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Articles |
a Departments of Anatomy and Cell Biology,
b Molecular and Integrative Physiology, and
c Obstetrics and Gynecology, Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is a 17-kDa, multi-functional hormone-like polypeptide that is produced by many types of cells in the ovary [1]. Immunoreactive TNF, mRNA, and bioactivity have been localized to numerous ovarian compartments in various animal species and humans [1]. TNF can inhibit gonadotropin-stimulated progesterone and androgen production by theca [2, 3] and also inhibit progesterone and aromatase (and estradiol secretion) in granulosa cells [4]. It is hypothesized that during early stages of follicular development, TNF from numerous cells of origin within the ovary acts on granulosa and thecal cells after binding with specific TNF receptors, modulates steroidogenesis [1], and participates in apoptosis of follicles [5] as they enter or attempt to enter the growing pool. The TNF receptor type used and the mechanism by which TNF exerts these actions on the ovary are unknown. TNF is known to have two receptors, p55 (type I; TNFRI) and p75 (type II; TNFRII). The extracellular domains of the receptors are similar, but the intracellular (cytoplasmic) domains are quite different [6]. The type I receptor is thought to mediate apoptosis and inhibitory functions induced by TNF, and the type II receptor is thought to mediate trophic functions including stimulating cell division. However, there are exceptions to the generalized functions of these receptors.
TNF exhibits multiple effects on ovarian cells; however, little is known about TNF receptor signaling in ovarian cells. TNF receptor knockout mice have been generated [7] and can be maintained as homozygous knockouts. This in itself might indicate normal fertility in the absence of TNF receptors. However, because TNF has been shown to have dramatic effects on ovarian cells, it was our interest to investigate various parameters of ovarian function and fertility in mice lacking the TNFRI. The results indicate that the presence of the TNF type I receptor is important for onset of puberty, ovarian responsiveness to gonadotropins, estrous cyclicity, and ovarian senescence. In addition, these studies demonstrate that the inhibitory action of TNF on gonadotropin-stimulated steroidogenesis is likely to be mediated through the type I receptor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TNFRI -/- mice on a C57BL6 background were obtained from Immunex [7] (Seattle, WA), C57BL6 mice were obtained from Harlan, Inc. (Indianapolis, IN), TNFRII null mice were obtained from Jackson Laboratories (Bar Harbor, ME), and all three were established as breeding colonies in our laboratory. All mice were provided food and water ad libitum, exposed to a 12L:12D schedule, and housed under pathogen-free conditions. Breeding pairs were established as follows: A single male and a single female were paired at the time of weaning and housed together continuously in a single cage. Animals were checked daily, and the date and number of pups born was carefully monitored. All weaning occurred on Day 20 of age with the exception of those mice assessed for ovulatory response to gonadotropin, which were weaned on Day 19. Pups were checked daily to assess for vaginal opening beginning on the day of weaning. Estrous cycles were determined by daily (0900 h) assessment of cells retrieved after vaginal lavage. For cyclicity studies, mice were housed four per cage and were assessed by vaginal lavage for 21 continuous days beginning at 2 or 6 mo of age. Day of the cycle was determined as originally described by Allen [8] on the basis of the majority of cells present in the vaginal lavage as follows, briefly: proestrus, presence of rounded nucleated cells; estrus, presence of cornified cells; diestrus, presence of leukocytes. The percentage of days spent in each phase was determined for each animal, and the means for a group were calculated. There were times in the 21 days when an animal was not in a cycle at all, and consequently, the number of cycles in a 21-day period cannot be derived simply by dividing the length of a cycle into 21. The data were transformed by the arc sine before analysis by ANOVA. All studies were approved by the Institutional Animal Care and Use Committee.
