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Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid
Research in Veterinary Science 89 (2010) 14–19
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Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid Anita Ganguly *, Indrajit Ganguly 1, Sanat K. Meur Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India
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Article history: Accepted 20 January 2010
Keywords: Inhibin Buffalo Follicular fluid Ovary Gel filtration chromatography Proteolysis
a b s t r a c t Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the anterior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspirated from surface follicle (with diameter P5 mm). A combination of techniques (viz., gel filtration, SDS– PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS–PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Inhibin is a gonadal-derived heterodimeric glycoprotein hormone, which selectively inhibits secretion of follicle stimulating hormone (FSH) from the anterior pituitary gland (Burger, 1988). Besides its action on FSH through hypothalamo–hypophyseal– ovarian axis, inhibin may also be playing an important role in regulation of folliculogenesis through autocrine and paracrine control (Findlay, 1993). The heterodimer is composed of an a subunit linked with either a bA or bB subunits by disulfide bonds to form active dimers known as inhibin A and inhibin B, respectively (Burger and Igarashi, 1988). Both a- and b-subunits of inhibin are generated by proteolytic cleavage of two independently synthesized large precursor molecules (Mason et al., 1985, 1986; Forage et al., 1986). In several species, the most predominant form of biologically active inhibin identified has a molecular weight (MW) of approximately 31–32 kDa (Robertson et al., 1985, 1986; Rivier et al., 1985; Fukuda et al., 1986; Miyamoto et al., 1985; Ling et al., 1985). In addition, inhibin of different MW has been isolated * Corresponding author. Present address: Department of Veterinary Biochemistry, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar, Haryana 125004, India. Tel.: +91 01662 289497; fax: +91 0651 2450838. E-mail address: [email protected] (A. Ganguly). 1 Present address: Animal Genetics and Breeding Section, Project Directorate on Cattle, Meerut, Uttar Pradesh 250001, India. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.01.014
from various species including cattle (Fukuda et al., 1986; Sugino et al., 1992). Ovarian follicular fluid (FF) is a rich source of inhibin (de Jong and Robertson, 1985). Treatment with follicular fluid (proteins) and its subsequent withdrawal increases FSH flux which in turns leads to greater ovulation rate and/or litter size in sheep and cattle (Findlay et al., 1993; O’Shea et al., 1994). It has also been reported that crude FF and highly purified bovine inhibin suppress FSH secretion in vivo (Ireland et al., 1983; Beard et al., 1990). In India, buffalo is the most important milk, meat and draught animal as compared to cattle and other farm animals. However, inhibin in buffalo follicular fluid (buFF) has not yet been purified or characterized. Initial studies in goat indicate a delayed onset of estrus and increased ovulation rate by direct administration of crude (Kumar, 1997) and partially purified (Ghosh et al., 2005) buFF. However, active immunization against 30 kDa and above buFF proteins does not affect the onset, duration of estrus or ovulation rate and large follicle population due to low antibody titre (Ghosh et al., 2005). Further, passive immunization against inhibin in cycling Murrah buffalo preferentially increases plasma FSH level (Chandrasekhar and Madan, 1998). A detailed study of characterization and biological activity of buFF proteins (viz., inhibin(s), activin and follistatin etc.) may further show the way to modulate ovarian function in farm animals. Accordingly, the present investigation was undertaken to explore the molecular nature of inhibin in buFF.
