scholarly journals Effect of base media, FSH and anti-Müllerian hormone (AMH) alone or in combination on the growth of pig preantral follicles in vitro

2021 ◽  
Vol 10 (15) ◽  
pp. e53101522488
Author(s):  
Rebeca Magalhães Pedrosa Rocha ◽  
Marcello Rubessa ◽  
Laritza Ferreira de Lima ◽  
Ana Flávia Bezerra da Silva ◽  
Rebecca Winters ◽  
...  
Keyword(s):  
Formation Rate ◽  
Follicular Development ◽  
Hormone Production ◽  
Steroid Production ◽  
Preantral Follicles ◽  
North Carolina State University ◽  
Base Medium ◽  
The Media ◽  
Survival And Growth

To compare the efficiency of North Carolina State University medium 23 (NCSU23) and Alpha Minimum Essential Medium (α-MEM) as a base medium, and to evaluate the effects of Anti-Müllerian Hormone (AMH) alone or in combination with Follicle Stimulating Hormone (FSH) on the in vitro development and steroid production of isolated porcine preantral follicles. Porcine secondary follicles were cultured in NCSU23 or α-MEM media for 4 days. Once α-MEM was determined to be the optimal culture medium, secondary follicles were cultured in α-MEM alone or supplemented with FSH (1.5 ng/mL), AMH (50 ng/mL) or the combination of the two hormones. Follicle development was evaluated by measuring follicular growth, morphology and hormone production. There was no difference between the media NCSU23 and α-MEM in terms of follicle survival and growth (P > 0.05). However, at day 2, the antrum formation rate tended to be (P < 0.074) higher in α-MEM than NCSU23. At day 4 of culture, the estradiol and progesterone secretion were higher in α-MEM than NCSU23 (P < 0.01), while the opposite was observed for testosterone (P < 0.01). The addition of AMH and/or FSH did not affect follicular survival and growth. Nevertheless, the secretion of estradiol and progesterone induced by FSH was reduced with AMH (P < 0.01). α-MEM is a more effective base medium than NCSU23 for the in vitro follicular development of pig preantral follicles and AMH reduces the steroid production induced by FSH.

scholarly journals High diluted and dynamised follicle stimulating hormone modulates steroid production in isolated porcine preantral follicles cultured in vitro

Homeopathy ◽  
2017 ◽  
Vol 106 (02) ◽  
pp. 87-92 ◽  
Author(s):  
Laritza F de Lima ◽  
Marcello Rubessa ◽  
Rebeca MP Rocha ◽  
Rebecca Winters ◽  
Derek J Milner ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Follicle Stimulating Hormone ◽  
Hormone Production ◽  
Steroid Production ◽  
Preantral Follicles ◽  
Testosterone Production ◽  
Progesterone Production ◽  
Vitro Development ◽  
Stimulating Hormone

Objective: This study investigated the effect of two different follicle stimulating hormone (FSH) preparations (diluted/dynamised and diluted) on the in vitro development and steroid production (estradiol, progesterone and testosterone) of isolated porcine preantral follicle after in vitro culture. Methods: Secondary follicles were cultured in Alpha Minimum Essential Medium (α-MEM+) supplemented with grain ethanol (AL – 0.2%, v/v), diluted/dynamised FSH (rFSH 6cH – 0.05 fg/mL) or diluted-only FSH (1.5 ng/mL) for 4 days. Follicle development was evaluated on the basis of follicular growth, morphology and hormone production. Results: The percentage of follicular integrity and extrusion were not affected by the treatments after culture. For all treatments, follicular diameter increased significantly from Day 0 to Day 4. On Day 2 of culture, the estradiol production was significantly higher in AL and diluted-only FSH treatments compared with diluted/dynamised FSH. However, diluted/dynamised FSH showed a significantly higher progesterone production on Day 2. Only on Day 4, the testosterone production was higher in the AL than diluted-only FSH treatments, but similar to diluted/dynamised FSH treatment. Except for diluted/dynamised FSH treatment, progesterone production increased (P < 0.05) from Day 2 to Day 4; only for AL treatment, a significant increase of testosterone production was observed during culture. Conclusion: Compared to control the diluted/dynamised FSH addition increased progesterone production but decreased the estradiol production after in vitro culture of isolated porcine preantral follicles. Taken together the results suggest that at least for progesterone production the mechanism of action of diluted/dynamised FSH differs from its vehicle.

