| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovary |
a Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and a GnRH analogue, respectively, and the preovulatory follicle was obtained at 0, 3.5, 6, 12, 18, or 24 h after GnRH treatment. RNase protection assays revealed a transient increase in levels of PR mRNA, which peaked at 6 h after GnRH and declined to the time 0 value by 12 h and a second increase at 24 h. The second objective was to investigate the mechanisms that regulate PR mRNA expression through in vitro studies on follicular cells of preovulatory follicles obtained before the LH/FSH surge. Theca and granulosa cells were isolated and cultured with or without a luteinizing dose of LH or FSH, progesterone, LH + progesterone, or LH + antiprogestin (RU486). Levels of PR mRNA increased in a time-dependent manner in granulosa cells cultured with LH or FSH and in theca cells cultured with LH, peaking at 10 h of culture. In contrast, progesterone (200 ng/ml) did not upregulate mRNA for its own receptor, and neither progesterone nor RU486 affected LH-stimulated PR mRNA accumulation. Furthermore, RU486 completely blocked LH-stimulated expression of oxytocin mRNA, indicating that PR induced by LH in vitro is functional. These results show that the gonadotropin surge induces a rapid and transient increase in expression of PR mRNA in both theca and granulosa cells of bovine periovulatory follicles followed by a second rise close to the time of ovulation and that the first increase in PR mRNA can be mimicked in vitro by gonadotropins but not by progesterone. These results suggest multiple and time-dependent roles for progesterone and PR in the regulation of periovulatory events in cattle.
follicular development, granulosa cells, ovary, progesterone receptor, theca cells
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The PR, a member of the nuclear transcription factor superfamily, mediates many of progesterone's actions on ovarian tissues [14], although other progesterone binding sites have been implicated in the mediation of nongenomic actions of progesterone on follicular cells [15–17]. In primate [18], rabbit [19], and porcine [20] ovaries, PR has been immunolocalized to theca cells of antral follicles and to granulosa cells of preovulatory follicles that either were obtained after the LH surge [18, 20] or were exposed to an ovulatory dose of hCG in vivo [19], whereas granulosa cells of small and medium-size antral follicles were consistently negative for PR [18–20]. In rats, in contrast, PR mRNA was localized by in situ hybridization to granulosa but not theca cells of preovulatory follicles; its expression was transient and tightly coupled to the preovulatory gonadotropin surge [21]. The transient induction of PR mRNA could be mimicked by ovulatory concentrations of LH and FSH in cultures of rat [9] or porcine [22] granulosa cells. Two forms of PR protein (A and B) have been identified by immunoblot analysis of cultured rat granulosa cells [9] and porcine ovaries [20].
In bovine ovaries, production of progesterone and oxytocin by preovulatory follicles increases in response to the gonadotropin surge, whereas production of estrogen and androgen declines [23–25]. Progesterone stimulates oxytocin secretion by bovine granulosa cells from preovulatory follicles obtained before the LH surge [26], and PR antagonists inhibit oxytocin production and oxytocin mRNA expression in cultured bovine granulosa cells obtained from slaughterhouse ovaries [11], suggesting at least one functional role for PR in bovine periovulatory follicles. PR mRNA has been detected in cultured bovine granulosa cells [11] and in the bovine corpus luteum (CL) [27]. However, nothing is known about the pattern of expression or regulation of the PR in bovine preovulatory follicles. In the present study, we determined when and where PR mRNA is expressed in bovine follicular cells in vivo during the periovulatory period (i.e., between the gonadotropin surge and ovulation), tested hypotheses about the regulation of expression of PR mRNA through in vitro studies on follicular cells of bovine preovulatory follicles obtained before the LH surge, and examined whether the PR induced in vitro is functional.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Holstein heifers exhibiting normal and regular estrous cycles were used in accordance with procedures approved by the Cornell University Animal Care and Use Committee. To induce regression of the CL and initiate a follicular phase, animals were injected with 25 mg of prostaglandin F2 (PGF2) (Lutalyse; Pharmacia and Upjohn Co., Kalamazoo, MI) on the evening of Day 6 or the morning of Day 7 of the estrous cycle (Day 0 = onset of estrus). Jugular blood samples were collected twice daily beginning on Day 6 of the estrous cycle. Plasma samples were stored frozen for later measurement of progesterone to verify that luteal regression had been induced. Thirty-six hours after PGF2 injection, 100 µg of a GnRH analogue (Cystorelin; Sanofi Animal Health, Overland Park, KS) was administered i.m. to induce the LH surge. This experimental protocol induces ovulation (about 29 h after GnRH injection) and a normal luteal phase [25]. Ovaries were examined daily by transrectal ultrasonography, beginning on Day 4 of the cycle, to monitor development of the preovulatory follicle as described previously [28].
