132. ACTIVIN A HAS A STIMULATORY EFFECT IN VITRO ON EARLY FOLLICLE DEVELOPMENT IN RAT OVARIES

2010 ◽  
Vol 22 (9) ◽  
pp. 50
Author(s):  
D. A. Cossigny ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Treatment Group ◽  
Real Time ◽  
Granulosa Cells ◽  
Combined Treatment ◽  
Activin A ◽  
Tunel Staining ◽  
Follicle Development ◽  
Primordial Follicles ◽  
Preantral Follicles

Activins are dimers of inhibin β subunits and are growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily (1). Both βA and βB subunits are highly expressed in rat granulosa cells, while theca cells express little or no β subunit mRNAs (2). Oocytes lack expression of either subunit (3, 4). Activin is suggested to facilitate the responsiveness of granulosa cells to FSH (5). We hypothesized that activin, with or without FSH, could enhance the transition from the primordial to later preantral stages of follicle development. In two independent experiments, day 4 rat ovaries (n = 3 from different rats per treatment) were randomly assigned and cultured (6, 7) for 10 days in DMEM/Hams F-12 media with either no additives, FSH (100 ng/mL), activin A (50 ng/mL), or both. Day 4 fresh ovaries were also used as controls. Media and treatments were refreshed every alternate day. Ovaries were fixed andsectioned, or placed into Ultraspec for RNA extraction and real-time PCR analysis. Follicle numbers were counted as described previously (7). The proportion of atretic follicles (TUNEL staining) was determined in 3 randomly selected sections per ovary. Primordial follicles in all treatment groups were approximately 20% of those in Day 4 fresh ovaries. Primary follicles increased significantly (P < 0.05) only in the combined treatment group, where preantral follicles increased significantly (P < 0.0001) only when treated with activin A alone. Activin A alone decreased the proportion of atretic follicles in the primary and preantral classes, where the combined treatment increased the proportion of atretic preantral follicles. Real-time analysis revealed that expression levels of follistatin, FSH receptor and activin βA and βB subunits were all expressed at significantly higher levels in the Activin A-only treated group (P < 0.05). In summary, there was no effect on primordial follicle activation by any treatment. Activin alone had a stimulatory effect in vitro on subsequent folliculogenesis, but in the presence of FSH its effect was counteracted shown by an increase in atresia. Reasons for an increase in atretic preantral follicles in the combined treatment group are unclear. These studies support a stimulatory role for activin A in early follicle development and confirm the in vivo effects of activin on folliculogenesis (4). NHMRC program grant # 494802 and Fellowship (# 441101) provided financial support. (1) Vale W et al. 1986. Nature 321: 776–779.(2) Meunier H et al. 1988. Proc Natl Acad Sci USA 85: 547–251.(3) Roberts V et al. 1993. Journal of Clinical Endocrinology & Metabolism 7: 1402–1410.(4) Sidis Y et al. 1998. Biology of Reproduction 59(4): 807–812.(5) Drummond A et al. 2002. Endocrinology 143 (4): 1423–1433.(6) Nilsson E et al. 2001. Molecular and Cellular Endocrinology 182 (2): 145–155.(7) Rosairo D et al. 2008. Reproduction 136: 799–809.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

scholarly journals The activin-follistatin system and in vitro early follicle development in goats

2006 ◽  
Vol 189 (1) ◽  
pp. 113-125 ◽  
Author(s):  
J R V Silva ◽  
T Tharasanit ◽  
M A M Taverne ◽  
G C van der Weijden ◽  
R R Santos ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Activin A ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Follicle Development ◽  
Cortical Tissue ◽  
Primary Follicle ◽  
Growth And Survival ◽  
Primordial Follicles

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

123. ACTIVIN A AND OVARIAN FOLLICLE DEVELOPMENT

2009 ◽  
Vol 21 (9) ◽  
pp. 42
Author(s):  
D. A. Cossigny (Rosairo) ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Ovarian Follicle ◽  
Combined Treatment ◽  
Rna Extraction ◽  
Activin A ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Follicle Development ◽  
Preantral Follicle ◽  
Primary Follicle ◽  
Preantral Follicles ◽  
Ovarian Follicle Development

