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Ovary |
a Department of Obstetrics & Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gonadotropin-releasing hormone, gonadotropin-releasing hormone receptor, ovary, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ovary also produces a GnRH-like peptide [6–8] and has GnRH receptors [8–10]. A GnRH-like peptide and an mRNA for mammalian GnRH have been reported in rat, monkey, and human ovarian tissues, and the presence of an ovarian receptor for GnRH in human granulosa cells has been described. The affinity of the ovarian receptor for mammalian GnRH or its analogues is greatly reduced compared with that of the pituitary's receptor. This finding is similar to that observed in the placenta [11]. The placental GnRH receptor has a much lower affinity for mammalian GnRH than does the pituitary receptor. Most studies of the activity of mammalian GnRH and its analogues in extrapituitary tissues have been plagued by low-affinity binding sites and the high concentration of mammalian GnRH required to produce tissue function. The limited activity of mammalian GnRH in the ovary can be partially explained by the presence of peptidases in the ovary [12]; however, even mammalian GnRH analogues with increased resistance to certain enzymatic degradation processes are still relatively inactive at the human ovarian receptor [10]. Overall, the data support the hypothesis that there is an active GnRH axis in the ovary but have led us to question the physiologic relevance of mammalian GnRH in extrapituitary reproductive tissues and the specificity of the ligand for the receptor in extrapituitary tissues and, relevant to these studies, in ovarian tissue.
The existence of multiple forms of GnRH in nonmammalian vertebrates has been recognized for many years, and these forms are thought to have various functions in different cells [13–15]. However, it was not until GnRH II (which was first defined in the chicken and thus named chicken II GnRH) was demonstrated in the brains of the tree shrew, musk shrew, and mole [16–19] that researchers recognized that multiple forms of GnRH exist in mammalian species. Shortly thereafter, the expression of the mRNA for GnRH II was reported in the human brain [20, 21] and the nonhuman primate brain [22]. The GnRH II receptor has been described in other human tissues [23]. However, Millar et al. [23] speculated that the function of the GnRH II receptor is vestigial because GnRH isoforms, other than the mammalian isoform, have limited to no ability to stimulate pituitary gonadotropin release [24].
We recently demonstrated that the human placenta produces GnRH II, which is released in a pulsatile fashion and binds the chorionic GnRH receptor with high affinity [11, 25, 26]. Previously, we characterized a very active postproline peptidase in human placentas [27, 28] and designed a GnRH II analogue that is stable in the presence of this peptidase. This GnRH II isoform and its analogue have high affinity for the placental GnRH receptor and have enhanced bioactivity associated with placental hormone regulation.
We hypothesized that the ovary also produces a different isoform of GnRH, which binds to the ovarian GnRH receptor with high affinity and has enhanced bioactivity associated with the regulation of ovarian function. The objective of these studies was to determine the production of GnRH II in the ovary and to examine the binding affinity and bioactivity of GnRH II and its stable analogues associated with ovarian function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mammalian GnRH, its analogue Buserelin, and chicken II GnRH (GnRH II) were purchased from Sigma Chemicals (St. Louis, MO) and Peninsula Laboratories (Belmont, CA). A GnRH II analogue, D-Arg(6) GnRH II-aza-Gly(10)-NH2, was synthesized by Peninsula Laboratories.
Baboon Tissues
Baboon ovaries were obtained at necropsy from animals 1, 2, and 3, who were 12, 12, and 30 yr of age. These tissues were used for the enzyme activity and endogenous GnRH II studies. Pituitaries were obtained from male baboons that were 8 and 9 yr of age. The tissues were obtained immediately following necropsy from animals housed at the Southwest Research Foundation for Biomedical Research (San Antonio, TX). Ovaries and pituitaries obtained at necropsy were immediately placed on ice and transported to the University of Texas Health Science Center. Different baboons undergoing in vitro fertilization (IVF) procedures were used for the granulosa cell culture studies. Granulosa cells were obtained at the time of egg retrieval. For IVF, the baboon was treated with Leuprolide (0.25 mg/day s.c.) for 8–15 days, with recombinant human FSH treatment (75 IU/day i.m., twice daily) for 8 days, and with combined FSH and LH (75 IU/day i.m., twice daily) for 3 days, followed the next day by 5000 IU hCG i.m. Eggs were retrieved 34 h later. Follicular aspirates were placed in modified human tubal fluid (Irvine Scientific, Irvine, CA) at 35°C and transported to the University of Texas Health Science Center, where the granulosa cells were removed from the egg and placed in culture.
