Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

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Immunohistochemical localization of steroidogenic enzymes and comparison with hormone production during follicle development in the pig

Reproduction Fertility and Development ◽

10.1071/rd99085 ◽

1999 ◽

Vol 11 (6) ◽

pp. 337 ◽

Cited By ~ 15

Author(s):

Ellen M. Shores ◽

Morag G. Hunter

Keyword(s):

Granulosa Cells ◽

Follicular Development ◽

Staining Intensity ◽

Immunohistochemical Localization ◽

Vital Role ◽

Steroidogenic Enzymes ◽

Hormone Production ◽

Theca Cells ◽

Enzyme Distribution ◽

Follicle Size

The steroidogenic enzymes, P450 aromatase (P450 arom ) and P450 17a-hydroxylase (P450 17a ), were precisely located within the healthy porcine follicle by immunohistochemistry. Enzyme distribution was examined throughout follicular development during natural oestrous cycles (n = 14 gilts) and was compared with steroid production by healthy whole and theca-only follicles. All follicles 2 mm or more in diameter were either fixed for immunohistochemistry (n = 380 of which 197 were assessed as healthy) or incubated as whole (n = 110) or theca-only (n = 110) follicles to measure steroidogenesis. P450 17a was confined to the theca layer. The number of positive cells and staining intensity increased with follicle size. P450 arom was consistently detected in the granulosa layer of follicles measuring 6 mm or more in diameter and those cells furthest from the antrum were most strongly stained. P450arom was also detected in the theca layer of these large follicles. Whole and theca-only follicles produced oestradiol and androstenedione, and the levels of both hormones increased with follicle size (P<0.001). Whole follicles produced more oestradiol (P<0.001), but less androstenedione (P = 0.01) than theca-only follicles of the same size. Although granulosa cells contained P450 arom and synthesized oestradiol, only theca cells contained P450 17a. Theca cells therefore provided granulosa cells with androgen substrate. In addition, theca cells possessed P450 arom , making them capable of independent oestradiol production, which may be required to trigger the LH surge. This study confirms the vital role of theca cells in follicular steroidogenesis in the pig.

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222 CHARACTERIZATION OF CO-CULTURES OF GRANULOSA CELLS AND THECA CELLS AS EXPLANTS OF BOVINE OVARY FOLLICULAR WALLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab222 ◽

2010 ◽

Vol 22 (1) ◽

pp. 269

Author(s):

L. P. Salles ◽

R. B. Vasconcelos ◽

I. Oliveira e Silva ◽

L. V. M. Gulart ◽

F. A. G. Torres ◽

...

Keyword(s):

Cytochrome P450 ◽

Estrous Cycle ◽

Granulosa Cells ◽

Internal Control ◽

Regulatory Protein ◽

Defined Medium ◽

Aromatase Activity ◽

Steroidogenic Enzymes ◽

Theca Cells

Granulosa cells (GC) and theca cells (TC) luteinize in culture because of the presence of serum in the medium and, as a consequence, the production of progesterone (P4) increases and that of 17β-estradiol (E2) decreases. The follicular phase of the bovine estrous cycle is characterized by the presence of E2, as well as aromatase activity, that is turned off in the atresia or in the luteal phase of the bovine estrous cycle. We propose to characterize 2 co-culture systems utilizing slices of follicular walls (explants) containing both GC and TC and to cultivate them in nondefined medium (NDM; TCM199+serum) or defined medium (DM; α MEM+polyvinyl alcohol). To prepare the explants, 80 follicles measuring 4 to 6 mm in diameter were selected. Concentrations of P4 and E2 were evaluated by RIA after 24, 48, and 72 h of culture and values were corrected by tissue weight (10 mg). Morphological analysis of the explants was stained with hematoxylin and eosin. The total mRNA from GC-TC explants was purified and the expression of the following genes was estimated by RT-PCR: bovine 3 beta-hydroxysteroid dehydrogenase (HSD3B1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1), 17|3-hydroxylase (CYP17), aromatase cytochrome P450 (CYP19), steroidogenic acute regulatory protein (StAR), follicle-stimulating hormone receptor (FSHr), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) as internal control. The results were analyzed by two-way ANOVA followedby Bonferroni least-significant tests. Microscopic analyses showed that cells cultured in NDM lost their polyhedral shape and acquired a fibroblast-like form. Defined medium maintained GC morphology with low cytoplasm:nucleus ratio and GC-GC contact, similar to in vivo cells. In NDM, the concentration of P4 increased as opposed to that of E2, which decreased after 48 h of culture. In DM, the concentrations of P4 and E2 were maintained in the first 48 h of culture. The level of P4 in NDM was higher than in DM during all periods tested. Our data show that CYP11A1 expression remains absent in NDM for follicles explants that initially did not express CYP11A1, whereas in DM the expression of this gene appears after 24, 48, or 72 h of culture, evidence that indicates rescue from atresia. It is important to notice that gene expression of steroidogenic enzymes in DM were higher than in NDM. Very low levels of CYP11A1 and CYP19 were observed in the NDM group. On the other hand, in the DM group there was a progressive increase of all the steroidogenic enzymes and FSHr, which reached a peak at 48 h of culture. In conclusion, our results suggest that DM showed a stimulating effect on steroidogenic activity of GC-TC co-culture similar to that observed in vivo for functional growing follicle, whereas in NDM the explants seemed to mimic luteinizing follicles. Grants from FAP-DF, Finatec, CNPq, and Capes.