Ovulatory Response to Gonadotropins
Mice received s.c. injections of 2.5 IU eCG on Day 19, 21, 25, or 55 of age. Fifty-two hours after injection, 2.5 IU hCG was administered s.c. On the following morning, ovaries and oviducts were carefully dissected, and the oviducts were irrigated with saline. The cumulus oocyte complexes were briefly exposed to hyaluronidase to remove cumulus granulosa cells, and the ova were counted. In addition, blood was collected and serum was prepared for subsequent RIA.
Dispersion and Culture of Ovaries
Ovaries were collected from mice killed by cervical dislocation on Day 26 of age, and cleaned of adhering tissues. Ovaries from 4–10 animals were pooled for a given experiment. Ovaries were incubated in 0.6 ml dispersion medium (collagenase [4 mg/ml]/deoxyribonuclease [DNase; 10 µg/ml], in Medium 199) in a humidified atmosphere of 5% CO2/95% air at 37°C for a total of 30 min. After 10-min periods, the cells were aspirated through progressively smaller needles (18-20-22 gauge) fitted onto a 1-cc syringe. After the final incubation, 1 ml complete medium (Medium 199 containing Hanks' salts, 25 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid buffer, 2 mM L-glutamine, 50 µg/ml streptomycin, 0.1% [w:v] BSA, 1.0% fetal bovine serum, and insulin:transferrin:selenium [ITS: insulin 5 µg/ml, transferrin 5 µg/ml, and selenium 5 ng/ml]) was added, and the cells were centrifuged and washed 2 additional times with complete medium. The cells were diluted in complete medium and enumerated with a hemocytometer. Cell viability was determined by the exclusion of trypan blue. For culture, 150 000 viable cells were plated per well of a 24-well plate in 0.5 ml complete medium and cultured overnight to allow for attachment. On the following day, medium was removed, and fresh medium and treatments (LH, 50 ng/ml, and/or TNF 10 ng/ml) were added; conditioned media were collected 48 h later and stored for steroid and cAMP determinations. Within each experiment, treatments were in replicates of 3–5, and each experiment was repeated 4 times. Data presented are means ± SEM of a single experiment and are representative of the findings of each independent experiment.
Steroid and cAMP Analysis
Serum progesterone and accumulation of progesterone and estradiol in culture medium was measured by RIA as previously described [9]. Cyclic AMP levels in medium were determined with a commercially available kit according to the manufacturer's directions as previously described [2].
Statistics
Data were analyzed by one-way ANOVA followed by least-significant difference. Differences were considered significant if P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaginal opening occurred on Day 23.8 ± 0.6 (n = 10) in TNFRI knockout mice and on Day 27.6 ± 0.2 (n = 11) in controls (P < 0.001 as determined by one-way ANOVA).
Ovulatory Response to Gonadotropin
Immature mice on Day 19 of age responded poorly to treatment with eCG and hCG to induce ovulation. The morning after hCG treatment, only 50% of these mice had ovulated, and those ovulating shed 1–3 ova. There was no difference in the response in TNFRI knockout mice and control C57BL6 mice at this age. In contrast, mice that had received injection of eCG on Day 21 or 25 of age ovulated in response to hCG administration 2 days later. TNFRI knockout mice shed significantly more ova compared to C57BL6 controls of the same age (Fig. 1A). The greater number of ovulations by TNFRI knockout mice was accompanied by higher serum levels of progesterone on both Day 21 and Day 25 (Fig. 1B). The increased ovulatory response in immature TNFRI knockout mice was no longer apparent in the mature animal. At Day 55 of age, similar numbers of ova were shed by TNFRI knockout and control mice, and serum progesterone levels the morning after ovulation were also similar (Fig. 1).
