Characterization of prohibitin in a newly established rat ovarian granulosa cell line.

Prohibitin is an evolutionary conserved protein that is associated with cellular differentiation, atresia, and luteolysis in the rat ovary. However, the specific cellular location and function of prohibitin in ovarian cells has not been clearly elucidated. To characterize the expression of prohibitin during cell proliferation, differentiation, and cell death, we have successfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but revert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were observed in the differentiated phenotype when compared with proliferating cells. Differentiated RGA-1 cells were found to express inhibin alpha- and beta-transcripts, as well as steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor proteins in a manner reminiscent of steroidogenic functional responses observed in primary differentiated granulosa cells. Prohibitin expression correlated well with the expression of these steroidogenic proteins. At 39 C, RGA-1 cells also displayed increases in p53 protein levels, indicative of growth arrest in the nonproliferating cells. Confocal and electron microscopic examinations revealed increased prohibitin localization to the mitochondria at 39 C, along with changes in mitochondrial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmembrane potential. In addition, cell fractionation studies demonstrated that the prohibitin protein was mainly localized to the mitochondrial membrane. Collectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulosa cells. Prohibitin expression may also be indicative of mitochondrial destabilization during apoptosis-related events.

Non-invasive exclusion of fetal aneuploidy in an at-risk couple with a balanced translocation.

A pregnant woman who was a carrier for a balanced chromosome translocation [46,XX, t(1;6) (p31;q14)] and who had had six miscarriages, declined invasive testing but agreed to non-invasive prenatal diagnosis by analysis of fetal cells in maternal blood. Monoclonal antibody (Mab) against the zeta (z) and gamma (gamma) chains of embryonic and fetal haemoglobin were used to identify fetal nucleated erythrocytes (FNRBC). There were no FNRBC detected at 7 weeks, one anti-z-positive FNRBC was detected at 11 weeks, and 12 anti-gamma-positive FNRBC were detected at 20 weeks. Fluorescent in-situ hybridization was performed using probes for chromosomes X, Y, 1 and 6 to identify fetal gender and the presence of an unbalanced chromosomal translocation. A tentative prenatal diagnosis was made of a female fetus disomic for chromosomes 1 and 6. A female infant with a 46,XX karyotype was born at term. This is the first attempt of exclusion of a chromosome translocation using fetal cells isolated from maternal blood. There is an advantage of using fetal cells isolated from maternal blood for non-invasive prenatal diagnosis in couples who have a history of multiple miscarriages due to a parental translocation, and who decline invasive testing in a pregnancy that continues to the second trimester.

Developmental expression and regulation of basic fibroblast growth factor and vascular endothelial growth factor in rat decidua and in a decidual cell line.

During pregnancy, the decidua is comprised of two separate tissues located either mesometrially or antimesometrially in the uterus. Trophoblast invasion takes place only in the mesometrial decidua, where extensive angiogenesis, essential for successful implantation, occurs. Both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been implicated in this phenomenon. The aim of this study was to determine whether the expression of both growth factors is intrinsic to decidua and occurs in the absence of conceptuses, whether their genes are expressed specifically in the mesometrial decidua, the site of angiogenesis, and whether both growth factors are developmentally and hormonally regulated. Decidual tissue was dissected from pseudopregnant rats and levels of both bFGF and VEGF mRNA were examined in mesometrial and antimesometrial decidua by semi-quantitative RT-PCR at different stages of pseudopregnancy. Although induction of decidualization triggered the mRNA expression of bFGF, VEGF mRNA expression remained unchanged. VEGF mRNA level was similar in both antimesometrial and mesometrial decidua, and remained constant throughout pseudopregnancy. In sharp contrast, bFGF mRNA was highly expressed in the mesometrial decidua at a time when extensive angiogenesis takes place in this tissue. Very little signal was observed in the antimesometrial decidua. To examine the regulation of these growth factors, we used a temperature-sensitive decidual cell line developed by transforming antimesometrial decidual cells with SV-40 tsA 209 mutant virus. These cells express both bFGF and VEGF mRNA. Because progesterone is necessary for decidualization and decidua secretes prolactin (PRL)-related hormones, we examined the role of these hormones on VEGF and bFGF mRNA expressions. Neither progesterone nor PRL had any effect on VEGF mRNA levels. However, bFGF mRNA expression was greatly stimulated by PRL. In conclusion, results of this investigation have revealed that bFGF, but not VEGF, mRNA becomes highly expressed in the mesometrial decidua, where angiogenesis occurs, and where trophoblasts, by invading decidual cells, may promote the release of bFGF. In addition, these results indicate that the locally secreted PRL-like hormone up-regulates the mRNA expression of bFGF.

