Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro

J.T. Jensen1,6, K.M. Schwinof2, M.B. Zelinski-Wooten2,3, M. Conti4, L.V. DePaolo5 and R.L. Stouffer2,3

1 Department of Obstetrics and Gynecology and Division of Reproductive Sciences, Oregon Regional Primate Research Center, Oregon Health & Science University, Portland, OR 97201, 2 Division of Reproductive Sciences, 3 Department of Physiology and Pharmacology, Oregon Regional Primate Research Center, Oregon Health & Science University, Beaverton, OR 97006, 4 Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305 and 5 Reproductive Sciences Branch, Center for Population Research, NICHD, NIH, Bethesda, MD, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates. METHODS: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis. RESULTS: At 24 h, 72 of 121 (60%) control oocytes progressed to GVBD compared with 9/34 (27%, P < 0.01), 4/36 (11.1%, P < 0.01) and 0/28 (0%, P < 0.01) oocytes incubated with ORG 9935 at 0.1, 0.5 and 1.0 µmol/l respectively. Similar results were achieved at 24 h with 1.0 µmol/l Cilostamide (2/24 oocytes, 8%, P < 0.01) and 100 µmol/l Milrinone (2/32, 6%, P < 0.01). In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 µmol/l Rolipram for 24 h (43/58, 74%) or 48 h (44/58, 76%). CONCLUSIONS: These experiments establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Thus, PDE3 inhibitors have potential use as contraceptives in primates.

Key words: contraception/meiosis/non-human primate/oocyte/phosphodiesterase 3 inhibitor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During fetal life in mammals, oogonia destined to become primary oocytes cease entering mitotic cycles, and initiate nuclear changes characteristic of prophase of the first meiotic division (Adashi, 1996Go). With the onset of puberty, gonadotrophin levels rise, and follicular development occurs. Low levels of gonadotrophins stimulate cAMP production in theca and granulosa cells of the follicle, and induce differentiation of pre-ovulatory follicles. Resumption of oocyte meiotic maturation, however, must await the pre-ovulatory LH surge. Thus, a fertilizable metaphase II (MII) stage oocyte is available at the time of ovulation. In addition, experiments in many species, including human and non-human primates, have revealed that, upon removal from the follicle, both denuded (granulosa-free) and cumulus-enclosed oocytes (CEO) are capable of spontaneously completing meiotic maturation in vitro (Edwards, 1965Go; Dekel et al., 1984Go; Wassarman, 1996Go; Schramm and Bavister, 1999Go).

Interestingly, the gonadotrophin surge leads to opposing changes in cAMP levels in ovarian somatic cells and oocytes. While cAMP levels rise in granulosa cells, a decrease in intracellular cAMP must occur in the oocyte prior to germinal vesicle breakdown (GVBD) and the resumption of meiosis (Tsafriri et al., 1996Go; Conti et al., 1998Go). Evidence from experiments in the mouse suggests that the common pathway for both LH-induced and spontaneous oocyte maturation involves cAMP-dependent protein kinase A (PKA) (Bornslaeger et al., 1986Go). High concentrations of cAMP in the oocyte activate PKA, and correlate with inhibition of the resumption of meiosis (Bornslaeger et al., 1986Go). Mouse oocyte–cumulus cell complexes cultured in the presence of non-selective inhibitors of phosphodiesterases (PDE), such as hypoxanthine, maintain high intrafollicular levels of cAMP, and remain meiotically arrested (Downs et al., 1989Go). Microinjection of PKA inhibitors induces meiotic maturation in hypoxanthine-arrested mouse oocytes (Bornslaeger et al., 1986Go). Blockade of c-mos, a critical factor in the mitogen-activated protein kinase (MAPK) pathway, has been shown to inhibit the progression of meiosis in hamster and human oocytes (Hashiba et al., 2001Go).