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Cyclicity
At 2 mo of age, cycle length was similar in TNFRI knockout and control mice (5.69 ± 0.26 vs. 5.88 ± 0.26 days, respectively); however, TNFRI null mice spent significantly more time in estrus and less time in diestrus than did controls (Table 1). Therefore, the number of cycles per mouse in a 21-day period was greater in TNFRI null mice. At 6 mo of age, a dramatic difference was observed in estrous cyclicity (Table 1). Only 40% of TNFRI null mice exhibited estrous cycles by 6 mo of age compared to 100% of controls. Of those TNFRI null mice exhibiting cycles, considerably less time was spent in estrus and more time in diestrus. This resulted in longer cycles in those TNFRI null mice with cycles than in controls (7.0 ± 1.1 days vs. 5.0 ± 0.2 days, respectively). Therefore, the number of cycles in a 21-day period was significantly reduced in TNFRI null mice compared to controls (Table 1). Interestingly, the percentage of time spent in each phase of the cycle for TNFRI null mice at 2 mo of age was not different when compared to that for C57BL6 controls at 6 mo of age.
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Fertility
Fertility of TNFRI null mice was compared to that of controls by determining the number of litters born and the percentage of pairs producing litters. All colonies were maintained in the animal facilities at the same time under identical conditions. The number of litters born per month over a 12-mo period for 10 breeding pairs delivering at least one litter was significantly lower for TNFRI null mice than for C57BL6 controls (Table 2). In addition, mice null for TNFRII had significantly more litters than TNFRI null mice, whereas the number of litters born to TNFRII null mice was not different from that for C57BL6 controls. A striking difference in overall infertility was also noted. Of 34 TNFRI null breeding pairs continuously housed for at least 12 mo, 17.7% never produced a single litter. This is approximately three times that seen in control C57BL6 (5.3%) and TNFRII null (5.9%) mice.
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Culture of Ovarian Dispersates
TNFRI null and control C57BL6 ovarian dispersates responded in vitro to LH treatment with increased cAMP, progesterone, and estradiol accumulation (Fig. 2). Treatment of ovarian cultures from controls with LH and TNF resulted in inhibition of LH-stimulated progesterone and estradiol accumulation in the media. In controls, although reduced steroid production was observed, TNF had no effect on LH-stimulated cAMP accumulation. TNF treatment of ovarian cultures derived from TNFRI null mice had no effect on LH-stimulated steroid or cAMP accumulation (Fig. 2). In addition, basal steroid and cAMP accumulation in TNFRI null and control cultures was not different. TNF treatment did have a tendency to increase steroid production in control cultures. However, this tendency was not statistically significant and was not a consistent finding.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initially, the onset of puberty in TNFRI null mice appears to be accelerated: vaginal opening, one measure of the onset of puberty, occurs early in comparison to that of controls. The mechanism for early vaginal opening in the TNFRI null mouse is not clear. Vaginal opening occurs as a result of increasing estradiol secretion and can be stimulated by injection of estradiol into immature mice or rats [10]. Whereas vaginal opening in the rat occurs simultaneously with the first ovulation, vaginal opening in the mouse may occur up to 10 days before the first vaginal cornification and the onset of cyclicity [11]. Vaginal opening is an apoptosis-mediated event [12]. Overexpression of Bcl2, an apoptosis inhibitor, results in the complete inhibition of vaginal opening, even when estradiol is administered exogenously [12]. Early vaginal opening in the TNFRI null mouse may be directly related to a loss of TNFRI-mediated signaling. TNF both stimulates and inhibits apoptosis in a cell- and development-specific fashion. In some systems, TNF stimulates the expression of the apoptosis inhibitor, FLIP (Fas-associated death domain protein-like interleukin-1-converting enzyme inhibitory protein) [13]. Thus, in the absence of TNFRI-mediated signaling, an inhibitor of apoptosis such as FLIP may not be expressed, resulting in apoptosis and vaginal opening. A direct role for TNF in the process of vaginal opening has not been addressed. Serum concentrations of TNF dramatically decline at vaginal opening and the first ovulation in the rat [14]. Early vaginal opening in the TNFRI null mouse may simply be a result of precocious follicle development and increased estradiol levels due to the lack of the inhibitory action of TNF on the ovary. Early increases in estrogen emanating from the ovary and acting on the vaginal epithelium would result in early opening. Serum estradiol levels throughout the peripubertal period were not investigated in the present study.