Expression of Wilms' tumor suppressor gene (WT1) in term human trophoblast: regulation by cyclic adenosine 3',5'-monophosphate.

The Wilms' tumor suppressor gene (WT1), which is deleted in some Wilms' tumors, encodes a zinc finger transcription factor. We studied WT1 messenger ribonucleic acid (mRNA) in human term placenta and cytotrophoblasts differentiating into syncytiotrophoblasts in vitro by RT-PCR. The results suggest that WT1 mRNA is expressed in the trophoblasts in a cell-specific fashion. WT1 mRNA expression has been observed to decline remarkably in trophoblast cells after 72 h, when these cells are morphologically differentiated into multinucleated syncytiotrophoblasts. As it is well known that cAMP as a second messenger plays a significant role in cellular proliferation and differentiation of placental cells, we examined the effect of 8-bromo-cAMP on WT1 mRNA expression in undifferentiated cytotrophoblasts and differentiated syncytiotrophoblasts. We observed that cAMP enhanced WT1 mRNA expression in cytotrophoblasts, but remained ineffective in altering WT1 mRNA in syncytiotrophoblasts. In summary, the results of this investigation demonstrate that the WT1 gene is developmentally regulated during trophoblast differentiation. An involvement of the cAMP-mediated system in regulating the WT1 gene in the trophoblast is suggested.

Preimplantation genetic diagnosis of chromosome balance in embryos from a patient with a balanced reciprocal translocation.

Duplications or deletions are present in a high percentage of the gametes produced by individuals carrying balanced translocations. Preimplantation genetic diagnosis was used to examine chromosome balance in embryos from a patient having a reciprocal translocation within the short arms of chromosomes 5 and 8 (46,XX,t(5;8)(p13;p23)). This woman has two sisters with the translocation unbalanced, resulting in a partial trisomy for chromosome 5 and partial monosomy for chromosome 8 (46,XX,-8, +der(8)t(5;8)(p13;p23)) with associated mental retardation and physical abnormalities. The patient and her husband desired to have children without the abnormal chromosome balance and wished to reduce the likelihood of spontaneous abortion or need for therapeutic abortion. Fluorescence in-situ hybridization (FISH) probes for the alpha-satellite region of chromosome 8 and for a region on the short arm of chromosome 5 (5p15.2) were tested initially on lymphocytes from the patient and her sisters. The hybridization signal for chromosome 5 was detected in the expected two copies for the patient and three copies for the sisters in 87% of the cells. Two hybridization signals for chromosome 8 were detected in 96% of the cells from all individuals. Additional probe testing was done using blastomeres from polyspermic embryos. The couple then proceeded with a stimulated in-vitro fertilization (IVF) cycle and biopsies were done on 13 embryos at the 7-10-cell stage using a method of zona drilling and fluid displacement. Diagnosis was possible on at least one blastomere for nine embryos. Three embryos had nuclei with three hybridization signals for chromosome 5, three had fewer than two signals for one or both chromosomes, one was mosaic, and two had two signals for each chromosome. The latter were transferred to the patient, but pregnancy was not achieved. The results demonstrate that preimplantation genetic diagnosis for patients with reciprocal translocations can be used to identify embryos having normal chromosome balance. The potential advantages and limitations of this approach are discussed.

Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique.