Collection of Follicles During the Periovulatory Period
The ovary bearing the preovulatory follicle was removed by colpotomy at 0 (36 h post-PGF2), 3.5, 6, 12, 18, or 24 h post-GnRH injection (n = 3 follicles/time point). The ovary was transported from the farm to the laboratory (
10 min) in ice-cold Eagle minimum essential medium (MEM) buffered with 25 mM Hepes, supplemented with penicillin (50 U/ml) and streptomycin (50 µg/ml) (all obtained from Gibco BRL, Grand Island, NY). The preovulatory follicle was dissected from the ovary as described previously [29]. The theca interna with adherent granulosa cells was peeled off the theca externa and surrounding stromal tissue. The resultant follicle wall preparation (theca interna with attached granulosa cells) was cut into small pieces and frozen in liquid N2 for later extraction of total RNA.
Collection of Follicles Before the LH Surge; Isolation and Culture of Theca and Granulosa Cells
The ovary bearing the preovulatory follicle was removed 36 h after PGF2 injection. The preovulatory follicle was dissected and processed as described previously [29]. Granulosa cells were scraped from the theca interna with a bent glass needle and counted using a hemacytometer. One tenth of the granulosa cells were snap-frozen in liquid N2 and used as the 0 h control. The remaining granulosa cells were distributed to 12-well plates (500 000 cells/well; Costar, Cambridge, MA). The theca interna tissue was cut into 60 pieces; 6 pieces were selected at random, snap-frozen in liquid N2, and used as the 0 h control, and the rest were distributed to 24-well plates (3 pieces/well; Costar, Corning, NY). Granulosa cells and pieces of theca interna were cultured in defined medium consisting of Eagle MEM, supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), L-glutamine (2 mM), nonessential amino acids (0.1 mM), insulin (27.6 mIU/ml), human transferrin (5 µg/ml), and cortisol (40 ng/ml).
To determine the effects of gonadotropins on the expression of PR mRNA, granulosa cells and pieces of theca interna were incubated in medium alone or with LH (100 ng/ml, NIH LH-S26) or FSH (100 ng/ml, NIH FSH-S17) for 4, 10, or 24 h. To determine the effects of progesterone on the expression of PR mRNA, granulosa cells were incubated for 10 h in medium alone or with progesterone (200 ng/ml; the approximate concentration in the follicular fluid 3.5 h post-GnRH [30, 31]), LH (100 ng/ml), LH + progesterone, or LH + RU486 (1 µM; this concentration blocked oxytocin mRNA expression in cultures of bovine granulosa cells [11]). To determine whether the increase in PR mRNA induced by LH in vitro is correlated with an increase in functional PR, granulosa cells were cultured in medium alone or with LH (100 ng/ml) or LH + RU486 (1 µM) for 48 h. At the end of each culture period, granulosa cells in two culture wells from each treatment group were subjected to a direct lysate RNase protection assay (RPA), and theca interna pieces were snap-frozen for later isolation of total RNA. Experiments were repeated with cells from three preovulatory follicles.
