Estradiol-17β and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4–6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17β (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH, 0.25 μg/mL) or luteinizing hormone (LH, 1 μg/mL). Addition of testosterone or androstenedione (0.5 μM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3′,5′-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.

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The Cultivation of Human Granulosa Cells

Acta Medica (Hradec Kralove Czech Republic) ◽

10.14712/18059694.2017.19 ◽

2008 ◽

Vol 51 (3) ◽

pp. 165-172 ◽

Cited By ~ 9

Author(s):

Lenka Brůčková ◽

Tomáš Soukup ◽

Jiří Moos ◽

Martina Moosová ◽

Jana Pavelková ◽

...

Keyword(s):

Growth Factors ◽

Granulosa Cells ◽

Follicular Fluid ◽

Doubling Time ◽

Ovarian Follicle ◽

Vitro Fertilization ◽

Thecal Cells ◽

Cell Doubling Time

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

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Comparison of the FSH-induced estradiol-17β production by bovine antral and mural granulosa cells cultured in vitro in a completely defined medium

Theriogenology ◽

10.1016/s0093-691x(05)80196-7 ◽

1994 ◽

Vol 41 (1) ◽

pp. 286

Author(s):

P. Rouillier ◽

J. Saumande ◽

M.-A. Sirard ◽

P. Matton ◽

L.A. Guilbault

Keyword(s):

Granulosa Cells ◽

Defined Medium ◽

Estradiol 17Β ◽

Cells Cultured

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A technique for superfusion of isolated granulosa cells. Dynamics of cAMP production and steroidogenesis in response to gonadotropins

Acta Endocrinologica ◽

10.1530/acta.0.1170497 ◽

1988 ◽

Vol 117 (4) ◽

pp. 497-506 ◽

Cited By ~ 4

Author(s):

Carl Johanson ◽

Viktor Johanson

Keyword(s):

Granulosa Cells ◽

Culture Medium ◽

Cell Damage ◽

Adenosine Monophosphate ◽

Camp Production ◽

Morphological Examination ◽

Ovarian Cells ◽

Temporal Relationships ◽

Polycarbonate Membranes ◽

Estradiol 17Β

Abstract. A superfusion model for isolated ovarian cells was developed and characterized in detail. Granulosa cells isolated from pre-ovulatory rat ovarian follicles were placed in superfusion (perifusion) chambers with a volume of 125 μl. Culture medium was pumped through the chambers, collected in 20-min fractions of 600 μl and analysed for cAMP and steroids. Viability was confirmed by morphological examination. The use of polycarbonate membranes to retain the cells in the chambers was abandoned since the membranes caused severe cell damage. The temporal relationships between gonadotropic stimuli and the release of cyclic 3':5'-adenosine monophosphate (cAMP) and steroids was investigated. Within 10 min FSH elicited transient increase in the release of cAMP and progesterone but had no effect on testosterone or estradiol-17β release. Amplitude and duration of the response in cAMP and progesterone release were correlated to concentration and length of the FSH pulse when these parameters were varied within the ranges 1–100 μg/l and 30–270 min, respectively. Compared with the cAMP response, the progesterone response peaked up to 30 min later and lasted 1 to 2 h longer but could not be extended to more than approximately 6 h, not even with longer FSH pulses. These results could indicate a development of desensitization.

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Serum deprivation affects secretory activity of cultured porcine ovarian follicles and granulosa cells and their response to hormones

Veterinární Medicína ◽

10.17221/112/2009-vetmed ◽

2009 ◽

Vol 54 (No. 10) ◽

pp. 455-460

Author(s):

A.V. Sirotkin

Keyword(s):