A modified embryo biopsy method was tested on four- and eight-cell stage mouse embryos and used on human embryos to obtain blastomeres for preimplantation genetic diagnosis. The biopsy method tested combines zona drilling and fluid displacement to force one or two cells through an opening in the zona pellucida of the cleavage-stage embryo. Rates of cell division and the percentage of mouse embryos forming blastocysts following biopsy at the eight-cell stage were not significantly different from those observed in unoperated control embryos. The percentage blastocyst formation was not significantly different in embryos biopsied at the four-cell stage and in control embryos, although cell division was significantly retarded following biopsy. 96% of the mouse blastomeres isolated at the eight-cell stage were recovered intact and 96% of those placed in culture underwent cell division. Survival and division of cells isolated at the four-cell stage were 92 and 84% respectively. Most of the cultured blastomeres cleaved several times and formed small trophoblast vesicles. Chromosomes were observed in 59% of blastomeres incubated in the presence of colcemid. In the initial use of this biopsy technique for human preimplantation genetic diagnosis, blastocyst formation was observed in 9 of 13 human embryos biopsied at the 7- to 10-cell stage. These findings support the use of this biopsy method as an alternative to aspiration techniques.

Transvaginal amniotic puncture for cytogenetic evaluation of missed abortions.

Transvaginal amniotic puncture (TAP) was performed on 20 consecutive missed abortions immediately prior to dilatation and evacuation and the cytogenetic results compared. The information received from products of conception (POC) and TAP was in concordance in only 5 of 20 (25%) cases. Tissue obtained from POC yielded cells in all instances. However, only 3 of 20 POC samples yielded findings other than normal female. In contrast, 92.8% of the conclusive diagnoses would have been achieved by TAP alone. These data strongly suggest that TAP is superior to POC for accurate cytogenetic assessment of missed abortion and should lead to a reevaluation of our current understanding and management of pregnancy loss.

Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line.

The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone (GH) modulates insulin-like growth factor I (IGF-I) and type I IGF receptor mRNA levels in the ovary of prepubertal GH-deficient rats.

In order to explore the potential role of growth hormone (GH) in modulating insulin-like growth factor I (IGF-I) gene expression in the prepubertal rat ovary, female rats were rendered GH deficient by neonatal administration of monosodium glutamate (MSG). One group of rats received vehicle and served as the control. At 21 days of age, MSG-treated rats received either GH or vehicle for 2 weeks. On days 21, 24, 28 and 31 animals were weighed and subsets were sacrificed for liver RNA extraction. The remaining animals were sacrificed at day 35 when livers and ovaries were collected, and serum was obtained for GH determinations. The IGF-I mRNA levels were estimated by Northern blots and corroborated further by slot-blot analysis. The MSG-treated rats had lower body weights (p < 0.01) and GH levels (p < 0.05) than controls. Growth hormone replacement significantly accelerated the weight gain of MSG-treated rats. At day 24 and thereafter, three RNA IGF-I species (7.5, 1.8 and 0.8-1.2 kB) were seen in the liver. In the ovary, at age 35 days, two major IGF-I mRNA species (7.5 and 0.8-1.2 kb) were seen. The MSG treatment consistently reduced the levels of both IGF-I mRNA species in the ovary. Growth hormone administration partially restored their expression, both in the liver and in the ovary. In addition, ovarian type IIGF receptor mRNA levels were increased in the MSG-treated rats when compared to controls. This trend was reversed by GH replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptors are present in human granulosa cells.