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2. Material and methods 2.1. Experimental sample 2.1.1. Whole follicular fluid Follicular fluid was aspirated from apparently healthy medium and large surface follicles (diameter P5 mm) of buffalo ovaries, collected from local abattoir using sterile 22 ga needle and syringe (5 ml). The pooled FF was centrifuged (3000 rpm for 10 min at 4 °C) to remove cellular debris. After a second spin (10,000 rpm for 15 min at 4 °C), the final supernatant was stored at 20 °C (<24 h) in the presence of protease inhibitor (1 mM phenylmethyl sulfonyl fluoride and 1 mM benzamidine) and 0.01% sodium azide. 2.1.2. Acetone powder The starting material for isolation and identification of inhibin was acetone powder. To precipitate proteins, chilled acetone was added drop wise to the whole FF with continuous stirring. The final lyophilized product was referred to as crude acetone powder. 2.2. Fractionation and visualization of different proteins To conduct gel filtration chromatography, acetone powder was dissolved in Tris–HCl buffer (pH 7.4). Supernatant containing 15 mg/ml protein was applied to a Sephadex-150 column (bed volume 240 ml) and fractionated by using Tris–HCl buffer (pH 7.4) at 4 °C containing 0.15 M NaCl and 0.02% sodium azide. Fractions of 3 ml were collected at a flow rate of 20 ml/h. All the fractions were monitored by absorption at 280 nm. Purity of fractions was checked by SDS–PAGE. The concentration of protein was estimated by spectrophotometric method of Lyne (1957) using the following formula: mg/ml = 1.55 A280 0.76 A260 as and when required. 2.3. Raising of antisera against synthetic porcine inhibin-a-subunit (1– 32 amino acids) in rabbit 2.3.1. Preparation of antigen Porcine inhibin /-subunit (1–32 amino acids) was conjugated with the Imject Maleimide-activated mariculture Keyhole limpet hemocyanin (mcKLH; PIERCE, Rockford, IL). Maleimide-activated mcKLH carrier protein was reconstituted by adding distilled water to make a 10 mg/ml solution. Activated 22 ll (0.0138 lmol) mcKLH was mixed with 500 lg (0.138 lmol hapten) porcine inhibin a-subunit and allowed to react for 2 h at room temperature. Cystein (0.0138 lmol) was added after hapten’s conjugation to quench any remaining active maleimide groups. Conjugate was purified by dialysis (10 K MW cut off) against PBS (pH 7.4) buffer at 4 °C to remove unreacted hapten as well as EDTA carried over from the activated mcKLH. Conjugate (500 ll) was obtained after dialysis. 2.3.2. Raising of hyperimmune sera Hyperimmune serum against conjugated hapten was raised in rabbit as described by Staros et al. (1986). The immunization was performed by mixing equal volumes of Freund’s adjuvant with 100 ll of conjugate solution at a dose of 100 lg of immunogen per injection. On day 0, the emulsion was injected s.c. at multiple points (5–6 sites) in the back region of the rabbit. Complete Freund’s adjuvant was used in the first injection, whereas incomplete adjuvant was used in booster injections. Booster injections were carried out on days 14, 28 and 42 after the primary immunization. At 1 week after the second booster dose, blood sample was obtained from the marginal ear vein of rabbit and tested for their reactivity with corresponding porcine inhibin /-subunit (1–32 amino acids) by indirect ELISA (Signorella and Hymer,
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1984). Pre-immune rabbit serum was used as a negative control. Rabbit was bled 10 day after the third immunization and every week thereafter. At each bleeding, serum sample was checked for antibody response. Bleeding was continued for as long as the antibody titre remained high (1:4000). Serum with good antibody titre (1:4000) was collected and stored at 20 °C for further use. 2.3.3. Indirect ELISA The optimum concentration/dilution of antigen, samples and conjugate were determined by the checkerboard method. Briefly, a 96 well microtitre plate was coated with antigen (porcine inhibin a-subunit: 1–32 amino acids, 20 ng/100 ll/well) dissolved in 0.1 M sodium carbonate bicarbonate buffer (pH 9.6). The plate was incubated overnight at 4 °C and washed thrice with washing buffer (PBS-T) to remove excess of antigen. The wells were blocked for nonspecific binding with 100 ll/well of 5% skim milk powder dissolved in washing buffer (PBS-T). The plate was incubated at 37 °C for 2 h and washed three times as described above. Serum sample was serially 10-fold diluted, using serum dilution buffer, with a starting dilution of 1:10. Each serum dilution was assayed in duplicate. All controls (Antigen, antibody, conjugate, hyperimmune serum) were added. The plate was incubated at 37 °C for 2 h. 100 ll optimally diluted (1:10,000) anti-goat HRPO conjugate (Bangalore Genei, India) was added to each well. The plate was incubated for 2 h at 37 °C and washed thrice with washing buffer (PBS-T). Bound peroxidase activity was visualized after adding 100 ll of substrate buffer containing o-phenylenediamine (OPD; Sigma) and 0.03% (v/ v) H2O2. After incubation in the dark at 37 °C for 30 min, the colour reaction was stopped by adding 100 ll of 1 N H2S04. The absorbance of each well was measured at 492 nm using an ELISA plate reader. 2.3.4. Purification of IgG using DEAE – cellulose chromatography (DE 52 Whatman) Hyperimmune plasma was centrifuged at 10,000g for 15– 20 min at 4 °C. Saturated ammonium sulphate was added drop wise into the collected supernatant (plasma) with continuous stirring to produce 45% final concentration. After 0.5–1.0 h of stirring, the solution was kept at 4 °C for overnight. The mixture was centrifuged at 3000g for 30 min at 4 °C. The supernatant was discarded and the precipitate was dissolved in 50% of the original volume in PBS (pH 7.4). When fully dispersed, the original volume was reconstituted by adding more PBS (pH 7.4) and exhaustively dialyzed using 10 K MW cut off against the sodium phosphate buffer, pH 8.0 (buffer used for DEAE – cellulose chromatography). Sample was applied to the column (2.5 5.0 cm) after the equilibration with sodium phosphate buffer (pH 8.0), and washed the ion exchanger with two volumes of sodium phosphate buffer. Unbound fractions were collected, which contain IgG, and absorbance was monitored at 280 nm. Fractions were pooled and protein concentration was estimated by the method of Lowry et al. (1951) as well as by spectrophotometric method of Lyne (1957) using following formula: mg/ml = 1.55 A280 0.76 A260. 2.4. SDS–PAGE and Western Blot Chromatographic fractions obtained from acetone powder of buFF were checked for the presence of proteins by monitoring their absorbance at 280 nm. The fractions showing the presence of different proteins were further analyzed by SDS–PAGE (Laemmli, 1970). The identification of inhibin(s) in pooled fractions was carried out by Western blot analysis (Sanchez et al., 1992) using hyperimmune sera raised against conjugated porcine inhibin asubunit.
