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a Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006
b Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201
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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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Rupture and luteinization of the periovulatory follicle appear to involve an increase in proteolytic activity [10]. Collagenolytic activity in the ovary increases within 90 min of the ovulatory bolus in rats [11], and injection of bacterial collagenase into rabbit preovulatory follicles rapidly induces rupture [12]. The matrix metalloproteinase (MMP) family of enzymes and their tissue inhibitors (TIMPs) increase in ovarian follicles following an ovulatory bolus, implicating these proteases in ovulation and luteinization [13–15]. As early as 1970, Rondell and coworkers [16] suggested a link between follicular steroid production and collagenolytic activity in rabbit follicle walls that was LH independent. More recently, the anti-progestin RU-486 was shown to block hCG induction of ovarian proteolytic activity in rats [17] and to reduce proteolytic inhibitor activity in human luteinized granulosa cells [18]. While these reports provide an association between progesterone and changes in protease activity in the follicle, little is known regarding a time course of expression for individual genes encoding proteolytic enzymes, their regulation by steroids, and hence progesterone's potential role in periovulatory processes in primate species.
In order to characterize the pattern and progesterone regulation of MMP and TIMP gene expression during the periovulatory interval in the primate follicle, granulosa cells were obtained from rhesus monkeys undergoing controlled ovarian stimulation before (0 h), 12, 24, or 36 h after administration of an ovulatory hCG bolus. This model provides multiple large preovulatory follicles that are biochemically similar to those of natural cycles [9] and are capable of ovulating [6]. In addition, the intrafollicular steroid milieu can be controlled using a 3ß-HSD inhibitor during the periovulatory interval [6], permitting analysis of local steroid actions on MMP and TIMP genes. The mRNA levels for MMP-1 (interstitial collagenase), -2 (gelatinase A), -3 (stromelysin-1), -7 (matrilysin), and -9 (gelatinase B) and TIMP-1 and -2 were determined in periovulatory monkey granulosa cells after progesterone ablation and replacement.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The general care and housing of rhesus monkeys at the Oregon Regional Primate Research Center was described previously [19]. Adult female rhesus monkeys exhibiting normal menstrual cycles of approximately 28 days were stimulated with recombinant human gonadotropins (rhFSH, 30 IU i.m. twice daily for 8 days; rhLH, 30 IU twice daily on Days 7 and 8; Laboratoires Serono SA, Aubonne, Switzerland) beginning 1–3 days after the onset of menses in order to promote the development of multiple preovulatory follicles. Monkeys also received a daily s.c. injection of the GnRH antagonist Antide (0800 h, 0.5 mg/kg BW in propylene glycol:water (1:1); Laboratoires Serono SA) throughout the stimulation protocol to prevent an endogenous LH surge. This or comparable protocols have been utilized by our research group to study folliculogenesis and periovulatory events in the primate, including the effects of steroid deprivation [6, 9]. Animals were assigned randomly to receive no ovulatory stimulus or 1000 IU rhCG (single injection i.m.; Laboratoires Serono SA) to initiate periovulatory events. Preovulatory follicles (4–7 mm) were aspirated using a 22-gauge needle during laparotomy of anesthetized animals either the morning after the last LH/FSH treatment (0 h) or 12, 24, or 36 h after administration of 1000 IU rhCG (n = 3–5 monkeys per time point). An additional group of monkeys (n = 3/time point) was stimulated in an identical fashion but also received the 3ß-HSD inhibitor Trilostane (TRL; Sanofi Research Division, Malvern, PA) orally (1 g in 8 ml orange Kool-Aid; Kraft General Foods, White Plains, NY) containing 1% (w:v) gum tragacanth; Sigma, St. Louis, MO) beginning 4 h prior to hCG administration and for every 12 h thereafter until the time of follicular aspiration. A third group of animals (n = 3/time point) received TRL plus the nonmetabolizable progestin R5020 (Promegestrone; DuPont/NEN, Boston, MA; 2.5 mg in sesame oil, s.c., once daily starting at the time of hCG). The TRL and TRL+R5020 groups were aspirated only at 12 and 36 h post-hCG, representing, respectively, the time point when follicular fluid progesterone is substantially increased [9] and the time point just prior to follicular rupture [1, 2].
