Identification of Hypoxanthine in Bovine Follicular Fluid

Identification of Hypoxanthine in Bovine Follicular Fluid ARJUN L. KADAM'

AND SAMUEL

s. KOIDE

Received November 11, 1988, from the Center for Biomedical Research, The Population Council, 7230 York Avenue, New York, NY 10027. Accepted for publication January 23, 1990. Abstract 0 Bovine follicular fluid (bFF) blocks the occurrence of spontaneous germinal vesicle breakdown (GVBD) of isolated mouse oocytes. One of the active factors was purified by ultrafiltration through a PM-10 membrane filter, gel filtration through Sephadex G-25, and HPLC using a reversed-phase ODs-3 column and a Superose 12 column. This factor was identified as hypoxanthine by gas chromatography and mass spectrometry. The purified substance and hypoxanthine showed the same retention time on HPLC, and identical UV absorbance and mass spectra. There are other oocyte maturation inhibitors in follicularfluid that should be identified.

Meiosis in mammalian oocytes is initiated during fetal development. Shortly after birth, the oocytes are arrested a t the dictyate stage of prophase I. This meiotic arrest is maintained throughout the growth period until the oocyte resumes meiotic maturation (germinal vesicle breakdown, GVBD) in response to the preovulatory surge of gonadotropins.' However, when oocytes at the germinal vesicle stage are removed from follicles and cultured, they undergo meiosis spontaneously,2*3suggesting that the environment in the follicle maintains them in the arrested state. In 1955,Chang' demonstrated that follicular fluid (FF) can block oocyte maturation in vitro. This finding, as well as subsequent observations,5.6 led to the concept that FF contains an oocyte maturation inhibitor (OMI).This OM1 was reported not to be species specific.7-10 Its level in FF appears to decline with growth and development of the follicles,llJ2 and it is produced by the follicular granulosa cells.13-17 The OM1 has been partially purified from FF of different species.7J1J@-20 There are reports, however, claiming that FF and granulosa cell factors do not block oocyte maturation in vitro.21-23 Recently, an oocyte maturation inhibitor was isolated from pig and mouse FF and identified as the purine hypoxanthine.24.26 In the present study, we found that bovine FF blocks the spontaneous GVBD of isolated mouse oocytes in vitro. One of the active fractions of bovine FF (bFF) was purified by ultrafiltration, gel filtration, and HPLC.Data are presented showing that the physicochemical properties of the active fraction are identical with hypoxanthine.

Experimental Section M a t e r i a l d e p h a d e x G-25 (bead size 20-80 pm) was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). Bovine serum albumin (BSA),3-isobutyl-1-methylxanthine (IBMX), hypoxanthine, sodium pyruvate, penicillin-G-sodium salt, streptomycin sulfate, Folin-Ciocalteu phenol reagent, mineral oil (light), and hepes were purchased from Sigma Chemical Company (St. Louis, MO), phenol red from Aldrich Chemical Company (Milwaukee, WI), trifluoroacetic acid (TFA, HPLC grade) from Pierce Chemical Company (Rockland, IL), and n-propanol from American Burdick and Jackson Company (Muskegon, MI). All other chemicals were of analytical grade and purchased from commercial sources. Culture Medium-Mouse oocytes were cultured in modified Krebs Ringer bicarbonate (KRB) solution containing 119.07 mM NaCl, 4.78 mM KCl, 1.71 mm CaCl,, 1.19 mM KH2P04,1.19 mM MgSO,, 25.07 mM NaHCO,, 0.25 mM sodium pyruvate, BSA (1mg/mL), penicillin aO22-3549/90/1200-7 077$0 1.00/0 0 7990, American Pharmaceutical Association