scholarly journals Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

Reproduction ◽  
2007 ◽  
Vol 133 (6) ◽  
pp. 1121-1128 ◽  
Author(s):  
Fiona H Thomas ◽  
Bruce K Campbell ◽  
David G Armstrong ◽  
Evelyn E Telfer
Keyword(s):  
Follicular Development ◽  
Follicle Development ◽  
Dependent Manner ◽  
Igf Binding Proteins ◽  
Preantral Follicles ◽  
Follicle Diameter ◽  
Igf I ◽  
Igf Binding ◽  
Development In Vitro

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.

scholarly journals Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

2021 ◽  
Author(s):  
C. Chetan Kumar ◽  
B. Rambabu Naik ◽  
A.V.N. Siva Kumar ◽  
A. Ravi ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Ovarian Function ◽  
Follicular Development ◽  
Oocyte Quality ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Optimum Level ◽  
Preantral Follicles ◽  
Positive Effects

Background: Melatonin, a powerful free radical scavenger and broad-spectrum antioxidant may directly affect ovarian function by regulating folliculogenesis, maintenance of follicular integrity, oocyte quality and maturation capacity. Therefore, we aimed to study effects of melatonin and its interaction with growth factors in sheep preantral follicles. Methods: The influence of different concentrations of Melatonin (5-500 pM) on in vitro culture of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiments I and II were conducted to standardize the optimum concentration of Melatonin that supports better development of preantral follicles. Experiment III was conducted with the optimum level of Melatonin derived in the Experiments I and II to evaluate the effect of melatonin at 100pM in combination with various growth factors. Result: Overall follicular development was found to be the best in the PFs’ cultured in medium supplemented with 100pM of Melatonin. Melatonin supplementation showed positive effects on the preantral follicular development in combination with different growth factors.

Follicular steroidogenesis in vitro in cyclic and PMSG/hCG-treated gilts

1991 ◽  
Vol 71 (4) ◽  
pp. 1257-1260
Author(s):  
T. Wiesak ◽  
R. T. Hardin ◽  
G. R. Foxcroft ◽  
M. G. Hunter
Keyword(s):  
Key Words ◽  
Follicular Development ◽  
Steroid Production ◽  
Functional Differences ◽  
Key Words Pig

Basal steroid production in vitro by comparable follicles from cyclic and PMSG/hCG-treated gilts was similar. Follicular responses to LH in vitro varied, however, confirming earlier evidence for functional differences in the pattern of follicular development in immature gilts in response to exogenous gonadotropins compared with naturally cyclic gilts. Key words: Pig, gonadotrophin, follicle, steroidogenesis, steroids

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.

136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 198
Author(s):  
M. Rubessa ◽  
R. Rocha ◽  
L. Lima ◽  
R. Winters ◽  
J. R. Figueiredo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Positive Trend ◽  
Minimal Essential Medium ◽  
Hormone Production ◽  
Chi Square ◽  
Preantral Follicles ◽  
Porcine Serum ◽  
Follicle Size ◽  
Α Level

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6

scholarly journals Histological study of capuchin monkey (Cebus apella) ovarian follicles

2004 ◽  
Vol 34 (3) ◽  
pp. 495-501 ◽  
Author(s):  
Sheyla Farhayldes Souza Domingues ◽  
Luiz Viana Diniz ◽  
Sonia Helena Costa Furtado ◽  
Otavio Mitio Ohashi ◽  
David Rondina ◽  
...  
Keyword(s):  
Qualitative Data ◽  
Histological Study ◽  
Follicular Development ◽  
Cebus Apella ◽  
Neotropical Primates ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
The Right