Tissue Extraction
Baboon ovaries were homogenized in 40 mM Tris buffer, pH 7.4, filtered through cheesecloth, and centrifuged at 1000 x g for 10 min at 4°C. The pellet was discarded, the resulting supernatant was centrifuged at 30 000 x g for 30 min, and the membrane pellet was collected and resuspended in Tris buffer with 0.3 M sucrose. Protein concentrations for both the resuspended membrane fraction and the cytosolic supernatant were determined using a protein assay (Bio-Rad, Hercules, CA). The supernatant was adjusted to 20% glycerol (4:1) and frozen at -20°C until assayed for enzymatic activity capable of degrading GnRH. The membrane fraction was also stored frozen (-20°C) until used for binding assays.
Enzyme Activity Assay
The enzymatic activity for the degradation of mammalian GnRH in three different ovarian cytosolic extracts was determined by incubating 100 µl of various concentrations of the ovarian cytosolic extract (0–280 µg protein/100 µl) with 100 µl of mammalian GnRH (0.00312 µM) and 100 µl of 0.05 M phosphate buffer, pH 7.2, for 16 h at room temperature. The reaction was stopped by heating for 10 min 70°C. The degraded GnRH (product formed) was calculated by subtracting the remaining GnRH from the starting concentration of GnRH. The mammalian GnRH was quantified using an RIA specific for that isoform of GnRH with <0.1% cross-reactivity for the GnRH II analogue [27].
The ability of this GnRH II analogue to compete with the degradation of mammalian GnRH in ovarian tissue was assessed. Various concentrations of mammalian GnRH (100 µl of 0.0000, 0.0094, 0.0188, 0.0375, and 0.0750 µM) were incubated in the presence or absence of 100 µl of various concentrations of GnRH II analogue (100 µl of 0.000, 0.075, 0.150, 0.0300, and 0.600 µM) and ovarian cytosolic extract (100 µl of 14–50 µg/100 µl). This amount of ovarian extract was predetermined to degrade approximately 50% of 0.00312 µM mammalian GnRH (the lowest concentration studied) under the conditions described. The remaining GnRH was measured using an RIA specific for mammalian GnRH. The Ks for the degradation of mammalian GnRH was calculated from the x-axis intercept using a Lineweaver-Burke double reciprocal plot of the concentration of the product formed versus the concentration of the substrate used. To determine the stability of this GnRH II analogue compared with that of mammalian GnRH, the inverse of the product formed in the presence of various concentrations of mammalian GnRH was plotted versus the concentration of GnRH II analogue. The inhibitor constant (Ki) for this GnRH II analogue was calculated from the average point of the converging lines for various concentrations of mammalian GnRH studied. This study was performed for three different baboon ovaries.
GnRH Receptor Binding Assay
Prior to use, ovarian membranes were diluted to 5000 µg protein/ml with Tris buffer containing 0.5% BSA and 50 U/ml bacitracin. Ovarian membranes (100 µl) were mixed with 100 µl of various concentrations of mammalian GnRH, Buserelin, GnRH II analogue, and radiolabeled GnRH II analogue (100 µl/tube, iodinated by the method of Hunter and Greenwood [29] to 100 000 cpm/100 µl). After incubation at room temperature for 4 h, the bound and free hormone were separated using polyethylene glycol (PEG) precipitation (0.5 ml of 25% PEG, molecular weight 8000) followed by centrifugation (3000 x g) for 10 min at 4°C. The membrane-bound I125-GnRH II analogue was counted to 2% efficiency using a Cobra gamma counter (Boston, MA). The binding affinity (Kd) for each GnRH isoform or analogue was calculated using the double reciprocal plot of bound versus free ligand. Each study was done for three different baboon ovarian tissues.