A significant developmental stage in ovarian folliculogenesis is the acquisition of gonadotropin sensitivity by ovarian follicles. Activin has previously been suggested to be involved in the responsiveness of granulosa cells to FSH (1). Therefore, the role of activin was investigated using a ‘physiological’ culture system to determine if pathways exist to transduce activin signals within the postnatal rat ovary. Organ cultures with day 4 whole ovaries were employed in order to assess the potential impact of Activin A on follicle growth and transition from the primordial through to the primary and later preantral stages of development. Ovaries were isolated and cultured for 10 days with the addition of supplemented DMEM/Hams F-12 media (2)and either FSH (100ng/ml), Activin A (50ng/ml), or a combination of the two. Media and treatments were refreshed every alternate day. At the end of the culture period, ovaries were fixed and sectioned, or placed immediately into Ultraspec for RNA extraction for future real-time PCR. Sections were used for morphological assessment and ovarian follicle counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labeling (TUNEL). Primary follicle numbers increased significantly (P<0.05) in the combined treatment group whereas, preantral follicle numbers increased significantly (P<0.0001) when treated with Activin A alone. This is consistent with a morphological appraisal of atresia where a decrease in atresia was found in primordial and primary follicles, supporting the primary follicle development data and Activin A treatment alone resulted in more healthy primary and preantral follicles than atretic ones. Therefore, a stimulatory role for Activin A both in the presence of FSH (primary follicle development) or alone (preantral follicle development) has resulted in more follicles making the transition from the primordial to primary stages, as well as to the later preantral stages.

scholarly journals The effects of FSH and activin A on follicle development in vitro

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Davina A Cossigny ◽  
Jock K Findlay ◽  
Ann E Drummond
Keyword(s):  
Combined Treatment ◽  
Primordial Follicle ◽  
Treated Group ◽  
Activin A ◽  
Pcr Analysis ◽  
Follicle Development ◽  
Development In Vitro ◽  
Rates Of Growth

Numerous studies have reported on the roles of activins in gonadal regulation; however, little is known about their specific roles in early folliculogenesis. Ovarian follicular growth was investigated in 10-day cultures of day 4 postnatal whole ovaries treated with activin A (ActA; 50 ng/ml), with or without FSH (100 ng/ml) in vitro. We hypothesized that treatment with ActA±FSH would affect rates of growth and atresia in follicles. None of the treatments affected primordial follicle activation, and antral follicles were not observed after 10 days in culture. Primordial follicle numbers from all treatment groups were ∼20% of those in day 4 fresh ovaries, indicating that activation had occurred. In the presence of ActA, preantral follicle numbers increased significantly (P<0.0001). ActA alone decreased the proportion of atretic follicles in the primary and preantral classes, whereas the combined treatment of ActA+FSH increased the proportion of atretic preantral oocytes. Real-time PCR analysis revealed that follistatin, FSH receptor, and activin βA and βB subunits were all expressed at significantly higher levels in the ActA-only treated group but not in the ActA+FSH group. Here, we report novel findings supporting the role of FSH in primordial follicle survival through an action on apoptosis and a stimulatory role of ActA in the primordial to primary and preantral stages of follicle development, suggesting an inhibitory action of activin on oocyte apoptosis.

scholarly journals Regulation of Ovarian Primordial Follicle Assembly and Development by Estrogen and Progesterone: Endocrine Model of Follicle Assembly

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3329-3337 ◽  
Author(s):  
Phillip Kezele ◽  
Michael K. Skinner
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Single Layer ◽  
Primordial Follicle ◽  
Follicle Development ◽  
Newborn Rat ◽  
Primary Follicle ◽  
Primordial Follicles

Abstract The assembly of the developmentally arrested primordial follicle and the subsequent transition of the primordial follicle to the primary follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian follicles do not proliferate and the primordial follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated follicle development. Abnormalities in primordial follicle assembly, arrest, and development (i.e. primordial to primary follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial follicle transition to the primary follicle was found to be 3 times greater in culture. This abnormal rate of primary follicle development in culture suggests the primordial follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary follicle transition. Approximately 60% of follicles make the primordial to primary follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial follicle assembly. All viable oocytes assembled into primordial follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial follicle assembly in vivo with 10% of the total follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial follicle assembly and primordial to primary follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial follicle assembly and early follicular development.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

scholarly journals Time- and Dose-Dependent Effects of 17 Beta-Estradiol on Short-Term, Real-Time Proliferation and Gene Expression in Porcine Granulosa Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Sylwia Ciesiółka ◽  
Joanna Budna ◽  
Karol Jopek ◽  
Artur Bryja ◽  
Wiesława Kranc ◽  
...  
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Hormone Receptor ◽  
Proliferative Activity ◽  
Cell Transformation ◽  
Luteinizing Hormone Receptor ◽  
Luteal Cell ◽  
Ovarian Activity ◽  
Short Term

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 μM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs.

