scholarly journals Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents

Reproduction ◽  
2005 ◽  
Vol 130 (2) ◽  
pp. 147-156 ◽  
Author(s):  
I Demeestere ◽  
J Centner ◽  
C Gervy ◽  
Y Englert ◽  
A Delbaere
Keyword(s):  
Oocyte Maturation ◽  
Culture Medium ◽  
Follicular Growth ◽  
Medium Effects ◽  
Paracrine Factors ◽  
Autocrine Factors ◽  
Preantral Follicles ◽  
Whole Process ◽  
Complex Process

Folliculogenesis is a complex process regulated by various paracrine and autocrine factors. In vitro growth systems of primordial and preantral follicles have been developed for future use of immature oocytes, as sources of fertilizable oocytes and for studying follicular growth and oocyte maturation mechanisms. Rodents were often chosen for in vitro follicular culture research and a lot of factors implicated in folliculogenesis have been identified using this model. To date, the mouse is the only species in which the whole process of follicular growth, oocyte maturation, fertilization and embryo transfer into recipient females was successfully performed. However, the efficiency of in vitro culture systems must still be considerably improved. Within the follicle, numerous events affect cell proliferation and the acquisition of oocyte developmental competency in vitro, including interactions between the follicular cells and the oocyte, and the composition of the culture medium. Effects of the acting factors depend on the stage of follicle development, the culture system used and the species. This paper reviews the action of endocrine, paracrine factors and other components of culture medium on in vitro growth of preantral follicles in rodents.

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.

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

STUDIES ON THE MECHANISM OF SECRETION OF CORTICOSTEROIDS BY THE ISOLATED PERFUSED ADRENAL OF THE RAT

1981 ◽  
Vol 91 (2) ◽  
pp. 313-323 ◽  
Author(s):  
C. P. SIBLEY ◽  
B. J. WHITEHOUSE ◽  
G. P. VINSON ◽  
C. GODDARD ◽  
E. McCREDIE
Keyword(s):  
Culture Medium ◽  
Ion Concentration ◽  
Acth Stimulation ◽  
Steroid Production ◽  
Simple Diffusion ◽  
Corticosterone Secretion ◽  
Complex Process ◽  
Doc Production ◽  
Different Levels

A technique for the perfusion of the rat adrenal cortex is described. With tissue culture Medium 199 the preparation was responsive in terms of steroid production to both ACTH and K+ ions. Production of corticosterone and 18-hydroxydeoxycorticosterone (18-hydroxy-DOC) was stimulated by ACTH when it was administered at rates between 5 μu./min and 5 mu./min. Increasing the K+ ion concentration of the perfusate from 3·6 to 5·4 and 8·9 mmol/l stimulated the production of aldosterone, 18-hydroxycorticosterone and deoxycorticosterone, although not of corticosterone or 18-hydroxy-DOC. This preparation has been used to study further the mechanism of secretion of corticosterone and 18-hydroxy-DOC. Thus, production of these two steroids was measured at different perfusion flows, varying between 0·1 and 0·6 ml/min, with different levels of ACTH stimulation. Corticosterone production was significantly (P < 0·001) increased by increasing flows both under control conditions and when ACTH was administered at constant rates of 50 μu./min or 1 mu./min. Production of 18-hydroxy-DOC was not affected by flow either under control conditions or with 50 μu. ACTH/min. However, when ACTH was administered at 1 mu./min, 18-hydroxy-DOC production was also significantly (P < 0·001) increased by flow. The results are consistent with those obtained in previous in-vitro studies and have been interpreted as suggesting that the main mechanism of corticosterone secretion is simple diffusion. In contrast, 18-hydroxy-DOC secretion, at least at sub-maximal levels of stimulation, appears to require a more complex process.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro

2017 ◽  
Vol 29 (6) ◽  
pp. 1144 ◽  
Author(s):  
R. M. P. Rocha ◽  
L. F. Lima ◽  
I. R. Brito ◽  
G. M. Silva ◽  
H. H. V. Correia ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cytochrome P450 ◽  
Hormone Receptors ◽  
Mrna Levels ◽  
Follicular Growth ◽  
Steroid Secretion ◽  
Preantral Follicles ◽  
Treatment Groups ◽  
Cytochrome P450 Family

