| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
Enhances Oocyte/Follicle Apoptosis in the Neonatal Rat Ovary1
a Department of Biological Sciences, Kent State University, Kent, Ohio 44242-0001
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(TNF) is a multifunctional cytokine present in oocytes and macrophages in the neonatal rat ovary. The presence of both TNF and its receptors in the neonatal rat ovary suggests a potential role for it in follicle assembly or oocyte atresia. Previous studies have provided support for effects of TNF on isolated granulosa and theca cells and intact follicles; however, to our knowledge, this is the first study to investigate the effects of TNF on the earliest stages of follicular development. Effects of TNF on oocyte/follicle number and apoptosis were investigated using an ovarian organ-culture system that supported assembly of primordial follicles in vitro. Ovaries were collected on the day of birth and treated with TNF (0, 0.1, 1.0, 10, or 50 ng/ml), a function-blocking TNF antibody (5 µg/ml), or control immunoglobulin (Ig) G. At 1 ng/ml, TNF decreased follicle and oocyte numbers during 3 days of culture, whereas higher (10 and 50 ng/ml) or lower (0.1 ng/ml) doses had no effect. Treatment with TNF antibodies increased the number of oocytes and follicles compared to nonspecific IgG control. To determine whether the decreased oocyte/follicle numbers were due to an apoptotic effect of TNF, apoptosis was examined by DNA laddering. At 1 ng/ml, TNF increased apoptotic DNA laddering twofold, with no significant effect from lower or higher doses. The cells undergoing apoptosis, as determined by in situ end-labeling, were oocytes, interstitial cells, and granulosa cells. These findings suggest that TNF may be involved in oocyte atresia that normally occurs during the perinatal period.
apoptosis, cytokines, follicular development, immunology, ovary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One potential factor that may play a role in these key events is tumor necrosis factor (TNF), which is a 17.3-kDa protein that was first identified as a product of activated macrophages [4]. It has been identified in ovarian cells of various species (reviewed in [5]) and, in the rat, has been localized in oocytes [6], macrophages [7], and granulosa cells [8]. The onset of TNF production in the oocyte occurs between Embryonic Day 20 and Day 2 postpartum [6]. Thus, the appearance of TNF coincides both with follicle assembly [1] and widespread oocyte atresia [9]. Although TNF and its receptors have been localized in the ovaries of several different species, no work, to our knowledge, has been done to identify a role for TNF in the early neonatal rat ovary.
The multiple activities of TNF are mediated by specific cell-surface receptors. Two distinct receptors of 55–60 kDa (TNF-RI) and 75–80 kDa (TNF-RII) are expressed at low levels by various cells [10]. The extracellular domains of these receptors exhibit some sequence homology, but their intracellular domains are entirely different, indicating that distinct intracellular signaling pathways are activated by binding of TNF. Several reports have shown that the signal through TNF-RI is necessary for cytotoxicity [11, 12], whereas that through TNF-RII, although poorly understood, has been shown to be involved in the growth and stimulation of lymphoid cells [11]. Both receptor subtypes have been detected by in situ hybridization [13] and reverse transcription-polymerase chain reaction [14] in rat ovaries on Embryonic Day 19 through Day 20 postpartum. Competitive receptor-binding assays have further confirmed the presence of specific ovarian TNF binding during this time period, supporting a potential role of TNF in early ovarian development [14].