Biopotency Studies
A cell culture system was used to determine the effect of the mammalian GnRH analogue Buserelin and GnRH II or GnRH II analogue on the release of progesterone. Baboon granulosa cells were dissected from the retrieved eggs of baboons undergoing IVF protocols. These baboons had not been exposed to any form of GnRH analogue for >13 days. Within 2 h, the cells were pelleted by centrifugation, resuspended in 10 ml of Medium 199 containing 10% fetal calf serum (FCS), PSF (100 U/ml penicillin, 100 µg/ml streptomycin, and 50 ng/ml fungizone), and ITS (10 µg/ml insulin, 5.5 µg/ml transferrin, and 6.7 ng/ml selenium), and counted (
48 x 106 cells). Cell viability assessed by trypan blue dye exclusion was >95%. The cells were then mixed with Cytodex beads (0.5 g). After overnight incubation at 37°C in a 95%:5% air:CO2 atmosphere, the medium was removed and replaced with 50 ml of Medium 199 containing PSF and ITS without FCS, and cells were plated in a 24-well Petri dish (2.0 x 106 cells/well [2 ml]). After a 2-h baseline incubation at 37°C in a humidified chamber with an atmosphere of 5% CO2 and 95% air, the medium was removed and replaced with test medium: Medium 199 containing PSF and ITS without FCS and with various concentrations of GnRH II or GnRH II analogue (0, 10-9, 10-8, 10-7, 10-6 M) or Buserelin (10-7 M). Each medium was tested in quadruplicate wells. Incubation continued at 37°C in a humidified chamber with an atmosphere of 5% CO2 and 95% air. Spent medium was collected and replaced after 2, 24, and 48 h of culture and stored frozen at -20°C until assayed for progesterone using a specific double-antibody procedure as described previously [30, 31]. In control wells, the release of GnRH II was also measured using a specific RIA for GnRH II. The GnRH II analogue was studied using two different baboon granulosa cell preparations.
Baboon pituitary cells were also studied using a similar cell culture system. Baboon pituitaries were minced, placed in 40 ml of Medium 199, and washed twice with 40 ml of Medium 199. Then the pituitary tissue fragments were shaken at 100 cycles/min in a Dubernoff water bath at 37°C in Medium 199 with 0.25% collagenase (Sigma) and 0.1% hyaluronidase (Sigma). The fragments were gently disrupted by suction five times through a sterile 10-ml pipet at 3- to 5-min intervals. After 30–60 min, the dispersed cells were decanted and diluted to 50 ml with culture medium. This mixture was then centrifuged at 225 x g for 10 min at room temperature, and the pelleted cells were resuspended in culture medium (10 ml). Cell viability was >95% as assessed using trypan blue. The cells were counted (40 x 106) and incubated overnight with 0.5 g Cytodex beads (Pharmacia, Piscataway, NJ) at 37°C. The cell-bead fraction was then washed with culture medium, resuspended in 50 ml of culture medium, and plated in a 24-well dish (2 ml/well). After a 2-h baseline incubation, the medium was removed and replaced with test medium: Medium 199 containing PSF and ITS without the FCS and with various concentrations of GnRH II analogue (0, 10-9, 10-8, 10-7, 10-6 M) or Buserelin (10-7 M). Each medium was studied in quadruplicate wells. Plates were incubated at 37°C in a humidified chamber with an atmosphere of 5% CO2 and 95% air. Spent medium was collected and replaced after 2, 24, and 48 h of culture and stored frozen at -20°C until assayed for baboon LH using a specific double-antibody procedure as described previously [32]. The GnRH II analogue was studied using two different baboon pituitary cell preparations.
Assays for GnRH II and GnRH II Analogue
Using a rabbit antiserum to GnRH II at a final dilution of 1:100 000, a specific and sensitive RIA for GnRH II was developed and implemented as previously described [25]. The standard used was GnRH II purchased from Peninsula Laboratories. GnRH II was radioiodinated by the method of Hunter and Greenwood [29], and 7.5 fmoles was added to each tube. The bound hormone was precipitated using magnetic beads coated with anti-rabbit gamma globulin (PolySciences, Warrington, PA). Assay sensitivity was 0.1 fmole/tube, and the intra- and interassay coefficients of variation were 4.5% and 5.9%, respectively. Cross-reactivity with mammalian, chicken I, herring, salmon, or lamprey I GnRH was <0.5%. Recovery of exogenous GnRH II was 106% ± 9%. A parallel dose response was observed for samples and standard, and GnRH II was stable in the samples for at least 24 h at room temperature and 4 mo at -20°C (the longest time tested).
A sensitive and specific RIA for GnRH II analogue was developed. The antiserum was generated in rabbits by Sigma-Genosys using the keyhole limpit hemocyanin conjugate of the analogue and was used at a final concentration of 1:50 000. The standard used was the GnRH analogue, D-Arg(6) GnRH II-aza-Gly(10)-NH2, synthesized by Peninsula Laboratories. The analogue was radioiodinated using the method of Hunter and Greenwood [29], and 10 fmoles was added to each tube. After incubation was overnight at 4°C, the bound hormone was precipitated with anti-rabbit gamma globulin-conjugated magnetic beads. Assay sensitivity was 7.7 fmoles/tube, and the intra-assay coefficient of variation was 3.0%. Cross-reactivity with chicken II, chicken I, mammalian, salmon, herring, or lamprey I GnRH was <0.5%.