The present study aimed to obtain quanti-qualitative data about the follicular ovarian population in Cebus apella females. Seven ovaries were obtained from 4 C. apella adult females. The ovaries were subjected to light microscopy. The number of preantral and antral follicles for each ovary was estimated using the Fractionator method. The preantral follicles were classified into primordial, transitional, primary and secondary follicles. Antral follicles were those that presented an antral cavity. All counted follicles were classified as normal or degenerated. The diameter of the follicles, oocytes and their nuclei were determined to accompany the follicular development. All results were represented as mean ± SE. The number of preantral follicles was 56,938 ± 21,888 and 49,133 ± 26,896 for the right and left ovaries, respectively. The percentage of normal follicles was 80 ± 4.95%. The follicular diameter ranged from 22 ± 0.5 µm to 61.2 ± 4.0 µm. Regarding the antral follicles, the number of normal and degenerate follicles per ovary were 60.0 ± 19.0 and 3 ± 1.8 follicles, respectively. The antral follicular diameter was 514.4 + 56.6 µm. In conclusion, the information obtained in this study can be used as a parameter for subsequent in vivo or in vitro studies about folliculogenesis in non-human neotropical primates of the C. apella species.

Effects of IAA in combination with FSH on in vitro culture of ovine preantral follicles

Zygote ◽  
2009 ◽  
Vol 18 (1) ◽  
pp. 89-92 ◽  
Author(s):  
Sonia H.F. Costa ◽  
Regiane R. Santos ◽  
Davide Rondina ◽  
Evelyn R. Andrade ◽  
Otávio M. Ohashi ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Follicle Stimulating Hormone ◽  
Follicular Development ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Viability Test ◽  
Preantral Follicles ◽  
High Concentrations ◽  
Indole 3 Acetic Acid

SummaryOvarian cortical fragments from five adult ewes were in vitro cultured for 1, 3 or 5 days in the presence of minimum essential medium either supplemented or not by follicle-stimulating hormone (FSH) (100 ng/ml) or indole-3-acetic acid (IAA) (10, 20, 40 or 100 ng/ml), alone or in combination. After in vitro culture, ovarian fragments were submitted to follicular isolation and viability test was performed using trypan blue. Addition of IAA (10 ng/ml) to a free-FSH medium resulted in the highest percentages of viable follicles, but was progressively deleterious in higher concentrations (20, 40 and 100 ng/ml) if in absence of FSH. Follicular development was observed only when FSH was added to an IAA-free medium. In conclusion, IAA at a concentration of 10 ng/ml increases follicular survival in vitro. However, at high concentrations (20, 40 or 100 ng/ml), this auxin may be deleterious to preantral follicles, the addition of FSH to the medium being necessary.

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.

Effect of LH and Estradiol-17β Supplementation at Different Time Points on In vitro Development of Preantral Follicles in Sheep

2020 ◽  
Author(s):  
L.S.S. Varaprasad Reddy ◽  
B.R. Naik ◽  
A.V.N. Sivakumar ◽  
B. Punyakumari ◽  
J. Suresh
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Follicular Development ◽  
Standard Medium ◽  
In Vitro Development ◽  
Preantral Follicles ◽  
Time Points ◽  
Vitro Development ◽  
Estradiol 17Β

Background: Ovarian follicular development and growth are controlled by many hormones and growth factors. Despite the fact that LH and estradiol-17β have been utilized for the in vitro culture of preantral follicles yet, the suitable time points of supplementation of LH and estradiol-17β is not known. Therefore this study aimed to investigate the influence of addition of LH and estradiol-17β at different time points on in vitro development of preantral follicles (PFs’) in sheep. Method: Preantral follicles isolated from the ovarian cortical slices using micro dissection method were cultured for six days in Bicarbonate buffered Tissue culture medium 199B (TCM 199B) or in a standard culture medium supplemented with LH (2 μg/ml) and estradiol-17β (5 ng/ml) at different points during the culture period. COCs isolated from the follicles at the end of six day culture in different treatments were subjected to in vitro maturation for additional 24h. Result: Supplementation of LH and estradiol-17β during last two days of the culture supported better proportion of PFs’ exhibiting growth whereas supplementation of LH and estradiol-17β during first two days of the culture supported better average increase in diameter and proportion of PFs’ exhibiting antrum formation at the end of six day culture. Further the oocytes in COCs isolated at the end of culture in these treatments and subsequently subjected to in vitro maturation (IVM) for 24hr developed at a higher frequency to MII (metaphase II) stage. Supplementation of LH and estradiol-17β to TCM 199B culture medium in early stages followed by standard medium alone in later stages supports better development of PFs’ in vitro. Following supplementation with LH and estradiol-17β for the first two days culture of PFs’ in standard medium appears to be advantageous for the development of preantral follicles in vitro.

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