Several investigators have demonstrated that TNF exerts a variety of effects on ovarian cells in vitro. These include inhibition of FSH-stimulated estrogen and progesterone production in the rat granulosa cells (reviewed in [5]). In addition, it has been shown to decrease LH-induced androgen production by rat theca-interstitial cells and to cause these cells to form compact clusters [15]. In addition to its effects on ovarian steroidogenesis, TNF can cause cytotoxicity in rat ovarian follicles [16] and induce apoptosis in cultured hen granulosa cells [17]. The objectives of the present study were to investigate the effects of TNF on follicle assembly and apoptosis in the neonatal rat ovary.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Waymouth medium MB752/1, pyruvic acid, penicillin/streptomycin, BSA (fraction IV), MgCl2, CaCl2, propidium iodide, and goat immunoglobulin (Ig) G were purchased from Sigma (St. Louis, MO). Costar Transwell membrane inserts (nontissue culture-treated; Nucleopore polycarbonate membrane; pore size, 3.0 µm; diameter, 24 mm) and six-well plates were obtained from Costar Corp. (Cambridge, MA). Fetal bovine serum was obtained from Mediatech (Herndon, VA). The JB-4 Plus embedding kit and embedding reagents were obtained from Polysciences (Warrington, PA). Hematoxylin, 100% ethanol, Hemo-D, and formaldehyde were obtained from Fisher Scientific (Pittsburgh, PA). Eosin was purchased from Labchem, Inc. (Pittsburgh, PA). Recombinant rat TNF and polyclonal goat anti-rat TNF IgG were purchased from Genzyme Diagnostic (Cambridge, MA). The DNA extraction kit and glycogen were purchased from Gentra Systems (Minneapolis, MN). Proteinase K, EDTA, and yeast tRNA were purchased from Gibco BRL (Rockville, MD). Terminal transferase kit was obtained from Boehringer Mannheim (Indianapolis, IN). The [32P]ddATP was purchased from Amersham (Arlington Heights, IL). Ammonium acetate was obtained from Matheson Coleman & Bell (Rutherford NJ). Ecolite scintillation fluid was purchased from ICN (Costa Mesa, CA). The In Situ Cell Death Detection kit (fluorescein) was obtained from Roche Diagnostic (Indianapolis, IN). Vectashield Mounting Media was purchased from Vector (Burlingame, CA).
Animals
Timed pregnant Sprague-Dawley rats were obtained from Zivic-Miller (Portersville, PA). The day of birth was considered to be Day 0 postpartum. Rats were provided with unlimited access to feed and water and were housed in a temperature- and light-controlled facility with lights-on at 0730 h and lights-off at 1930 h. Neonates were anesthetized with Metophane (methoxyflurane; Mallinckrodt Veterinary, Inc., Mundelein, IL) and decapitated. The ovaries were removed and cleaned quickly in a sterile environment. Ovaries for histological examinations and in situ end-labeling were cultured for 3 days, then processed through a graded series of ethanol. Ovaries for DNA extractions were immediately frozen at -70°C at the end of the culture period. All animal procedures were conducted in accordance with protocols approved by the Kent State University Animal Care and Use Committee and in accordance with the Guiding Principles for the Care and Use of Research Animals as set forth by the Society for the Study of Reproduction.
Ovary Culture System
The effects of TNF were evaluated using an ovary culture system that is very similar to those previously established to study early stages of folliculogenesis in the rat [18], mouse [19], cow [20], and baboon [21]. Ovaries were removed on Day 0 postpartum and immediately placed in ice-cold Waymouth medium MB752/1. Tissue adhering to the ovary was cut away using the beveled edge of a 21-gauge needle. Each ovary was cleanly bisected with a scalpel blade, and the two hemiovaries were transferred to a Costar Transwell membrane insert. Culture medium (1.5 ml of Waymouth MB752/1 supplemented with 0.23 mM pyruvic acid, 50 mg/L of streptomycin sulfate, 75 mg/L of penicillin G, 3 mg/ml of BSA [fraction IV], and 10% [v/v] fetal bovine serum) was placed in the culture dish compartment below the membrane, and the ovaries were covered by a thin film of medium. Hemiovaries were incubated at 37°C in 95% air and 5% CO2 for up to 3 days. The media were changed every 48 h.
