Mark P. McLean

Placental-derived regulators and the complex control of luteal cell function.

Publisher Summary The corpus luteum is a transient ovarian endocrine gland that results from a rapid growth and differentiation of follicular theca and granulosa cells. Several types of corpora lutea are formed during the reproductive life of the female rat. They differ in size, life span, and steroidogenic output. While corpora lutea of the cycle and pseudopregnancy do not have a clearly defined role, the corpus luteum of pregnancy is absolutely crucial for normal fetal development and survival in every mammalian species. In the rat, this gland is initially the same size as that of pseudopregnancy; however, instead of involuting, it increases in size and in steroidogenic capacity and remains functional for approximately 20 days. The capacity of this corpus luteum to secrete progesterone and to sustain a normal pregnancy is tightly regulated by hormones generated by the pituitary, the placenta, and by itself. This chapter reviews the hormonal interrelationships that regulate the corpus luteum of pregnancy in the rat, focusing primarily on placental secretion of androgen and the role of placental steroids in luteal steroidogenesis and growth.

STEROL REGULATORY ELEMENT-BINDING PROTEIN-1A BINDS TO CIS ELEMENTS IN THE PROMOTER OF THE RAT HIGH DENSITY LIPOPROTEIN RECEPTOR SR-BI GENE

The high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), is critical for cholesterol transport and a potential target for hypercholesterolemic drugs. Thus, elucidation of the mechanism underlying regulation of the HDL receptor SR-BI gene is essential. It has been previously shown that there is a correlation between depletion in ovarian cholesteryl ester content and increased HDL receptor SR-BI expression in response to hormonal stimulation. We wanted to determine whether the levels of mature sterol response element-binding protein-1a (SREBP-1a), a key protein in the transcriptional regulation of several genes by sterols, are affected under these conditions. Thus, Western blot analysis was carried out. Consistent with the possibility that SREBP-1a may be involved in the regulation of the HDL receptor SR-BI gene, we found that mature SREBP-1a levels increased up to 11-fold in the ovary after treatment with 50 U hCG. This increase in mature SREBP-1a protein levels correlated...

Transcriptional repression of the rat steroidogenic acute regulatory (StAR) protein gene by the AP-1 family member c-Fos.

PGF2alpha, working via protein kinase C, may inhibit transcription of the StAR gene through negative regulatory factors. Administration of PGF2alpha increased c-Fos mRNA with a corresponding reduction in StAR mRNA. A search of the rat StAR promoter revealed three putative AP-1 elements at bp positions -85, -187 and -1561, which demonstrated specific binding of c-Fos by mobility shift assays. Co-transfection of c-Fos with the p-1862 StAR promoter caused a reduction in luciferase activity in the presence or absence of cAMP. Mutation of all three AP-1 sites in the p-1862 StAR promoter abolished c-Fos repression. Mutation of the proximal AP-1 site in the p-1862 StAR promoter reduced SF-1 mediated induction. This study is the first to demonstrate that c-Fos represses StAR gene transcription and adds to the current knowledge on the complex relationship that exists between SF-1 and c-Fos in the regulation of StAR activity.

Repression of the rat steroidogenic acute regulatory (StAR) protein gene by PGF2α is modulated by the negative transcription factor DAX-1

The steroidogenic acute regulatory protein (StAR) is thought to mediate the rapid increase in steroid hormone biosynthesis by facilitating cholesterol transport to the inner mitochondrial membrane. Recent studies indicate that StAR gene expression is enhanced by gonadotropins, whereas prostaglandin F2α (PGF2α) appears to suppress both basal and gonadotropin-stimulated StAR mRNA levels. While studies have dem-onstrated that steroidogenic factor 1 (SF-1) mediates transcriptional activation of the StAR gene, the mechanism for the reduction in StAR expression requires analysis. Recent studies have shown that DAX-1 (Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X-chromosome, gene-1), a negative transcription factor, inhibits transcription of reporter genes in vitro. To determine whether DAX-1 could negatively regulate expression of the StAR gene, approx 2 kb of the rat StAR promoter was linked to a luciferase reporter gene (creating p-1862 StAR) and cotransfected into Y1 adrenal tumor cells and HTB9 human bladder carcinoma cells with vectors which encode DAX-1 and SF-1. Luciferase levels were significantly increased in both cell types when SF-1 was present. In contrast, when DAX-1 was cotransfected with the StAR promoter, Y1 adrenal and HTB9 cell luciferase activities were reduced to levels that were 57% and 24% of basal promoter levels, respectively. Furthermore, when dibutyryl-cAMP (dbcAMP) was added to the DAX-1 expressing cells, cAMP responsiveness was repressed 50% and 75% in Y1s and HTB9s respectively, relative to the non-DAX-1 expressing dbcAMP-treated cells. The inhibition of StAR gene transcription by DAX-1 was dose-dependent reducing transcription to 6% of control levels. Consistent with the possibility that PGF2α regulates ovarian StAR expression via DAX-1, Western blot analysis indicated a three- and fivefold increase in rat ovarian DAX-1 levels at 2 and 4 h following PGF2α injection (250 μg). The increase in DAX-1 protein corresponded to a 50% reduction in StAR mRNA levels concomitant with a 39% reduction in serum progesterone levels. Truncation of the DAX-1 protein at the C-terminal end caused a loss of inhibition of trans-criptional activity. Deletion of bp-95 to-50 within the StAR promoter, a proposed DAX-1 binding site, did not alter the ability of wild-type DAX-1 to inhibit transcription. In a mammalian two-hybrid system, cotransfection of DAX-1 and SF-1 caused a 25-fold induction in luciferase activity demonstrating that these proteins interact in the two-hybrid assay. This study is the first to demonstrate that the rat StAR promoter is regulated by DAX-1 and that DAX-1 reduces StAR promoter responsiveness to cAMP. The enhanced level of DAX-1 following PGF2α administration is consistent with DAX-1 having a role in controlling both basal, gonadotropin-stimulated, and PGF2α-mediated StAR gene expression. These results imply that DAX-1 has an important role in regulating ovarian steroidogenesis by repressing StAR transcription.

Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary.

Steroidogenic acute regulatory (StAR) protein is thought to mediate the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating cholesterol transport to the inner mitochondrial membrane where the P450 side-chain cleavage enzyme (P450scc) is located. Since cholesterol delivery is the regulated step in steroidogenesis and is dependent onde novo protein synthesis, StAR mRNA levels were examined in response to the tropic hormones, pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The results of this investigation revealed that major StAR mRNA transcripts of 3.4 and 1.6 kb and a less abundant transcript of 1.2 kb were detected in the adrenal, ovary, and testis. Within the ovary, StAR mRNA levels were regulated by PMSG and hCG. The two major transcripts were increased in the immature rat ovary following PMSG administration and in the ovary, 8 d after ovulation, in response to stimulation by hCG. Serum progesterone levels were increased following hCG treatment in parallel with the enhanced expression of StAR. Following PMSG treatment, ovarian StAR transcripts at 3.4 and 1.6 kb were each increased twofold. In the ovary, 8 d following ovulation, basal ovarian StAR mRNA levels were elevated up to sixfold relative to the preovulatory StAR mRNA levels. Even with the enhanced basal level of StAR mRNA within the ovary 8 d postovulation, hCG administration still resulted in a 2.5- and 7-fold increase in the 3.4 and 1.6 kb (p<0.025) transcripts, respectively, and a 58% increase in serum progesterone. In contrast to the dramatic alterations in StAR mRNA expression following hormonal stimulation, P450scc mRNA levels remained unchanged in response to hCG stimulation. The levels of serum progesterone paralleled the change in ovarian StAR mRNA in all experiments. This study provides the first evidence that StAR mRNA expression in the rat ovary is mediated by gonadotropins, further supporting its important role in the regulation of steroid hormone biosynthesis.

Effects of cigarette smoke on fertilization and embryo development in vivo

OBJECTIVE To determine the effects of smoking on eggs and subsequent embryo development by maternal exposure to cigarette smoke. DESIGN Mice were exposed to cigarette smoke or cigarette smoke condensate (CSC) for 4 weeks and then examined for development and telomere function of embryos in vitro after fertilization. In addition, the effects of continuous smoke on embryo development and telomere length were determined by treating mice for 4 weeks, followed by continuous exposure to cigarette smoke or CSC after fertilization. SETTING Laboratory study. ANIMAL(S) CD1 mice. INTERVENTION(S) Mice were exposed to cigarette smoke or CSC. MAIN OUTCOME MEASURE(S) The percentage (rate) of blastocyst development, quality of embryos assessed by total cell number, apoptosis, Oct4 expression (a molecular marker of embryonic stem cells), telomere length and loss, and chromosomal instability were compared between smoke- and CSC- treated mice and sham-treated mice. RESULT(S) Mice exposed to cigarette smoke or CSC for 4 weeks exhibited increased egg fragmentation or delayed fertilization, thus reducing development to blastocysts in vitro. Fragmented eggs showed increased reactive oxygen species. Mice exposed to smoke or CSC showed increased apoptosis and altered expression of Oct4 in developed embryos. The effects of smoke or CSC on embryo development showed a dose-dependent relationship to exposure time. Exposure to smoke or CSC beginning 4 weeks before fertilization altered expression of Oct4 and increased apoptosis in blastocysts. Notably, the rate of abnormal embryos significantly increased in the smoke and CSC groups. Smoke and CSC shortened telomeres in embryos, but their telomere shortening was not enough to induce major chromosome abnormalities in mice, which have unusually long telomeres. CONCLUSION(S) Together, the whole animal exposure model shows that cigarette smoke induces oxidative stress, telomere shortening, and apoptosis, and compromises embryo development in vivo.