Intracellular levels of cAMP levels are a function of the rate of cAMP synthesis by adenylyl cyclase and the rate of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDE). Eleven distinct families, or isoforms, of PDE have been described based on their substrate specificity, mode of regulation and kinetic properties (Reid, 1999Go). Selective inhibitors of many PDE families are now available, and some are approved agents with therapeutic use in humans.

Divergence of PDE isoform expression exists in ovarian cell types. In mammals studied to date, the PDE4 enzyme is expressed in follicular somatic cells, while PDE3 exists in the oocyte (Tsafriri et al., 1996Go). This observed compartmentalization suggests the basis for a novel contraceptive strategy. Selective inhibition of the PDE3 enzyme should maintain elevated cAMP levels and prevent meiotic maturation of the oocyte without effecting maturation of the ovarian somatic cells. Thus, ovulation of a non-fertilizable, immature oocyte should occur with the subsequent development of a functional corpus luteum and normal ovarian cyclicity. This strategy has been proposed and tested in rodents (Wiersma et al., 1998Go). Although these studies demonstrated proof of the concept, whether it applies to primates remains unknown. The specific aim of our study is to investigate the feasibility of PDE3 inhibitors as human contraceptives by testing the hypothesis that PDE3 inhibitors selectively block the spontaneous resumption of meiosis of rhesus monkey oocytes in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals and ovarian stimulation
The general care and housing of rhesus monkeys at the Oregon Regional Primate Research Center has been described previously (Wolf et al., 1990Go). After a period of observation to establish normal menstrual cycles of ~28 days, adult female rhesus monkeys received recombinant human gonadotrophins (rFSH, 30 IU i.m. twice daily for 8 days; rLH, 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 pre-ovulatory follicles. To prevent an endogenous LH surge, these monkeys also received a daily s.c. injection of the GnRH antagonist Antide [0800 h, 0.5 mg/kg body weight in propylene glycol:water (1:1); Laboratoires Serono SA] throughout the stimulation protocol. Follicular growth was monitored using serum steroid concentrations and ultrasonography performed on days 6–7 of stimulation (Zelinski-Wooten et al., 1997Go). This or comparable protocols using urinary gonadotrophin preparations typically result in ~18 large follicles that do not ovulate unless stimulated (VandeVoort et al., 1989Go; Zelinski-Wooten et al., 1994Go; Hibbert et al., 1996Go). No ovulatory stimulus was provided.

Oocyte collection and in-vitro maturation
On the morning after the last LH/FSH treatment, large (>4 mm) antral follicles from both ovaries of a single animal were aspirated using a 22-gauge needle during laparoscopy of anaesthetized animals (Hibbert et al., 1996Go). Pooled follicular contents were collected in a physiological buffered solution, Tyrode’s albumin lactate pyruvate medium (TALP)–HEPES with 0.3% bovine serum albumin (BSA). Oocytes were separated from follicular fluid and non-luteinized granulosa cells using a dissecting microscope, and scored for meiotic stage (Zelinski-Wooten et al., 1991Go). Those that retained a small amount of adherent granulosa cells were briefly (30 s) treated in 1% hyaluronidase (Sigma, St Louis, MO, USA) in TALP–HEPES with 0.3% BSA, and then washed in plain media prior to culture. Only denuded oocytes were studied, therefore the nuclear membrane (germinal vesicle) was readily visible.

Normal-appearing oocytes with intact germinal vesicles (GV) were identified, and transferred to 100 µl microdrops of media (TALP with 0.3% BSA) with or without various doses of one of three PDE3 inhibitors; Cilostamide (Sigma), Milrinone (Sigma) or ORG 9935 (Organon, Oss, The Netherlands), or a selective PDE4 inhibitor Rolipram (Sigma). Prior to, and following the addition of oocytes, the microdrops were equilibrated under mineral oil (Sigma) at 37°C in a humidified CO2 atmosphere.