Histology
Ovaries were fixed in neutral buffered formalin (4% [w/v] formaldehyde in PBS) for 24 h. The ovaries were processed through a graded series of ethanol and JB-4 Plus infiltrating solution and then embedded in plastic blocks. Serial sections (thickness, 5 µm) were cut from each ovary, followed by staining with hematoxylin-and-eosin according to well-established procedures [22]. Morphological characteristics were recorded for every fifth section using the following criteria: 1) number of naked oocytes, 2) number of primordial follicles, and 3) number of primary follicles. Total follicle counts for each ovary were obtained by adding the values obtained from each of the two hemiovaries. Only apparently healthy oocytes and follicles were counted, because degenerating oocytes were difficult to distinguish. Criteria for "healthy" follicles were an intact oocyte, granulosa cells without a granular appearance, and nuclei that were not condensed. Follicles were then classified as naked oocytes (oocytes not completely enclosed by a layer of granulosa cells), primordial follicles (oocytes surrounded by a single layer of flattened granulosa cells), or primary follicles (oocytes surrounded by a cuboidal layer of granulosa cells).
Effects of Exogenous TNF and TNF Antibodies
Ovaries from neonatal rats (Day 0) were collected, and hemiovaries were cultured and incubated at 37°C in 95% air and 5% CO2 for 1–3 days. In the TNF dose-response experiment, the ovaries were treated with various doses of TNF (0.1, 1, 10, and 50 ng/ml) or with a medium control. In the TNF antibody experiment, ovaries were cultured with medium containing an antibody to TNF (polyclonal goat anti-rat TNF IgG, 5 µg/ml) to reduce active, endogenous TNF within the ovaries. The control group was treated with an equivalent concentration of goat IgG (5 µg/ml).
DNA Extraction
The DNA was isolated from three to six cultured ovaries using a DNA Isolation kit (Gentra Systems) according to manufacturer's instructions with the following modifications: To obtain a maximum yield, 1.5 µl of Proteinase K solution (20 mg/ml) was added to lysed cells. Because the tissue weight was generally less than 50–100 mg and the DNA yield was expected to be low (<1 µg), 0.5 µl of glycogen solution (20 mg/ml) was added to aid in DNA precipitation.
3' End-Labeling
The DNA was end-labeled using a protocol described by Tilly and Hsueh [23]. Briefly, the reaction mixture (total volume, 50 µl) consisted of DNA template (1 µg in a volume of 29 µl), 10 µl of 5x reaction buffer, 5 µl of CoCl2, 5 µl of [32P]ddATP (50 µCi), and 1 µl (25 U) of terminal transferase enzyme. The reaction was allowed to proceed for 60 min at 37°C and was terminated by addition of 5 µl of 0.25 M EDTA (pH 8.0). Labeled DNA was precipitated twice, resuspended in 40 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and stored at -20°C until electrophoretic gel analysis.
Gel Electrophoresis and Quantitation of Radioactivity
Half of each labeled sample (20 µl) was run on a 2% (w/v) agarose gel for 3.5 h at 50 V. The gel was dried under vacuum for 2 h at room temperature. The dried gel was then sealed in plastic wrap and exposed to autoradiographic film overnight at room temperature. After the film was developed, the amount of radioactivity was measured using methods previously described [24]. Low-molecular-weight bands (<15 kilobases [kb]) were identified by comparison with DNA size standards and excised from each lane. Gel pieces were placed in vials containing Ecolite scintillation fluid, and radioactivity was measured.
In Situ End-Labeling (TUNEL)
The ovaries were treated with either medium control or TNF (1 ng/ml). Ovaries were fixed in neutral buffered formalin solution for 24 h. The ovaries were then processed through a graded series of ethanol and Hemo-D (xylene substitute) using standard procedures [22] and embedded in paraffin blocks. Serial sections were cut (thickness, 5 µm). The DNA was end-labeled using a TUNEL staining kit according to the manufacturer's directions and optimized for the ovarian tissue. Briefly, the sections were deparaffinized by immersing the slides in Hemo-D for two 10-min periods at room temperature. The samples were rehydrated by immersing the slides in 100% ethanol for two 10-min periods at room temperature, followed by rehydration through graded ethanol washes (90%, 70%, and 50%) for 5 min each at room temperature. The samples were incubated in Proteinase K for 5 min at 37°C, then washed twice in PBS for 10 min each time. Samples used for positive controls were then equilibrated for 15 min in DNase buffer (20 mM Tris, 2 mM calcium chloride, and 5 mM magnesium chloride), followed by incubation in DNase solution (100 µg/ml) for 15 min at 37°C. All samples were then washed twice in PBS for 5 min each time. Tissues were incubated with 45 µl of fluorescein-labeled nucleotides and 5 µl of terminal deoxynucleotidyl transferase enzyme (TdT) for 60 min at 37°C; however, for negative-control tissues, TdT was omitted. Tissues were then washed in PBS (four washes of 10 min each), followed by a 15-min incubation with propidium iodide (1 µg/ml) to counterstain cell nuclei. Tissues were then rinsed twice for 10 min each time in PBS, covered with 20 µl of Vectashield Mounting Media, and examined under a fluorescence microscope.