Generation of the Plasmid Containing Bovine cDNA for PR
A 325-base pair (bp) bovine PR cDNA fragment was generated by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA (1 µg) isolated from a bovine CL obtained on Day 7 of the estrous cycle was reverse transcribed at 37°C for 1 h using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and a random hexamer primer (Promega). First-strand cDNA samples were amplified using oligonucleotide primer pairs (5'-CCCACAGGAGTTTGTCAAGCTC-3', 5'-TAACTTCAGACATCATTTCCGG-3') based on the previously reported sequence of ovine PR cDNA (GenBank accession number Z66555) within the region coding for the steroid-binding domain of the receptor. Amplification consisted of a preincubation at 94°C for 5 min before adding Taq polymerase (Perkin-Elmer, Foster City, CA) and then 35 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec. A PCR product of the predicted size was cloned into the pGEM-T Easy Vector and sequenced using BIG Dye Terminator chemistry and AmpliTaq-FS DNA polymerase (ABI 377 Automated DNA Sequencer; Applied Biosystem Division, BioResource Center/DNA Sequencing Facility, Cornell University, Ithaca, NY). The 325-bp bovine PR gene fragment was 96%, 95%, 94%, and 94% similar to the sheep, pig, rhesus monkey, and human PR cDNA sequences, respectively (BLAST database; www.ncbi.nlm.nih.gov/blast; [32]).
Quantification of PR mRNAs and Oxytocin
Total RNA was extracted from follicle wall samples, cultured theca interna, bovine CL, myometrium from a pregnant cow, and bovine spleen using Trizol reagents (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. Aliquots of 10–15 µg total RNA were used for Northern blot analysis as described previously [33] with the following modifications. To synthesize radiolabeled riboprobes, plasmids containing bovine cDNAs for PR and 18S rRNA [34] were linearized with NcoI and EcoRI, respectively, and transcribed using [-32P]CTP (10 mCi/ml; DuPont New England Nuclear, Boston, MA) and SP6 and T3 RNA polymerase, respectively. Membranes were hybridized sequentially with 106 cpm 32P-labeled antisense riboprobe for PR and 5 x 103 cpm 32P-labeled antisense riboprobe for 18S rRNA per milliliter of ULTRAhyb hybridization buffer (Ambion, Austin, TX). After hybridization at 68°C for at least 16 h, excess probe was removed by three washes at room temperature in 2x saline-sodium citrate (SSC) and 0.1% SDS for 5 min and two stringent washes in 0.1x SSC and 0.1% SDS at 65°C for 60 min. The membrane was exposed to Fuji Medical x-ray film (Fisher Scientific, Pittsburgh, PA) for 24 h at -80°C with one intensifying screen and subsequently exposed to a phosphorimaging plate (Fuji Medical Systems, Stamford, CT). Radioactivity in each band was quantified with a Fuji BAS1000 PhosphorImager (Fuji, Tokyo, Japan). The relative levels of PR mRNA were calculated by dividing the intensity of the PR mRNA band by the intensity of the corresponding 18S rRNA band.
RPAs were carried out according to the instructions for the RPA II kit (Ambion). Samples of total RNA (1-2 µg) were hybridized with excess amounts of 32P-radiolabeled antisense riboprobes for bovine PR and 18S rRNA for 12–15 h at 45°C. The protected fragments were fractionated by electrophoresis through a 6% denaturing polyacrylamide gel containing 7 M urea. After electrophoresis, the gel was covered with plastic wrap and exposed to Fuji Medical x-ray film and then to a phosphorimaging plate. Radioactivity in each band was quantified by phosphorimaging, as described above.
Lysate RPAs were carried out according to the instructions for the Direct Protect lysate RPA kit (Ambion). Granulosa cell lysates were hybridized with excess amounts of 32P-radiolabeled antisense riboprobes for bovine PR and 18S rRNA or for bovine oxytocin and 18S rRNA overnight at 37°C. Bovine cDNA for oxytocin (a gift from Dr. D. Richter, University of Hamburg, Hamburg, Germany) was linearized with AvaII and transcribed using [-32P]CTP and T3 RNA polymerase. The protected fragments were fractionated, and the relative levels of mRNA for PR or oxytocin were quantified by phosphorimaging as described above.