Hormone and prostaglandin F2α regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in human corpora lutea

Steroidogenic acute regulatory (StAR) protein mediates the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating transport of cholesterol into the inner mitochondrial membrane. Although our laboratory has recently reported on the hormonal regulation of StAR mRNA in the rat ovary, the same regulation in the human corpus luteum requires analysis. To this end, a human StAR complementary DNA (cDNA) probe of 858 bp was generated using reverse transcriptase-PCR and RNA from human corpora lutea. The StAR sequence was confirmed by dideoxy chain-termination sequence analysis. Northern blot analysis using the StAR cDNA probe on human corpora lutea mRNA showed that the probe hybridized to a major 1.6-kb transcript and a minor 4.4-kb transcript. Examination of corpora lutea of different luteal phases revealed that the basal expression of the 1.6-kb transcript was significantly more abundant in the early (days 15–19) luteal phase than in the middle (days 20–23) or late (days 24–28) phases. To examine the hormonal regulation of StAR mRNA, corpora lutea were treated in vitro with increasing concentrations of human chorionic gonadotropin (hCG) or prostaglandin F2α (PGF2α). Following hCG stimulation, both 1.6- and 4.4-kb StAR transcripts were increased. A statistically significant increase of 2.2- and 1.8-fold in the 1.6-kb transcript was seen with hCG concentrations of 50 and 100 mIU/mL, respectively. This increase was coupled with a significant elevation in media progesterone levels. In contrast, PGF2α treatment significantly decreased both StAR messenger ribonucleic acid (mRNA) expression and media progesterone levels at concentrations of 500 and 5000 ng/mL. This investigation demonstrated that StAR mRNA is regulated by tropic hormones and prostaglandins in the human corpus luteum. The parallel change in StAR mRNA in conjunction with a change in progesterone levels further supports StAR’s putative role in the regulation of steroidogenesis.

Telomere shortening and DNA damage of embryonic stem cells induced by cigarette smoke

Embryonic stem cells (ESCs) provide a valuable in vitro model for testing toxicity of chemicals and environmental contaminants including cigarette smoke. Mouse ESCs were acutely or chronically exposed to smoke components, cigarette smoke condensate (CSC), or cadmium, an abundant component of CSC, and then evaluated for their self-renewal, apoptosis, DNA damage and telomere function. Acute exposure of ESCs to high dose of CSC or cadmium increased DNA damage and apoptosis. Yet, ESCs exhibited a remarkable capacity to recover following absence of exposure. Chronic exposure of ESCs to low dose of CSC or cadmium resulted in shorter telomeres and DNA damage. Together, acute exposure of ESCs to CSC or cadmium causes immediate cell death and reduces pluripotency, while chronic exposure of ESCs to CSC or cadmium leads to DNA damage and telomere shortening. Notably, a sub-proportion of ESCs during passages is selected to resist to smoke-induced oxidative damage to telomeres.

ELECTRON TRANSFER FROM CYTOCHROME B5 TO CYTOCHROME C

The reaction of cytochromeb5 with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.

Expression and hormonal regulation of the high-density lipoprotein (HDL) receptor scavenger receptor class B type I messenger ribonucleic acid in the rat ovary.

Since cholesterol delivery to the ovary is an essential regulated step in steroidogenesis, mRNA levels for the Scavenger Receptor Class B Type I (SR-BI), a putative high-density lipoprotein receptor (HDL-R), were examined in response to tropic hormones and the luteolytic agent prostaglandin F2α (PGF2α). For this, the rat SR-BI cDNA was isolated and cloned. The results of this investigation revealed that a single SR-BI mRNA transcript of 2.4 kb was highly expressed in the rat adrenal, ovary, and testis. The SR-BI transcript was increased (twofold) in the immature rat ovary following pregnant mare’s serum gonadotropin (PMSG) administration and in the ovary, 8 d after ovulation, in response to stimulation by human chorionic gonadotropin (hCG). In the ovary 8 d following ovulation, basal ovarian SR-BI mRNA levels were elevated up to sixfold relative to the preovulatory SR-BI mRNA levels. Even with the enhanced basal level of SR-BI mRNA within the ovary, hCG administration still resulted in a 2.5- (p<0.025) and sevenfold (p<0.01) increase in the 2.4-kb transcript, 3 and 6 h postinjection, respectively. This increase corresponded to a 58% increase in serum progesterone. In contrast, when PGF2α was administered, SR-BI mRNA levels were significantly reduced (3.5-fold; p<0.01) in concert with a fourfold reduction (p<0.001) in serum progesterone secretion. Furthermore, PGF2α blocked the hCG-induced increase in SR-BI mRNA levels when administered 30 min prior to hCG injection. The results of this study demonstrate that SR-BI mRNA levels are dramatically increased following exposure to gonadotropins in the ovary, whereas PGF2α exposure significantly reduced SR-BI mRNA levels.