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

1999 ◽  
Vol 11 (6) ◽  
pp. 337 ◽  
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.

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

Reproduction ◽  
2002 ◽  
pp. 651-661 ◽  
Author(s):  
HA Garverick ◽  
G Baxter ◽  
J Gong ◽  
DG Armstrong ◽  
BK Campbell ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Gnrh Agonist ◽  
Follicular Growth ◽  
Steroidogenic Enzymes ◽  
Lh Receptor ◽  
Theca Cells ◽  
Follicular Wave ◽  
Follicle Size ◽  
Bovine Somatotrophin

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.

scholarly journals ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells

2017 ◽  
Vol 312 (3) ◽  
pp. C341-C353 ◽  
Author(s):  
Yongjie Xiong ◽  
Huatao Chen ◽  
Pengfei Lin ◽  
Aihua Wang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Er Stress ◽  
Granulosa Cells ◽  
Physiological Function ◽  
Steroid Hormone ◽  
Follicular Development ◽  
S Phase ◽  
Mrna Levels ◽  
Hormone Production ◽  
Protein Levels

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 ( Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein 4 ( Igfbp4) expression and decreased hyaluronan synthase 2 ( Has2), prostaglandin-endoperoxide synthase 2 ( Ptgs2), and prostaglandin F receptor ( Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

scholarly journals Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 969-981 ◽  
Author(s):  
Padmasana Singh ◽  
Amitabh Krishna ◽  
Rajagopala Sridaran
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Cell Types ◽  
Physiological Significance ◽  
Rt Pcr ◽  
Releasing Hormone ◽  
Theca Cells ◽  
Gonadotrophin Releasing Hormone

GnRH I and its receptors have been demonstrated in the ovaries of various vertebrates, but their physiological significance in reproductive cascade is fragmentary. Bradykinin is a potent GnRH stimulator in the hypothalamus. In the present study, the presence of GnRH I and its receptor, and bradykinin and its receptor in the ovaries of non-mammalian vertebrates were investigated to understand their physiological significance. GnRH I immunoreactivity in the ovaries of fish, frog, reptile and bird were mainly found in the oocyte of early growing follicles and granulosa cells and theca cells of previtellogenic follicles. Vitellogenic follicles showed mild GnRH immunoreactivity. GnRH I-receptor and bradykinin were localized in the same cell types of the ovaries of these vertebrates. The presence of GnRH I, GnRH I-receptor and bradykinin in the ovaries of these vertebrates was confirmed by immunoblotting. The presence of GnRH I mRNA was demonstrated in the ovary of vertebrates using RT-PCR. The ovaries of reptiles and birds showed significantly higher intensity of immunoreactivity for GnRH I-receptor as compared with the fish and amphibian. This may have a correlation with the higher yolk content in the ovary of reptile and bird. These results suggest the possibility of GnRH I and bradykinin as important regulators of follicular development and vitellogenesis in the vertebrate ovary.

scholarly journals Enhanced serum oestrogen levels and highly steroidogenic, luteinized atretic follicles in the ovaries of the Djungarian hamster (Phodopus sungorus) kept under a short photoperiod from birth

2002 ◽  
pp. 701-710 ◽  
Author(s):  
R Van Den Hurk ◽  
G Dijkstra ◽  
FH De Jong
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
High Serum ◽  
Short Photoperiod ◽  
Phodopus Sungorus ◽  
Hormone Production ◽  
Serum Progesterone ◽  
Antral Follicles ◽  
Light Regimes ◽  
Design And Methods

OBJECTIVE: In contrast to the elaborate information available on the effects of the photoperiod on the testes of hamsters, little is known about the influence on their ovaries. This study aimed to describe the ovarian follicular development and steroid hormone production in Djungarian hamsters kept from birth under a short daylight regime. DESIGN AND METHODS: Female Djungarian hamsters (Phodopus sungorus) were kept under two different light regimes: (i) 16 h light:8 h darkness (long daylight; LD) and (ii) 4 h light:20 h darkness (short daylight; SD). They were killed at 28, 56 and 80 days after birth; blood and ovaries were collected. Ovaries were either fixed in Bouin's solution or frozen. Fixed material was dehydrated, embedded in paraffin, serially sectioned at 5 microm and stained with haematoxylin and eosin, whereafter all healthy and atretic follicles were classified and counted. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) was histochemically demonstrated in 10 microm sections of frozen ovaries. Serum oestradiol-17beta and progesterone levels were determined by RIA. RESULTS: The numbers of healthy preantral and antral follicles were higher in LD than in SD hamsters. Antral follicles did not significantly differ in number during development in LD hamsters, but they were completely absent from 80-day-old SD animals. In LD animals the number of apoptotic preantral follicles dramatically increased with age. In SD animals the numbers of apoptotic antral follicles strongly decreased with age, whereas numerous non-apoptotic follicles with luteinized granulosa cells and a degenerated oocyte appeared, and in increasing numbers with age. During development, moderate 3beta-HSD activity was present in interstitial cells, theca cells of healthy follicles, and in both theca and granulosa cells of degenerating follicles. Strong enzyme activity was found in the hypertrophied granulosa cells of luteinized atretic follicles. Mean serum progesterone values varied from 2 to 6 nmol/l and were not different in LD and SD hamsters. Mean serum oestradiol levels varied from 132 to 542 and 325 to 2353 pmol/l in LD and SD hamsters respectively. The highest oestradiol levels were found in SD animals at day 28 of development. CONCLUSIONS: Folliculogenesis was dramatically disturbed in Djungarian hamsters raised under a short photoperiod. These animals developed high serum oestradiol levels and numerous luteinized atretic follicles with highly steroidogenic granulosa cells, which appear to be the source of the increased serum oestradiol levels.