Statistical Analysis
Values in all figures are given as the mean ± SEM. For histomorphometric analyses, the oocyte and follicle numbers obtained from each ovary were considered to be independent observations (n = number of ovaries/group). For in situ end-labeling experiments, ovaries were pooled, and each experiment using a different ovarian pool was considered to be an independent observation (n = number of experiments). Statistical analysis was performed by one-way ANOVA, followed by the Fisher least-significant-difference test. Results were considered to be significantly different at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and TNF Antibodies on Follicular Development
Exogenous TNF (0.1, 1, 10, and 50 ng/ml) was added to cultured hemiovaries for 1–3 days. On Day 3, the only treatment significantly affecting naked oocyte numbers was 1 ng/ml (Fig. 1A). This dose significantly (P < 0.05) decreased the number of naked oocytes compared to all other treatment groups. In terms of primordial follicle number on Day 3 (Fig. 1B), the only two groups that were significantly different from each other were those receiving 1 and 50 ng/ml. A significant decrease was observed in primary follicle number at 1 and 10 ng/ml compared to both control and 0.1 ng/ml (Fig. 1C). This effect was diminished in the group receiving 50 ng/ml, which was not different from the control or the 0.1 ng/ml groups. Effects of TNF after shorter periods of culture were also evaluated and showed a similar significant decrease of oocytes in ovaries treated with 1 ng/ml on Day 1 (control vs. TNF, 2264 ± 185 vs. 1088 ± 76, P < 0.05) and on Day 2 (control vs. TNF, 2008 ± 107 vs. 1355 ± 56, P < 0.05). Other doses of TNF had no effect on oocyte numbers after 1 or 2 days of culture. Primordial and primary follicle numbers were not significantly different at any dose of TNF on Days 1 and 2.
|
Figure 2 shows representative ovaries after 3 days of organ culture with the varying TNF treatments. Both cultured (Fig. 2B) and age-matched, noncultured (Fig. 2A) control ovaries contained follicles at various stages of development. Naked oocytes as well as primordial and primary follicles were present at this time (Fig. 2, A, B, G, and H). In both cultured and noncultured, age-matched control ovaries, primordial follicles were first apparent on Day 1 (data not shown). Ovaries treated with TNF (Fig. 2, C–F and I–L) also contained the same spectrum of naked oocytes and primordial and primary follicles. However, ovaries treated with 1 ng/ml of TNF contained fewer follicles and oocytes (Fig. 2D), and many cells that appeared to be shrunken, condensed, and darkly stained.
|
A neutralizing TNF antibody was used to evaluate the effects of endogenous TNF on follicle number. Following 1 day of culture with TNF antibody, no significant difference was observed in naked oocyte numbers between the control IgG and the TNF antibody group. On both Days 2 and 3, the addition of TNF antibody significantly increased (P < 0.05) the number of naked oocytes compared to control (Fig. 3A). Antibody addition also significantly increased (P < 0.05) the number of primordial follicles on Days 2 and 3 compared to control while having no effect on Day 1 (Fig. 3B). Primary follicles were absent on Day 1 of culture and significantly increased on Days 2 and 3 (P < 0.05) by antibody treatment (Fig. 3C).