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Fig. 1. Gel filtration of acetone powder (AP) on a Sephadex G-150 column (2.5 49 cm). The column was equilibrated with 0.01 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl, and 0.02% sodium azide. Fractions of 2.0 ml were collected at flow rate of 20 ml/h. Fractions were monitored at 280 nm. Nos. I, II and III represent the pooled fractions from acetone powder after its gel filtration. Fractions were pooled according to their electrophoretic pattern shown in Fig. 2.
2.4.1. Molecular weight determination by SDS–PAGE A molecular weight ladder of standard proteins was run adjacent to the sample lanes to determine the MW of the purified fraction. To determine the relative mobility (Rm) of a protein: its migration distance from the top of the Separating Gel to the center of the protein band was divided by the migration distance of the tracking dye from the top of the Separating Gel. The relative mobility (Rm) was calculated and a standard curve for protein markers was plotted with log values of MW of each marker against its relative mobility. The molecular weight of unknown protein was estimated from the calibration curve.
Rm ¼
Distance of protein migrating after destaining Distance of tracking dye migration
3. Results 3.1. Fractionation, visualization and immunoreactivity of proteins from buFF Chromatographic fractions, obtained from acetone powder of buFF, are presented graphically (Fig. 1). The SDS–PAGE electrophoretic patterns of the fractions showing the presence of proteins (Fig. 2) revealed two major forms (MW 129 and 63 kDa). Fractions from acetone powder were then pooled, after checking their electrophoretic patterns, into three pools. The immunoreactivity of pooled fractions for inhibin, when checked by Western blot, revealed two major forms (MW 129 and 63 kDa) of inhibin (Fig. 3). 3.2. Molecular weight of protein
2.4.2. Proteolytic digestion The high and intermediate MW forms of inhibin, isolated from gel filtration chromatography of acetone powder using Sephadex G-150 column, were reduced by b-mercaptoethanol treatment using Laemmli sample buffer at a final concentration of 1% SDS and 655 mM b-mercaptoethanol. The reduced sample was then separated by SDS–PAGE, transferred to a PVDF membrane and immunoreactivity of the fractions was determined by Western blot. The high molecular weight form was further digested with trypsin (0.02% trypsin for 24 h at 37 °C) and electrophoresed on SDS–PAGE, transferred on PVDF membrane and analyzed for their cross reaction with hyperimmune sera.
Fig. 2. Non-reducing SDS–PAGE (12.5%) of fractions obtained from acetone powder by Sephadex G-150 column. Lanes 1–13: showing the elution pattern of different fractions.
Molecular weight of purified fractions was calculated from standard curve between Rm and log MW of markers of protein
Fig. 3. Western blot of fractions obtained from acetone powder by Sephadex G-150 column. Antibody against inhibin a- (1–32 amino acids) detected a high molecular weight inhibin as well as of intermediate molecular weight form of inhibin under non-reducing condition. Lane 1: contain fraction no. I (mentioned in Fig. 1); Lane 2: contain fraction no. II (mentioned in Fig. 1); Lane 3: contain fraction no. III (mentioned in Fig. 1).
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Fig. 4. Calibration curve between relative mobility (Rm) vs log molecular weight of prestained protein molecular weight marker (MBI, Fermentas, Germany). The markers had MW of 118, 85, 48, 32, 26, 19 kDa. Molecular weight of samples was calculated from the linear trendline equation: y = 0.9545x + 2.1674.