Granulosa Cells ◽

Ovarian Follicle ◽

Secretory Activity ◽

Ovarian Follicles ◽

Serum Deprivation ◽

Ovarian Cells ◽

Igf I ◽

Granulose Cells ◽

Vitro Model

The aim of the present study is to understand the hormonal mechanisms of the effect of malnutrition on ovarian follicle functions. For this purpose, we examined the effect of malnutrition/serum deprivation, addition of metabolic hormones and gonadotropin (IGF-I, leptin and FSH) and their combination on the release of progesterone (P4), testosterone (T), estradiol (E2) and insulin-like growth factor I (IGF-I) by cultured whole ovarian follicles and on P4 and IGF-I output by cultured granulosa cells isolated from porcine ovaries. It was observed that in ovarian follicles cultured with nutrients/serum addition of IGF-I reduced release of P4, but not of T or E2. Exogenous leptin reduced output of E2, but not of P4 or T, and increased IGF-I output. No significant effect of FSH on release of steroid hormones by isolated follicles was found. Serum deprivation did not affect release of P4, but reduced output of T and E2, and promoted IGF-I release by cultured ovarian follicles. Addition of hormones failed to prevent the effect of malnutrition on the secretory activity of cultured ovarian follicles. In cultured granulose cells, all the tested hormones promoted release of both P4 and IGF-I. Food restriction/serum deprivation reduced both P4 and IGF-I output. Additions of either IGF-I, leptin and FSH prevented the inhibitory action of malnutrition on both P4 and IGF-I release. The present observations (1) confirm the involvement of the hormones IGF-I, leptin and FSH in the control of secretory activity of ovarian cells, (2) demonstrate, that both isolated ovarian granulosa cells and whole follicles cultured in the absence of serum nutrients could be an adequate in-vitro model for studying the effect of malnutrition on ovarian secretory functions, and (3) suggest, that malnutrition could affect ovarian functions through changes in the release of ovarian hormones.

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224 SILDENAFIL ACCELERATES SPERM PENETRATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab224 ◽

2013 ◽

Vol 25 (1) ◽

pp. 260

Author(s):

H. Funahashi ◽

Q.-S. Wu

Keyword(s):

Adenosine Monophosphate ◽

Defined Medium ◽

Fluorescence Assay ◽

Chemically Defined Medium ◽

Sperm Capacitation ◽

Boar Sperm ◽

Sperm Penetration ◽

Type 3 ◽

Chlortetracycline Fluorescence Assay

Sperm capacitation, a cyclic-adenosine monophosphate-dependent phenomenon, is an important initiation step for penetration into oocytes. In porcine IVF, the use of caffeine, as a nonspecific phosphodiesterase (PDE) inhibitor, is common to accelerate sperm capacitation and penetration. The objective of this study was to examine the effects of PDE inhibitors (cilostamide, rolipram, and sildenafil as PDE type 3-, type 4-, and type 5-specific inhibitors, respectively) on the capacitation of boar sperm and the penetration into porcine oocytes in the absence of caffeine and other capacitation inducers in a chemically defined medium. After washing sperm samples collected from an ejaculated sperm-rich fraction of different individual Berkshires, the sperm were resuspended in capacitation inducer-free (theophylline- and adenosine-free) PGM-tac4 (mPGM-tac) at 5 × 105 cells mL–1. The suspension was cultured in mPGM-tac nonsupplemented or supplemented with 2.5 mM cilostamide, rolipram, or sildenafil for 90 min at 39°C in an atmosphere of 5% CO2 in air, and then the capacitation status was assessed by chlortetracycline fluorescence assay. Other sperm suspensions were used to co-culture with denuded in vitro-matured oocytes in the same medium for 8 h in an atmosphere of 5% CO2 in air and fixed, and sperm penetration was then examined. Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In our result from the chlortetracycline fluorescence assay, although the incidence of intact sperm was significantly reduced in the presence of rolipram (54.3%) and sildenafil (52.7%) as compared with controls (66.7%), there were no differences in capacitated sperm among experimental groups (24.3 to 34.3%). The incidence of acrosome-reacted sperm was higher in the presence of cilostamide (17.3%) than in the others (9.0 to 13.0%). High sperm penetration was observed only in the presence of sildenafil (76.6%) as compared with the control (0%) or the presence of rolipram (4.4%) or cilostamide (1.8%). These results demonstrate that inhibition of PDE type 5, but not PDE type 3 and type 4, significantly accelerates the penetration of boar sperm into the oocytes in a capacitation inducer-free chemically defined medium, whereas inhibition of PDE type 3 may induce an acrosome reaction.