Early onset of follicle development might also explain the enhanced ovulatory response to exogenous gonadotropin in TNFRI null mice. It is hypothesized that TNF has a modulatory role in follicular development [1, 15]. In rats, TNF levels are elevated in the ovaries just before vaginal opening and the first ovulation, after which ovarian TNF levels decrease [14]. In addition, administration of eCG to immature rats resulted in rapid reduction in ovarian TNF levels [14]. Those data imply that the role of TNF in the prepubertal ovary is to retard the onset of follicular development. The data presented here for the mouse support this hypothesis. If TNF inhibits prepubertal follicular maturation, then loss of the functional TNFRI would prevent the action of TNF. Although heightened sensitivity of the ovary to gonadotropins was apparent in immature ovaries, response to gonadotropin administration in the TNFRI knockout adult mice was not different from that of controls. It appears that, in adults, unknown mechanisms corrected for the heightened response to gonadotropin in young mice. Although gonadotropin levels were not measured in the present study, it may be that these levels in TNFRI knockout adult mice have compensated for the lack of TNF and are lower than in control mice. Lower gonadotropin levels might be one mechanism to control follicular development and ovulation in highly sensitive ovaries (those that lack TNFRI), especially in the adult.
Another significant finding in this study is the early onset of senescence in TNFRI knockout mice. Only 40% of animals exhibited estrous cycles at 6 mo of age. Those TNFRI null mice with estrous cycles at 6 mo of age had cycles comparable to the estrous cycles previously described for C57BL6 mice 15 mo and older [11]. Therefore, TNFRI null mice 6 mo of age, normally the time of peak reproductive cyclicity [11], had already undergone senescence. Early onset of senescence is a likely explanation for reduced litters produced by TNFRI null mice. This finding provides insight into potential mechanisms of premature ovarian failure in women. Serum levels of TNF are reduced in women with premature ovarian failure compared to age-matched normally cycling women [16]. Similarly, in mice, absence of TNFRI and thus TNF signaling capability results in early reproductive senescence, likely to be associated with a lack of ovarian cyclicity since estrous cycles were disrupted, although it is not known if those ovaries contained fewer oocytes. Therefore, in both the human and the mouse it appears that TNF is a common factor in premature aging of the reproductive system. The precise mechanisms whereby TNF modulates reproductive aging are not known. Possibly, the TNFRI null mouse will provide a useful model for investigating the mechanisms of premature ovarian aging and the role of TNF in this process.
In addition to early senescence of estrous cyclicity, TNFRI null mice at 2 mo of age exhibited estrous cycles not different from those of controls at 6 mo of age. Therefore, it appears that mature cyclicity was attained at an earlier age in TNFRI null mice. This observation is likely to be related to early vaginal opening and heightened responsiveness of the ovary to gonadotropin stimulation in the TNFRI null mice. As already discussed, it has been hypothesized that TNF inhibits follicular development in the immature ovary. Early establishment of mature cyclicity is another measure of accelerated puberty in the TNFRI null mouse and provides additional data to support this hypothesis.
Estrous cyclicity, as determined by vaginal cytology, is a reflection of the function of the hypothalamic-pituitary-ovarian axis and the resultant cyclicity of ovarian steroids. Early senescence associated with the loss of estrous cyclicity is probably a measure of ovarian follicular development and ovulation. Although ovulation during natural cycles was not assessed in the present study, it is likely that the TNFRI null mice underwent early ovarian senescence, and this is probably the explanation for the reduced numbers of litters born to TNFRI null females. It is also possible that TNFRI null males have reduced reproductive capacity. In the present study, TNFRI null females were mated only with TNFRI null males, and thus altered reproductive capacity of the females and males cannot be separated. However, the abnormal characteristics of the estrous cycles of the TNFRI null females appear to be the most likely explanation for the reduced litters born to TNFRI null females.