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 2-Hydroxy-4-Methylselenobutanoic Acid (HMSeBA) Promote Follicle Development By Antioxidant Pathway in Gilts

2021 ◽  
Author(s):  
Yanpeng Dong ◽  
Sirun Chen ◽  
Yalei Liu ◽  
Zimei Li ◽  
Xinlin Jia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antioxidant Capacity ◽  
Granulosa Cells ◽  
Body Weight Gain ◽  
Control Diet ◽  
Negative Control ◽  
Follicle Development ◽  
Selenium Content ◽  
Total Selenium

Abstract Background Dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation can exert antioxidant effects in poultry, pigs and weaned pigs. However, it is unknown whether HMSeBA could improve the development of follicle by anti-oxidize effects in gilt. This study was conducted to evaluate the effects of dietary HMSeBA supplementation on the follicle development in gilt. A total of 36 gilts were randomly fed the control diet (CON, negative control), Na2SeO3 diet containing 0.3 mg Se/kg (positive control) or the HMSeBA diet containing 0.3 mg Se/kg from weaning to the 19th day after the second estrus. In another study, the effect of HMSeBA on the cells viability, proliferation, release of 17βestradiol (E2 ) and antioxidant capacity were investigated in the mouse ovarian granulosa cells in vitro. Results Results showed that HMSeBA group increased the average daily body weight gain (ADG) and decreased the ratio of feed: gain during day 120 to 176 in gilts ( P < 0.05). The selenium (HMSeBA and Na 2 SeO 3 ) increased the weight of uterine at the third estrus. There was no effect of HMSeBA on the number of large follicles (diameter >5mm), but HMSeBA decreased the gene expression of growth differentiation factor-9 ( GDF-9 ) and bone morphogenetic protein-15 ( BMP-15 ) in cumulus-oocyte complexes (COCs). HMSeBA group increased the total selenium content in serum ( P < 0.05) and liver ( P < 0.01) and tended to increase the total selenium content in ovary ( P = 0.08). HMSeBA group decreased the malondialdehyde (MDA) concentration in the serum, liver and ovary ( P < 0.05), increased the total antioxidant capacity (T-AOC) in the liver, thioredoxin reductase (TrxR) in the ovary ( P < 0.05) and increased the activity of GPx in the serum, liver and ovary ( P < 0.05). Na 2 SeO 3 supplementation decreased MDA and increased the T-AOC in liver, increased the T-SOD and TrxR in the ovary compared with control. At the transcription level, HMSeBA group increased the glutathione peroxidase 2 ( GPx2 ) and TrxR1 ( P < 0.05) expression in the liver, and increased the GPx1 expression ( P < 0.05) in the ovary of gilts compared with Na2SeO3 treatment. Besides, HMSeBA group increased the expressions of superoxide dismutase 1 ( SOD1 ) and Thioredoxin l ( Trx1 ) in the liver. In vitro experiment, HMSeBA improved granulosa cells’ proliferation and E2 secretion ( P < 0.05). HMSeBA and Na 2 SeO 3 both increased the T-AOC and decreased MDA in granulosa cells in vitro. Meanwhile, HMSeBA increased T-SOD, GPx, glutathione reductase (GR) and TrxR activity in granulosa cells in vitro. In addition, HMSeBA up-regulated SOD2 and GPx1 gene expression in the granulosa cells in vitro.Conclusion These results demonstrate directly, HMSeBA was more conducive to absorption and storage of selenium in the liver and ovary in gilt, and beneficial to exert the effect of HMSeBA on the antioxidant function in the liver and ovary of gilt. Moreover, HMSeBA has stronger antioxidant capacity in granular cells in vitro , which is more conducive to promoting follicle development. Therefore, the new type of organic selenium, HMSeBA, could be potentially useful for the control of reproductive processes in gilt.

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