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Theca: the forgotten cell of the ovarian follicle

Reproduction ◽

10.1530/rep-10-0094 ◽

2010 ◽

Vol 140 (4) ◽

pp. 489-504 ◽

Cited By ~ 246

Author(s):

J M Young ◽

A S McNeilly

Keyword(s):

Granulosa Cells ◽

Developmental Stages ◽

Vascular System ◽

Ovarian Follicle ◽

Primary Follicle ◽

Diverse Range ◽

Theca Cells ◽

Structural Support ◽

Highly Active ◽

Thecal Cells

Theca cells function in a diverse range of necessary roles during folliculogenesis; to synthesize androgens, provide crosstalk with granulosa cells and oocytes during development, and provide structural support of the growing follicle as it progresses through the developmental stages to produce a mature and fertilizable oocyte. Thecal cells are thought to be recruited from surrounding stromal tissue by factors secreted from an activated primary follicle. The precise origin and identity of these recruiting factors are currently not clear, but it appears that thecal recruitment and/or differentiation involves not just one signal, but a complex and tightly controlled combination of multiple factors. It is clear that thecal cells are fundamental for follicular growth, providing all the androgens required by the developing follicle(s) for conversion into estrogens by the granulosa cells. Their function is enabled through the establishment of a vascular system providing communication with the pituitary axis throughout the reproductive cycle, and delivering essential nutrients to these highly active cells. During development, the majority of follicles undergo atresia, and the theca cells are often the final follicular cell type to die. For those follicles that do ovulate, the theca cells then undergo hormone-dependent differentiation into luteinized thecal cells of the corpus luteum. While the theca is an essential component of follicle development and ovulation, we do not yet fully understand the control of recruitment and function of theca cells, an important consideration since their function appears to be altered in certain causes of infertility.

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Treatment with cyclic adenosine monophosphate modulators prior to in vitro maturation alters the lipid composition and transcript profile of bovine cumulus–oocyte complexes and blastocysts

Reproduction Fertility and Development ◽

10.1071/rd17335 ◽

2018 ◽

Vol 30 (10) ◽

pp. 1314 ◽

Cited By ~ 6

Author(s):

Eduardo M. Razza ◽

Mateus J. Sudano ◽

Patricia K. Fontes ◽

Fernanda F. Franchi ◽

Katia Roberta A. Belaz ◽

...

Keyword(s):

Lipid Content ◽

Lipid Composition ◽

Transcriptional Profiling ◽

Ovarian Follicle ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Cumulus Cells ◽

Membrane Lipid ◽

Unsaturated Lipids

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus–oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2 h and 16 transcripts after 24 h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

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Involvement of miRNAs in equine follicle development

Reproduction ◽

10.1530/rep-13-0107 ◽

2013 ◽

Vol 146 (3) ◽

pp. 273-282 ◽

Cited By ~ 43

Author(s):

S N Schauer ◽

S D Sontakke ◽

E D Watson ◽

C L Esteves ◽

F X Donadeu

Keyword(s):

Cell Survival ◽

Granulosa Cells ◽

Follicular Fluid ◽

Ovarian Follicle ◽

Follicle Development ◽

Previous Evidence ◽

Ovarian Follicle Development ◽

Fluid Levels ◽

Follicle Selection

Previous evidence fromin vitrostudies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels ofCYP19A1andLHCGR(P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets,PTEN,RASA1, andSMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

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