Daily blood samples were obtained from unanesthetized animals by saphenous venipuncture from the beginning of gonadotropin treatment. Serum estradiol and progesterone concentrations were determined using specific RIAs [19], and follicular growth was monitored using serum steroid levels and ultrasonography performed on Days 6–7 of stimulation [20]. In animals receiving TRL, with or without R5020, serum samples collected the morning after hCG/TRL administration and for the remainder of the study period were chromatographed to separate progesterone from pregnenolone prior to RIA as described previously [20]. Serum concentrations of bioactive LH were determined for the 3 days prior to and including the day of follicle aspiration using an in vitro mouse Leydig cell bioassay [21] to confirm the absence of an endogenous LH surge.
Follicle Aspiration and Granulosa Cell Preparation
Granulosa cells were obtained by follicle aspiration during laparotomy of anesthetized animals. Cells were removed from follicular fluids by centrifugation at 277 x g for 15 min (4°C), and the resulting follicular fluid was aliquoted into volumes of 25–100 µl and stored at -80°C. The cell pellet was resuspended in Tyrode's albumin lactate pyruvate (TALP)-Hepes, oocytes were removed for use in other studies, and the remaining aspirate was enriched for granulosa cells as described by Chaffin et al. [9]. In brief, cells were centrifuged at 190 x g (10 min, 4°C) and resuspended in Ham's F-10 medium (Life Technologies, Grand Island, NY). The resuspension was layered onto a gradient of 40% Percoll (Sigma) and 60% Hanks' Balanced Salt Solution with 0.1% BSA and centrifuged at 470 x g for 30 min at 4°C. The resulting layer of granulosa cells was resuspended in Ham's F-10, cell numbers were determined using a hemacytometer, and cell viability was assessed by trypan blue exclusion.
Total RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
In order to maximize the amount of information obtainable from limited numbers of granulosa cells, an RT-PCR assay was employed. This assay, while highly sensitive, does not detect multiple splice variants. Total RNA was isolated from 104-105 granulosa cells using the Trizol reagent (BRL, Gaithersburg, MD) according to manufacturer's instructions. Quality and quantity of RNA were determined by electrophoresis of samples against known concentrations of total ovarian RNA in a 2% agarose gel stained with ethidium bromide. Granulosa cell RNA (0.5–1.0 µg in 10 µl) was treated with RNase-free DNase I (BRL) for 15 min at room temperature to remove contaminating genomic DNA, and DNase I was subsequently inactivated by the addition of 1 µl of 25 mM EDTA for 15 min at 65°C. Reverse transcription (RT) was carried out for 2 h at 37°C in a 20-µl reaction volume using the 10-µl DNase I reaction, single-strength RT buffer (50 mM Tris-Cl, pH 8.3, 40 mM KCl, 6 mM MgCl2), 1 mM dithiothreitol (DTT), 25 pmol oligo(dT) primer (Promega Biotech, Madison, WI), and 200 units of Molony murine leukemia virus reverse transcriptase (BRL); then the reverse transcriptase was heat inactivated at 94°C for 5 min.