Stock solutions of the PDE inhibitors were prepared by dissolving the compounds in dimethylsulphoxide (DMSO; Sigma) at a concentration of 10 mmol/l. These stock solutions were diluted in TALP media to produce the final desired concentration of each agent. All four inhibitors were tested at 1.0 µmol/l. Milrinone and Rolipram were tested at 100 µmol/l. Lower concentrations of Cilostamide (0.5 µmol/l) and ORG 9935 (0.5 and 0.1 µmol/l) were also analysed. The final concentrations of DMSO in solution ranged from 0.001 to 1% in the treatment groups. Oocytes were transferred to fresh media after 24 h. At least three different animals typically contributed oocytes exposed to each concentration of inhibitor, except for 0.5 µmol/l Cilostamide where only two animals were studied. Control data reflect the pooled results from 12 animals stimulated collectively 18 times.

An inverted microscope at x200 magnification was used to assess the development of oocytes at 1, 24 and 48 h of incubation. Only grossly normal oocytes with intact germinal vesicles at the 1 h observation are included in the analysis. The primary outcome measurement was the number of oocytes that resumed meiosis by 24 and 48 h of observation. The objective criterion for resumption of meiosis was germinal vesicle breakdown (GVBD), indicating progression to the metaphase I (MI) stage.

Granulosa cell preparation and culture
After oocyte removal, enriched preparations of granulosa cells from individual monkeys (n = 3) were obtained as previously described (Brannian and Stouffer, 1993Go). The granulosa cells were plated on fibronectin (Sigma)-coated 48- or 96-well plastic plates (Nalge Nunc, Denmark) at 50 000 or 20 000 cells/well and cultured in Dulbecco’s modified Eagle’s medium–Ham’s F-12 plus ITSA [insulin (2 mg/l), transferrin (5 mg/l), H2SeO3 (0.25 nmol) and aprotinin (25 g/l); Sigma] medium containing 25 µg/ml human low density lipoprotein (Sigma) with and without the addition of one of the PDE inhibitors or DMSO. Cells were incubated at 37°C in a 5% CO2–95% air environment. Media were collected after 24 h and frozen at –20°C until assayed for progesterone. Cells were cultured in triplicate for each treatment group, and each experiment used a cell preparation from one monkey. In order to compare between experiments, control results were pooled, and the results for each inhibitor were analysed as a percentage of controls.

Statistical analysis
Frequencies of GVBD in oocytes of the total cohort collected during control and PDE inhibitor exposure at 24 and 48 h were analysed with Fisher’s exact test. Comparisons of frequencies of GVBD between 24 and 48 h within each group were by the McNemar test. Progesterone levels were analysed with a one-way analysis of variance. All the tests applied two-sided statistics with alpha set at 0.05 and analyses were performed using SPSS for Windows (version 10.0).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 539 oocytes was recovered. Of these, 420 (78%) were normal in appearance and immature (displaying an intact GV) at the 1 h observation (Figure 1AGo), and included in the study.



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Figure 1. Photomicrographs of representative oocytes exposed to treatments. (A) A typical germinal vesicle (GV)-intact oocyte at 1 h observation. (BF) Oocytes representative of treatment groups at 24 h. (B) Control oocyte with germinal vesicle breakdown (GVBD) at metaphase I. (C) Rolipram 100 µmol/l oocyte with GVBD at metaphase II. Note the polar body at 6 o’clock. (D) Milrinone 100 µmol/l oocyte with intact GV. (E) Cilostamide 1.0 µmol/l oocyte with intact GV. (F) ORG 9935 1.0 µmol/l oocyte with intact GV.

 
At 24 h, 60% of oocytes incubated in the absence of a PDE inhibitor (controls) underwent spontaneous germinal vesicle breakdown (Table IGo; Figure 1BGo). Incubation with the selective PDE4 inhibitor Rolipram produced no significant effect on the spontaneous maturation of oocytes (Table IGo; Figure 1CGo). Resumption of meiosis was observed in 71% and 74% of oocytes exposed to Rolipram at concentrations of 1.0 and 100 µm respectively. Furthermore, compared with the lower dose, there was no significant difference in maturation in the presence of the higher concentration of Rolipram.


