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R L Robker

Publications and source records attributed to R L Robker.

10 recordsLinked to original sources
PubMed2000

Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases.

Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and/or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.

ADAM Proteins↗
PubMed2000

Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat.

Mammalian ovulation is a dynamic process that requires degradation of the collagenous connective tissue in the thecal layers of a mature follicle. In this reverse transcription-polymerase chain reaction differential display study, gonadotropin-primed immature rats were used to detect ovarian expression of a relatively new type of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) that is known to cleave extracellular matrix in acutely inflamed tissues. Immature Wistar rats were primed with 10 IU eCG s. c., and the temporal pattern of expression of the ADAMTS-1 gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation by injecting the primed animals with 10 IU hCG s.c. The differential display data, Northern analyses, and in situ hybridization micrographs all showed significant up-regulation of ADAMTS-1 gene expression by 8 h after hCG administration. The in situ data indicated that the ADAMTS-1 mRNA was in the granulosa layer of mature follicles. Expression reached a peak at 12 h and remained elevated at 24 h after hCG. ADAMTS-1 gene expression was impaired by the antiprogesterone agent epostane, but this inhibition could be overcome by exogenous progesterone. ADAMTS-1 expression was not affected when ovulation was blocked by treatment of the animals with the anti-eicosanoid agent indomethacin. In conclusion, the temporal pattern of expression of this gene, and its apparent regulation by progesterone, suggests that ADAMTS-1 has a significant role in the inflammatory events of the ovulatory process.

ADAM Proteins↗
PubMed2000

Induction of early growth response protein-1 gene expression in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin.

Granulosa cells in a mature ovarian follicle have an abundance of LH/hCG receptors that respond rapidly to an ovulatory surge in gonadotropins. Within minutes, membrane signal transduction sets in motion metabolic changes that lead to follicular rupture. This study provides evidence that the initial ovarian response to such an ovulatory stimulus includes induction of the immediate-early transcription factor gene for early growth response protein-1 (Egr-1). Immature Wistar rats were primed with 10 IU equine CG (eCG), sc, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG, sc. Ovarian RNA was extracted at 0, 0.5, 1, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display for random detection of gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified complementary DNAs confirmed that it was part of a gene that was significantly up-regulated within 1 h after the ovaries had been stimulated by hCG. Maximum transcription was at 4 h after hCG, and expression declined to 0 h control levels by 24 h after hCG. Subcloning and sequence analysis revealed that the complementary DNA matched the gene for Egr-1. In situ hybridization indicated that the Egr-1 messenger RNA was in the granulosa layer of mature follicles. Western blotting confirmed the temporal pattern of Egr-1 expression detected by differential display, Northern analysis and in situ hybridization. The Egr-1 protein is approximately 84 kDa. In conclusion, the data show that expression of the zinc finger transcription factor Egr-1 is an early event in the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone.

Androstenols↗
PubMed1998

Molecular mechanisms of ovulation and luteinization.

Ovulation is a complex process initiated by the mid-cycle surge of luteinizing hormone (LH). Once initiated, a cascade of events occurs that culminates in the release of a fertilizable oocyte. The complex series of events involves specific ovarian cell types, diverse signaling pathways and temporally controlled expression of specific genes. This review will focus on several genes shown to control the ovulation process.

Animals↗
PubMed1998

Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1.

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.

Animals↗
PubMed1998

Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol.

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.

Adenosine Monophosphate↗
PubMed1997

Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge.

Aromatase (CYP19) mRNA is induced by follicle-stimulating hormone (FSH) in granulosa cells of preovulatory follicles and subsequently is rapidly diminished as a consequence of the luteinizing hormone (LH) surge. Primary cultures of rat granulosa cells were used to identify some of the cellular mechanisms by which FSH increases and LH decreases steady-state levels of aromatase mRNA. Induction of aromatase mRNA by FSH was increased by cycloheximide but was blocked by alpha-amanitin and the C-kinase activators gonadotropin-releasing hormone (GnRH) and phorbol 12-myristate 13-acetate (PMA). In contrast, the decrease in steady-state levels of aromatase mRNA by LH was mimicked by A-kinase (forskolin) and C-kinase (PMA or GnRH) activators. The decrease in aromatase mRNA was associated with decreased amounts of mRNA and protein for steroidogenic factor-1 (SF-1), a nuclear orphan receptor that binds and trans-activates the aromatase promoter, and with the A-kinase subunit type II (RII beta), which is required for mediating cAMP action in these cells. The down-regulation of aromatase, SF-1, and RII beta by each kinase activator and alpha-amanitin was prevented by cycloheximide when the drug was added in combination with the activator. If, however, cycloheximide was added 2 h after PMA (or LH), the drug did not prevent the rapid loss of mRNA. When granulosa cells were transfected with an aromatase CAT transgene, CAT activity was stimulated 10- to 20-fold by FSH and forskolin but not by PMA. Taken together, these results indicate that the A-kinase but not the C-kinase pathway can trans-activate the aromatase gene in immature granulosa cells, whereas the C-kinase, as well as A-kinase pathways, mimic the LH surge to decrease aromatase mRNA in preovulatory cells. By increasing degradation of aromatase mRNA and by inhibiting transcription, the LH surge rapidly terminates the granulosa cell pattern of gene expression while reprogramming the cells to express genes associated with ovulation and luteinization.

Amanitins↗
PubMed1996

Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Animals↗
PubMedDate not supplied

Ovulation: a multi-gene, multi-step process.

The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.

ADAM Proteins↗