Recent studies failed to detect estrogen receptors in primate follicles. This study was initiated to determine whether estrogen receptor (ER) messenger ribonucleic acid (mRNA) is present in human granulosa cells and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time of oocyte retrieval for assisted reproduction was extracted, and complementary DNA synthesis was performed by the reverse transcriptase-polymerase chain reaction. Oligonucleotide primers were used to amplify basepairs 570-852 in the B- and C- domains of the ER mRNA. Southern blotting was performed and confirmed that the amplified DNA fragment identified in granulosa cells represented ER. By reverse transcriptase-polymerase chain reaction, mRNA for the ER is clearly identified in primary human granulosa cells obtained at the time of oocyte retrieval. To expand these studies and determine whether functional ER were present in human granulosa cells in culture, a simian virus-40-transformed human granulosa cell line was studied. Cells were transfected with a plasmid containing as estrogen response element up-stream from the bacterial reporter gene chloramphenicol acetyltransferase (CAT). In transfected cells, CAT activity is inducible by estradiol if endogenous functional ER are present. In these studies, the transfection analysis confirmed that functional, transcriptionally competent ER are present in a human granulosa cell line, with a 4- to 5-fold enhancement of CAT activity demonstrated after the addition of estradiol compared to that in nonhormone-treated cells. In conclusion, ER mRNA is present in human granulosa cells. Functional ER are also demonstrated in a transformed human granulosa cell line. We hypothesize that low, but biologically significant, amounts of ER protein are present in human granulosa cells, which are not routinely detectable by standard assays.

Fertilization and implantation.

Recent advances in seemingly remote areas of investigation, i.e. yeast cell cycle research and DNA amplifications, have opened spectacular avenues for understanding reproduction. The new insights on the single cell and subcellular level of processes, such as egg maturation, sperm-egg interaction and implantation enhance, immensely, the power of assisted fertilization. These techniques, have become the mainstay of infertility therapy. This review focuses on the recent developments in these areas.

Gamma delta T cells in human decidua.

OBJECTIVE: Our purpose was to assess the presence and frequency of gamma delta T cells in the decidua of term and first-trimester pregnancies. STUDY DESIGN: Term and first-trimester placentas were obtained from normal subjects. Frozen sections and cell suspensions were prepared from decidual tissue and stained with monoclonal antibodies to T cell markers. Cell sorter analysis was performed. RESULTS: gamma delta T cells in term decidual cell preparations were enriched 2.4-fold compared with peripheral blood. Immunohistochemical staining of term decidual tissue demonstrated many gamma delta + and CD3+ T cells, fewer CD8+ cells, and rare CD4+ cells. In contrast, first-trimester decidua was found to be devoid of gamma delta + T cells, by both cell sorter analysis and immunohistochemical methods. CONCLUSIONS: Term, but not early, decidua harbors a resident T-cell population that is significantly enriched in gamma delta T cells compared with blood. These lymphocytes may provide an added defense mechanism against infection during the peripartum.

Regulatory effect of antiphospholipid antibodies on signal transduction: a possible model for autoantibody-induced reproductive failure.

OBJECTIVES: The broad range of reproductive processes apparently affected by antiphospholipid antibodies suggests that antiphospholipid antibodies have to exert their clinical effects at every basic cellular level. Because phospholipids constitute essential components of every cell membrane, and because signal transduction processes are dependent on second messengers with phospholipid epitopes, we investigated the hypothesis that interference with signal transduction processes by antiphospholipid antibodies could represent such a basic cellular process. STUDY DESIGN: To test this hypothesis we established an in vitro placental culture system in which the stimulation of explants by phospholipase A2 and phospholipase C in the presence of normal human serum resulted in a significant increase in human chorionic gonadotropin secretion. RESULTS: When normal human sera were replaced with antiphospholipid antibody-containing sera from two women with established histories of reproductive failure, this increase in human chorionic gonadotropin production was inhibited 42% to 44% under phospholipase A2 stimulation and by 34% to 39% under phospholipase C stimulation. The hypothesis that antiphospholipid antibodies can interfere with signal transduction processes was further strengthened by the observation that antiphospholipid antibody-positive sera contained significantly higher immunoglobulin A antibody reactivity toward the second messenger inositol 1,4,5-triphosphate than control sera (p less than 0.049). CONCLUSIONS: These observations suggest the possibility that if present at excessive levels antiphospholipid antibodies may exert their adverse effect on reproductive processes through the interception of signal transduction processes.