scholarly journals Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Mélodie Diot ◽  
Maxime Reverchon ◽  
Christelle Ramé ◽  
Yannick Baumard ◽  
Joëlle Dupont
Keyword(s):  
Granulosa Cells ◽  
Specific Inhibitor ◽  
Follicular Development ◽  
Rt Pcr ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Theca Cells ◽  
Protein Levels ◽  
Progesterone Secretion ◽  
Ovulatory Follicles

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

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

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.

scholarly journals The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 329-339 ◽  
Author(s):  
Leon J Spicer ◽  
Satoko Sudo ◽  
Pauline Y Aad ◽  
Lora Shuo Wang ◽  
Sang-Young Chun ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Mrna Abundance ◽  
Rt Pcr ◽  
Theca Cells ◽  
Qrt Pcr ◽  
Hedgehog Proteins ◽  
Patched 1 ◽  
And Function ◽  
First Time

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarianIhhandDhhtranscripts decreased 4–24 h after hCG versus 0–48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mousePtch1andSmotranscripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system withIhhmRNA abundance in granulosa cells was greater, whereasSmoandPtch1mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance ofGli1andPtch1mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreasedIhhmRNA abundance in bovine granulosa cells. The expression and regulation ofIhhtranscripts in granulosa cells andPtch1mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

Development of the membrana granulosa of bovine antral follicles: structure, location of mitosis and pyknosis, and immunolocalization of involucrin and vimentin

1999 ◽  
Vol 11 (1) ◽  
pp. 37 ◽  
Author(s):  
I. L. van Wezel ◽  
R. J. Rodgers ◽  
M. Krupa
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Follicular Development ◽  
Staining Intensity ◽  
Keratinocyte Differentiation ◽  
Follicular Epithelium ◽  
Intermediate Filament Protein ◽  
Basal Cells ◽  
Filament Protein ◽  
Dying Cells

The membrana granulosa of the ovarian follicle is termed the ‘follicular epithelium’, yet there have been no studies considering its epithelial nature and how it changes during follicular development. Therefore, these issues were investigated using histology (n = 45 ovaries), considering its structure and the location of proliferating and dying cells, and drawing analogies with other epithelia. Additionally, differences between the layers of granulosa cells were demonstrated by immunohistochemistry (n =7 ovaries). The structure of the membrana granulosa differed between follicles. Six arbitrary classifications were designed based on these structures, 80 follicles were allocated (n = 13 ovaries) to these classes and the follicular diameters were then measured. For the first time, differences in membrana granulosa structure were shown to correspond to follicle size. Follicles in classes 1–3, where basal granulosa cells were columnar with nuclei positioned basally in the cell, were all ≤3 mm in diameter. All follicles larger than 3 mm had either columnar basal cells with nuclei positioned centrally (class 4), or had rounded basal cells (class 5), and all follicles >5 mm had only rounded basal cells. In all these classes, cells in the middle zone were rounded; cells aligning the antrum were often flattened. Irrespective of follicle class, cell proliferation and cell death were shown to be predominantly in the middle portions, rather than the most antral or most basal portions, of the membrana granulosa of healthy and atretic follicles. Involucrin, a marker of keratinocyte differentiation, was localized to the suprabasal region of the membrana granulosa of healthy follicles, particularly in the second and third cellular layers in from the follicular basal lamina. Conversely, the staining intensity for the intermediate filament protein vimentin was lowest in this region, and greatest in the more antral and basal regions. In atretic follicles, there was widespread staining for involucrin and vimentin throughout the membrana granulosa. In conclusion, the membrana granulosa is highly structured, and alters with follicular development. Layers in the membrana granulosa can differ in terms of cell shape, and differ in proliferation and gene expression. In the light of the current work, and an associated study, it is proposed that proliferation occurs in the middle layers, and that granulosa cells then progress basally or antrally, the latter undergoing terminal differentiation.

scholarly journals Liver Receptor Homolog-1 Regulates the Transcription of Steroidogenic Enzymes and Induces the Differentiation of Mesenchymal Stem Cells into Steroidogenic Cells

Endocrinology ◽  
2009 ◽  
Vol 150 (8) ◽  
pp. 3885-3893 ◽  
Author(s):  
Takashi Yazawa ◽  
Yoshihiko Inanoka ◽  
Tetsuya Mizutani ◽  
Mayu Kuribayashi ◽  
Akihiro Umezawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Leydig Cells ◽  
Steroid Hormone ◽  
Vital Role ◽  
Human Mscs ◽  
Steroidogenic Enzymes ◽  
Hormone Production ◽  
Steroidogenic Cells ◽  
Immunohistochemical Studies

Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.

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

Reproduction ◽  
2002 ◽  
pp. 819-826 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document