G (50 IU/mL), streptomycin (50 pg/mL), 10.0 mM hepes buffer (pH 7.41, and phenol red (10 pg/mL). The medium was filtered through a sterilization filter unit (Nalgene Company, Rochester, NY) before use. Substances to be tested for biological activity were dissolved in the culture medium and assayed for inhibitory activity with isolated mouse oocytes. Assay for Maturation Inhibiting Activity-Prepubertal Swiss mice (Nelson-Collins strain) of 15-20-g body weight were purchased from Charles River Breeding Laboratories (Wilmington, MA). Ova. ries were dissected from mice immediately after cervical dislocation and placed in the culture medium (KRB) at 35 "C. The oocytes were liberated from the follicles by teasing with a sterile needle (25 gauge) visualized under a stereomicroscope. Approximately 20 to 30 oocytes can be obtained from a single female mouse. Adhering cumulus oophorus cells were stripped off the oocyte by drawing the sample in and out of a sterile Pasteur pipette. Denuded full-grown oocytes with an intact germinal vesicle (GV) were selected by visual examination under a light microscope. Each group contained 15 to 20 oocytes in 0.2 mL of culture medium under light paraffin oil and was cultured at 37 0.5 "C in an atmosphere of 95% air and 5% C02 saturated with water. After 2-3 h of incubation, oocytes were examined for the presence or absence of an intact GV. The oocytes were scored as immature (intact GV) or mature (dissolution of GV). The occurrence of spontaneous GVBD in oocytes from different females varied from 80-95%. Because control, untreated oocytes (80% or greater) underwent spontaneous GVDB within 3 h after isolation, this value was used as control GVBD. The percentage of inhibition of oocyte maturation was calculated according to the following formu1a:le

-

*

% inhibition =

% oocyte GVBD (control) - % oocyte GVBD (expt) % oocyte GVBD (control)

x 100

(1)

The values shown in the figures are the mean of three replicate experiments. In the assay, IBMX (0.2 mM) was used as the positive control and the medium alone (KRB) was used as the negative control. Collection of Bovine Follicular Fluid (bFFGFrozen ovaries from adult cows were purchased fom A.P. Roth Company (Lansdale, PA). The ovaries were thawed and washed several times with physiological saline solution (0.154 M NaCl). Follicular fluid (FF)was collected from ovarian follicles by needle aspiration under vacuum suction. The pooled FF was centrifuged at 3000 rpm for 15 min at 4 "C to separate cellular debris. The supernatant was processed immediately. Purification of a Maturation Inhibiting Substance from Bovine Follicular Fluid-Ultrafiltration-Two hundred milliliters of bFF were filtered through a PM-10 membrane (Amicon Corporation, Danvers, MA) fixed in a 200-mL Amicon pressure cell under N2. The filtration was carried out for -24 h. The filtrate (165 mL) was lyophilized. Gel Filtration on Senhadex G-25-The lvouhilized PM-10 filtrate was resuspended in distilled water (6 mLj and centrifuged at 3000 rpm for 10 min. The supernatant was applied to a Sephadex G-25 column (1.5 x 75 cm) equilibrated with 0.01 M ammonium carbonate (pH 7.8). The column was eluted with the same buffer at 4 "C at a flow rate of 30 mL/h, and fractions Of 3 mL/tube were collected. The pooled fractions (see Figure 1)were lyophilized. Each fraction was assayed for oocyte maturation inhibitory activity as described above. High-Performance Liquid Chromatography-The active fraction Journal of Pharmaceutical Sciences I 1077 Vol. 79, No. 12, December 1990

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1078 / Journal of Pharmaceutical Sciences Vol. 79,No. 12, December 7990

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(GF,) obtained on Sephadex G-25 column was dissolved in 0.1% TFA in water (2 mg/mL). Insoluble materials were removed by microfuge centrifugation. The supernatant was injected into a Partisil-10 O D s 3 column (4.6 x 250 mm; Whatman Inc., Clifton, NJ). A short precolumn (5 x 60 mm) packed with an octadecyl-coated pellicular support (C0:pell ODS, Whatman, Clifton, NJ) was used as a guard column. The column was eluted with a linear gradient (see Figure 2) of 0.1% TFA in water and 0.1% TFA in water:n-propanol(50:50) at a flow rate of 1 mumin. Fractions were collected by measuring absorbance at 280 nm using a Waters model 481 LC spectrophotometer detector. Each pooled fraction was lyophilized, and lyophilized residue was dissolved in culture medium and assayed for inhibitory activity. The active fraction was further purified by a second reversed-phase HPLC assay on a Partisil-10 ODs-3 column. The lyophilized active fraction from HPLC and reference hypoxanthine were resuspended in pH 6.8 0.1 M ammonium acetate (1 mglmL). After microfuge centrifugation, the supernatant was injected into a Superose 12 HR 10130 column (10 x 300 mm; Pharmacia Inc., Piscataway, NJ) and eluted with pH 6.8 0.1 M ammonium acetate at a flow rate of 1 mumin. The maximum back pressure on the column was 3.0 MPa. Fractions of 1 mutube were collected. Ultraviolet Spectrophotometry-The highly purified active fraction and reference hypoxanthine were dissolved in water at a concentration of 1mg/mL, and a4justed to two different pHs (10.3 and 3.5).The UV absorbance spectrum was determined with a Uvikon 310 spectrophotometer attached to a Uvikon recorder 21 (Kontron Instruments, Everett, MA). Mass Spectrometry (MS)and Gas Chromatography fGS)-The samples (unknown active fraction and reference hypoxanthine) were subjected to mass spectrometric analysis in both underivatized and silylated form. Both electron ionization (EI) and chemical ionization (CI) spectra26 were obtained with a VG 70-250 magnetic sector GC-MS instrument. Isobutane was used as the reactant gas in the CI determinations. The underivatized samples were introduced via the