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Table I. Effects of phosphodiesterase (PDE) inhibitors on spontaneous resumption of meiosis by macaque oocytes in vitro
 
Inhibition of GVBD during exposure to PDE3 inhibitors (Figure 1D–FGo) was dose dependent, and varied between compounds (Table IGo). Progressively fewer oocytes matured in ORG 9935 concentrations of 0.1 µmol/l (27%, P < 0.01) and 0.5 µmol/l (11%, P < 0.01). Moreover, spontaneous maturation of oocytes was not observed among the cohort incubated with 1.0 µmol/l ORG 9935.

The inhibition of spontaneous maturation at 24 h produced by 0.5 and 1.0 µm Cilostamide (8–9%) was comparable with 0.5 µmol/l ORG 9935, and significantly different from control (P < 0.01). However, a similar dose of Milrinone (1.0 µmol/l) failed to achieve a significant reduction in spontaneous maturation over controls (41%). A high concentration of Milrinone (100 µmol/l) was necessary to achieve equivalent results (6.3%, P < 0.01).

Incubation for an additional 24 h resulted in few additional mature oocytes (Table IGo). Only the control and 1.0 µmol/l Milrinone groups showed a significant increase in GVBD from 24 to 48 h (P < 0.05). There was no change in the dose-response or relative potency of the PDE3 inhibitors at 48 h. Notably, exposure to 1.0 µmol/l ORG 9935 completely suppressed the spontaneous maturation of oocytes.

Compared with controls, there was no significant difference in progesterone levels produced by granulosa cells cultured for 24 h in concentrations of PDE3 inhibitors that inhibited GVBD (data not shown). Progesterone production by the three PDE3 inhibitors averaged 91% of control values, a non-significant change.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
While resumption of mammalian oocyte meiosis in peri-ovulatory follicles in vivo is triggered by the midcycle gonadotrophin surge, immature oocytes removed from the follicular milieu will spontaneously resume meiosis when cultured in vitro provided they have accomplished their final growth (Wassarman, 1996Go). Intracellular cyclic AMP plays a pivotal role in nuclear maturation. Agents that increase cAMP by stimulation of adenyl cyclase (such as forskolin), (Urner et al., 1983Go; Racowsky, 1984Go) or by inhibition of degradation by phosphodiesterase (such as dibutyryl cAMP or isobutyl methyl xanthine) (Cho et al., 1974Go; Sirard and First, 1988Go) prevent spontaneous resumption of meiosis in vitro. Research using selective PDE inhibitors in the rat led to the current understanding of opposing changes in cAMP levels in follicular cells and the oocyte involving differential expression and regulation of PDE4 and PDE3 isoforms respectively (Tsafriri et al., 1996Go). This differential expression provides the basis for the current study.

Our experiments are the first to establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block the spontaneous resumption of meiosis of primate oocytes in vitro. Spontaneous resumption of meiosis was not observed when oocytes were incubated in the presence of 1.0 µmol/l ORG 9935, and <10% underwent GVBD in the presence of 1.0 µmol/l Cilostamide or 100 µmol/l Milrinone. In contrast, incubation in up to 100 µmol/l Rolipram, a PDE4 inhibitor, did not affect spontaneous GVBD. These data are very similar to the results observed in the rodent (Wiersma et al., 1998Go). As different forms of PDE have been identified in the oocyte (PDE3) and follicular cells (PDE4) of rodents (Conti et al., 1998Go) our results suggest that the same compartmentalization of PDE isoforms exists in primates. However, this concept awaits experimental verification in the primate ovary.

