scholarly journals Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo

Reproduction ◽  
1996 ◽  
Vol 108 (2) ◽  
pp. 185-191 ◽  
Author(s):  
A. C. Hynes ◽  
M. T. Kane ◽  
J. M. Sreenan
Keyword(s):  
Molecular Mass ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Partial Purification ◽  
Inhibitory Effects

Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid

1980 ◽  
Vol 95 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Knut Nordenström ◽  
Anita Sjögren ◽  
Lars Hamberger
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Bicarbonate Buffer ◽  
Ovulatory Follicles

Abstract. Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 μg/rat) or 0.154 m NaCl, 2 h prior to sacrifice. Granulosa cells were isolated from the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulosa cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FF1 which interacts with the sensitivity of the isolated preovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

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.

scholarly journals HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy

2021 ◽  
Vol 9 ◽  
Author(s):  
Zonghao Tang ◽  
Renfeng Xu ◽  
Zhenghong Zhang ◽  
Congjian Shi ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Ovarian Follicle ◽  
Hypoxia Inducible Factor ◽  
Follicular Development ◽  
B Cell Lymphoma ◽  
Underlying Mechanism

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

The role of intraovarian regulators in the aetiology of polycystic ovarian syndrome

1996 ◽  
Vol 5 (3) ◽  
pp. 151-168 ◽  
Author(s):  
Ghanim Almahbobi ◽  
Alan O Trounson
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Aromatase Activity ◽  
Lh Receptor ◽  
Follicular Maturation ◽  
Developmental Capacity ◽  
Follicle Selection

The present review demonstrates that the availability of bioactive FSH and LH in PCOS is normal and that granulosa cells of PCO are not apoptotic and instead hyperexpress functional FSH receptors and may possess intact aromatase activity. Consequently, these cells respond excessively to exogenous FSH stimulation and produce high amounts of oestradiol both in vivo and in vitro. The altered developmental capacity of follicles from PCO in vivo is most likely due to the abnormal follicular milieu of PCO and the culminating effects of intrafollicular inhibitors and stimulators. The failure of ovarian oestradiol production and follicular maturation to dominance in vivo may be due to a mechanism that interferes with the function of FSH, such as intraovarian steroids and growth factors. It has previously been shown that EGF and TGFα have inhibitory actions on follicular development, aromatization and LH receptor formation. In contrast, EGF enhances early follicular recruitment and growth. Therefore, it is hypothesized that EGF/TGFα may have a causal relationship in the mechanisms of anovulatory infertility in women with PCOS. Thus, an aberration in the regulation of follicular fluid EGF and/or TGFα may result in reduced numbers of granulosa cells, cessation of follicle selection and ultimately in the creation and maintenance of PCOS. The exact mechanism by which the hyperfunction of EGF/TGFα occurs and the trigger for this hyperactivity in the ovary remain to be determined. An experimental animal model may be required to assist such investigations in the future.

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants

2006 ◽  
Vol 18 (4) ◽  
pp. 403 ◽  
Author(s):  
Kenneth P. McNatty ◽  
Stephen Lawrence ◽  
Nigel P. Groome ◽  
Mohammed F. Meerasahib ◽  
Norma L. Hudson ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Point Mutations ◽  
Follicular Development ◽  
Ovis Aries ◽  
Ovulation Rate ◽  
Active Immunisation ◽  
Signalling Molecules ◽  
Stage Of Development

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on 3H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19–40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

scholarly journals 170 FOLLICULAR FLUID CONCENTRATION AND OOCYTE QUALITY FROM TOGGENBURG GOATS FED WITH UREA

2009 ◽  
Vol 21 (1) ◽  
pp. 184
Author(s):  
E. A. M. Amorim ◽  
L. S. Amorim ◽  
C. A. A. Torres ◽  
J. D. Guimãres ◽  
J. F. Fonseca ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Urea Concentration ◽  
Follicular Aspiration ◽  
Randomized Design ◽  
Fluid Concentration

Protein and urea concentrations impair oocyte and embryo development in vivo and in vitro through an unclear mechanism. A possible way to understand this process is to determine the concentration of hormones and metabolites in follicular fluid associated with normal development. The objective of this study was to determine the effect of dietary urea levels on follicular fluid concentration of hormones and metabolites and oocyte quality. A trial was conducted with 9 nonpregnant and nonlactating Saanen goats, which had been distributed in a randomized design and fed with diets with 0 (n = 4) and 2.4% of urea in the total dry matter (DM) of the diet (n = 5). Before follicle aspiration by laparotomy, the goats were synchronized by inserting intravaginal sponges containing 60 mg of acetate medroxyprogesterone (Progespon®, Sintex) for 10 days followed by 125 μg of cloprostenol (Ciosin® Coopers) 48 h before the removal of the sponge. The sponge was removed immediately before the follicular aspiration. The follicular development was stimulated with 70 mg of NIH-FSH-P1 (Folltropin V® Vetrepharm) i.m., and 300 IU of eCG i.m., (Novormon® Sintex) given 36 h before the follicular aspiration. Fluid from the 2 lartest follicles of each ovary were analyzed to determine the concentration of estradiol, progesterone, and testosterone by quimioluminesence, and glucose and urea concentrations were measured by enzymatic kit. The other follicles in each ovary were aspired with new needles and syringes and the oocyte quality was recorded. Oocytes were classified according to cytoplasma aspect and number of granulosa cells: Class A (dark cytoplasm and uniform aspect) with 3 (AMG) and <3 layers of cumulus cells (AmG); class B (cytoplasm with color alterations, desuniform aspect and vacuoles) with 3 (BMG) and <3 layers of cumulus cells (BmG); number of partially denuded oocytes (PD) and number of denuded oocytes (DO). Data were analyzed by ANOVA and treatment difference separated by SNK test. Follicular fluid estradiol concentration was lower in goats fed with urea (4.02 ± 0.16; 4.97 ± 0.18 ng mL–1; P < 0.05), progesterone concentration did not differ between treatments (2.48 ± 0.58; 3.37 ± 0.52 ng mL–1; P > 0.05), testosterone concentration was lower in the control animals (1.17 ± 0.48; 3.20 ± 0.43 ng mL–1; P < 0.05). The glucose (91.44 ± 3.60; 84.78 ± 5.58 mg dL–1) and urea concentration (23.04 ± 1.06; 18.00 ± 2.35) were greater in the animals treated with 2.4% compared with 0% of urea (P < 0.05), respectively. The number of oocytes in the different categories was not affected by treatment (P > 0.05): AMG 1.20 ± 1.09 v. 0.50 ± 0.57, AmG 4.20 ± 2.16 v. 3.50 ± 3.10, BMG 0.40 ± 0.54 v. 0.25 ± 0.50, BmG 1.40 ± 0.54 v. 1.75 ± 1.25, DO 10.20 ± 3.76 v. 11.50 ± 5.44, in the 0 and 2.4% of urea groups respectively. Only the number of PD (1.60 ± 0.54 v. 3.50 ± 1.91) recovered from animals treated with 2.4% was greater than in controls (P < 0.05). The hormone and metabolites concentration in follicular fluid as well as the oocyte quality was affected by the urea concentration of the diet. Supported by grant from: CNPq, FAPEMIG, Shering Plough®, Tecnopec®, Carbogel®.