|
3' End-Labeling
To determine if TNF induced apoptosis in neonatal ovaries, DNA end-labeling was done. End-labeled DNA from all groups demonstrated a laddering pattern (Fig. 4), evident from the appearance of DNA fragments differing in size by 180–200 base pairs. Although laddering occurred in all groups, approximately twofold more DNA laddering was observed in the treatment group receiving 1 ng/ml on Day 3 compared to control (P < 0.05) (Fig. 5). No other dose was significantly different from control.
|
|
In Situ End-Labeling
To determine which cells were undergoing apoptosis, in situ end-labeling was done. The negative controls (lacking TdT) exhibited no nonspecific staining, as expected (data not shown). Results of in situ end-labeling revealed that the cell types undergoing apoptosis in the control and TNF (1 ng/ml) treatment groups were oocytes, granulosa cells, and interstitial cells (Fig.6). The most intense staining appeared to be within the oocytes and interstitial cells, with occasional staining in granulosa cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and its receptors in the ovary during the early neonatal period indicates a potential role for this factor in key events such as follicle assembly and atresia. The results of these experiments strongly suggest that TNF may play an important role in the process of oocyte atresia that occurs during the perinatal period in rats. The significant findings of this research were 1) TNF (at 1 ng/ml) decreased naked oocytes and primary follicles; 2) treatment with TNF antibodies significantly increased naked oocytes, primordial follicles, and primary follicles compared to control; 3) TNF (at 1 ng/ml) increased DNA laddering (apoptosis) compared to control; 4) apoptosis occurred primarily in oocytes and interstitial cells; and 5) more extensive apoptotic staining was observed in ovaries treated with TNF. These findings are very important, because they demonstrate that TNF can induce apoptosis in the neonatal rat ovary.
TNF is a known inducer of programmed cell death in both nonovarian tissues (as discussed in [25]) and in ovarian tissues, such as intact follicles [16], and isolated rat [26] and hen [17] granulosa cells. The apoptotic signaling of TNF through TNF-RI may occur by multiple pathways (reviewed in [27]). The intracellular portion of the TNF-RI is known as TRADD (TNF-receptor associated death domain). Binding of TNF to TNF-RI results in the assembly of various adaptor proteins to the intracellular death domain, with subsequent activation of caspase 8, an initiator caspase, and downstream effector caspases, such as caspase 3 [28, 29] The binding of TNF to TNF-RI is also known to activate sphingomyelinases, resulting in ceramide production [30] and apoptosis in several cancer cell lines [31–33]. Furthermore, TNF-stimulated ceramide production has been shown to induce apoptosis in ovarian cells [16, 17, 26]. This effect appeared to be mediated by ceramide, because treatment of follicles or granulosa cells with ceramide or an analogue also resulted in apoptosis. Apoptotic signaling may also involve calcium release, because the endonuclease responsible for that DNA cleavage that is characteristic of apoptosis is calcium- and magnesium-dependent [34, 35]. Additionally, TNF has been shown to increase levels of intracellular calcium in hen granulosa cells [36], and rat granulosa cells exposed to elevated magnesium and calcium underwent apoptosis [37]. Thus, TNF may be important in causing calcium release to activate the endonuclease necessary for apoptosis.
In the current study, the effects of TNF varied dramatically depending on the dose. A consistent pattern of response was seen whereby lower (0.1 ng/ml) and higher (10 and 50 ng/ml) concentrations had no effect on either oocyte/follicle numbers or apoptosis, whereas an intermediate dose (1 ng/ml) significantly reduced oocyte/follicle numbers and stimulated apoptosis. Several reports have shown that signaling through the TNF-RI is necessary for cytotoxicity [11, 12], whereas signaling through the TNF-RII has been shown to be involved in growth and stimulation [11]. Differential binding to the two receptors at different doses could be involved in the dose-related effects observed during the current study, because TNF-RI and TNF-RII are both present in the rat ovary as early as Embryonic Day 19 [14]. The TNF-RI has a higher binding affinity for soluble TNF than does TNF-RII [38], as illustrated by the long half-life of TNF-TNF-RI complexes (33.2 min) compared to those of TNF-RII (half-life, 1.1 min).