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The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽

10.1530/rep-10-0142 ◽

2010 ◽

Vol 140 (2) ◽

pp. 295-303 ◽

Cited By ~ 8

Author(s):

Jennifer L Juengel ◽

Lisa J Haydon ◽

Brigitta Mester ◽

Brian P Thomson ◽

Michael Beaumont ◽

...

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Cell Function ◽

Antral Follicle ◽

Brushtail Possum ◽

Follicular Growth ◽

Theca Cells ◽

Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

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Cell-specific expression of aromatase and LH receptor mRNAs in rat ovary

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0090309 ◽

1992 ◽

Vol 9 (3) ◽

pp. 309-312 ◽

Cited By ~ 40

Author(s):

P.F. Whitelaw ◽

C.D. Smyth ◽

C.M. Howles ◽

S.G. Hillier

Keyword(s):

Cytochrome P450 ◽

Granulosa Cells ◽

Direct Evidence ◽

Specific Expression ◽

Lh Receptor ◽

Paracrine Regulation ◽

Receptor Mrna ◽

Rat Ovary ◽

Per Se ◽

Receptor Mrnas

ABSTRACT Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.

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LH receptor mRNA and cytochrome P450 side-chain cleavage expression in bovine theca and granulosa cells luteinized by LH or forskolin

Domestic Animal Endocrinology ◽

10.1016/s0739-7240(97)00085-4 ◽

1998 ◽

Vol 15 (2) ◽

pp. 103-114 ◽

Cited By ~ 19

Author(s):

Roni Mamluk ◽

David Wolfenson ◽

Rina Meidan

Keyword(s):

Cytochrome P450 ◽

Granulosa Cells ◽

Side Chain ◽

Lh Receptor ◽

Side Chain Cleavage ◽

Receptor Mrna ◽

Chain Cleavage

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225 FOLLICULAR DIAMETER, OVULATION RATE, AND LH RECEPTOR GENE EXPRESSION IN NELLORE COWS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab225 ◽

2010 ◽

Vol 22 (1) ◽

pp. 270 ◽

Cited By ~ 1

Author(s):

R. A. L. Simões ◽

R. A. Satrapa ◽

F. S. Rosa ◽

M. Piagentini ◽

A. C. S. Castilho ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Internal Control ◽

Receptor Gene ◽

Rna Extraction ◽

Sao Paulo ◽

Ovulation Rate ◽

São Paulo ◽

Lh Receptor ◽

Theca Cells

The aim of the present experiment was to verify the relationship among follicular diameter, ovulation rate, and gene expression of LH receptor (LHR) isoforms in order to know whether these aspects could or could not influence ovulation rates in Nellore cows. In Experiment 1, at a random stage of the estrous cycle (Day 0), Nellore cows (n = 53) received a progesterone intravaginal device (1.0 g, Primer®, Tecnopec, São Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB; i.m. Estrogin®; Farmavet, São Paulo, Brazil). On Day 8, PGF2 (150 μg d-cloprostenol; Prolise® ARSA S.R.L., Buenos Aires, Argentina) was administered i.m. and the device was removed. Twenty-four hours after device removal, cows were treated i.m. with EB (1.0 mg) and, 48 h afterwards, ovulation was determined by ultrasonography (US; Aloka 900, Tokyo, Japan). Three days after ovulation, follicular growth was observed daily by US and cows were randomly allocated into 3 groups according to follicular diameter (mm) [G1 (7.0-8.0), G2 (8.1-9.0), and G3 (9.1-10.0)] to receive 6.25 mg of LH (i.m. Lutropin®-V, Bioniche, Belleville, Ontario, Canada), which corresponds to twice the minimum ovulatory dose (3.12 mg) as determined in a preliminary experiment. The results were analyzed by logistic regression (PROC GEN MOD, SAS Institute, Cary, NC). The ovulation rates were 9 (2/21), 36 (8/22), and 90% (9/10) for G1, G2, and G3, respectively. There were significant differences when comparing G1 v. G3 (P < 0.01), G2 v. G3 (P < 0.02), and G1 v. G2 (P < 0.03). In Experiment 2, granulosa and theca cells from Nellore cows were recovered from follicles obtained in a local abattoir and submitted to total RNA extraction and expression of LHR isoforms (LHR-B3, LHR-B4, LHR-B5, and LHR-B6) by semiquantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPD) as the internal control. Follicles were dissected, measured with a paquimeter, and allocated in 3 groups according to follicular diameter (mm): A (8.0-9.0), B (9.1-10.0), and C (10.1-11.0). Considering that follicles measured with paquimeter are on average 1.0 mm larger than those measured by US, Groups A, B, and C correspond to Groups G1, G2, and G3 (Experiment 1). In order to select only nonatretic (healthy) follicles, the E2/P4 >1.0 ratio was used. Therefore, from a total of 400 ovaries, only 5, 4, and 4 granulosa (n = 13) and 7, 8, and 8 theca samples (n = 23) from Groups A, B, and C, respectively, were obtained. The data were analyzed by ANOVA and Pearson’s correlation. There were no significant differences in total LHR expression (LHR-B3 + LHR-B4 + LHR-B5 + LHR-B6) in theca cells from Groups A, B, and C. However, in granulosa cells, follicles from Group A had lower LHR expression (16.5; mRNA LHR/mRNA GAPD) compared with Group C (37.6; P < 0.05). There was a positive correlation between expression of LHR-B5 and LHR-B6 isoforms and an increase in follicular diameter. In conclusion, these preliminary results indicate that ovulatory capacity in Nellore cattle is related to an increase in follicular diameter and LHR expression in granulosa cells. R. A. L. Simões, R. A. Satrapa, and A. C. S. Castilho are recipients of fellowship and funding from FAPESP (São Paulo, Brazil).

