Extracellular-like matrices and leukaemia inhibitory factor for in vitro culture of human primordial follicles

2017 ◽  
Vol 29 (10) ◽  
pp. 1982 ◽  
Author(s):  
Assiel J. Younis ◽  
Galit Lerer-Serfaty ◽  
Dana Stav ◽  
Bethsabee Sabbah ◽  
Tzippy Shochat ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertility Restoration ◽  
Tobacco Plant ◽  
Follicular Development ◽  
Inhibitory Factor ◽  
Leukaemia Inhibitory Factor ◽  
Follicular Growth ◽  
Primordial Follicles ◽  
Human Primordial Follicles

The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen–thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10 ng mL–1 and 100 ng mL–1), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17β-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.

scholarly journals Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

2018 ◽  
Vol 38 (12) ◽  
pp. 2284-2288
Author(s):  
Camila Bizarro-Silva ◽  
Suellen M. González ◽  
Isabela Búfalo ◽  
Andressa G. Lindquist ◽  
Fabiana D. Sarapião ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Follicular Development ◽  
Significance Level ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Medium Exchange ◽  
Significant Difference

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.

Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Larissa Zamparone Bergamo ◽  
Denis Vinicius Bonato ◽  
Camila Bizarro-Silva ◽  
Francieli Gesleine Capote Bonato ◽  
Tamires Korchovei Sanches ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ascorbic Acid ◽  
Culture Medium ◽  
Bos Taurus ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
Bos Taurus Indicus ◽  
Bonferroni Test ◽  
And Oxidative Stress

Summary The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher’s exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

scholarly journals The Role of Androgens in Mammals Folliculogenesis

2018 ◽  
Vol 44 (1) ◽  
pp. 15
Author(s):  
Livia Brunetti Apolloni ◽  
Jamily Bezerra Bruno ◽  
Benner Geraldo Alves ◽  
José Ricardo de Figueiredo
Keyword(s):  
Androgen Receptor ◽  
In Vitro Culture ◽  
Steroid Hormones ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Follicular Growth ◽  
Preantral Follicles ◽  
Ovarian Cells

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized.Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects of substances as androgens on follicular development have been evaluated. Androgens are steroid hormones produced in theca cells (TC) that are fundamental for follicular growth. These cells provide all the androgens required by the developing follicles for conversion into estrogens by the granulosa cells (GC). Androgens receptors (AR) are localized in cell cytoplasm of all follicular categories, being more expressed in preantral follicles. The androgen pathway initiates through its connection to its receptor, making a complex androgen-AR, that in the nucleus helps on the process of gene transcription related with follicular survival. This mechanism is androgen receptor genomic activity. In addition to genomic action, there is an androgen receptor non-genomic activity. This occurs through activation of AR and its interaction with different signaling molecules located on the cell membrane, triggering events that aid in the follicular development. Regardless of the androgens actions, ovarian cells of several species subjected to in vitro culture have shown the importance of these hormones on the follicle development. Recent studies demonstrated that androgens addition on the culture medium stimulated the activation of preantral follicles (bovine and caprine), antrum formation (swine), survival (non-primate), and oocyte maturation (antral follicles; bovine). Also, some studies suggest that the addition of these hormones on in vitro culture is dose-dependent and species-specific.Conclusion: This review shows the role of androgens in different stages of follicular development and its action as a substrate for steroidogenesis and transcription of genes related to follicular survival and oocyte maturation. However, when these hormones should be added during in vitro follicular culture and which concentration is required remains unclear, being necessary more studies to elucidate these aspects.

Culture of goat preantral follicles in situ associated with mesenchymal stem cell from bone marrow

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Camila Arrivabene Neves ◽  
Lucilene dos Santos Silva ◽  
Camila Ernanda Sousa de Carvalho ◽  
Marina Silva Carvalho ◽  
José Lindenberg Rocha Sarmento ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Culture Medium ◽  
Reduced Glutathione ◽  
Follicular Growth ◽  
Morphological Classification ◽  
Nitrite Concentration ◽  
Preantral Follicles ◽  
Rate Of Activation

