| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
Department of Obstetrics & Gynecology and Cooperative Reproductive Science Research Center,3
Department of Microbiology Biochemistry and Immunology,4 Morehouse School of Medicine, Atlanta, Georgia 30310
Department of Obstetrics & Gynecology and Cellular & Molecular Medicine,5 University of Ottawa and Ottawa Health Research Institute, Ottawa, Canada K1Y 4E9
Departments of Animal Sciences6
Obstetrics and Gynecology,7 University of Missouri-Columbia, Columbia, Missouri 65211-5300
Departement de Chimie-Biologie,8 Section de Biologie-Medicale, Universite du Quebec a Trois-Rivieres, Trois-Rivieres, Quebec, Canada G9A 5H7
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
apoptosis, follicular development, granulosa cells, oocyte development, ovary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A growing body of evidence has implicated prohibitin in mitochondrial structure, function, and inheritance [10, 21–27]. Prohibitin is predominantly localized to the inner mitochondrial membrane of rat granulosa cells [10]. Both rat and human prohibitin possesses a short transmembrane helix near their N-termini that may be integrated into the mitochondrial membranes. It has been speculated that prohibitin, as an inner mitochondrial membrane protein, may control ion transport and calcium-dependent ATP production [3]. If this hypothesis is correct, then prohibitin may play a similar role in regulating the granulosa cell steroidogenic machinery, because the rate-limiting step of steroidogenesis is also located in the inner mitochondrial membrane. The potential involvement of prohibitin in both the cell cycle and mitochondrial function is paramount to the fate of the ovarian follicle. Studies in our laboratory have demonstrated that increased prohibitin expression occurs during ovarian development [8]. More recently, we have shown that this increased prohibitin expression correlates with granulosa cell differentiation, mitochondrial structure and function, as well as the early stages of apoptosis in an immortalized granulosa cell model [10].
Previously, we have shown in related studies [8] that prohibitin expression in the ovarian tissue is age- and stage-regulated, suggesting a growth-regulatory role of prohibitin in the rat ovary. The object of the present study was to examine the spatial pattern of prohibitin during folliculogenesis and oocyte development using established in vivo models [28–32] for the induction of follicular development and atresia in the mammalian ovary. Our findings show that prohibitin is expressed in granulosa cells, theca-interstitial cells, and the oocyte in a follicular stage-dependent manner. Prohibitin expression is associated with cytodifferentiation, is upregulated by gonadotropin, and is low during cell proliferation. The intracellular localization of prohibitin is dependent on the cellular health status.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Unless otherwise stated, all reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO). Enhanced chemiluminescence Western blotting detection kit was purchased from Amersham (Arlington Heights, IL). Polyclonal antiprohibitin antibody and preimmune rabbit serum were purchased from Neomarkers (Fremont, CA), monoclonal antiproliferating cell nuclear antigen (anti-PCNA) antibody from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anticholesterol side-chain cleavage cytochrome P450 (P450scc) was from Chemicon International, Inc. (Temecula, CA). Goat anti-mouse immunoglobulin (Ig) G (H+L) conjugated to Alexa Fluor 488, goat anti-rabbit IgG conjugated to Alexa Fluor 594, and 4',6'-diamidino-2-phenylindole were purchased from Molecular Probes (Eugene, OR).
Animal and Cell Preparations
Female Sprague-Dawley rats (age, 23 days) were separated into three groups (n = 15 rats/group). In the first group, animals were injected s.c. with saline, followed 24 h later by normal (preimmune) rabbit serum (NRS; 0.1 ml). In the second group, animals were injected s.c. with 15 IU of eCG, followed 24 h later by NRS (0.1 ml). In the third group, animals were injected s.c. with 15 IU eCG, followed 24 h later by anti-eCG (0.1 ml). In all groups, animals were killed with an overdose of pentobarbital 24 h after the last hormone/antibody injection [28, 29]. Subsequently, ovaries were excised, cleared of adhering fat, weighed, and fixed either in 10% neutral buffered formalin for immunohistochemical detection of prohibitin, PCNA, or P450scc or for in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of apoptotic cells nuclei (n = 5 rats/group). In addition, granulosa cells from each group (n = 10 rats/group) of animals were harvested by follicle puncture as previously described [9], washed, and resuspended in 10 mM Hepes buffer (pH 7.4) containing 1 mM EGTA and 2 mM PMSF. Cells were processed for Western blot analyses.
