scholarly journals Expression and Localization of Serum/Glucocorticoid-Induced Kinase in the Rat Ovary: Relation to Follicular Growth and Differentiation1

Endocrinology ◽  
2000 ◽  
Vol 141 (1) ◽  
pp. 385-395 ◽  
Author(s):  
Tamara N. Alliston ◽  
Ignacio J. Gonzalez-Robayna ◽  
Patricia Buse ◽  
Gary L. Firestone ◽  
JoAnne S. Richards
Keyword(s):  
In Situ Hybridization ◽  
Subcellular Localization ◽  
Granulosa Cells ◽  
Corpora Lutea ◽  
Pregnant Rats ◽  
Western Blots ◽  
Stromal Compartment ◽  
Luteal Cells ◽  
Preovulatory Follicles

Abstract Expression of serum/glucocorticoid-inducible kinase (Sgk), one member of an inducible serine/threonine kinase family, is induced by FSH/cAMP in rat granulosa cells cultured in defined medium. The FSH-stimulated pattern of sgk expression is biphasic, and transcriptional activation of the sgk gene depends on an intact Sp1/Sp3 binding site within the proximal promoter. To determine whether sgk was expressed in a hormone-dependent and physiologically relevant manner in vivo, the cellular levels of sgk messenger RNA (mRNA) and protein as well as the subcellular localization of this kinase were analyzed in ovaries containing follicles and corpora lutea at specific stages of differentiation. To stimulate follicular development and luteinization, hypophysectomized (H) rats were treated with estradiol (E; HE) and FSH (FSH; HEF) followed by hCG (hCG; HEF/hCG). To analyze Sgk in functional corpora lutea, PRL was administered to HEF/hCG rats, or ovaries of pregnant rats were obtained on day 7, 15, or 22 of gestation. In situ hybridization indicated that sgk mRNA was low/undetectable in granulosa cells of H and HE rats. An acute injection (iv) of FSH to HE rats rapidly increased sgk mRNA at 2 and 8 h. Sgk mRNA was also elevated in granulosa cells of preovulatory follicles of HEF rats and in luteal cells of HEF/hCG and pregnant rats. Northern blots and Western blots confirmed the in situ hybridization data, indicating that the amount and cellular localization Sgk protein were related to that of sgk mRNA. When the subcellular localization of this kinase was analyzed by immunohistochemistry, Sgk protein was nuclear in granulosa cells and some thecal cells of large preovulatory follicles. In contrast, Sgk protein was cytoplasmic in luteal cells as well as some cells within the stromal compartment. Intense immunostaining was also observed in oocytes present in primordial follicles, but not in growing follicles. Collectively, these results show that FSH and LH stimulate marked increases in the cellular content of Sgk, as well as dramatic changes in the subcellular distribution of this kinase. The specific nuclear vs. cytoplasmic compartmentalization of Sgk in granulosa cells and luteal cells, respectively, indicates that Sgk controls distinct functions in proliferative vs. terminally differentiated granulosa cells.

Estrogen Receptor-β mRNA Expression in Rat Ovary: Down-Regulation by Gonadotropins

1997 ◽  
Vol 11 (2) ◽  
pp. 172-182 ◽  
Author(s):  
Michael Byers ◽  
George G. J. M. Kuiper ◽  
Jan-Åke Gustafsson ◽  
Ok-Kyong Park-Sarge
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Adult Rats ◽  
Mature Adult ◽  
Preovulatory Follicles