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50 ng mL–1 AMH and/or 100 ng mL–1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0–9) and second (Days 10–18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH + FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P < 0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P > 0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

scholarly journals Orphan Nuclear Receptor NR4A1 Is a Negative Regulator of DHT-Induced Rat Preantral Follicular Growth

2012 ◽  
Vol 26 (12) ◽  
pp. 2004-2015 ◽  
Author(s):  
Kai Xue ◽  
Jia-yin Liu ◽  
Bruce D. Murphy ◽  
Benjamin K. Tsang
Keyword(s):  
Receptor Antagonist ◽  
Nuclear Receptor ◽  
Mrna Abundance ◽  
Orphan Nuclear Receptor ◽  
Follicular Growth ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Preantral Follicles

Abstract Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5α-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G1/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

scholarly journals 316EFFECTS OF ESTRADIOL-17² AND PROGESTERONE SUPPLEMENT ON THE RESUMPTION OF MEIOSIS OF CANINE OOCYTES MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 277
Author(s):  
M.K. Kim ◽  
Y.H. Fibrianto ◽  
H.J. Oh ◽  
G. Jang ◽  
K.S. Lee ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Linear Models ◽  
Uv Light ◽  
Germinal Vesicle ◽  
Domestic Animal ◽  
Estradiol 17Β

In the bitch, oocytes are ovulated at the germinal vesicle (GV) stage and mature in the isthmus of the oviduct around 3 days after ovulation, it is not known what elements trigger the release of this meiotic arrest. Canine IVM has shown limited success with maturation rates, usually around 20% (MII) (Farstad W, 2000 Anim. Reprod. Sci. 60–61, 375–387). Estrogen and progesterone are suggested to play a significant role in causing oocyte resumption of meiosis and progression to MII stage. The purpose of this study was to investigate the role of estradiol-17β (E2) and progesterone (P4) during in vitro maturation of canine oocytes in serum-free tissue culture medium (TCM)-199. Canine oocytes collected from bitches were categorized into three groups based on estrous stages, follicular, luteal, or anestrus, at routine ovariohystrectomy. Oocytes were cultured in vitro in TCM-199 supplemented with E2, P4 or E2+P4 according to experimental design at 39°C in 5% CO2 and O2. After 72h of maturation culture, oocytes were denuded, fixed in a 3.7% paraformaldehyde solution for 10min, stained with Hoechst 33342 in glycerol, and observed under the UV light. Three groups of oocytes were cultured in TCM-199 supplemented with different concentrations (0, 0.1, 1.0 or 2.0μgmL−1) of E2 (Experiment 1, n=898, replications: 5) or P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 2, n=734, replications: 5). Multiple comparisons were implemented using Generalized Linear Models in the SAS 8.12 program. The rates of oocyte maturation to MII stage were higher (P&lt;0.05) in follicular stage oocytes cultured with 2μgmL−1 E2 (17.9%) compared to other supplement groups (0 to 7.6%). No differences (P&lt;0.05) in rate of MII stage oocytes among P4 supplement groups were observed. In Experiment 3, to investigate the combined effects of E2 and P4 on in vitro maturation, three groups of oocytes were cultured in TCM-199 supplemented with 2μgmL−1 E2 and various concentration of P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 3, n=1613, replications: 5). The rate of oocyte maturation to MII stage (11.5%) was higher (P&lt;0.05) in follicular stage oocytes cultured with 2μgmL−1 E2+2.0μgmL−1 P4 supplement compared to other supplement groups (0 to 6.4%). In conclusion, the present study demonstrated that E2 supplement in the culture medium increased maturation of canine oocyte to MII stage and that supplement of P4 alone did not promote oocyte maturation. However, P4 supplemented with E2 further promoted oocyte maturation in the follicular stage compared to E2 supplement alone, indicating that P4 acts synergistically with E2 on canine oocyte maturation in the presence of E2. From our results, we conclude that canine oocytes are exposed to high levels of P4 during maturation due to the preovulatory luteinization of canine follicles which gives rise to high intrafollicular as well as intratubal P4 concentrations-this is very different from the situation in oocytes from other domestic animal species. This study was supported by Biogreen 21-1000520030100000.

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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