(Fig. 4). SDS–PAGE of the final products under non-reducing condition revealed bands with a MW of 129 and 63 kDa (Fig. 5A and 5B). 3.3. b-Mercaptoethanol reduction, tryptic digestion and immunoreactivity of inhibin The two forms of inhibin, one of high molecular weight (present in fraction no. I, Fig. 1) and another of intermediate molecular weight (present in fraction no. III, Fig. 1), were reduced by b-mercaptoethanol and electrophoresed on SDS–PAGE. The intermediate form of inhibin did not reduce and produced a single band in SDS– PAGE under reducing condition while the high molecular weight form resolved into four components of 63, 43, 29 and 20 kDa (Fig. 6). Out of them, only the 63, 29 and the native 129 kDa were immunoreactive after transferral to PVDF membrane (Fig. 7). A total of five fractions (63, 54, 39, 29 and 25 kDa) were obtained when high molecular weight form was digested by trypsin (Fig. 8A). However, only the 63 and 29 kDa bands were immunoreactive (Fig. 8B). 4. Discussion Inhibin in buFF has not previously been purified and subsequently characterized. In the present investigation we are report-
Fig. 6. SDS–PAGE of high and intermediate molecular weight forms of inhibin isolated from buffalo follicular fluid in the presence of b-mercaptoethanol (reducing conditions). Samples were electrophoresed on 12.5% polyacrylamide gel. M: molecular weight standards; Lanes 1 and 2: high molecular form of inhibin reduced into four components 63, 43, 29, 20 kDa; Lane 3: intermediate molecular weight form did not reduce.
ing different molecular forms of inhibins from the buffalo ovarian FF for the first time. Hyperimmune serum to porcine inhibin detected two major inhibins of different MW in buFF. The major one is a high MW form (129 kDa) and the other one is of interme-
Fig. 5. Molecular weight of purified inhibin from buffalo FF. (A) SDS–PAGE of high molecular weight form of inhibin isolated from buffalo follicular fluid under non-reducing condition. Samples were electrophoresed on 12.5% polyacrylamide gel. Calculated molecular weight of high molecular weight form of inhibin was 129 ± 2.26 kDa. (B) Intermediate form of inhibin showing MW of 63.23 ± 0.81 kDa.
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Fig. 8. Tryptic digestion of high molecular weight form of inhibin. (A) SDS–PAGE (12.5%) of high molecular weight form of inhibin after its tryptic digestion. M: molecular weight standards; Lane 1: high molecular form of inhibin reduced into five components 63, 54, 39, 29, 25 kDa. (B) Western blot of high molecular weight form of inhibin after its tryptic digestion. Out of five components (mentioned in 8A) only 63 and 29 kDa had shown immunoreactivity with antibody against inhibin a(1–32 amino acids).
Fig. 7. Western blot of high molecular weight form of inhibin under reducing conditions. Lanes 1 and 2: showing that out of four components (mentioned in Fig. 6) only 63, 29, and native 129 kDa had shown immunoreactivity with antibody against inhibin a- (1–32 amino acids).
diate MW (63 kDa) with relative OD ratio 1.8:1.4 at 280 nm. The high MW form, upon trypsin digestion produced several fragments; the fragment resembling a MW of 63 kDa did not show similar behaviour as that of a 63 kDa fraction of acetone powder. On reduction and subsequent SDS–PAGE, this intermediate form did not cleave whereas 129 kDa fraction yielded five fractions. Out of these five fractions, 63, 29 kDa and native 129 kDa protein interacted with the antibody in Western blot. This indicates that there is very little proteolytic cleavage/processing inside follicular fluid of the large precursor of inhibin. There is evidence that ovine FF contain a monomeric polypeptide of 65 kDa that resembles albumin in a number of biochemical properties like molecular weight, isoelectric pH and N-terminal amino acid, but differs in bioactivity and CD-spectra. It suggests that ovine FF has a molecule, which acts like inhibin and is not a cleavage product of the high MW form of inhibin (Kumar et al., 1992). van Dijk et al. (1984) have also isolated a 65 kDa non-reducible inhibin from bovine follicular fluid. In bovine follicular fluid, Robertson et al. (1985) found a 58 kDa species as predominant form of inhibin, which was processed to a 31 kDa form when incubated in the presence of serum (McLachlan et al., 1986) or after acid treatment (Robertson et al., 1986). Therefore, it is quite possible that the enzymatic cleavage of native inhibins, produced by granulosa cells in buffalo, takes place during circulation. In bovine follicular fluid, the larger 105 kDa form of inhibin is processed successively to produce the lowest molecular