PCR was performed in a 75-µl volume made up of two parts. Part A contained an empirically determined amount of the RT reaction dictated by the specific PCR primer set, 7.5 µl of 10-strength Taq buffer (Promega Biotech), 1–3 mM MgCl2, 2 µl of 10 mM dNTPs, and 3 U of Taq DNA polymerase (Promega) in a 60-µl volume. Part B contained 15 µl of the experimental + internal standard primers, the concentrations of which were determined as part of the validation process. The mixture of parts A and B was overlaid with 40 µl of mineral oil, and the PCR reaction was performed in a thermal cycler (MJ Research, Watertown, MA) for an empirically determined number of cycles for denaturing at 94°C for 30 sec, annealing at 60°C for 1 min, and primer extension at 72°C for 1 min. Aliquots of each PCR reaction (20 µl) were electrophoresed through a 2% agarose gel stained with 0.1 µg/ml ethidium bromide. Gels were visualized on a UV transilluminator and photographed using 667 Polaroid (Cambridge, MA) film, and the photographs were analyzed by densitometry. All values were normalized to the internal standard ß2-microglobulin (ß2MG) or cyclophilin; no apparent changes were observed in granulosa cell expression for either standard between time points after administration of hCG. The choice of internal standard was based on the ability of the experimental primers to be validated against one or the other set of standard primers (see below). In order to conserve limited samples, RNA from TRL+R5020-treated animals was not assayed unless significant differences were observed between control and TRL groups.
Validation of the RT-PCR assay was performed using RNA from granulosa cells aspirated 27 h after hCG during routine in vitro fertilization (IVF) laparoscopy ([22]; data not shown). In brief, the amount of coamplified product for experimental and internal standard primer sets was linear and parallel with increasing amount of cDNA, and both sets of primers were in the exponentially increasing phase relative to the number of cycles. To control for between-assay variability, total RNA from granulosa cells of three monkeys was combined and reverse-transcribed as described above to form a pool that was amplified in triplicate during each PCR with the appropriate set of primers. Within-assay variability calculated using the triplicate pool samples typically ranged from < 1 to 12%. Sequence analysis confirmed the identity of the PCR products. Because data for each set of primers was collected in 2–3 rounds of PCR reactions, the pool triplicates were also used to normalize data between reactions.
Oligonucleotide PCR Primers
Oligonucleotides used for PCR were synthesized by the Molecular Biology Core Laboratory at the Oregon Regional Primate Research Center. Table 1 lists the primer sequences with the corresponding optimal MgCl2 concentration.
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Statistical Analysis
In order to test for heterogeneity of variance, data were subjected to a Bartlett's chi-square test and subsequently transformed (to log+2) prior to one-way ANOVA, followed by Newman-Keuls test for comparison between means. Because TRL and TRL+R5020 data were collected at only 12 and 36 h post-hCG, comparisons were made between treatments within a time point by separate one-way ANOVAs. Differences were considered significant at p < 0.05, and values are presented as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Periovulatory Changes in the Expression of MMPs
Interstitial collagenase (MMP-1) mRNA was undetectable in granulosa cells prior to the administration of hCG but increased (p < 0.05) to appreciable levels by 12 h post-hCG, remaining at this level for the duration of the periovulatory interval (Fig. 2). Granulosa cells aspirated 12 h after hCG+TRL expressed 3.5-fold less mRNA (p < 0.05) than control (hCG alone) counterparts, an effect that was fully reversed in animals receiving hCG+TRL plus the progestin R5020. Likewise, 36 h after hCG+TRL administration, interstitial collagenase mRNA was 43-fold less (p < 0.05) than in the time-matched controls. The replacement of progesterone with R5020 resulted in interstitial collagenase mRNA levels that were not different from those in controls.
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Gelatinase A (MMP-2) mRNA was detectable in granulosa cells from two of three monkeys prior to hCG and increased 5-fold within 12 h of hCG (p < 0.05; Fig. 3, top). Although not significant, there was tendency for gelatinase A mRNA to increase throughout the periovulatory interval, while steroid depletion with TRL did not change the levels at either 12 or 36 h post-hCG. Gelatinase B (MMP-9) mRNA, however, did not increase until 36 h after the ovulatory bolus, at which time levels were 34-fold higher than at 24 h (Fig. 3, bottom). Only one of three animals aspirated at 24 h post-hCG had detectable gelatinase B mRNA, supporting the idea that levels may decline between 0 and 24 h after hCG. The administration of hCG+TRL reduced gelatinase B mRNA at 36 h by 3-fold, but message levels from animals receiving progestin replacement did not return to control values.