These results readily explain why most cellular responses are dominated by TNF-RI, even when considerable numbers of TNF-RII are coexpressed [32]. It is feasible that concentrations of 1 ng/ml of TNF in the current study could exhibit preferential binding to TNF-RI, inducing cytotoxicity, whereas higher concentrations of TNF could bind to both receptors and, thereby, initiate an antiapoptotic or survival pathway. A second possibility is that the calcium- and magnesium-dependent endonuclease responsible for apoptosis may be affected differently by TNF depending on the dose. Studies by Soboloff et al. [36] showed that TNF (at 10 ng/ml) mobilized calcium from intracellular stores, whereas higher concentrations (100 ng/ml) had no effect on calcium release. Third, TNF at higher doses may activate nuclear factor kappa B (NF-
B). This transcription factor has been shown to protect cells from apoptosis, whereas inhibition of this transcription factor has been shown to enhance cell death [39]. At higher doses (10 and 50 ng/ml), TNF may exert an antiapoptotic effect compared to the 1 ng/ml dose by activating NF-B. Recent studies by Xiao and Tsang [40] demonstrated that TNF (at doses of 5–20 ng/ml) prevented ovarian surface epithelial cell apoptosis by activation of NF-B. Additionally, TNF has been shown to activate NF-B by stimulating expression of the apoptosis-inhibitor FLIP (FLICE-inhibitory peptide) in granulosa cells [40], murine microglia cells [41], and lymphoma cells [42]. Finally, the inability of higher doses of TNF to stimulate apoptosis may be due to the presence of soluble TNF receptors, because elevated concentrations of TNF stimulate shedding of soluble TNF receptors [43], providing protection against the apoptotic effect of TNF [44, 45].
Apoptosis is a normal characteristic of ovarian physiology. This was partially reflected in the detection of DNA laddering in all ovarian DNA examined, including noncultured controls. Beaumont and Mandl [46] demonstrated that there are waves of germ cell degeneration in fetal and neonatal rat ovaries, with a reduction in germ cell numbers by 64% between Embryonic Day 18.5 and Day 2 postpartum. The current study supports the hypothesis that oocyte apoptosis during the perinatal period may be stimulated by endogenous ovarian TNF. This finding is supported by localization of TNF in neonatal rat oocytes [6] and identification of TNF receptors in oocytes, interstitial cells, and granulosa cells [13]. In the present study, exogenous TNF decreased oocyte and follicle numbers in culture at a dose that stimulated apoptosis. Conversely, treatment of ovaries with TNF antibodies significantly increased oocyte and follicle numbers. Oocyte and follicle numbers were significantly increased by TNF antibodies on Days 2 and 3 of culture, but not after 1 day of culture. Neutralization may have been ineffective on Day 1 because ovarian TNF increases between Days 0 and 2 [6]. That the TNF antibody resulted in increased follicle numbers suggested that endogenous concentrations of TNF are inhibitory, despite the lack of effect of higher concentrations of exogenous TNF. It is thus possible to speculate that TNF may be a factor involved in the normal physiological death of ovarian cells.
Because significant apoptosis occurred at the 1 ng/ml dose, it was of interest to identify which cells were undergoing apoptosis. Apoptotic cells were primarily oocytes and interstitial cells, with occasional TUNEL staining in the granulosa cells of primordial or primary follicles. These results are in agreement with the appearance of hematoxylin-stained sections from ovaries treated with 1 ng/ml of TNF that exhibited numerous cells within the interstitium that appeared to be condensed, with darkly staining nuclei. It is important to note that apoptotic staining was observed in both the control and the TNF-treated ovaries, because cell death occurs as a natural physiological process.