weight form of 32 kDa inhibin through the smaller 95 and 55 kDa forms (Sugino et al., 1992). Ireland et al. (1994) demonstrated that amount of most forms of bovine inhibin and a subunits in bovine FF and serum were markedly greater than the 32–34 kDa. It supports that the low molecular weight form, mature form of the bovine inhibin, is a minor by-product of intrafollicular processing of the bovine inhibin precursor. The results of the present investigation also indicate very little proteolytic cleavage/processing of large precursor (129 kDa) of inhibin inside the ovarian follicle. The 29 kDa protein showing positive reaction in Western blot in the present investigation clearly indicates that the native protein from FF may get processed either in FF or during circulation by proteolytic cleavage. The remaining forms isolated and identified by Western blot do not carry much resemblance with the other form of inhibins from different species. However, the 29 kDa fraction, obtained either by tryptic cleavage or b-mercaptoethanol reduction, has definite resemblance to 30–32 kDa forms of inhibin reported in literature (Robertson et al., 1985). In fact, research on the multiple forms of inhibin has appeared mostly in last decade. For instance, Sunderland et al. (1996) identified seven different MW forms of inhibin (>160, 110, 77, 68, 58, 49, 34, and 29 kDa) in individual follicles during all the stages of follicle development. Good et al. (1995) isolated nine different biologically and immunologically active molecular variants of inhibins from bovine follicular fluid (pro alpha C – 29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and >160 kDa). A high proportion of the total activity is present in higher molecular weight forms of inhibin in equine follicular fluid (90, 56, and 32 kDa) than with porcine inhibin (Moore et al., 1994). It now appears that not only among species but also within same species many different forms of inhibin are available from the same source i.e. follicular fluid, because of both biochemical
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and physiological reasons. FF is mainly a transudate of blood plasma; however, it also contains many substances contributed by the granulosa cells. They include inhibins along with growth factors, vitamins, proteoglycans and follicle regulatory proteins etc. (McNatty, 1978). Further, the FF used for isolation of inhibin (s) by different workers has been collected in different ways, at different stages of estrous cycle, at different ages and at different reproductive health of the animals. In most of the cases, the source of isolation or the follicular fluid is a mixture of substances, which are in the state of a dynamic change both due to physiological state and biochemical reactions. If, within a given species, the FF is collected exclusively from a particular size of follicle, which is healthy or non-atretic, at a particular stage of the cycle, perhaps, some uniform results could be obtained. In conclusion, the present study revealed two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements Financial assistance provided to A. Ganguly in the form of Senior Research Fellowship (IVRI) is duly acknowledged. Authors are grateful to Dr. A.K. Mathur, Principal Scientist, Project Directorate on Cattle, Meerut, UP 250001, India for critical reading of the manuscript. References Beard, A.J., Castillo, R.J., McLeod, B.J., Glencross, R.G., Knight, P.J., 1990. Comparison of the effects of crude and highly purified bovine inhibin (Mr 32000 form) on plasma concentration of FSH and LH in chronically ovariectomized prepubertal heifers. Journal of Endocrinology 125, 21–30. Burger, H.G., 1988. Inhibin: definition and nomenclature, including related substances. Journal of Endocrinology 117, 159–160. Burger, H.G., Igarashi, M., 1988. Inhibin: definition and nomenclature including related substances. Journal of Clinical Endocrinology and Metabolism 66, 885– 886. Chandrasekhar, T., Madan, M.L., 1998. Passive immunization against inhibin raises the plasma concentration of FSH in cycling Murrah buffaloes. Indian Journal of Animal Sciences 68 (2), 131–134. de Jong, F.H., Robertson, D.M., 1985. Inhibin: 1985 update on action and purification. Molecular and Cellular Endocrinology 42, 95–103. Findlay, J.K., 1993. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biology of Reproduction 48, 15–23. Findlay, J.K., Xiao, S., Shukovski, L., Michel, U., 1993. Novel peptides in ovarian physiology: inhibin, activin, and follistatin. In: Adashi, E.Y., Leung, P.C.K. (Eds.), The Ovary. Comprehensive Endocrinology, Revised Series, Series Editor, L. Martini. Raven Press, New York, pp. 413–432. Forage, R.G., Ring, J.M., Brown, R.W., Mclnerney, B.V., Cobon, G.S., Gregson, R.P., Robertson, D.M., Morgan, F.J., Hearn, M.T.W., Findlay, J.K., Wettenhall, R.E.H., Burger, H.G., de Kretser, D.M., 1986. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proceeding of the National Academy of Sciences, USA 83, 3091–3095. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K., Matsuo, H., 1986. Isolation of bovine follicular fluid inhibin of about 32-kDa. Molecular and Cellular Endocrinology 44, 55–60. Ghosh, J., Yadav, M.C., Maity, S.K., Meur, S.K., 2005. Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus). Theriogenology 63 (1), 179–189.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid Anita Ganguly *, Indrajit Ganguly 1, Sanat K. Meur Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India
a r t i c l e
i n f o
Article history: Accepted 20 January 2010
Keywords: Inhibin Buffalo Follicular fluid Ovary Gel filtration chromatography Proteolysis
a b s t r a c t Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the anterior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspirated from surface follicle (with diameter P5 mm). A combination of techniques (viz., gel filtration, SDS– PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS–PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Inhibin is a gonadal-derived heterodimeric glycoprotein hormone, which selectively inhibits secretion of follicle stimulating hormone (FSH) from the anterior pituitary gland (Burger, 1988). Besides its action on FSH through hypothalamo–hypophyseal– ovarian axis, inhibin may also be playing an important role in regulation of folliculogenesis through autocrine and paracrine control (Findlay, 1993). The heterodimer is composed of an a subunit linked with either a bA or bB subunits by disulfide bonds to form active dimers known as inhibin A and inhibin B, respectively (Burger and Igarashi, 1988). Both a- and b-subunits of inhibin are generated by proteolytic cleavage of two independently synthesized large precursor molecules (Mason et al., 1985, 1986; Forage et al., 1986). In several species, the most predominant form of biologically active inhibin identified has a molecular weight (MW) of approximately 31–32 kDa (Robertson et al., 1985, 1986; Rivier et al., 1985; Fukuda et al., 1986; Miyamoto et al., 1985; Ling et al., 1985). In addition, inhibin of different MW has been isolated * Corresponding author. Present address: Department of Veterinary Biochemistry, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar, Haryana 125004, India. Tel.: +91 01662 289497; fax: +91 0651 2450838. E-mail address: [email protected] (A. Ganguly). 1 Present address: Animal Genetics and Breeding Section, Project Directorate on Cattle, Meerut, Uttar Pradesh 250001, India. 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.01.014
from various species including cattle (Fukuda et al., 1986; Sugino et al., 1992). Ovarian follicular fluid (FF) is a rich source of inhibin (de Jong and Robertson, 1985). Treatment with follicular fluid (proteins) and its subsequent withdrawal increases FSH flux which in turns leads to greater ovulation rate and/or litter size in sheep and cattle (Findlay et al., 1993; O’Shea et al., 1994). It has also been reported that crude FF and highly purified bovine inhibin suppress FSH secretion in vivo (Ireland et al., 1983; Beard et al., 1990). In India, buffalo is the most important milk, meat and draught animal as compared to cattle and other farm animals. However, inhibin in buffalo follicular fluid (buFF) has not yet been purified or characterized. Initial studies in goat indicate a delayed onset of estrus and increased ovulation rate by direct administration of crude (Kumar, 1997) and partially purified (Ghosh et al., 2005) buFF. However, active immunization against 30 kDa and above buFF proteins does not affect the onset, duration of estrus or ovulation rate and large follicle population due to low antibody titre (Ghosh et al., 2005). Further, passive immunization against inhibin in cycling Murrah buffalo preferentially increases plasma FSH level (Chandrasekhar and Madan, 1998). A detailed study of characterization and biological activity of buFF proteins (viz., inhibin(s), activin and follistatin etc.) may further show the way to modulate ovarian function in farm animals. Accordingly, the present investigation was undertaken to explore the molecular nature of inhibin in buFF.
