIDAT PROBIOTIK DALAM MENGHADAPI Aeromonas hydrophila PADA MEDIA BUDIDAYA The Study of Bacillus firmus as Probiotic Candidate in Supressing Aeromonas hydrophila in Culture Media

2016 ◽  
Vol 11 (2) ◽  
pp. 111
Author(s):  
Alfabetian Harjuno Condro Haditomo ◽  
Angela Mariana Lusiastuti ◽  
Widanarni Widanarni
Keyword(s):  
Aeromonas Hydrophila ◽  
Pathogenic Bacteria ◽  
Culture Media ◽  
Inhibition Zone ◽  
Probiotic Bacteria ◽  
Bacterial Disease ◽  
Second Phase ◽  
Bacillus Firmus ◽  
Culture Methods

ABSTRAK   Pengendalian penyakit bakterial yang umum dilakukan dengan pemakaian antibiotik atau  bahan kimia sudah tidak diperbolehkan lagi karena menimbulkan patogen yang resisten  terhadap bahan kimia tersebut, terlebih jika penggunaan tidak sesuai dengan anjuran yang diberikan. Dampak negatif terhadap kesehatan konsumen berupa residu antibiotik juga menjadi pertimbangan yang harus diperhatikan. Manipulasi terhadap populasi mikroba yang berada di perairan guna pencegahan sebelum terjadinya serangan bakteri yang bersifat mematikan perlu dilakukan sebagaimana konsep probiotik sebagai biokontrol. Tujuan penelitian ini adalah menguji kandidat probiotik dalam menekan atau menghambat bakteri patogen Aeromonas hydrophila. Penelitian ini dilaksananakan dalam dua tahap. Tahap pertama adalah tahap pengujian bakteri kandidat probiotik secara in vitro menggunakan metode zona hambat dan kultur bersama pada media agar.  Tahap kedua adalah uji tentang bakteri kandidat probiotik dengan patogen pada media budidaya. Hasil terbaik penelitian tahap pertama pada  uji kultur bersama antara kandidat probiotik B. firmus dengan A. hydrophila pada skala in vitro adalah dengan penambahan probiotik  B. firmus sebanyak 108 cfu/ml. Sedangkan pada penelitian tahap kedua didapatkan hasil berturut-turut perlakuan D dengan tingkat kelangsungan hidup (SR) mencapai 90%, perlakuan C dengan SR 75%, perlakuan A dengan SR 50% dan perlakuan K dengan SR 50%.   Kata kunci: Bacillus firmus, probiotik, Aeromonas hydrophila, media budidaya   ABSTRACT  Controlling bacterial disease with the use of antibiotics or chemicals is no longer allowed as it results in pathogens that are resistant to the chemicals, especially when not in accordance with the recommendations provided. The negative impactsof the antibiotics residues on the consumers’ health  also need to be considered. Manipulation of microbial populations present in the waters as preventation before the lethal attack of bacteria needs to be done which is in accordance with the concept of probiotics as biocontrol.The purpose of this study was to test the probiotic candidates in suppressing or inhibiting pathogenic bacteria Aeromonas hydrophila. This study was conducted in two stages. The first stage was to test a candidate probiotic bacteria in vitro using culture methods and inhibition zone on the media together. The second stage wasto test candidate probiotic bacteria to pathogens on the cultivation media. The best results in the first phase of the research is shared culture test between probiotic candidate B. FIRMUS with A. hydrophila on vitro scale is the addition of the probiotic B. FIRMUS 108 cfu / ml. While in the second phase of the research results obtained successively: treatment D with a survival rate (SR) reaches 90%, treatment C with SR 75%, treatment A with SR 50% and treatment K with SR 50%. Keywords: Bacillus FIRMUS, probiotics, Aeromonas hydrophila, media cultivation

Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models

2003 ◽  
Vol 31 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Hanna Tähti ◽  
Heidi Nevala ◽  
Tarja Toimela
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Three Dimensional ◽  
Brain Barrier ◽  
Culture Methods ◽  
Blood Brain ◽  
Capillary Endothelial Cells ◽  
In Vitro Bbb Model

The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.

scholarly journals Serum deprivation affects secretory activity of cultured porcine ovarian follicles and granulosa cells and their response to hormones

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 455-460
Author(s):  
A.V. Sirotkin
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Secretory Activity ◽  
Ovarian Follicles ◽  
Serum Deprivation ◽  
Ovarian Cells ◽  
Igf I ◽  
Granulose Cells ◽  
Vitro Model

The aim of the present study is to understand the hormonal mechanisms of the effect of malnutrition on ovarian follicle functions. For this purpose, we examined the effect of malnutrition/serum deprivation, addition of metabolic hormones and gonadotropin (IGF-I, leptin and FSH) and their combination on the release of progesterone (P<sub>4</sub>), testosterone (T), estradiol (E<sub>2</sub>) and insulin-like growth factor I (IGF-I) by cultured whole ovarian follicles and on P<sub>4</sub> and IGF-I output by cultured granulosa cells isolated from porcine ovaries. It was observed that in ovarian follicles cultured with nutrients/serum addition of IGF-I reduced release of P<sub>4</sub>, but not of T or E<sub>2</sub>. Exogenous leptin reduced output of E<sub>2</sub>, but not of P<sub>4</sub> or T, and increased IGF-I output. No significant effect of FSH on release of steroid hormones by isolated follicles was found. Serum deprivation did not affect release of P<sub>4</sub>, but reduced output of T and E<sub>2</sub>, and promoted IGF-I release by cultured ovarian follicles. Addition of hormones failed to prevent the effect of malnutrition on the secretory activity of cultured ovarian follicles. In cultured granulose cells, all the tested hormones promoted release of both P<sub>4</sub> and IGF-I. Food restriction/serum deprivation reduced both P<sub>4</sub> and IGF-I output. Additions of either IGF-I, leptin and FSH prevented the inhibitory action of malnutrition on both P<sub>4</sub> and IGF-I release. The present observations (1) confirm the involvement of the hormones IGF-I, leptin and FSH in the control of secretory activity of ovarian cells, (2) demonstrate, that both isolated ovarian granulosa cells and whole follicles cultured in the absence of serum nutrients could be an adequate in-vitro model for studying the effect of malnutrition on ovarian secretory functions, and (3) suggest, that malnutrition could affect ovarian functions through changes in the release of ovarian hormones.

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