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Matrilysin (MMP-7) mRNA was expressed in periovulatory granulosa cells at relatively low abundance compared to the other MMPs. Prior to hCG, only two of four monkeys had detectable levels, but by 12 h, matrilysin mRNA increased 12-fold over that in 0-h hCG samples (p < 0.05; Fig. 4). By 24 h after hCG, matrilysin mRNA decreased to a point midway between the 0- and 12-h values (p < 0.05), increasing again at 36 h. Coadministration of TRL resulted in a 3-fold reduction in mRNA by 36 h post-hCG (p < 0.05); moreover, progestin replacement further reduced matrilysin mRNA levels relative to TRL treatment alone (p < 0.05). Stromelysin-1 (MMP-3) mRNA was not detectable in granulosa cells from any animal at any time point (Fig. 5). However, stromelysin-1 mRNA was present in RNA obtained from granulosa cells aspirated during laparoscopy (versus the laparotomies used in the current study) of monkeys undergoing controlled ovarian stimulation.
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TIMP-1 and -2 mRNA
TIMP-1 mRNA was expressed at high levels in periovulatory granulosa cells following hCG. TIMP-1 mRNA was detectable in two of three animals prior to the administration of hCG, and levels increased 16-fold by 12 h post-hCG (Fig. 6, top). By 24 h after hCG, TIMP-1 mRNA decreased to an intermediate level between the 0- and 12-h values (p < 0.05) and remained at this level near the end of the periovulatory interval. At 12 h post-hCG, TIMP-1 mRNA was reduced by TRL (p < 0.05), and levels were partially recovered by R5020 administration (p < 0.05 vs. control and TRL). TIMP-2 mRNA was undetectable in granulosa cells aspirated prior to hCG but increased (p < 0.05; Fig. 6, bottom) within 12 h of the ovulatory stimulus and remained at this level until 36 h; at this time the TIMP-2 mRNA level was 3-fold greater than at 12–24 h (p < 0.05; Fig. 6, bottom). Steroid depletion after TRL administration reduced TIMP-2 mRNA at both 12 and 36 h after hCG (p < 0.05); notably, progestin replacement further reduced message levels at both time points (p < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The preovulatory follicle contains three distinct layers of connective tissue (reviewed in [23]): 1) the outermost epithelial layer, in which collagen type IV predominates; 2) the tunica albuginea and theca externa, which consists primarily of collagen types I and III, whose fibrils provide structural support and tensile strength to the follicle wall [24, 25]; and 3) the basal lamina, which separates the vascular theca and avascular granulosa layers and is composed mainly of collagen type IV, laminin, fibronectin, and proteoglycan. The varied patterns of mRNA expression across the periovulatory interval support the notion that different MMPs play diverse roles during breakdown and reorganization of the various tissue layers for the purpose of follicle rupture, neovascularization and differentiation of the granulosa cell layer, and loss of tissue compartmentalization in the developing corpus luteum.
Among the different patterns of mRNA expression, interstitial collagenase, gelatinase A, and matrilysin increased early (within 12 h) following the hCG stimulus. These findings are consistent with reports that interstitial collagenase and gelatinolytic activity in rat and rabbit ovaries and human follicular fluid was elevated following an ovulatory stimulus [11, 13–15, 26–30]. Interstitial collagenase, which degrades collagen types I and III, may play a role in follicle rupture; for example, the apex of the periovulatory follicle expresses substantial pro-interstitial collagenase and collagenolytic activity [15, 31]. The role of interstitial collagenase in the antrum of the follicle is less apparent. It is unlikely that this enzyme degrades the basal lamina, as this layer does not contain significant amounts of collagen types I or III. Interstitial collagenase, however, is required for angiogenesis in vitro [32], suggesting a role for this enzyme in vascularization of the luteinizing follicle. Studies detailing the time course of MMP-1 activity in follicular fluid will further clarify this system.