Porcine germinal vesicle (GV)-stage oocytes were collected from slaughterhouse ovaries and were matured and fertilized in vitro as described previously [30, 33]. Briefly, the oocyte-cumulus complexes (OCCs) were aspirated from ovaries, washed, and matured for 22 h at 39°C and 5% CO2 serum-free modified tissue-culture medium (TCM) 199 (Gibco, Grand Island, NY) supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml of epidermal growth factor, 0.5 µg/ml of FSH, 0.5 µg/ml of LH, 0.1% polyvinyl alcohol (w/v), 75 µg/ml of penicillin G, and 50 µg/ml of streptomycin sulfate. The OCCs were then washed in modified TCM 199 without FSH or LH and matured for an additional 20 h. Matured oocytes were stripped of cumulus cells and fertilized in 50-µl drops of modified Tris-buffered medium consisting of 113.1 mM NaCl, 3 mM KCL, 7.5 mM CaCl2, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, 2 mM caffeine, and 0.2% (w/v) BSA. Cryopreserved boar semen was thawed in 10 ml of Dulbecco PBS (Gibco) supplemented with 0.1% (w/v) BSA, and spermatozoa were washed two times by centrifugation (1000 x g for 4 min) and added to fertilization drops to a final concentration of 5 x 105 spermatozoa/ml.
Porcine Embryo Heat Shock Treatments
Oocyte collection, maturation, and fertilization were performed as described above with the following modifications: First, oocytes were matured in the presence of gonadotropins (LH and FSH; concentrations as described above) for the entire 40- to 44-h maturation period. Second, 1 x 106 spermatozoa/ml were used for the production of in vitro fertilized embryos. Embryos were removed from the fertilization microdrops and immediately placed in North Carolina State University-23 (NCSU-23) embryo culture medium at 5% CO2 for approximately 15 h at 39°C, then half the embryos were heat shocked at 42°C for 9 h while the remaining half were maintained at 39°C. After the 9-h heat shock period, all embryos were incubated at 39°C for continued culture.
Nuclear Transfer
Porcine nuclear transfer (NT) was performed as described by Park et al. [34]. Briefly, the cumulus-free oocytes were enucleated in TCM/BSA medium supplemented with 7.5 µg/ml of cytochalasin B. The first polar body and the surrounding cytoplasm were removed with the aid of a beveled pipette (inner diameter, 
25–30 µm). Enucleated oocytes were kept and micromanipulated in TCM/BSA until NT. A Nikon Diaphot (Nikon Corporation, Tokyo, Japan) equipped with a 40x objective and a pair of Narishige micromanipulators (Narishige International USA, East Meadow, NY) was used for micromanipulations. Donor fibroblasts (fetal fibroblasts were isolated from a 35-day-old pig fetus) were selected according to their size and shape (small cells with smooth membranes). A single cell was transferred into the perivitelline space with the same pipette as used for enucleation. Cytoplast-fibroblast complexes were placed between two electrodes (1 mm apart), overlaid with fusion medium (0.3 M mannitol, 1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes), and aligned manually. Fusion/ activation was achieved by two pulses of 1.2 kV/cm for 30 µsec as measured by the BTX-optimizer (BTX, San Diego, CA). Fused embryos were cultured for up to 6 days in 500 µl of NCSU-23 supplemented with 4 mg/ ml of BSA.
In all experiments, the zona pellucida was removed from porcine oocytes and embryos by a brief pronase (Protease; Sigma) treatment and then processed for immunofluorescence as described previously [33]. All animal handling procedures in the present study were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health and the U.S. Department of Agriculture.