SummaryThis study aims to develop an in vitro co-culture system of in situ goat preantral follicles with bone marrow-derived mesenchymal stem cells (BM-MSC), evaluating the influence of these cells on follicular growth, rate of activation and morphologically normal follicles. Fragments of ovarian cortex were cultured for 1 or 7 days in the presence of BM-MSC (BM-MSC+) and absence of BM-MSC (BM-MSC−). Histological sections of the fragments were analysed and data were obtained regarding morphological classification, survival rate of morphologically normal follicles and rate of follicular activation. Culture medium on days 1 and 7 was also sampled for nitrite concentration and reduced glutathione activity. There was a reduction (P < 0.05) in the percentage of morphologically normal follicles in the BM-MSC+ compared with the fresh control only on the seventh day of culture. When comparing treatments, on the seventh day of culture, a higher rate of morphologically normal preantral follicles was observed in BM-MSC+ (P < 0.05). In both treatments, primordial and developing follicle rates were similar to the fresh control (P > 0.05). When comparing treatments with each other, as well as with the fresh control, no differences were observed in follicular diameter (P > 0.05) or nitrite concentration (P > 0.05). The concentration of reduced glutathione was lower on the seventh day of co-culture in both treatments (P < 0.05). In conclusion, co-culture had no influence on follicular or oocyte development. However, it was critical to maintain the survival of preantral follicles during 7 days of culture.

scholarly journals Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†

2019 ◽  
Vol 102 (2) ◽  
pp. 511-520
Author(s):  
Yanrong Kuai ◽  
Xiaobo Gao ◽  
Huixia Yang ◽  
Haiyan Luo ◽  
Yang Xu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Crop Production ◽  
Follicular Development ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Serum Progesterone ◽  
Primordial Follicles ◽  
Progesterone Production

Abstract Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300 mg/kg by daily gavage for 7 days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

scholarly journals In vitro development of mouse fetal germ cells into mature oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Wei Shen ◽  
Lan Li ◽  
Zhaodai Bai ◽  
Qingjie Pan ◽  
Mingxiao Ding ◽  
...  
Keyword(s):  
Genomic Imprinting ◽  
Germ Cells ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Primordial Follicles ◽  
Ovarian Cells ◽  
Fetal Mice ◽  
Vitro Development

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish anin vitroexperimental model that allows one to study such mechanisms. Mouse follicular development has been studiedin vitroover the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were culturedin vitrofor 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were maturedin vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula–blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normallyin vitro.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

scholarly journals Changes in the transcriptome of bovine ovarian cortex during follicle activation in vitro

2015 ◽  
Vol 47 (12) ◽  
pp. 600-611 ◽  
Author(s):  
M. Y. Yang ◽  
J. E. Fortune
Keyword(s):  
Bovine Genome ◽  
Follicular Development ◽  
Cellular Growth ◽  
Primary Follicle ◽  
Primordial Follicles ◽  
Tensin Homolog ◽  
Genechip Array ◽  
Pten Mrna ◽  
Follicle Activation

The signals that regulate activation, a key transition in ovarian follicular development, are still not well understood, especially in nonrodent species. To gain insight into the regulation of this transition in cattle, we combined a microarray approach with an in vitro system in which ovarian cortical pieces cultured in control medium are enriched for primordial follicles, whereas pieces cultured with insulin are enriched for primary follicles. Total RNA was extracted from cultured cortical pieces, and then transcripts were identified and analyzed using the Affymetrix Bovine Genome GeneChip array. Around 65% of the transcripts in the bovine GeneChip were detected in cultured cortical pieces. Comparison between pieces cultured with or without insulin generated 158 differentially expressed transcripts. Compared with controls, 90 transcripts were upregulated and 68 were downregulated by insulin. These transcripts are involved in many biological processes and functions, but most are associated with cellular growth or cell cycle/cell death. The transcript encoding ubiquitin-conjugating enzyme E2C (UBE2C) was significantly upregulated during follicle activation, and Ingenuity Pathways Analysis revealed that UBE2C can interact with the tumor suppressor phosphatase and tensin homolog (PTEN). Both PTEN mRNA and protein were lower in cortical pieces cultured with insulin than in controls. In addition, FOXO3a, a downstream effector of PTEN signaling, underwent nuclear-cytoplasmic shuttling during primordial to primary follicle development in bovine fetal ovaries, further suggesting the involvement of the PTEN pathway in follicle activation in cattle. Genes and pathways identified in this study provide interesting candidates for further investigation of mechanisms underlying follicle activation.

Oocyte-secreted factros regulate granulosa cell steroidogenesis

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 317-321 ◽  
Author(s):  
Barbara C. Vanderhyden
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Inhibitory Factor ◽  
Preovulatory Follicles ◽  
Loss Of Contact

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.

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