Abstract We have examined the expression and regulation of the two estrogen receptor (ERα and ERβ) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ERα and ERβ genes as single (∼6.5-kb) and multiple (ranging from ∼1.0-kb to ∼10.0-kb) transcripts, respectively. ERα mRNA is expressed at a level lower than ERβ mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ERβ mRNA levels were decreased. Examination of ERβ mRNA expression at the cellular level, by in situ hybridization, showed that ERβ mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ERβ mRNA was observed in a subset of corpora lutea, and that the decrease in ERβ mRNA during proestrous evening is attributable, at least in part, to down-regulation of ERβ mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ERα mRNA in the ovary. Although whole ovarian content of ERα mRNA was clearly detected by RT-PCR, no apparent modulation of ERα mRNA levels was observed during the estrous cycle. Examination of ERα mRNA expression at the cellular level, by in situ hybridization, showed that ERα mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERβ mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERβ mRNA. In situ hybridization for ERβ mRNA shows that small, growing, and preovulatory follicles express ERβ mRNA in the granulosa cells. The preovulatory follicles contain ERβ mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERβ mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERβ mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERβ mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ERα and ERβ genes, although ERβ is the predominant form of estrogen receptor in the ovary, 2) ERβ mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ERβ mRNA. These results clearly implicate the physiological importance of ERβ in female reproductive functions.

scholarly journals Ovarian characteristics in sheep with multiple fecundity genes

Reproduction ◽  
2017 ◽  
Vol 153 (2) ◽  
pp. 233-240 ◽  
Author(s):  
Kenneth P McNatty ◽  
Derek A Heath ◽  
Zaramasina Clark ◽  
Karen Reader ◽  
Jennifer L Juengel ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Total Mass ◽  
Total Cell ◽  
Ovulation Rate ◽  
Corpora Lutea ◽  
Preovulatory Follicles ◽  
Luteal Tissue ◽  
In The Wild ◽  
The Mean ◽  
The One

Ewes heterozygous for combinations of the Inverdale (FecXI; I+), Booroola (FecB; B+) and Woodlands (FecX2W; W+) mutations have ovulation rates higher than each mutation separately. The aims of the experiments described herein were to examine the ovarian phenotypes in I+B+ and I+B+W+ ewes and to compare these with the appropriate ++ (controls), I+ and BB animals available for this study. The mean ± s.e.m. ovulation rates in the ++ (n = 23), I+ (10), I+B+ (7), I+B+W+ (10) and BB (3) animals were 1.8 ± 0.1, 2.5 ± 0.2, 6.6 ± 1.0, 9.6 ± 0.9 and 9.7 ± 0.9 respectively. The maximum number of granulosa cells per follicle in the ++ and I+ genotypes was accumulated after exceeding 5 mm diameter, whereas in I+B+, I+B+W+ and BB animals, this was achieved when follicles reached >2–3 mm. The number of putative preovulatory follicles, as assessed from those with LH-responsive granulosa cells, 24 h after the induction of luteolysis, was higher (P < 0.01) in the I+B+ and I+B+W+ compared to the ++ and I+ genotypes. The median follicular diameters of these follicles in the ++, I+, I+B+, I+B+W+ and BB genotypes were 6, 5, 3, 3 and 3 mm respectively. The total number of granulosa cells in the putative preovulatory follicles when added together, and total mass of luteal tissue, did not differ between the genotypes. Thus, despite large ovulation rate differences between animals with one or more fecundity genes, the total cell compositions over all preovulatory follicles and corpora lutea, when added together, are similar to that from the one or two such follicles in the wild types.

Localization of ovine follistatin and α and βA inhibin mRNA in the sheep ovary during the oestrous cycle

1994 ◽  
Vol 12 (2) ◽  
pp. 181-193 ◽  
Author(s):  
D J Tisdall ◽  
N Hudson ◽  
P Smith ◽  
K P McNatty
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Indirect Evidence ◽  
Oestrous Cycle ◽  
Corpora Lutea ◽  
Α Subunit ◽  
Ovarian Follicle Development ◽  
Antral Follicles

ABSTRACT The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F2α (PGF2α)-induced follicular phase. Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF2α. Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia. These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.

scholarly journals Expression and Localization of Secreted Frizzled-Related Protein-4 in the Rodent Ovary: Evidence for Selective Up-Regulation in Luteinized Granulosa Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (10) ◽  
pp. 4597-4606 ◽  
Author(s):  
Minnie Hsieh ◽  
Sabine M. Mulders ◽  
Robert R. Friis ◽  
Arun Dharmarajan ◽  
JoAnne S. Richards
Keyword(s):  
Chorionic Gonadotropin ◽  
Granulosa Cells ◽  
Human Chorionic Gonadotropin ◽  
Follicular Development ◽  
Corpora Lutea ◽  
Related Protein ◽  
Rich Domain ◽  
And Function ◽  
Periovulatory Follicles