In contrast to interstitial collagenase, the periovulatory increase in granulosa cell gelatinase A mRNA may have a direct role in follicle rupture. Puistola et al. [29] reported that type IV collagenolytic (i.e., gelatinase A) activity increases in human follicular fluid near the time of ovulation and decreases soon thereafter. Gelatinolytic activity also increases in the rat ovary 4–8 h following the ovulatory gonadotropin stimulus [14]. Ewes immunized against the N-terminal portion of inhibin alpha do not ovulate, presumably due in part to a reduction in gelatinase A activity [33]. Therefore, gelatinase A degrades collagen type IV in the basal lamina as part of the process of follicle rupture. Gelatinase A is also localized to capillary endothelial cells of growing vessels [30] and plays a prominent role in endothelial cell invasion [32], suggesting that the increase in mRNA may also be related to vascularization of follicle.
Stromelysin-1 and matrilysin are broad-spectrum proteases that digest proteoglycans, fibronection, laminin, and elastins. The novel finding that matrilysin mRNA is expressed by periovulatory granulosa cells in a biphasic pattern suggests that this protease has multiple roles. The basal lamina of the follicle is rich in fibronection, collagen type IV, and proteoglycans, all targets of active matrilysin. Stromelysin-1 mRNA, on the other hand, was undetectable by PCR in granulosa cells at any time before or after hCG. There are no reports of stromelysin expression within the follicle; however, Curry et al. [14] reported proteoglycanase-like activity in rat ovarian homogenates with 8 h of hCG, and Nothnick et al. [34] detected proteoglycanase activity in rat corpora lutea. In the current study, aspiration during laparoscopy yielded follicular cells expressing stromelysin-1 mRNA, while granulosa cells collected by laparotomy did not. These differences most likely reflect the more rigorous laparoscopic aspiration, in which a single needle puncture is used to aspirate several follicles, as compared to the gentler aspiration during laparotomy by which follicles were individually aspirated. Thus, it seems reasonable to conclude that either theca-interstitial cells or blood contamination is the source of stromelysin-1 mRNA in laparoscopic aspirates.
While the other MMP mRNAs in macaque granulosa cells increased within 12 h of hCG, gelatinase B mRNA did not increase until much later at 36 h post-hCG. Gelatinase B is expressed in theca cells of the follicle, differentiating granulosa cells in culture, and luteal cells of the developing corpus luteum in rodents and domestic animals [30, 35]. This enzyme may therefore facilitate the remodeling and vascularization that accompanies the formation of the early corpus luteum, luteal maintenance, and regression [34–36]. However, a role for gelatinase B in follicle rupture cannot be excluded.
In the current study, TIMP-1 and -2 mRNA increased between 0 and 12 h after hCG administration and, while TIMP-1 declined at 24 h, TIMP-2 mRNA levels increased again at 36 h. TIMP-1 mRNA has been localized to granulosa-lutein cells and TIMP-2 mRNA to theca-lutein cells of the human corpus luteum [36], and both TIMP-1 and TIMP-2 are expressed in nonprimate follicles [37–39]. In the rat [13, 37, 40], sheep [41], and human [42], TIMP activity and mRNA increase following the ovulatory stimulus. TIMP-1 has high affinity for several MMPs, while TIMP-2 binds latent and active forms of gelatinase A (reviewed in [43]); thus the late rise in TIMP-2 may be associated with postovulatory control of gelatinase A activity.