- 1 ~FR-V

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FR-II

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TIME (mln) 100

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Flgure l-chromatographic elution profile of (A) bovine follicular fluid PM10 filtrate (bFF-PM-1OF) on Sephadex G-25, and (B) maturation inhibiting potency of the eluted fractions. Key: (C) negative control; (IBMX) positive control.

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Flgure 2-(A) The HPLC elution profile of the active bFF G-25 fraction (GF,) using Partisil-10ODs-3 column. (B) The inhibitory potency of bFF fractions (major peaks) separated by HPLC. Key: (C) negative control; (IBMX) positive control.

desorption CI probe,27 which was heated at a rate of 50 "Us. The trimethylsilylated samples were introduced via the gas chromatograph (HP-57901, which was equipped with a 0.31 mm x 30 m capillary column (liquid phase: 0.52 M crosslinked 5% phenyl methylsilicone, HP) and connected to the MS by a heated direct capillary interface. The derivatized unknown sample and the reference hypoxanthine samples were injected independently and were also coinjected in order to validate their identity. The GC parameters were as follows: Injector temperature, 250 "C; GC-MSinterface temperature, 250 "C; column temperature, 120 "C at the time of injection, followed by 1 min isothermal period, then programmed up to 250 "C at a rate of 15 "C/min, and held isothermally for 10 min. The He carrier gas was maintained at 7 psi with a split ratio of 1:6. The split valve was closed after the injection for 6 a. The relevant MS parameters were as follows: scanning mode with alternate EVCI scans between mlz 600 and 60 at a rate of 1 wscan; down scam, 0.55/decade (= 1 dscan) was taken.

Results Gel Chromatography-The substance in bFF that blocks spontaneous GVBD of isolated mouse oocytes was purified by

ultrafiltration on a PM-10 membrane and gel filtration on a Sephadex G 2 5 column. Four fractions (GF,, GF,, GF,, GF,) were collected (Figure 1A). Fractions GF, and GF, showed 81.3 and 41% inhibition of GVBD, respectively (Figure 1B). Fraction GF, possessed the highest inhibitory activity and showed a dose-dependent inhibition of GVBD (Figure 3). High-Performance Liquid Chromatography-The GF, fraction was purified on a reversed-phase HPLC ODs-3 column (Figure 2A). The tubes comprising the major protein peaks were collected into five fractions (I, 11, 111, IV, V). Fraction I (FR-I), a t a concentration of 200 pg/mL by weight, showed 82.7%inhibition of GVBD, while fraction IV (FR-IV) inhibited 40% of GVBD, and the other three fractions (11,111, V) had slight inhibitory activity (Figure 2B). The major active fraction, designated as HPLC-FR-I, showed dose-dependent inhibition of GVBD at concentrations ranging from 100to 200 pg/mL (Figure 4). The active HPLC-FR-I was purified by a second chromatography assay on a Partisill0 ODs-3 column and separated into five subfractions (Il,.I,, I, I,, and I,; Figure 5). Only fraction I, showed appreciable inhibitory activity. At a concentration of 100 pg/mL, GVBD was inhibited by 75%(Figure 5). The HPLC-FR-I, and reference hypoxanthine were applied to the same Partisil-10 ODs-3 column and eluted with a linear gradient of 0.1% TFA in water and 0.1% TFA in water:n-propano1(50:50).Both compounds eluted with a 20% gradient and had the same retention time of 8 min (Figures 6A and 6B). When they were applied separately to a Superose 12 column and eluted with 0.1 M ammonium acetate (pH 6.81, both substances had the same retention time of 28 min (Figure 7), indicating that the FF component (HPLC-FR-I,) and hypoxanthine are related, if not identical substances. Ultraviolet Absorbance Spectrum-The absorbance spectra of the purified FF substance (HPLC-FR-I,) and reference hypoxanthine were analyzed. The spectra of both substances showed a maximum at 250 nm and a minimum at 220 nm (Figure 8).No significant shiR in the spectra was observed at pH 3.5. Gas Chromatography and Mass Spectra-The retention time of the bis-trimethylsilyl derivative of hypoxanthine and HPLC-FR-I, was 757 min. The mass spectrometric analysis of the purified FF substance (HPLC-FR-I,) and reference hypoxanthine showed a single prominent ion in the EI spectra of underivatized samples with a molecular ion at rnlz 136. The