Statistical Analyses
All data are presented as means ± SEM. Data were tested for homogeneity of variance by a Hartley test, and log transformations were performed on data sets that had heterogeneous variances. All data were analyzed by ANOVA using the general linear model procedure of SAS (Carey, NC). The model included effects of time of tissue collection for comparisons of levels of PR mRNA across time from GnRH administration or effects of treatment in vitro on levels of mRNA for oxytocin. A model statement testing for effects of time of culture or treatment in vitro was used on levels of PR mRNA in cultured theca and granulosa cells. If ANOVAs revealed significant effects of treatments or time of culture, the means of treatment groups or means after various times of culture were compared using a Duncan multiple range test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Multiple PR gene transcripts of 8, 4, and 1.6 kilobases (kb) were detected in total RNA from bovine follicle wall obtained 6 h after GnRH injection, Day 7 CL, and myometrial tissue from a pregnant cow but not in total RNA from spleen, which has been reported to be negative for PR mRNA [27] (Fig. 1). To determine whether PR mRNA is induced or upregulated in periovulatory follicles by the gonadotropin surge in vivo, total RNA was isolated from bovine periovulatory follicle wall obtained at various times after GnRH injection and subjected to Northern blot analysis (Fig. 2). PR mRNA was detected in total RNA isolated from follicle wall obtained at 3.5, 6, or 24 h after GnRH injection; 8- and 4-kb transcripts were the most abundant, and upon longer exposure, a faint 1.6-kb transcript was evident. The samples were also analyzed by RPAs to increase the sensitivity of detection and quantification for PR mRNA. Like the Northern blots, results from RPAs revealed a triphasic pattern of change in the levels of PR mRNA, which initially peaked at 6 h after GnRH injection (15-fold increase relative to 0 h; P < 0.01), declined to time 0 values by 12 h, and then increased again by 24 h (6-fold increase relative to 0 h; P < 0.05; Fig. 3).
|
|
|
Expression of PR mRNA in Theca and Granulosa Cells Cultured with Gonadotropins
To determine whether the effects of the gonadotropin surge in vivo on PR mRNA expression can be mimicked in vitro, theca and granulosa cells isolated from preovulatory follicles obtained before the LH surge were cultured with or without a luteinizing dose of LH or FSH. Levels of PR mRNA in granulosa cells cultured in medium alone did not change between 0 and 10 h and then decreased between 10 and 24 h (P < 0.05; Fig. 4). Both LH and FSH increased PR mRNA expression in a time-dependent manner in granulosa cell cultures (P < 0.05). The highest expression was observed at 10 h (3.1-fold and 4-fold higher with LH and FSH, respectively, compared with 0 h levels), and expression declined to 0 h levels by 24 h of culture. PR mRNA levels were higher in granulosa cells cultured with LH or FSH than in control cultures at all culture times (P < 0.05).
|
Levels of PR mRNA in theca cells cultured in medium alone did not change throughout the culture period (P > 0.05; Fig. 5). LH increased PR mRNA expression in a time-dependent manner in theca cell cultures (P < 0.05). Expression peaked at 10 h (2.5-fold higher than 0 h levels) and declined to levels in control cultures by 24 h. As expected, FSH had no effect on the levels of PR mRNA in theca cells at any time of culture (P > 0.05).
|
Expression of PR mRNA in Granulosa Cells Cultured> with Progesterone or RU486
In cattle, the rapid rise in intrafollicular progesterone after the LH surge precedes the first increase in PR mRNA expression. To determine whether this rise in intrafollicular progesterone at the time of the gonadotropin surge has a role in the upregulation of PR mRNA in bovine granulosa cells after the LH surge in vivo, the effect of progesterone or the PR blocker RU486 on expression of PR mRNA was tested. As expected, LH stimulated accumulation of PR mRNA (P < 0.05; Fig. 6). In contrast, progesterone at a concentration similar to that in bovine follicular fluid did not affect the levels of PR mRNA in cultured granulosa cells (P > 0.05). In addition, neither progesterone nor RU486 had any effect on LH-stimulated PR mRNA accumulation in cultured granulosa cells (P > 0.05).
|
Expression of Oxytocin mRNA in Granulosa Cells: Effects of RU486 In Vitro
To determine whether the increase in PR mRNA induced by LH is correlated with an increase in functional PR, the effect of RU486 on expression of oxytocin mRNA was determined. As expected, oxytocin mRNA was detected in granulosa cells cultured in control medium or with LH. The addition of LH increased the levels of oxytocin mRNA by about 40% over levels in control cultures (P < 0.05; Fig. 7). Increases in the levels of oxytocin mRNA in granulosa cells cultured with LH were completely inhibited by RU486 (P < 0.05; Fig. 7).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Multiple transcripts (8, 4, and 1.6 kb) of the PR gene were observed in the bovine tissues examined in this study. Similar heterogeneity of PR mRNA has been reported in primate [35], rat [9, 21], and sheep [36] tissues. For instance, multiple transcripts of the PR gene ranging from 11- to 3.1-kb size were detected in cultured rat granulosa cells [9], and in primates luteinizing granulosa cells expressed 12-, 2.7-, and 0.7-kb PR transcripts [35].