The expression of several MMP and TIMP genes in macaque granulosa cells during the periovulatory interval decreased following steroid ablation. Interestingly, the effect of TRL administration varied temporally for individual genes, suggesting that MMP and TIMP gene regulation by steroids is dependent upon the duration of exposure to the ovulatory bolus of hCG. Notably, the mRNA levels for interstitial collagenase and TIMP-1 were increased by progestin replacement by 12 h post-hCG. Interstitial collagenase is a principal target for TIMP-1 inhibition [43], and thus it is reasonable to conclude that these two genes are regulated in tandem by progesterone [40]. On the contrary, the reduction in mRNAs for matrilysin, gelatinase B, and TIMP-2 following TRL treatment were not restored by progestin replacement. The actions of TRL on these genes may be nonspecific, although this is unlikely as matrilysin and gelatinase B were affected at only one time point. It is more likely that the depletion of estrogen and/or androgens following TRL administration was responsible for the reduced expression of these genes. In cultures of human granulosa-lutein cells, for example, estradiol-17ß increases the amount of gelatinase A in media [44]. Estrogen and androgen replacement following steroid ablation may help clarify the role of these hormones, as will the localization and regulation of steroid receptors [8].
The variety of mRNA expression patterns and regulation suggests that MMPs and TIMPs play diverse roles in ovarian biology. In addition to follicle rupture and angiogenesis, MMPs and TIMPs may be involved in, for example, steroidogenesis [45], growth promotion [46], generation of ovarian growth hormone-binding proteins [47], oocyte-cumulus interactions [48], and the fibroblast growth factor system [49, 50]. While collagenolytic enzymes most likely play a critical role in ovulation and luteinization [11, 12, 16, 26], other proteolytic systems such as the plasmin-generating system may also be important [51]. Progesterone and other steroids may therefore be involved in a variety of signaling systems leading not only to rupture but also to the formation of the corpus luteum.
In conclusion, an ovulatory stimulus given to rhesus monkeys undergoing controlled ovarian stimulation results in an early (
12 h) increase in granulosa cell mRNAs for interstitial collagenase, gelatinase A, matrilysin, TIMP-1, and TIMP-2, while gelatinase B mRNA does not increase until just prior to follicle rupture ( 36 h post-hCG). These mRNA data do not provide information regarding protein levels or activity; however, future studies will address the question of protease activity and histologic localization of the specific genes examined in the current study. The variety of time-course expression profiles and steroid regulation during the periovulatory interval suggests that the various MMPs and TIMPs have specific, tightly controlled roles in follicle rupture and luteinization. Finally, progesterone up-regulation of interstitial collagenase and TIMP-1 mRNA could be a critical mechanism whereby progesterone promotes ovulation and luteinization of the primate follicle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Richard L. Stouffer, Division of Reproductive Sciences, Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5563; stouffri{at}ohsu.edu
Accepted: February 3, 1999.
Received: November 9, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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during ovulation in the gonadotropin-primed immature rat. Endocrinology 1990; 127:259–263.[Abstract]-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown. Biol Reprod 1991; 45:334–342.[Abstract]This article has been cited by other articles:
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  B. L Dozier, K. Watanabe, and D. M Duffy Two pathways for prostaglandin F2{alpha} synthesis by the primate periovulatory follicle Reproduction, July 1, 2008; 136(1): 53 - 63. [Abstract] [Full Text] [PDF]
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 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]
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 J. R. A. Sherwin, A. M. Sharkey, P. Cameo, P. M. Mavrogianis, R. D. Catalano, S. Edassery, and A. T. Fazleabas Identification of Novel Genes Regulated by Chorionic Gonadotropin in Baboon Endometrium during the Window of Implantation Endocrinology, February 1, 2007; 148(2): 618 - 626. [Abstract] [Full Text] [PDF]