Statistical Analyses
The binding affinities for different analogues or isoforms were compared using the Student t-test. Significant difference in response to the various isoforms or analogues of GnRH was determined using a one-way ANOVA for the effect of treatment across doses and a two-way ANOVA for different treatments and for the comparison of different tissues. To determine points of significant variance for a particular treatment or dose, the data were tested for homogeneity using the Bartlett test and, if necessary, were log-transformed prior to statistical analysis. The Student-Newman-Keuls test was used to determine the points of significant difference compared with untreated tissues. A P value of <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The binding affinity of the the two analogues were compared. The GnRH II analogue had 24 times higher affinity than the mammalian GnRH analogue Buserelin. Table 1 lists the average Kd observed for three different baboon ovarian membrane preparations for Buserelin and the GnRH II analogue. The binding kinetics for this GnRH II analogue demonstrated the presence of two binding sites for GnRH II in the baboon ovary. Using linear line regression analysis for 6–60 nM GnRH II analogue, a high affinity (41 ± 3 nM) was observed. For analogue concentrations of 60–1000 nM of analogue, a low-affinity binding site (319 ± 69 nM) was observed. In contrast, the mammalian GnRH analogue Buserelin had only one apparent binding site for concentrations of 6–1000 nM, with a low-affinity Kd of 987 ± 255 nM (Fig. 2).
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The biopotency of GnRH II was compared with that of the mammalian GnRH analogue Buserelin. The basal release of progesterone after the first 2 h of culture in basal medium was used to normalize the progesterone release from each well to its response to treatment. Addition of GnRH II to quadruplicate wells at various concentrations (10-8–10-6 M) resulted in a significant inhibition of progesterone release from the baboon granulosa cells after 22 h of incubation (Fig. 3). Buserelin decreased progesterone, but not to a significant degree.
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In similar studies, the effect of the GnRH II analogue was compared with that of the mammalian GnRH analogue Buserelin. After 22 h of incubation with GnRH II analogue at concentrations of 10-8–10-6 M, progesterone was suppressed to 36% ± 3% of the untreated control wells. Continued exposure to this GnRH II analogue for 46 h resulted in a further inhibition of progesterone release from the baboon granulosa cells using concentrations of 10-9–10-6 M (Fig. 4). However, addition of Buserelin (10-7 M) to the medium had no significant effect on the release of progesterone release after 22 or 46 h of incubation.
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The GnRH II content of the three different ovarian tissue extracts was 4, 8, and 14 pmoles/mg protein. The release of GnRH II from baboon granulosa cells in vitro increased over time in culture producing 12.8 pmoles day-1 well-1.
The GnRH II analogue at low doses (10-9–10-8 M, Fig. 5) produced a small, insignificant decrease of LH release from baboon pituitary cell cultures after incubation for 24 or 48 h with 10-9 M. Only at a much higher concentration of the GnRH II analogue was the basal release of LH sustained or increased, but the release was not significantly different from that of the controls. Using the same pituitary cells, Buserelin (10-7 M) produced no change or only a small, insignificant inhibition of LH release.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of a GnRH-like substance in the ovary was first described in 1986 [6, 7]. An ovarian gene for mammalian GnRH, which is identical to the gene in the hypothalamus for mammalian GnRH, has been defined [8]. Steroid regulation of this gene has also been described in cultured granulosa cells [34]. Inhibitory activity of mammalian GnRH and its analogues on rat and human ovarian progesterone production also has been demonstrated [9, 35]. When long-acting GnRH analogues are administered during the early or midluteal phase, a reduction of progesterone results [36]. Although these mammalian GnRH analogues are known to downregulate the pituitary receptor and LH release, the progesterone deficiency following short-term use of GnRH or its analogues in IVF protocols may also be the result of a direct action of GnRH analogue on the ovary. Devreker et al. [37] reported that the use of long-acting GnRH analogues in IVF patients impaired implantation rates despite exogenous hCG stimulation. This observation also supports the hypothesis that analogues act directly at the corpus luteum prior to and during pregnancy to inhibit granulosa cell function.
However, other data do not support the hypothesis that mammalian GnRH plays a significant role in the ovary. Very high concentrations of mammalian GnRH or its analogues were needed to bind to the receptor or to produce progesterone inhibition. In many studies of the ovarian receptor, the affinity of mammalian GnRH was low (10-6) [10, 38, 39]. Only after cells are in culture for 4 days have low concentrations produced a response [40, 41]. In addition, the presence of the mammalian GnRH peptide in the ovary could not be demonstrated at the concentration needed for biological activity [42]. Although the limited activity of mammalian GnRH can be partially explained by the presence of ovarian enzymatic activity that degrades GnRH, mammalian GnRH analogues that are more resistant to enzymatic degradation are still relatively inactive at the ovarian receptor.