Western Blot Analysis
Granulosa cell extracts containing 50 µg of protein from different experimental groups were subjected to both one- and two-dimensional gel electrophoresis as described previously [8–10]. In brief, proteins separated by 12% SDS-PAGE were transferred onto 0.2-µm nitrocellulose membranes (Sigma) using the Royal Genie electrophoretic blotter (Idea Scientific, Minneapolis, MN) at 350 mA for 5 h. Blots were incubated for 1 h in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dried milk and subsequently (overnight at 4°C) with polyclonal antiprohibitin antibody (1:2000; Neomarkers). Membranes were incubated with the appropriate secondary antibody for 2 h at room temperature (RT), and antibody binding was detected by chemiluminescence (Amersham).
Immunofluorescence Microscopy
The procedure used for immunofluorescence microscopy of rat ovarian sections and porcine oocytes, zygotes, and blastocysts has been described in detail previously [8–10, 33]. Mouse monoclonal PCNA, polyclonal rabbit antiprohibitin, and polyclonal anti-rat cytochrome P450scc antibodies were used at a dilution of 1:200. The specificity of the antibodies was verified by incubating ovarian sections and oocytes, zygotes, and blastocysts without primary antibodies as well as with NRS. After thorough rinsing, sections were mounted in glycerol containing 50 µg/ml of n-propyl gallate and then examined using either an Olympus BX41 microscope equipped with an Optronics MagnaFire digital camera and Prior Proscan motorized driven stage (Olympus, Melville, NY) or a Nikon Eclipse 800 microscope and CoolSnap HQ RTE/CCD 1217 digital camera (Roper Scientific, Tucson, AZ).
For digital image capturing, the exposure time was adjusted using sections incubated without the primary antibody to minimize any auto or nonspecific fluorescence recording without compromising the actual signal. The signal obtained after such a background correction was considered to be an antigen-specific signal. For each image, specific antibody staining was merged with nuclear staining (blue) using Soft Imaging System software (Soft Imaging System Corp., Lakewood, CO) and MetaMorph 4.6 software (Universal Imaging Corp., Downington, PA) that caused virtually no pixel shifting during image merger and resulted in shades of red, green, and blue. To verify the reproducibility of the data, immunofluorescence localization studies were repeated at least three times per serially sectioned ovary using rat ovarian tissues from different animals (n = 15), porcine oocytes (n = 30), zygotes (n = 40), and blastocysts (n = 20 of different quality). Representative photomicrographs were arranged using Adobe PhotoShop (Adobe, San Jose, CA) without any further adjustment to maintain the true nature of the findings.
In Situ Localization of Apoptotic Cells: TUNEL
Ovaries fixed in 10% neutral buffered formalin were dehydrated through a graded series of ethanol, cleared in xylene, embedded in paraffin, and sectioned (section thickness, 4 µm). Sections mounted on positively charged slides (ProbeOn Plus; Fisher Scientific, Pittsburgh, PA) were deparaffinized, hydrated, and treated with 20 µg/ml of proteinase K (37°C, 30 min), and endogenous peroxidase activity was removed by treatment with 3.0% hydrogen peroxide (room temperature [RT], 10 min). A commercially available kit (ApopTag; Intergen, Purchase, NY) was used for the detection of 3'-OH DNA ends in the sections. After washing with distilled water three times for 10 min each, the sections were incubated in the equilibration buffer of the kit for 20 sec at RT. Then, sections were incubated at 37°C for 1 h in a moist chamber with 60 µl of the working buffer containing terminal deoxynucleotidyl transferase, digoxigenin-11-dUTP. The reaction was stopped by incubating the sections in a blocking buffer containing sodium citrate and NaCl at 37°C for 30 min. After rinsing with PBS four times for 15 min each, sections were incubated with antidigoxigenin antibody conjugated to horseradish peroxidase at RT for 30 min. After incubation with the antibody, the peroxidase activity was examined by exposing the sections to a solution containing 0.05% diaminobenzidine and 0.01% H2O2 in Tris buffer (pH 7.6) for 3–6 min at RT. The sections were counterstained with 1% methyl green. For control experiments, the enzyme incubation step was omitted.