We restricted our experiments to oocytes obtained by laparoscopic aspiration following in-vivo exposure to gonadotrophins for several reasons. The purpose of our paper was to investigate the effects of PDE3 inhibitors on spontaneous nuclear maturation of oocytes removed from pre-ovulatory follicles, not the effects of the compounds on in-vitro maturation of oocytes from immature follicles. In the human and macaque, culture conditions required for the maturation of oocytes obtained from immature follicles (such as those obtained from untreated ovaries), are more complex than those for immature oocytes obtained from animals primed with gonadotrophins but not treated with an ovulatory stimulus (Alak and Wolf, 1994Go; Schramm and Bavister, 1999Go). Primary oocytes from immature follicles must undergo a series of biochemical changes to become meiotically competent, events completed by oocytes exposed to gonadotrophins in vivo. Typically, upon aspiration, oocytes obtained from gonadotrophin-stimulated macaques prior to an ovulatory stimulus are at the GV stage and undergo spontaneous nuclear maturation to the MII stage in culture without further gonadotrophin support (Schramm and Bavister, 1994Go; 1999Go).

In our stimulation protocols, FSH alone is administered for 6 days followed by 2 days of combined FSH/LH administration. Almost all of the oocytes recovered from this stimulation protocol are denuded of most cumulus cells. In our study, 78% of recovered oocytes were normal in appearance with an intact GV remaining 1 h after isolation; only these apparently healthy oocytes were studied. Other protocols may be associated with a greater percentage of CEO. Zheng and colleagues studied the effects of various culture media on meiotic maturation, fertilization, and embryo development of cumulus-enclosed oocytes obtained following an 8 day regimen of recombinant FSH alone, and noted that 50–76% progressed to MII (Zheng et al., 2001Go). Denuded oocytes were not studied. Using an 8 day porcine FSH stimulation protocol, Schramm and Bavister obtained two distinct populations of oocytes (Schramm and Bavister, 1994Go). More than half (56%) of the recovered oocytes were cumulus-enclosed, while 44% were denuded. These researchers noted that the developmental potential of CEO was superior to denuded oocytes (79 versus 52% completing nuclear maturation).

Our experiments were designed to isolate the effect of PDE inhibitors on the oocyte. It is possible that the results would have been different if CEO had been studied, as granulosa cell–oocyte interactions were not assessed in our study design. Although generalization of the in-vitro effect of PDE3 inhibitors on denuded oocytes to the CEO of the developing follicle of primates awaits confirmation by in-vivo studies, these effects have been demonstrated in rodents (Wiersma et al., 1998Go).

Although our experiments did not specifically address reversibility of the effect of PDE3 inhibitors, it is doubtful that exposure to the agents results in a non-specific toxic effect. Experiments in the rat found no difference in spontaneous GVBD between oocytes incubated overnight in control media and those exposed to Milrinone or Cilostamide for 3 h prior to transfer to control media (Tsafriri et al., 1996Go). In our experiments, these agents produced a significant, dose-dependent decrease in the rate of GVBD with total suppression of GVBD obtained with the highest concentration (1.0 µmol/l) of ORG 9935. We chose GVBD as the primary outcome for our study, as it is the first morphological change seen with resumption of meiosis. Progression to GVBD in vitro can occur without overall competence to continue meiosis to a fertilizable MII stage (Hay et al., 1976Go; Racowsky and Kaufman, 1992Go; Gilchrist et al., 1997Go). Extrusion of the first polar body signifying development to MII was not a requirement for maturation in this study. Although this later stage indicates healthy progression of normal meiosis, and typically occurred in our control and PDE4 inhibitor-exposed oocytes (e.g. Figure 1CGo) it does not provide additional information regarding reinitiation of meiosis, the primary outcome of this study.