scholarly journals FOXO1 mediates hypoxia-induced G0/G1 arrest in ovarian somatic granulosa cells via activating the TP53INP1-p53-CDKN1A pathway

Development ◽  
2021 ◽  
Author(s):  
Chengyu Li ◽  
Zhaojun Liu ◽  
Gang Wu ◽  
Ziyu Zang ◽  
Jia-Qing Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Granulosa Cells ◽  
Nuclear Translocation ◽  
Follicular Development ◽  
Inhibitory Effect ◽  
G1 Phase ◽  
G1 Arrest ◽  
Female Reproduction

The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling, we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Actually, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributed to the suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings might provide avenues for the clinical treatment of human infertility caused by impaired follicular development.

scholarly journals β-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization

2010 ◽  
Vol 24 (8) ◽  
pp. 1529-1542 ◽  
Author(s):  
Heng-Yu Fan ◽  
Annalouise O'Connor ◽  
Manami Shitanaka ◽  
Masayuki Shimada ◽  
Zhilin Liu ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Integration Site ◽  
Gonad Development ◽  
Follicular Development ◽  
Underlying Mechanism ◽  
Female Fertility ◽  
Female Gonad ◽  
Tumor Virus

Abstract Wingless-type mouse mammary tumor virus integration site family (WNT)/β-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1(Ex3)fl/fl;Cyp19-Cre (Ctnnb1(Ex3)gc−/−), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1(Ex3)gc−/− mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1fl/fl;Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.

ALTERATIONS IN GRANULOSA CELL AROMATASE ACTIVITY ACCOMPANYING PREOVULATORY FOLLICULAR DEVELOPMENT IN THE RAT OVARY WITH EVIDENCE THAT 5α-REDUCED C19 STEROIDS INHIBIT THE AROMATASE REACTION IN VITRO

1980 ◽  
Vol 84 (3) ◽  
pp. 409-419 ◽  
Author(s):  
S. G. HILLIER ◽  
AGNES M. J. VAN DEN BOOGAARD ◽  
L. E. REICHERT ◽  
E. V. VAN HALL
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Hormonal Regulation ◽  
Follicular Development ◽  
Biologically Active ◽  
Aromatase Activity ◽  
Adult Rat ◽  
Oestradiol Production

Locally produced androgens and oestrogens are thought to be important factors in the hormonal regulation of follicular development. In the present study the relationship between follicular maturity and granulosa cell aromatase activity has been examined in vitro. Granulosa cells harvested from the largest antral follicles in adult rat ovaries produced negligible amounts of immunoreactive oestradiol when incubated for 3 h in vitro irrespective of the day of the oestrous cycle upon which they were obtained. However, the addition of aromatizable C19 steroid substrate (testosterone, androstenedione or 19-hydroxyandrostenedione) to the incubation medium resulted in time- and concentration-dependent increases in oestradiol production which were related to the level of follicular maturity attained in vivo. By measuring oestradiol production using testosterone (10−7 mol/l) as substrate, the aromatase activity of granulosa cells obtained on the first day of vaginal dioestrus was shown to be only a fraction (less than 5%) of that observed for cells obtained on the morning of pro-oestrus. Cells obtained on the second day of dioestrus displayed an intermediate level of activity which remained approximately five times lower than that of granulosa cells at pro-oestrus. These observations, therefore, establish the induction or activation of granulosa cell aromatase activity as a correlate of normal preovulatory follicular development. However, intrafollicular androgen/oestrogen ratios may also be influenced by quantitative and/or qualitative alterations in the C19 steroidal substrate available for the aromatase reaction. Thus, the naturally occurring non-aromatizable 5α-reduced androgen metabolites, 5α-dihydrotestosterone and 5α-androstanedione, proved to be potent competitive inhibitors of the granulosa cell aromatase reaction in vitro. In this respect each of these biologically active androgens was more effective than 1-enetestololactone, an established C19 steroidal aromatase inhibitor. Since C19 steroid 5α-reductase is known to be an ovarian enzyme, it is suggested that by affecting the androgenic/oestrogenic composition of the hormonal milieu, local alterations in the activity of this enzyme may be an additional determinant of preovulatory follicular development and function.

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