Statistical Analysis
Experiments were repeated a least three times, and representative chemiluminescence was first scanned using a Power Macintosh computer (G3; Apple Computer, Inc., Cupertino, CA) equipped with a ScanJet 6100C scanner (Hewlett-Packard Co., Greeley, CO). Quantification of the scanned images was performed according to the NIH Image version 1.61 software (National Institutes of Health, Bethesda, MD). Data are expressed as the mean ± SEM of three experiments. Statistical analysis was performed by one-way ANOVA using SPSS version 11.0 software (SPSS, Chicago, IL).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We previously demonstrated that the administration of eCG to immature female rats followed by an antibody against the gonadotropin induces follicular atresia and granulosa cell apoptosis [28, 29]. To determine whether prohibitin protein content in granulosa cells in vivo is modulated by gonadotropin, cells were isolated from rats treated with saline, eCG plus NRS, or eCG plus anti-eCG and analyzed by Western blot analysis. The results revealed the presence of immunoreactive proteins corresponding to prohibitin protein content in extracts of rat ovarian granulosa cells (Fig. 1A). Prohibitin content was significantly higher in granulosa cells isolated from the group treated with eCG plus NRS compared to the group treated with saline (P = 0.0375) and with eCG plus anti-eCG (P = 0.0425). Densitometric analyses of prohibitin levels on one-dimensional gels revealed an increase greater than twofold in prohibitin expression in granulosa cells isolated from ovaries of rats treated with eCG plus NRS (Fig. 1A).
|
Samples of the respective protein extracts were also utilized in two-dimensional Western blot analyses to determine whether changes in prohibitin isoforms occur during gonadotropin stimulation. Two polypeptide species from the 30-kDa protein (Fig. 1B) were delineated. The amount of the acidic isoform (Fig. 1B, arrowhead) of prohibitin content increased more than twofold in granulosa cells treated with eCG plus NRS compared to those treated with saline (P = 0.0312). Consistent with its effect on the induction of apoptosis in granulosa cells, withdrawal of gonadotropin (treatment with eCG plus anti-eCG) induced a decrease in the acidic isoform of the protein content 48 h after treatment.
Spatial Expression Pattern of Prohibitin During Induction of Follicular Maturation and Atresia
We previously demonstrated in a conditionally immortalized rat granulosa cell line that prohibitin content decreased during cell proliferation but increased in cells undergoing differentiation and at early stages of apoptosis. To determine whether these finding were physiologically relevant in vivo, the cellular distribution pattern of prohibitin expression in the ovarian follicles and the influence of gonadotropic stimulation and withdrawal were examined in ovaries of immature rats treated with eCG plus NRS and with eCG plus anti-eCG. As summarized in Table 1, prohibitin and PCNA (a cofactor of DNA polymerase 
and cyclin-cdk complexes and used as a marker for cellular proliferation) were immunolocalized in both granulosa and theca-interstitial cells in rat ovary 48 h following eCG treatment when follicular development was observed. Prohibitin immunostaining was found in the cytoplasm of granulosa and theca-interstitial cells, whereas PCNA localized in the nuclei within the preantral follicles (Fig. 2). At this stage of development, prohibitin immunoreactivity was higher in granulosa cells than in theca-interstitial cells. Cells with intense PCNA staining (Fig. 2D, arrow) showed low immunostaining for prohibitin (Fig. 2A, arrowhead). Some cells showed weak signal intensity for PCNA in the nucleus but exhibited increased prohibitin expression in the cytoplasm. The PCNA immunoreactivity was also evident in oocytes of preantral follicles (Fig. 2, D and G). Interestingly, some cells expressed neither prohibitin nor PCNA.
|
|
Similar developmental patterns for prohibitin and PCNA immunoreactivity were observed in granulosa cells from early and large antral follicles. Moreover, a gradient-like spatial expression pattern of prohibitin was evident, with mural granulosa cells being most intensely stained and granulosa cell layers closer to the antral cavity exhibiting lower immunoreactivity (Fig. 2, B, C, E, F, H, and I). As the follicles matured, prohibitin was more abundantly expressed in the theca-interstitial cells (Fig. 2, B and H).