Exploitation of the compartmentalization of ovarian PDE isoforms as a novel contraceptive strategy was first proposed and tested in rodents (Wiersma et al., 1998Go). Treatment of mouse oocytes with selective PDE3 inhibitors in vitro prevented GVBD and spontaneous resumption of meiosis. Administration of the PDE3 inhibitor ORG 9935 in vivo to FSH–HCG-treated rats did not interfere with ovulation, but yielded immature (GV intact) oocytes. The uteri of gonadotrophin-stimulated female rats treated systemically with ORG 9935, and examined at 1 or 5 days after mating, contained only immature oocytes (GV intact) with no sign of fertilization or formation of preimplantation blastocysts. In fertility experiments, chronic treatment of female rats with ORG 9935 resulted in no adverse effects on ovulation, estrous cycles, or mating behaviour, but only one viable fetus was noted in six animals, compared with a mean of 14 per control animal (Wiersma et al., 1998Go). Thus, PDE3 inhibitors have potential as oocyte-specific contraceptives without altering other events in the ovarian cycle. Although confirmation of a lack of effect of PDE3 inhibitors on ovulation, cycle length, and hormonal function in primates awaits in-vivo study, our in-vitro observations demonstrated a selective effect on the oocyte to prevent reinitiation of meiosis without altering progesterone production by rhesus granulosa cells.

Although the previously cited rodent studies (Wiersma et al., 1998Go) demonstrate proof of concept, and the current experiments on macaque oocyte maturation in vitro are encouraging, further studies are warranted to determine whether the contraceptive mechanism of PDE3 inhibitors applies to primates, or is unfeasible and impractical due to side-effects. Potential concerns regarding PDE3 inhibitors as anti-fertility agents include their effects on non-ovarian tissues. In the study of Wiersma and colleagues, treatment with ORG 9935 induced a significant increase in heart rate (Wiersma et al., 1998Go). The PDE3 isoform is expressed in respiratory smooth muscle (Billington et al., 1999Go), platelets, cardiac ventricular myocytes, coronary smooth muscle (Cone et al., 1999Go), pancreatic islet cells (El-Metwally et al., 1997Go) and adipocytes (Moberg et al., 1998Go).

As there appears to be extensive alternative splicing found for most PDE genes, efforts to characterize the expression of, and to sequence, PDE3 isoforms within the primate oocyte are ongoing. The human PDE3A and PDE3B genes have been cloned, and extensive studies have been performed to understand their patterns of expression (Degerman et al., 1997Go). PDE3B functions in the hormonal regulation of lipolysis and glyconeogenesis, while the PDE3A form is involved in the regulation of myocardial and smooth muscle contractility (Degerman et al., 1997Go). The PDE3A isoform is also expressed in the rat and mouse oocyte (Wiersma et al., 1998Go). Although the message for the full length, transmembrane form of PDE3A exists in the rodent oocyte, the evidence suggests that truncated soluble variants of this molecule are the active form (Conti, 2000Go; Shitsukawa et al., 2001Go). It remains to be determined whether the pharmacological properties of the oocyte forms of PDE3A are sufficiently unique to allow for the creation of novel PDE3 inhibitors with increased specificity for the oocyte, and negligible pharmacological effects on other physiological processes at contraceptive dosages.

Our results provide additional evidence of the potential of PDE3 inhibitors as novel, non-coitally related contraceptives free from hormone-related side-effects. Further work is necessary in the non-human primate model to document the efficacy and safety of systemic administration of PDE3 inhibitors prior to contraceptive trials in women. Development of a practical application of this technology could have important implications for controlling fertility.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This research was supported by NICHD/NIH as a Collaborative Research Initiative of the Specialized Cooperative Centers Program in Reproduction Research through cooperative agreement (U54 HD18185; U54 HD 031398), the Women’s Reproductive Health Research Scholar Career Development Program HD 01243-03 (Dr Jensen), and NCRR RR00163. The Serono Group generously donated recombinant gonadotrophins and Antide for these studies. ORG 9935 was provided as a gift of NV Organon through the kind assistance of Ursula M.Rose, PhD. The authors wish to thank the following ORPRC resources. John Fanton, DVM, Darla Jacobs, and the staff of the Division of Animal Resources, for excellent surgical and animal care, David Hess, PhD and the Endocrine Services Core laboratory for steroid assays, and the Assisted Reproduction Technology Core laboratory for training.


    Notes
 
6 To whom correspondence should be addressed. E-mail: jensenje{at}ohsu.edu. Back


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 Discussion
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Submitted on February 15, 2002; accepted on April 4, 2002.