TNF exerts its function through binding to two different transmembrane receptors, TNFRI(p55) and TNFRII(p75). These receptors are type I membrane glycoproteins with the N terminus located on the exterior of the cell. They are also related on the basis of a highly conserved cysteine-rich repeat in the extracellular domain; however, the intracellular domains exhibit little similarity [6]. It is generally believed that TNFRI is responsible for the majority of the biologic actions of TNF [17]. This hypothesis is supported by several studies defining specific cellular actions to TNFRI and not TNFRII using TNFR knockout mice [5]. Litters born to TNFRI null mice, TNFRII null mice, and C57BL6 mice, all colonies maintained by our laboratory, were compared to determine the significance of the TNF receptors to the findings observed in the present study. On the basis of the numbers of litters born, TNFRII null mice and C57BL6 mice exhibited similar reproductive capacity. In contrast, TNFRI null mice exhibited significantly reduced reproductive capacity compared to both C57BL6 mice and TNFRII null mice. This data indicated that the reduced reproductive capacity observed in the TNFRI null mice is specifically related to TNF signaling through TNFRI and not TNFRII. TNFRII null mice did not exhibit reduced reproductive capacity (Table 2).
Results from the culture experiments further indicate a role for TNFRI in the mechanism of TNF action. LH treatment of ovarian dispersates from TNFRI null mice resulted in increased secretion of progesterone and estradiol, and increased accumulation of cAMP in the culture media. This result mirrored that seen in the ovarian dispersates from C57BL6 controls. Thus, ovarian dispersates from TNFRI null mice have the capacity to respond to stimulation by LH. As previously demonstrated in other species, TNF treatment of ovarian cells inhibits responsiveness to gonadotropin [2, 18–22]. The same result was observed in the present study with C57BL6 mouse ovarian cells. TNF inhibited LH-stimulated (and FSH-stimulated; data not shown) steroid accumulation in the culture media. In contrast to the effects of TNF on control ovarian dispersates, the effects of TNF treatment on gonadotropin-stimulated TNFRI null ovarian dispersates were not different from those of gonadotropin treatment alone. Thus, in the absence of TNFRI, TNF did not alter gonadotropin-stimulated steroid production. These data provide further evidence that TNF mediates its action on ovarian cells via TNFRI.
The results obtained for the normal C57BL6 mouse in the present study appear most similar to those seen using human granulosa cells taken during the menstrual cycle [20, 21]; i.e., TNF inhibited gonadotropin-stimulated progesterone and estradiol secretion without affecting cAMP [20]. This action of TNF probably occurs at post-cAMP sites, since cAMP concentrations were not affected by TNF treatment [21]. We hypothesize that TNF inhibition of gonadotropin-stimulated estradiol secretion is mediated by alteration in steroidogenic factor 1 and cAMP response element-binding protein phosphorylation and/or binding to the aromatase promoter [23, 24].
This study describes the reproductive phenotype of TNFRI null mice. Overall reproductive capacity is reduced in TNFRI null mice compared to control C57BL6 mice and TNFRII null mice. These studies provide additional evidence for the role of TNF in normal ovarian function and provide further insights into additional potential roles of TNF in the onset of puberty and the regulation of reproductive senescence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Katherine F. Roby, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 7180; kroby{at}kumc.edu
Accepted: August 10, 1999.
Received: April 5, 1999.
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REFERENCES |
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D.-S. Son, K. Y. Arai, K. F. Roby, and P. F. Terranova Tumor Necrosis Factor {alpha} (TNF) Increases Granulosa Cell Proliferation: Dependence on c-Jun and TNF Receptor Type 1 Endocrinology, March 1, 2004; 145(3): 1218 - 1226. [Abstract] [Full Text] [PDF]
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K. F. Roby Alterations in Follicle Development, Steroidogenesis, and Gonadotropin Receptor Binding in a Model of Ovulatory Blockade Endocrinology, June 1, 2001; 142(6): 2328 - 2335. [Abstract] [Full Text] [PDF]
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