To determine whether prohibitin expression patterns correlated with the differentiated status of the cells, prohibitin and P450scc were immunolocalized in ovarian tissue sections from rats treated with eCG plus NRS. As shown in Figure 3A (see Table 1), prohibitin immunostaining was predominantly confined to the granulosa cells, whereas the theca-interstitial cells showed low staining patterns within the preantral follicles. In contrast, immunofluorescence labeling for P450scc was confined to the belt-like regions of the interstitial cells (Fig. 3B). The theca cells showed low immunoreactivity in the preantral follicles for this protein (Fig. 3A), whereas the granulosa cells were clearly devoid of P450scc (Fig. 3B).
|
As the follicle developed toward early and large antral stages, the theca-interstitial cells showed more intense fluorescent signals for prohibitin and P450scc, respectively (Fig. 3, C–F). The granulosa cells of early antral follicles revealed a gradient-like expression pattern for prohibitin, whereas immunostaining for P450scc in mural granulosa cells was evident (Fig. 3, C and D). Interestingly, not all granulosa cells from early antral follicles exhibited immunostaining for prohibitin. As the follicles matured to the large antral follicle stage, granulosa cells in the follicular wall, but not those surrounding the oocyte, expressed P450scc (Fig. 3F). A gradient-like pattern of prohibitin expression was also observed in the large antral follicle (Fig. 3E).
To determine the prohibitin expression patterns associated with follicular atresia and cell death, ovarian serial sections from rats treated with eCG plus anti-eCG antiserum were localized by in situ TUNEL and prohibitin immunohistochemistry, respectively. Consistent with our previous reports [28, 29, 34, 35], apoptotic granulosa cells were present in preantral, early antral, and large antral atretic follicles (Fig. 4, A, C, and E, and Table 1). Prohibitin immunostaining was evident in preantral, early antral, and large antral atretic follicles (Fig. 4, B, D, and F). Interestingly, prohibitin appeared to be translocated from the cytoplasm to the nucleus in some cells of follicles undergoing atresia and was markedly visible in early and large antral follicle cells (Fig. 4, B, D, and F, arrow). This observation is not an artifact, because we focused at higher magnification on the focal plane intersecting the nuclear compartment.
|
Localization of Prohibitin in Isolated Germinal Vesicle Oocytes and In Vitro Fertilized and Cloned Embryos
The rat and mouse prohibitin genes are functional homologues of human prohibitin. This gene is highly conserved, because comparative analysis of the amino acid sequences indicates a single amino acid substitution between these three species. We have determined that the protein expression patterns of prohibitin in rat ovarian follicles are similar to that observed in porcine follicles (data not shown). Considering the ease of isolating large numbers of pig ova and the difficulty of the same in rats, the porcine model, to which both in vitro fertilization (IVF) and NT procedures have routinely and successfully been applied (for reviews, see [30–32, 36, 37]), was used in the present study to provide a rationale for future use of porcine model in studies of prohibitin during early embryo development. To gain further insight regarding the expression of prohibitin during normal and aberrant oogenesis and preimplantation embryonic development, standard immunofluorescence procedures on whole-cell mounts (as opposed to the processing of tissue sections) were conducted. Prohibitin was readily detectable in normal GV-stage oocytes (Fig. 5, A and D), albeit at low levels. A distinct, speckled pattern of prohibitin immunoreactivity was detected in the GV-stage oocytes that also showed abnormal condensation of chromatin, suggestive of atresia (Fig. 5, B and C). Association of prohibitin with oocyte chromatin was not seen in metaphase II oocytes after in vitro or in vivo maturation (Fig. 5D). Similarly, neither the male nor the female pronuclei of zygotes fertilized in vitro (Fig. 5E) displayed significant prohibitin immunoreactivity. However, the zygotes and embryos arrested at the 2-cell stage after the apoptosis-inducing heat shock treatment displayed the pattern of nuclear prohibitin accumulation similar to that seen in defective GV-stage oocytes (Fig. 5F). This nuclear expression pattern was more pronounced in the embryos that failed to develop normally after NT (Fig. 5, G and H). In contrast, low levels of nuclear immunoreactivity were observed in embryos that reached the blastocyst stage at Day 5–6 after NT (Fig. 5I) or IVF (not shown) and displayed an acceptable morphology.