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 M. Tesone, R. L. Stouffer, S. M. Borman, J. D. Hennebold, and T. A. Molskness Vascular Endothelial Growth Factor (VEGF) Production by the Monkey Corpus Luteum During the Menstrual Cycle: Isoform-Selective Messenger RNA Expression In Vivo and Hypoxia-Regulated Protein Secretion In Vitro Biol Reprod, November 1, 2005; 73(5): 927 - 934. [Abstract] [Full Text] [PDF]
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 D. M. Duffy, C. L. Seachord, and B. L. Dozier An Ovulatory Gonadotropin Stimulus Increases Cytosolic Phospholipase A2 Expression and Activity in Granulosa Cells of Primate Periovulatory Follicles J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5858 - 5865. [Abstract] [Full Text] [PDF]
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 D. M. Duffy, C. L. Seachord, and B. L. Dozier Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle Hum. Reprod., June 1, 2005; 20(6): 1485 - 1492. [Abstract] [Full Text] [PDF]
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C. L. Seachord, C. A. VandeVoort, and D. M. Duffy Adipose Differentiation-Related Protein: A Gonadotropin- and Prostaglandin-Regulated Protein in Primate Periovulatory Follicles Biol Reprod, June 1, 2005; 72(6): 1305 - 1314. [Abstract] [Full Text] [PDF]
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M. C. Peluffo, K. A. Young, and R. L. Stouffer Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2327 - 2335. [Abstract] [Full Text] [PDF]
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D. M. Duffy, B. L. Dozier, and C. L. Seachord Prostaglandin Dehydrogenase and Prostaglandin Levels in Periovulatory Follicles: Implications for Control of Primate Ovulation by Prostaglandin E2 J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1021 - 1027. [Abstract] [Full Text] [PDF]
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M. Cherian-Shaw, R. Das, C. A. VandeVoort, and C. L. Chaffin Regulation of Steroidogenesis by p53 in Macaque Granulosa Cells and H295R Human Adrenocortical Cells Endocrinology, December 1, 2004; 145(12): 5734 - 5744. [Abstract] [Full Text] [PDF]
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 K. A. Young, B. Tumlinson, and R. L. Stouffer ADAMTS-1/METH-1 and TIMP-3 expression in the primate corpus luteum: divergent patterns and stage-dependent regulation during the natural menstrual cycle Mol. Hum. Reprod., August 1, 2004; 10(8): 559 - 565. [Abstract] [Full Text] [PDF]
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L. J. Bakke, Q. Li, C. A. Cassar, M. P.D. Dow, J. R. Pursley, and G. W. Smith Gonadotropin Surge-Induced Differential Upregulation of Collagenase-1 (MMP-1) and Collagenase-3 (MMP-13) mRNA and Protein in Bovine Preovulatory Follicles Biol Reprod, August 1, 2004; 71(2): 605 - 612. [Abstract] [Full Text] [PDF]
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Y.-X. Liu, K. Liu, Q. Feng, Z.-Y. Hu, H.-Z. Liu, G.-Q. Fu, Y.-C. Li, R.-J. Zou, and T. Ny Tissue-Type Plasminogen Activator and Its Inhibitor Plasminogen Activator Inhibitor Type 1 Are Coordinately Expressed during Ovulation in the Rhesus Monkey Endocrinology, April 1, 2004; 145(4): 1767 - 1775. [Abstract] [Full Text] [PDF]
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K. A. Young and R. L. Stouffer Gonadotropin and Steroid Regulation of Matrix Metalloproteinases and Their Endogenous Tissue Inhibitors in the Developed Corpus Luteum of the Rhesus Monkey During the Menstrual Cycle Biol Reprod, January 1, 2004; 70(1): 244 - 252. [Abstract] [Full Text] [PDF]
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K. A. Young, C. L. Chaffin, T. A. Molskness, and R. L. Stouffer Controlled ovulation of the dominant follicle: a critical role for LH in the late follicular phase of the menstrual cycle Hum. Reprod., November 1, 2003; 18(11): 2257 - 2263. [Abstract] [Full Text] [PDF]
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C. L. Chaffin, R. S. Brogan, R. L. Stouffer, and C. A. VandeVoort Dynamics of Myc/Max/Mad Expression during Luteinization of Primate Granulosa Cells in Vitro: Association with Periovulatory Proliferation Endocrinology, April 1, 2003; 144(4): 1249 - 1256. [Abstract] [Full Text] [PDF]
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