A similar pattern of rapid yet transient induction of PR mRNA by LH has been also observed in rat preovulatory follicles and cultured granulosa cells [9, 21] and in porcine granulosa cells [22]. In the eCG-primed rat ovary, PR mRNA was highly expressed in granulosa cells of preovulatory follicles, and its expression peaked at 5 h and declined by 12 h after hCG administration in vivo [21]. This time course of accumulation of PR mRNA after hCG injection was recapitulated in rat granulosa cells cultured with a luteinizing concentration of LH [9]. In cultured porcine granulosa cells, LH increased the level of PR mRNA in a time-dependent manner; it reached a peak at 3 h of culture and declined by 12 h [22]. These observations, together with results from the present study, suggest that the mechanism regulating the transient increase in PR mRNA in response to the LH surge is conserved across species and the stimulatory effect of gonadotropins on transcription of the PR gene is quite rapid but short lived. Previous studies have shown that LH receptor in rat granulosa cells is rapidly downregulated within several hours after the preovulatory gonadotropin surge [37] or injection of ovulatory doses of LH [38] or hCG [39]. Therefore, the early transient expression of PR mRNA may be associated with rapid downregulation of LH receptors and/or desensitization of the associated second messenger system after the preovulatory gonadotropin surge.
The current results also provide evidence for species differences between rats and cattle in that a second increase in PR mRNA, close to the time of ovulation, was observed in bovine preovulatory follicles but not in rat preovulatory follicles [21]. The pattern of progesterone production by preovulatory follicles is also quite different between these two species. Progesterone concentrations in follicular fluid of bovine periovulatory follicles show a triphasic pattern of change during the periovulatory period [30, 31], similar to the profile of PR mRNA expression in bovine follicle wall samples, whereas in the fluid of rat preovulatory follicles progesterone increases within 1 h of injection of LH and then remains elevated through the periovulatory period [40]. Rhesus monkeys appear to exhibit yet a third pattern in that an early transient rise in PR mRNA was observed within 12 h after an ovulatory stimulus, followed by a second increase between 24 and 36 h (ovulation expected at 36–38 h) [41], whereas progesterone concentrations in the follicular fluid increased dramatically between 0 and 12 h and then were maintained at that level at 24 and 36 h [42].
The coordinate changes in progesterone concentrations in follicular fluid and the levels of PR mRNA in follicular tissue in cattle suggest differential roles for progesterone in periovulatory follicular development in the early versus late periovulatory period. At present, it is difficult to pinpoint the specific targets of early versus later actions of progesterone on bovine follicular cells. However, in previous studies, concentrations of oxytocin [31, 43] and prostaglandins [44] in bovine follicular fluid increased dramatically close to the time of ovulation, coincident with the second dramatic rise in progesterone and PR mRNA. In addition, there is experimental evidence that progesterone stimulates production of oxytocin by bovine granulosa cells in vitro [26], and a blocker of progesterone biosynthesis (isoxazol) inhibits synthesis of PGF2 by blocking the conversion of PGE2 to PGF2 in periovulatory follicles of the ewe in vivo [2]. Therefore, the first increase in progesterone and PR mRNA may ensure the dramatic, simultaneous increases in follicular production of oxytocin, prostaglandins, and/or other important players in the ovulatory process during the periovulatory period, whereas the second rise in progesterone and PR mRNA may play an important role in the subsequent formation of the CL in cattle. This latter suggestion is supported by reports that PR and its mRNA have been detected in CLs of cattle [27] and primates [35, 45] but not in the CLs of cycling rats [21], a species that does not exhibit a second rise in PR mRNA and does not develop fully functional CLs in the absence of pregnancy or pseudopregnancy. Further studies will be needed to determine whether the expression of PR protein in bovine follicular cells follows the triphasic pattern of change observed for PR mRNA during the periovulatory period.