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2. Material and methods 2.1. Experimental sample 2.1.1. Whole follicular fluid Follicular fluid was aspirated from apparently healthy medium and large surface follicles (diameter P5 mm) of buffalo ovaries, collected from local abattoir using sterile 22 ga needle and syringe (5 ml). The pooled FF was centrifuged (3000 rpm for 10 min at 4 °C) to remove cellular debris. After a second spin (10,000 rpm for 15 min at 4 °C), the final supernatant was stored at 20 °C (<24 h) in the presence of protease inhibitor (1 mM phenylmethyl sulfonyl fluoride and 1 mM benzamidine) and 0.01% sodium azide. 2.1.2. Acetone powder The starting material for isolation and identification of inhibin was acetone powder. To precipitate proteins, chilled acetone was added drop wise to the whole FF with continuous stirring. The final lyophilized product was referred to as crude acetone powder. 2.2. Fractionation and visualization of different proteins To conduct gel filtration chromatography, acetone powder was dissolved in Tris–HCl buffer (pH 7.4). Supernatant containing 15 mg/ml protein was applied to a Sephadex-150 column (bed volume 240 ml) and fractionated by using Tris–HCl buffer (pH 7.4) at 4 °C containing 0.15 M NaCl and 0.02% sodium azide. Fractions of 3 ml were collected at a flow rate of 20 ml/h. All the fractions were monitored by absorption at 280 nm. Purity of fractions was checked by SDS–PAGE. The concentration of protein was estimated by spectrophotometric method of Lyne (1957) using the following formula: mg/ml = 1.55 A280 0.76 A260 as and when required. 2.3. Raising of antisera against synthetic porcine inhibin-a-subunit (1– 32 amino acids) in rabbit 2.3.1. Preparation of antigen Porcine inhibin /-subunit (1–32 amino acids) was conjugated with the Imject Maleimide-activated mariculture Keyhole limpet hemocyanin (mcKLH; PIERCE, Rockford, IL). Maleimide-activated mcKLH carrier protein was reconstituted by adding distilled water to make a 10 mg/ml solution. Activated 22 ll (0.0138 lmol) mcKLH was mixed with 500 lg (0.138 lmol hapten) porcine inhibin a-subunit and allowed to react for 2 h at room temperature. Cystein (0.0138 lmol) was added after hapten’s conjugation to quench any remaining active maleimide groups. Conjugate was purified by dialysis (10 K MW cut off) against PBS (pH 7.4) buffer at 4 °C to remove unreacted hapten as well as EDTA carried over from the activated mcKLH. Conjugate (500 ll) was obtained after dialysis. 2.3.2. Raising of hyperimmune sera Hyperimmune serum against conjugated hapten was raised in rabbit as described by Staros et al. (1986). The immunization was performed by mixing equal volumes of Freund’s adjuvant with 100 ll of conjugate solution at a dose of 100 lg of immunogen per injection. On day 0, the emulsion was injected s.c. at multiple points (5–6 sites) in the back region of the rabbit. Complete Freund’s adjuvant was used in the first injection, whereas incomplete adjuvant was used in booster injections. Booster injections were carried out on days 14, 28 and 42 after the primary immunization. At 1 week after the second booster dose, blood sample was obtained from the marginal ear vein of rabbit and tested for their reactivity with corresponding porcine inhibin /-subunit (1–32 amino acids) by indirect ELISA (Signorella and Hymer,
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1984). Pre-immune rabbit serum was used as a negative control. Rabbit was bled 10 day after the third immunization and every week thereafter. At each bleeding, serum sample was checked for antibody response. Bleeding was continued for as long as the antibody titre remained high (1:4000). Serum with good antibody titre (1:4000) was collected and stored at 20 °C for further use. 2.3.3. Indirect ELISA The optimum concentration/dilution of antigen, samples and conjugate were determined by the checkerboard method. Briefly, a 96 well microtitre plate was coated with antigen (porcine inhibin a-subunit: 1–32 amino acids, 20 ng/100 ll/well) dissolved in 0.1 M sodium carbonate bicarbonate buffer (pH 9.6). The plate was incubated overnight at 4 °C and washed thrice with washing buffer (PBS-T) to remove excess of antigen. The wells were blocked for nonspecific binding with 100 ll/well of 5% skim milk powder dissolved in washing buffer (PBS-T). The plate was incubated at 37 °C for 2 h and washed three times as described above. Serum sample was serially 10-fold diluted, using serum dilution buffer, with a starting dilution of 1:10. Each serum dilution was assayed in duplicate. All controls (Antigen, antibody, conjugate, hyperimmune serum) were added. The plate was incubated at 37 °C for 2 h. 100 ll optimally diluted (1:10,000) anti-goat HRPO conjugate (Bangalore Genei, India) was added to each well. The plate was incubated for 2 h at 37 °C and washed thrice with washing buffer (PBS-T). Bound peroxidase activity was visualized after adding 100 ll of substrate buffer containing o-phenylenediamine (OPD; Sigma) and 0.03% (v/ v) H2O2. After incubation in the dark at 37 °C for 30 min, the colour reaction was stopped by adding 100 ll of 1 N H2S04. The absorbance of each well was measured at 492 nm using an ELISA plate reader. 2.3.4. Purification of IgG using DEAE – cellulose chromatography (DE 52 Whatman) Hyperimmune plasma was centrifuged at 10,000g for 15– 20 min at 4 °C. Saturated ammonium sulphate was added drop wise into the collected supernatant (plasma) with continuous stirring to produce 45% final concentration. After 0.5–1.0 h of stirring, the solution was kept at 4 °C for overnight. The mixture was centrifuged at 3000g for 30 min at 4 °C. The supernatant was discarded and the precipitate was dissolved in 50% of the original volume in PBS (pH 7.4). When fully dispersed, the original volume was reconstituted by adding more PBS (pH 7.4) and exhaustively dialyzed using 10 K MW cut off against the sodium phosphate buffer, pH 8.0 (buffer used for DEAE – cellulose chromatography). Sample was applied to the column (2.5 5.0 cm) after the equilibration with sodium phosphate buffer (pH 8.0), and washed the ion exchanger with two volumes of sodium phosphate buffer. Unbound fractions were collected, which contain IgG, and absorbance was monitored at 280 nm. Fractions were pooled and protein concentration was estimated by the method of Lowry et al. (1951) as well as by spectrophotometric method of Lyne (1957) using following formula: mg/ml = 1.55 A280 0.76 A260. 2.4. SDS–PAGE and Western Blot Chromatographic fractions obtained from acetone powder of buFF were checked for the presence of proteins by monitoring their absorbance at 280 nm. The fractions showing the presence of different proteins were further analyzed by SDS–PAGE (Laemmli, 1970). The identification of inhibin(s) in pooled fractions was carried out by Western blot analysis (Sanchez et al., 1992) using hyperimmune sera raised against conjugated porcine inhibin asubunit.