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immunohistochemical analyses revealed that prohibitin was highly expressed in the cytoplasm and perinuclear region of granulosa cells of some, but not all, preantral follicles. This pattern of prohibitin immunostaining appeared to be inversely related to that of PCNA expression and remained consistent during subsequent stages of follicular growth. This apparent heterogeneous expression pattern of prohibitin may be indicative of a diverse population of growing cells. These observations support the notion that prohibitin may be a negative regulator of cell growth [4, 38–41] and, possibly, could exert antiproliferative activity. Based on these observations, prohibitin expression patterns likely correlated with those of P450scc in granulosa cells as the follicles matured, suggesting that prohibitin may mediate establishment of the steroidogenic machinery of the follicle during development. In support of this contention is the observation that, at the preantral stage of follicular development, prohibitin expression in the theca-interstitial cells was low or undetectable and that these cells were devoid of P450scc. As the follicles matured toward the preovulatory stage, increased prohibitin expression was observed in the theca-interstitial cells, which also had increased P450scc enzyme activity. These findings are in agreement with those of previous studies concerning the expression of this steroidogenic factor during follicular development in the rat ovary [42]. Our results raise the interesting possibility that induction of prohibitin expression may initiate events involved in differentiated action of gonadotropin in the ovary. Additionally, these results may also reflect increased metabolic activity within the mitochondria. In this context, Coates et al. [13] have reported that prohibitin is highly expressed in cells showing a particular reliance on mitochondrial metabolism, including those in prostate and melanoma tumors. In support of this result, we observed a strong immunostaining pattern for prohibitin, primarily in the cytoplasmic or perinuclear region of epithelial cells in papillary serous ovarian carcinoma and endometrioid adenocarcinoma (unpublished results). Whether prohibitin plays a directed role in granulosa cell differentiation or mitochondrial metabolism awaits further investigation.
Apoptosis in rat atretic follicles is confined primarily to granulosa cells. Irrespective of the stage of follicular maturation, theca cells in all atretic follicles were TUNEL-negative. Interestingly, prohibitin was highly expressed in theca cells, and its immunoreactivity progressively increased with follicular maturation. The expression of prohibitin in theca cells was much higher than that in granulosa cells. Although speculative, increased expression of prohibitin at this stage likely may be preventing theca cells from undergoing apoptosis. To address this issue, we are currently utilizing a recombinant adenovirus vector to constitutively overexpressed prohibitin to determine whether this gene product has a role in delaying apoptosis. Alternatively, prohibitin may be involved in other, as-yet-unidentified physiological process, such as theca cell differentiation. Studies by Fusaro et al. [43] demonstrated that overexpression of prohibitin in Ramos B cells blocked apoptosis induced by camptothecin, a topoisomerase-I inhibitor. This result suggests that prohibitin may, in part, be a cell survival or antiapoptotic factor in granulosa cells. Whether this regulatory mechanism exists in the antiapoptotic action for prohibitin in the ovary remains to be determined. We observed translocation of prohibitin from the cytoplasm to the nucleus in granulosa cells of atretic follicles, although the significance of this observation is not immediately apparent. The mechanism by which prohibitin interacts with established cell death pathways to regulate apoptosis is also not known.