In cattle, progesterone concentrations in follicular fluid are elevated <2 h after the peak of the LH surge, decline during the midperiovulatory period, and then increase again close to the time of ovulation [30, 31]. Because the early rise in intrafollicular progesterone after the LH surge precedes the first increase in PR mRNA and there is a low level of PR mRNA present before the surge, we examined whether progesterone affects expression of mRNA for its own receptor in follicular cells of bovine preovulatory follicles. Data from the present study showed that progesterone at a concentration (200 ng/ml) similar to that found in follicular fluid 3.5 h after GnRH injection (1.5 h after the LH peak in the blood) did not affect the levels of PR mRNA in granulosa or theca cells cultured for 10 h (results for theca cells not shown; based on results from duplicate theca cell cultures from one animal). We also tested the possibility that progesterone could augment the stimulatory effects of LH on PR mRNA expression in follicular cells by culturing cells with LH + progesterone or LH + RU486. Results from these experiments indicate that progesterone is not involved in the early transient increase in PR mRNA expression in bovine preovulatory follicles after the LH surge. Previous studies on the regulation of PR expression by its ligand in granulosa cells have generated conflicting results. For example, in vitro studies with rat [9] and porcine [22] granulosa cells have shown that progesterone has no effect on expression of PR or its mRNA, respectively, in those species. In contrast, treatment of rhesus monkeys in vivo with trilostane, an inhibitor of progesterone synthesis, prevented the transient increase in PR mRNA in granulosa cells of periovulatory follicles after hCG administration [41]. Trilostane treatment also reduced hCG-stimulated expression of PR protein (but not mRNA) in cultured granulosa cells [46], implying that progesterone may play a role in the upregulation of PR in primate periovulatory follicles. Although results of the present study suggest that the early rise in intrafollicular progesterone is not required for the first increase in PR mRNA in bovine follicular cells after the LH surge, whether progesterone facilitates the second increase in PR mRNA and/or has a regulatory effect on expression of PR protein, as shown in primate granulosa cells, remains to be determined.
In cattle, oxytocin has been recognized as a valuable indicator for complex, yet finely tuned, cellular and molecular changes occurring in granulosa cells during luteinization [33]. We reported previously that oxytocin stimulates progesterone secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH surge [47, 48]. Conversely, steroids affected oxytocin secretion by granulosa cells. For instance, progesterone stimulated oxytocin secretion by cultured bovine granulosa cells of preovulatory follicles, whereas estradiol had a biphasic, concentration-dependent effect on oxytocin secretion [26]. High concentrations of estradiol inhibited oxytocin secretion, whereas low concentrations of estradiol stimulated its secretion in vitro [26]. In addition, PR antagonists inhibited the induction of oxytocin and its mRNA in luteinizing bovine granulosa cells from follicles obtained at a slaughterhouse [11]. In the present study, we also showed that treatment with a PR antagonist (RU486) completely blocked LH-stimulated oxytocin mRNA expression in granulosa cells from bovine preovulatory follicles obtained before the LH surge. In preliminary experiments, we found that this inhibitory effect could be reversed by medroxyprogesterone acetate (data not shown). Consistent with that result are the previous findings of Lioutas et al. [11], who reported that the inhibitory effect of RU486 on oxytocin mRNA in cultured bovine granulosa cells could be reversed by addition of medroxyprogesterone acetate but not by dexamethasone, indicating that RU486 acts as a blocker of PR but not of glucocorticoid receptor in bovine granulosa cells from large antral follicles. Taken together, results from the current study show that the PR induced by LH in vitro is functional and support the idea of an autocrine positive feedback system in bovine granulosa cells in which progesterone stimulates oxytocin production and oxytocin further increases progesterone production during the periovulatory period.
In summary, the gonadotropin surge induced a rapid and transient increase in PR mRNA expression in bovine periovulatory follicles, followed by a later increase close to the time of ovulation. The increases in PR mRNA were concomitant with increases in follicular production of progesterone. The early transient rise in PR mRNA level could be mimicked in vitro by exposing granulosa cells to a luteinizing dose of LH or FSH and by exposing theca cells to LH, but not to progesterone. In addition, an antiprogestin completely inhibited LH-stimulated oxytocin mRNA expression in bovine granulosa cells, showing that the gonadotropin-induced increase in PR mRNA is functionally linked to a previously documented effect of progesterone. Further studies will be needed to identify other target genes for activated PR in bovine granulosa and theca cells and hence to identify other players in the cascade of events during the ovulatory process and luteinization in cattle.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: J.E. Fortune, T6-012B VRT, Cornell University, Ithaca, NY 14853. FAX: 607 253 3476; jf11{at}cornell.edu
3 Current address: Department of Obstetrics and Gynecology, University of Kentucky, Lexington, KY 40536-0293
4 Current address: Department of Animal Science, Iowa State University, Ames, IA 50011-3150
Received: 18 February 2002.