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150

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BFF,HPLC-FR-I (pg/ml) Figure &Inhibition of spontaneous GVBD of isolated mouse oocytes at varying concentrationsof FR-1.Of the fractionsseparated by gel filtration on Sephadex G-25 (Figure 2). FR-1 possessed the highest inhibitory potency. 100

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BFF.HPLC(FR-I)FRACTIONS (100 Pglml)

Figure +Inhibition of spontaneous GVBD by bFF HPLC-FR-I fractions obtained from second chromatography assay on a Partisil-10 ODs-3 column. Fraction I, had the highest inhibitory potency. Key: (C) negative control; (IBMX) positive control. CI spectrum had a protonated molecular ion at d z 137 (Figure 9). The EI spectra of the silylated derivative (Figure 10) showed a prominent parent molecular ion of the bistrimethylsilyl-hypoxanthineM+ a t rnlz 280 (rel. int., 50%), two major fragments at rnlz 265 [(M-CH,)+, loo%]and rnlz 73 [(CH,), Si'], and a minor fragment at rnlz 206 [M-(CH,),Si HI+. The isobutane CI spectrum was dominated by the protonated molecular ion a t rnlz 281. The mass spectra of the FF substance, reference hypoxanthine, and a mixture of FF substance plus hypoxanthine were identical (Figure 10).

Discussion

BFF, FRACTION-OF, ()lg/ml) Figure &Inhibition of spontaneous GVBD of isolated mouse oocytes at varying concentrations of GF,. Of the fractions separated by gel filtration on Sephadex G-25 (Figure l), GF, showed the highest inhibitory potency.

Bovine follicular fluid (bFF) contains several substances that inhibit the spontaneous GVBD of isolated mouse oocytes in vitro. In the present study, one of these substances was identified as hypoxanthine. This conclusion is based on the findings that the substance and hypoxanthine have the same retention time on HPLC (Figure 61, identical UV absorbance (Figure 8), the same mass spectra (Figures 9 and lo), and equivalent biological activity. In addition to hypoxanthine, other fractions (fractions GF, and HPLC-PR-IV)inhibited the spontaneous maturation of mouse oocytes. The active subJournal of Pharmaceutical Sciences I 1079 Vol. 79, No. 12, December 1990

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stances in these fractions need to be purified and identified. In a study comparing the potencies of known substances in FF with maturation inhibitory activities, cyclic adenosine pyrophosphate (cAPP) was the most potent agent in blocking the spontaneous GVBD of isolated mouse oocytes when combined 1080 I Journal of Pharmaceutical Sciences Vol. 79, No. 12, December 1990

with dibutyryl cAMP.28 The cAPP was 100 times more potent than hypoxathine. Our impression is that meiotic arrest induced by FF is not due to a single substance but rather to a mixture of several factors. It should be pointed out that frozen ovaries were the source

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identified a factor in bFF that blocks progesterone-induced maturation of Xenopus oocytes.33

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Flgure +Mass spectra (El and CI) of underivatized FF factor (bFFHPLC-FR-I,) and reference hypoxanthine. The method is described in the text. Key: (El) electron ionization; (CI) chemical ionization.