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Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes

Reproduction ◽

10.1530/rep.0.1230819 ◽

2002 ◽

pp. 819-826 ◽

Cited By ~ 13

Author(s):

L Abdennebi ◽

AS Lesport ◽

JJ Remy ◽

D Grebert ◽

C Pisselet ◽

...

Keyword(s):

Breeding Season ◽

Splice Variants ◽

Follicular Development ◽

Receptor Expression ◽

Physiological Control ◽

Lh Receptor ◽

Theca Cells ◽

Follicle Size ◽

Exon 9 ◽

Exon 11

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.

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Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis†

Biology of Reproduction ◽

10.1093/biolre/ioz167 ◽

2019 ◽

Author(s):

P Asiabi ◽

E C R Leonel ◽

E Marbaix ◽

M M Dolmans ◽

C A Amorim

Keyword(s):

Cytochrome P450 ◽

Luteinizing Hormone ◽

Granulosa Cells ◽

Hydroxysteroid Dehydrogenase ◽

Expression Intensity ◽

Theca Cells ◽

Perilipin 2 ◽

Cytochrome P450c17

Abstract The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2, play a role in the cascade after luteinizing hormone–choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 μm), and large (>3000 μm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.

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Hormonal and Nutritional Stretegies to Optimize Reproductive Function and Improve Fertility of Dairy Cattle during Heat Stress in Summer

10.32747/1994.7568773.bard ◽

1994 ◽

Author(s):

David Wolfenson ◽

William W. Thatcher ◽

Rina Meidan ◽

Charles R. Staples ◽

Israel Flamenbaum

Keyword(s):

Heat Stress ◽

Pregnancy Rate ◽

Granulosa Cells ◽

Gnrh Agonist ◽

Follicular Development ◽

Low Fertility ◽

Small Scale ◽

Plasma Progesterone ◽

Theca Cells ◽

Days Open

The BARD program includes two main parts. In the first, experiments were conducted to complete our understanding of the mechanisms responsible for the impairment of reproductive functions under heat stress. Experiments focused on follicular development and function, since results obtained in our previous BARD project indicate that the preovulatory follicle is susceptible to heat stress. The theca cells, sensitive to thermal stress, produced less androgen during the summer, as well as during the autumn. Similarly, luteinized theca cells obtained from cows in summer produced much less progesterone than in winter. Granulosa cells and luteinized granulosa cells were less susceptible to heat stress. A delayed effect of heat stress on follicular development, on suppression of dominance and on steroid production by theca and granulosa cells was noted. This may be related to the low fertility of cows during the cool months of autumn. In the second part, experiments were conducted aiming to improve fertility in summer. The timed AI program was developed using two injections of GnRH coupled with PGF2a. It was found effective in improving reproductive performance in lactating cows. Limitations induced by heat stress on estrus detection were eliminated with the timed AI management program. Replacing the second injection of GnRH with hCG instead of GnRH agonist increased plasma progesterone levels post ovulation but did not improve fertility. Use of the timed AI program in summer, shortened days open and increased the net revenue per cow, however, it did not protect the embryo fiom temperature-induced embryonic mortality. Incorporation of a GnRH-agonist implant into the timed AJ program was examined. The implant increased plasma progesterone and LH concentrations and altered follicular dynamics. The use of a GnRH-implant enhanced pregnancy rate in cows with low body conditions. In a timed embryo transfer experiment, the use of fresh or frozen in vitro produced embryos was compared in the summer to improve fertility. The use of flesh embryos (but not frozen ones) improved pregnancy rate, however, substantial embryonic death occurred between 21 and 45 days. The timed AI program, which is now being used commercially, shortened days open, and increased pregnancy rate during summer. Other approaches which were found to improve fertility in small-scale studies, need to be tested again in large-scale field trials.

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