First decision: 4 March 2002.
Accepted: 3 July 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-hydroxylase, in bovine preovulatory follicles. Biol Reprod 2001 64:1797-1805-hydroxylase, but not P450 aromatase. Endocrinology 1995 136:5102-5110[Abstract]This article has been cited by other articles:
|
|
 |
|
  Q. Li, F. Jimenez-Krassel, A. Bettegowda, J. J Ireland, and G. W Smith Evidence that the preovulatory rise in intrafollicular progesterone may not be required for ovulation in cattle J. Endocrinol., March 1, 2007; 192(3): 473 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Teilmann, C. A. Clement, J. Thorup, A. G. Byskov, and S. T. Christensen Expression and localization of the progesterone receptor in mouse and human reproductive organs J. Endocrinol., December 1, 2006; 191(3): 525 - 535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Bridges, C. M. Komar, and J. E. Fortune Gonadotropin-Induced Expression of Messenger Ribonucleic Acid for Cyclooxygenase-2 and Production of Prostaglandins E and F2{alpha} in Bovine Preovulatory Follicles Are Regulated by the Progesterone Receptor Endocrinology, October 1, 2006; 147(10): 4713 - 4722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jo and T. E. Curry Jr. Luteinizing Hormone-Induced RUNX1 Regulates the Expression of Genes in Granulosa Cells of Rat Periovulatory Follicles Mol. Endocrinol., September 1, 2006; 20(9): 2156 - 2172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Banerjee and C. M Komar Effects of luteinizing hormone on peroxisome proliferator-activated receptor {gamma} in the rat ovary before and after the gonadotropin surge Reproduction, January 1, 2006; 131(1): 93 - 101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Lohrke, T Viergutz, and B Kruger Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function J. Mol. Endocrinol., December 1, 2005; 35(3): 531 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A Stoops, R. D Pairan, and T. L Roth Follicular, endocrine and behavioural dynamics of the Indian rhinoceros (Rhinoceros unicornis) oestrous cycle Reproduction, December 1, 2004; 128(6): 843 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jo and T. E. Curry Jr Regulation of Matrix Metalloproteinase-19 Messenger RNA Expression in the Rat Ovary Biol Reprod, December 1, 2004; 71(6): 1796 - 1806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Li, L. J Bakke, J R. Pursley, and G. W Smith Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles Reproduction, November 1, 2004; 128(5): 555 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Quirk, R. G. Cowan, and R. M. Harman Progesterone Receptor and the Cell Cycle Modulate Apoptosis in Granulosa Cells Endocrinology, November 1, 2004; 145(11): 5033 - 5043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jo, M. C. Gieske, C. E. Payne, S. E. Wheeler-Price, J. B. Gieske, I. V. Ignatius, T. E. Curry Jr., and C. Ko Development and Application of a Rat Ovarian Gene Expression Database Endocrinology, November 1, 2004; 145(11): 5384 - 5396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Brown, D. Boerboom, N. Bouchard, M. Dore, J. G. Lussier, and J. Sirois Human Chorionic Gonadotropin-Dependent Regulation of 17{beta}-Hydroxysteroid Dehydrogenase Type 4 in Preovulatory Follicles and Its Potential Role in Follicular Luteinization Endocrinology, April 1, 2004; 145(4): 1906 - 1915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jo, L. E. Thomas, S. E. Wheeler, and T. E. Curry Jr Membrane Type 1-Matrix Metalloproteinase (MMP)-Associated MMP-2 Activation Increases in the Rat Ovary in Response to an Ovulatory Dose of Human Chorionic Gonadotropin Biol Reprod, April 1, 2004; 70(4): 1024 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
|
|
| |||||