of the FF and that hypoxanthine could have resulted from degradative processes.32 In conclusion, one of the factors in bFF that blocks spontaneous maturation of isolated mouse oocytes has been identified as hypoxanthine. Its role under in vivo conditionsneeds to be ascertained. W e recently have also

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Aspects of Reproductive Physiolo y . Proceedings of the 7th Annual Brook Lodge Workshop on Broblems in Re roductive Biology; Spillman, C. H.; Wilks, J. M., Ed.; Elsevier: fiew York, 1977; pp 3659. 11. Stone S.L.; Pomerantz, S. H.; Schwartz-Kripner, A.; Channing, C. P. Biol. Reprod. 1978,19, 585-592. 12. Tsafriri, A.; Weinstein, Y.; Gar-Ami, S.; Channing, C. P.; Pomerantz, S. H.; Lindner, H. R. In Research on Steroids, Vol. 8; Conti, E., Ed.; Academic: London, 1979;pp 193-198. 13. Tsafriri, A.; Pomerantz, S. H.; Channing, C. P. Biol. Reprod.

1976,14,511-516. 14. Sato, E.; Ishibashi,T. Jpn. J . Zootech. Scz. 1977,48,22-26. 15. Sato, E.; Furubayashi, R.; Ishibashi, T. Jpn. J . Anim. Reprod. 1978,24,59-62. 16. Sato, E.; Koide, S. S. Diferentiation 1984,26,59-62. 17. Anderson. L. D.; Stone, S. L. In 62nd Annual Meeting of the Endocrine Society, Washington, D.C., 1980. 18. Pomerantz, S.H.;Channing, C. P; Tsafriri, A. In Peptides: Structure and Biological Functions; Gross, E.; Meienhofer, J., Eds.; Pierce Chemical: Rockford, IL, 1979;pp 765-774. 19. Channing, C. P.; Bac, I. H.; Stone, S. L.; Anderson, L. D.; Edelson, S.; Fowler, S. C. P. Mol. Cell Endocrinol. 1981,22,359-370. 20. Down, S.M.;Eppig, J. J. Endocrinology 1984,114,418427. Journal of Pharmaceutical Sciences I 1081 Vol. 79, No. 12, December 7990

21. Leibfried, L.; First, N. Bwl. Reprod. 1980,23,705-709. 22. Racowsky, C.; McGaughey, R. W. J. Reprod. Fertil. 1982, 66, 505-512. 23. Fleming, A. D.; Khalil, W.; Armstrong, D. T. J. Reprod. Fertil. 1983,69,665-670. 24. Downs, S. M.; Coleman, D. L.; Ward-Bailey, P. F.; Eppig, J. J. P m .Natl. Acad. Sci. USA 1985,82, 454-458. 25. Ep ig, J.J.; Ward-Bailey, P. F.; Coleman, D. L. Biol. Reprod. 19i5,33,1041-1049. 26. Munson. M. S.B.: Field. F. H. J. Am. Chem. SOC.1966. 88. 2621-2630. 27. Cotter, R. J.Anal. Chem. 1980,52,1589A. 28. Sato, E.;Koide, S. S Endocrine Res. 1987,13,399-405. 29. Nandedkar, T.D.; Kadam, A. L.; Moodbidri, S. M. Znt. J . Fertil. 1988,33,52-59.

1082 I Journal of Pharmaceutical Sciences Vol. 79, No. 12, December 1990

30. Leibfried, L.;First, N. Biol. Reprod. 1980,23,699-704. 31. Sato, E.; Ishibashi, T.; Iritani, A. In Zntraovarian Control Mechanism; Channing, C. P.; Segal, S. J., Eds.; Plenum: New York, 1982;pp 161-173. 32. Saugstad, 0. D.Pediatr. Res. 1988,23,143-150. 33. Kadam, A. L.;,Koide, S . S. Abstracts of the Proceedings of the Endocrine Soctety, Pedratr. Res. 1989.

Acknowledgments We thank Dr. A. Bencsath of the Rockefeller University Mass Spectrometric Biotechnology Research Resource for the mass spectrometric and gas chromatographic determinations re orted in this study. The study was supported by grant GA PS 8!12 from the Rockefeller Foundation.