In light of these problematic findings and our similar findings for the human placenta [25], we hypothesized that more than one isoform of GnRH, which is not identical to mammalian GnRH, is active in the ovary and placenta. This hypothesis was based on the finding that multiple isoforms for the GnRH molecule exist in nonvertebrate species and have various functions in different cells [13–15]. GnRH evolved >500 million yr ago, prior to the time of the evolution of vertebrates. The mammalian isoform of GnRH evolved 350 million yr ago and was thought to be the only form expressed in mammals. In the mid-1990s, Dellovade et al. [17] and King et al. [18] described GnRH II in the brain of the tree shrew, musk shrew, and mole, demonstrating that two different forms of GnRH existed in a mammalian species. Recently, the expression of the gene for GnRH II in the shrew [16] and guinea pig [43] has been described. These findings were followed by the discovery of GnRH II in the rhesus monkey brain [22, 44] and the human brain [20, 21]. Subsequently, the chicken II receptor was identified in a variety of human tissues [23], although the investigators speculated that the receptor's function is vestigial. Recently, we described the pulsatile release of GnRH II from human placentas and the presence of high-affinity specific placental receptors for GnRH II. In addition, the bioactivity studies of GnRH II analogues associated with placental hCG and progesterone demonstrated the receptor binding activity of GnRH II and the fact that its analogues could affect human extrapituitary cellular activity [25].
The studies described here have provided the first direct evidence of the GnRH II peptide presence and release from ovarian tissue. This second isoform of GnRH has specific high-affinity bioactive receptors in the primate ovary. In a recent study, Kang et al. [41] reported the expression of mRNA for GnRH II in human granulosa cells and regulation of its mRNA, which is consistent with our results. We also demonstrated cytosolic enzymatic activity, which rapidly degrades mammalian GnRH with kinetics similar to those of the placental postproline peptidase, which degrades both mammalian GnRH and GnRH II. The existence of the GnRH II receptor [23] in the ovarian tissue and our findings of two binding sites for GnRH II support the hypothesis that this isoform of GnRH is active in regulating ovarian function. We further hypothesize that progesterone inhibition produced by this GnRH II analogue acts via a specific ovarian GnRH receptor, as at the placenta. The relative lack of effect of mammalian GnRH on ovarian function may reflect both its limited affinity for the ovarian receptor or its rapid degradation in the placenta. The finding of enhanced receptor binding and bioactivity of our GnRH II analogue supports our hypothesis that the GnRH II isoform is a potent and active form of GnRH in the ovary. We also suggest that the hormonal effect of GnRH II or its analogues depends on the frequency and duration of treatment and can affect ovarian function. In contrast, pituitary regulation of LH in the baboon was not significantly affected by the long-term exposure to GnRH II analogue.
We demonstrated the production and release of GnRH II from ovarian tissue and the competence of the ovary to degrade mammalian GnRH. High-affinity binding sites exist in the ovary for GnRH II and its analogue, and both GnRH II and its analogue affect ovarian progesterone production. The recognition that multiple forms of GnRH are expressed and regulate physiologic functions in nonmammalian species is not novel, but the information on their high-affinity binding sites and bioactivity in primate ovarian tissue is new. GnRH II may be the initiator of true paracrine GnRH-like activity in extrahypothalamic tissues, such as the ovary. In addition, our ongoing studies indicate that chicken II GnRH is active in other extrahypothalamic tissues [25], which is consistent with the widespread expression of the GnRH II receptor [23]. Specific GnRH II analogues may be useful for the site-specific regulation of ovarian function and may have a limited effect on pituitary function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Theresa M. Siler-Khodr, Department of Obstetrics & Gynecology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., Room 416E, San Antonio, TX 78229. FAX: 210 567 3013; silerkhodr{at}uthscsa.edu
Received: 11 January 2002.
First decision: 8 February 2002.
Accepted: 11 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 0.01250 µM; 
, 0.00625 µM; 
, 0.00312 µM) by the cytosolic ovarian extracts when coincubated with various concentrations of GnRH II analogue (0–0.200 µM) for three different baboon ovaries. The ability of this GnRH II analogue to compete with the degradation of mammalian GnRH was determined from the inverse plot of the degraded mammalian GnRH produced versus the concentration of initial mammalian GnRH substrate. Ki was calculated from the point of convergence for each concentration of GnRH for each of the three ovarian extracts studied