Based on the ovarian localization of TNF and its receptors [6, 13], it can be hypothesized that endogenous TNF could exert its apoptotic actions in an autocrine as well as a paracrine interaction between oocytes, interstitial cells, and granulosa cells. Studies in the human have demonstrated a similar pattern of TNF/receptor localization. Naz et al. [47] demonstrated TNF localization in human oocytes and cumulus granulosa cells from aspirated follicles, and TNF immunostaining has been identified in oocytes of human primordial follicles [48]. In the early stages of follicular atresia, immunoreactive TNF was observed in the degenerating human oocyte and the surrounding granulosa and theca cells [49]. Intense TNF immunostaining has also been observed in oocytes within atretic follicles in the mouse, particularly those in advanced stages of atresia [49]. Thus, oocytes and macrophages produce TNF, and it can likely affect oocyte, interstitial cell, and granulosa cell targets—the cells that were undergoing apoptosis in the current study.
In conclusion, the results of both application of exogenous TNF and neutralization studies supported the hypothesis that TNF inhibited oocyte/follicle numbers. This inhibitory effect is likely due to an apoptosis-stimulating effect. Some unanswered questions await further investigation. Why did some cells die whereas others were unaffected by the cytotoxic effects of TNF, and which signal transduction pathways are triggered by TNF activation during this critical period in follicular development? An important point that has become evident from the current study is that TNF may act differently depending on the dosage, making it important for investigators to evaluate their results carefully as a function of dose.
|
FOOTNOTES |
|---|
1 Supported by NIH grant 1-R15-HD34252-01 (to J.L.M.) and by Sigma Xi Grants-in-Aid of Research (to L.J.M.). 
2 Correspondence: Jennifer L. Marcinkiewicz, Box 5190, Department of Biological Sciences, Kent State University, Kent, OH 44242-0001. FAX: 330 672 3713; jmarcink{at}kent.edu
Accepted: September 19, 2001.
Received: April 16, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
receptor in the rat ovary. Biol Reprod 1998; 58: (suppl 1): 153 (abstract 263) induces clustering in ovarian theca-interstitial cells in vitro. Endocrinology 1992; 131:2503-2513[Abstract]B. Science 1996; 274::784-787B-mediated FLICE-like inhibitory proteins (FLIP). In: Program of the XIIIth Ovarian Workshop; 2000; Madison, WI. Abstract 45 This article has been cited by other articles:
|
|
 |
|
  D. J. Skarzynski, I. Woclawek-Potocka, A. Korzekwa, M. M. Bah, K. Piotrowska, B. Barszczewska, and K. Okuda Infusion of Exogenous Tumor Necrosis Factor Dose Dependently Alters the Length of the Luteal Phase in Cattle: Differential Responses to Treatment with Indomethacin and L-NAME, a Nitric Oxide Synthase Inhibitor Biol Reprod, April 1, 2007; 76(4): 619 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Greenfeld, K. F. Roby, M. E. Pepling, J. K. Babus, P. F. Terranova, and J. A. Flaws Tumor Necrosis Factor (TNF) Receptor Type 2 Is an Important Mediator of TNF alpha Function in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 224 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R Greenfeld, J. K Babus, P. A Furth, S. Marion, P. B Hoyer, and J. A Flaws BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries Reproduction, January 1, 2007; 133(1): 107 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E Nilsson, J. Stanfield, and M. K Skinner Interactions between progesterone and tumor necrosis factor-{alpha} in the regulation of primordial follicle assembly. Reproduction, December 1, 2006; 132(6): 877 - 886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. R. Kezele, J. M. Ague, E. Nilsson, and M. K. Skinner Alterations in the Ovarian Transcriptome During Primordial Follicle Assembly and Development Biol Reprod, January 1, 2005; 72(1): 241 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Skarzynski, M. M. Bah, K. M. Deptula, I. Woclawek-Potocka, A. Korzekwa, M. Shibaya, W. Pilawski, and K. Okuda Roles of Tumor Necrosis Factor-{alpha} of the Estrous Cycle in Cattle: An In Vivo Study Biol Reprod, December 1, 2003; 69(6): 1907 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Abdo, S. Hisheh, and A. Dharmarajan Role of Tumor Necrosis Factor-Alpha and the Modulating Effect of the Caspases in Rat Corpus Luteum Apoptosis Biol Reprod, April 1, 2003; 68(4): 1241 - 1248. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