Treatment of immature rats with eCG stimulated follicular maturation and increased prohibitin expression. One- and two-dimensional Western blot analyses have shown not only significant increases in prohibitin protein levels but an elevation in the more acidic isoform of the protein in gonadotropin-stimulated granulosa cells. On the basis of the electrophoretic studies, the prohibitin protein clearly is differentially processed on gonadotropin stimulation. These results are consistent with posttranslational modification by phosphorylation of prohibitin, as previously reported by Thompson et al. [8–10]. The physiological role of these isoforms is unknown, and whether their presence and differential response to gonadotropic stimulation accounts for the multifunctional nature of this protein remains to be determined. Nonetheless, caution should be exercised in assessing the potential role and regulation of ovarian prohibitin expression, because measuring total prohibitin content may have limited physiologic significance. Potentially, the acidic isoform of the protein could well be the active form of prohibitin. Currently, we are investigating the site of phosphorylation of prohibitin using high-performance liquid chromatography/mass spectrometry techniques.
The accumulation of prohibitin was observed in the aberrant embryos after NT and IVF followed by heat shock. Whereas cytoplasmic fragmentation and developmental arrests are common in mammalian preimplantation embryos raised in vitro, they may not always be paralleled by immediate activation of the obligatory apoptotic pathways. Both apoptotic and antiapoptotic molecules are expressed by the embryo undergoing fragmentation [44, 45], yet such embryos do not appear to die quickly and may not show signs of nuclear DNA fragmentation until several days after embryonic cleavage-arrest [32]. Such a paradox could be explained by a dual role of prohibitin as an antiproliferative and cell survival factor, which could prevent further cleavage of defective embryos and delay activation of the apoptotic pathways in the arrested embryos, respectively. Sequestration of prohibitin in the nuclear compartment of aberrant embryos is compatible with the proposed role of prohibitin in transcriptional control [20].
At this stage, data to ascribe an antiproliferative or cell survival role to prohibitin in the ovaries are inconclusive. However, these results suggest a potential multifunctional role for prohibitin in the regulation of follicle cell growth and differentiation during follicular maturation and atresia. The changes in prohibitin expression and localization may be reflective of its antiproliferative activities, although the functional implications of these observations are currently unknown. The specific role of this protein may be dependent on the cell type examined, its phosphorylation status, and its cellular concentration [46]. In addition, prohibitin may also be involved in modulating the growth of granulosa and theca-interstitial cells, development of their steroidogenic potential, and possibly, participation in antiapoptotic mechanisms governing both follicular and embryo development. Thus, this protein may play an important role in determining the developmental fate of these cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Winston E. Thompson, Department of Obstetrics & Gynecology, Cooperative Reproductive Science Research Center, Morehouse School of Medicine, 720 Westview Drive Southwest, Atlanta, GA 30310. FAX: 404 752 1754; thompsw{at}msm.edu
Received: 9 October 2003.
First decision: 3 November 2003.
Accepted: 2 March 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  I. Chowdhury, W. Xu, J. K. Stiles, A. Zeleznik, X. Yao, R. Matthews, K. Thomas, and W. E. Thompson Apoptosis of Rat Granulosa Cells after Staurosporine and Serum Withdrawal Is Suppressed by Adenovirus-Directed Overexpression of Prohibitin Endocrinology, January 1, 2007; 148(1): 206 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Peng, R. Mehta, S. Wang, S. Chellappan, and R. G. Mehta Prohibitin is a novel target gene of vitamin d involved in its antiproliferative action in breast cancer cells. Cancer Res., July 15, 2006; 66(14): 7361 - 7369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Rastogi, B. Joshi, G. Fusaro, and S. Chellappan Camptothecin Induces Nuclear Export of Prohibitin Preferentially in Transformed Cells through a CRM-1-dependent Mechanism J. Biol. Chem., February 3, 2006; 281(5): 2951 - 2959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Hussein Apoptosis in the ovary: molecular mechanisms Hum. Reprod. Update, March 1, 2005; 11(2): 162 - 178. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