Secreted frizzled-related protein-4 (sFRP-4) belongs to a family of soluble proteins that have a Frizzled-like cysteine-rich domain and function as modulators of Wnt-Frizzled (Fz) signals. As several Wnts and Fz are expressed at defined stages of follicular development in rodent ovaries, these studies were undertaken to evaluate the hormone-regulated expression and localization of sFRP-4. In the mouse ovary, the expression of sFRP-4 mRNA was up-regulated in granulosa cells of large antral follicles after human chorionic gonadotropin administration and was also elevated in corpora lutea, as determined by RT-PCR and in situ hybridization analyses. In hypophysectomized rat ovaries, sFRP-4 expression was similarly induced by human chorionic gonadotropin and further up-regulated by PRL. PRL also stimulated the secretion of sFRP-4 protein from luteinized rat granulosa cells in culture. Therefore, regulation of sFRP-4 by LH and PRL may be important for modulating Fz-1, which is known to be expressed in periovulatory follicles, and Wnt-4/Fz-4, which are expressed in corpora lutea.

scholarly journals Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization.

1989 ◽  
Vol 37 (1) ◽  
pp. 1-5 ◽  
Author(s):  
G M Jonakait ◽  
M Rosenthal ◽  
J I Morrell
Keyword(s):  
In Situ Hybridization ◽  
Tyrosine Hydroxylase ◽  
Messenger Rna ◽  
Sympathetic Ganglia ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Reserpine Treatment ◽  
Catecholaminergic Cells ◽  
Th Mrna

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.

scholarly journals Relationship between FoxO1 protein levels and follicular development, atresia, and luteinization in the rat ovary

2003 ◽  
Vol 179 (2) ◽  
pp. 195-203 ◽  
Author(s):  
F Shi ◽  
PS LaPolt
Keyword(s):  
Chorionic Gonadotropin ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Follicular Development ◽  
Immunohistochemical Analysis ◽  
Female Rats ◽  
Corpora Lutea ◽  
Protein Levels ◽  
Physiological Processes ◽  
Preovulatory Follicles

FoxO1 is a transcription factor implicated in a growing number of physiological processes, including apoptosis, cell cycle progression, and insulin signaling. Recent findings indicate that FSH and growth factors influence ovarian functions in part through regulation of FoxO1. The present study utilized immunohistochemical analysis to determine the ovarian localization and regulation of FoxO1 protein levels in neonatal rats, immature rats during gonadotropin-induced follicular development, ovulation, and luteinization, and in spontaneously developing ovarian cysts of aging rats. In postnatal rats, FoxO1 immunoreactivity was very faint in ovaries of 5- and 10-day-old females. In contrast, strong immunoreactivity was observed in granulosa cells of larger developing follicles at 25 days of age. To stimulate follicle development, immature female rats received equine chorionic gonadotropin (eCG) followed 52 h later by an ovulatory dose of human chorionic gonadotropin (hCG). Prior to gonadotropin treatment, moderate FoxO1 immunoreactivity was observed in granulosa cells of small follicles. Subsequently, treatment with eCG markedly decreased FoxO1 protein levels in granulosa cells of healthy antral and preovulatory follicles. Interestingly, FoxO1 staining was observed in cumulus and antral, but not mural granulosa cells of preovulatory follicles. Induction of ovulation and luteinization with hCG further decreased ovarian FoxO1 levels, with no staining evident in corpora lutea. At all time points, the most intensive FoxO1 staining was observed in granulosa cells of atretic follicles, with predominantly nuclear localization. Similarly, while FoxO1 levels were low in granulosa cells of preovulatory follicles in proestrous rats, FoxO1 staining was intense in granulosa cells of spontaneously developing cystic follicles in aged, acyclic females. Together, these findings indicate that FoxO1 is expressed in a regulated, cell-specific manner during ovarian follicular development, atresia and luteinization, suggesting roles in these physiological processes.

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