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Fig. 1. Gel filtration of acetone powder (AP) on a Sephadex G-150 column (2.5 49 cm). The column was equilibrated with 0.01 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl, and 0.02% sodium azide. Fractions of 2.0 ml were collected at flow rate of 20 ml/h. Fractions were monitored at 280 nm. Nos. I, II and III represent the pooled fractions from acetone powder after its gel filtration. Fractions were pooled according to their electrophoretic pattern shown in Fig. 2.
2.4.1. Molecular weight determination by SDS–PAGE A molecular weight ladder of standard proteins was run adjacent to the sample lanes to determine the MW of the purified fraction. To determine the relative mobility (Rm) of a protein: its migration distance from the top of the Separating Gel to the center of the protein band was divided by the migration distance of the tracking dye from the top of the Separating Gel. The relative mobility (Rm) was calculated and a standard curve for protein markers was plotted with log values of MW of each marker against its relative mobility. The molecular weight of unknown protein was estimated from the calibration curve.
Rm ¼
Distance of protein migrating after destaining Distance of tracking dye migration
3. Results 3.1. Fractionation, visualization and immunoreactivity of proteins from buFF Chromatographic fractions, obtained from acetone powder of buFF, are presented graphically (Fig. 1). The SDS–PAGE electrophoretic patterns of the fractions showing the presence of proteins (Fig. 2) revealed two major forms (MW 129 and 63 kDa). Fractions from acetone powder were then pooled, after checking their electrophoretic patterns, into three pools. The immunoreactivity of pooled fractions for inhibin, when checked by Western blot, revealed two major forms (MW 129 and 63 kDa) of inhibin (Fig. 3). 3.2. Molecular weight of protein
2.4.2. Proteolytic digestion The high and intermediate MW forms of inhibin, isolated from gel filtration chromatography of acetone powder using Sephadex G-150 column, were reduced by b-mercaptoethanol treatment using Laemmli sample buffer at a final concentration of 1% SDS and 655 mM b-mercaptoethanol. The reduced sample was then separated by SDS–PAGE, transferred to a PVDF membrane and immunoreactivity of the fractions was determined by Western blot. The high molecular weight form was further digested with trypsin (0.02% trypsin for 24 h at 37 °C) and electrophoresed on SDS–PAGE, transferred on PVDF membrane and analyzed for their cross reaction with hyperimmune sera.
Fig. 2. Non-reducing SDS–PAGE (12.5%) of fractions obtained from acetone powder by Sephadex G-150 column. Lanes 1–13: showing the elution pattern of different fractions.
Molecular weight of purified fractions was calculated from standard curve between Rm and log MW of markers of protein
Fig. 3. Western blot of fractions obtained from acetone powder by Sephadex G-150 column. Antibody against inhibin a- (1–32 amino acids) detected a high molecular weight inhibin as well as of intermediate molecular weight form of inhibin under non-reducing condition. Lane 1: contain fraction no. I (mentioned in Fig. 1); Lane 2: contain fraction no. II (mentioned in Fig. 1); Lane 3: contain fraction no. III (mentioned in Fig. 1).
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Fig. 4. Calibration curve between relative mobility (Rm) vs log molecular weight of prestained protein molecular weight marker (MBI, Fermentas, Germany). The markers had MW of 118, 85, 48, 32, 26, 19 kDa. Molecular weight of samples was calculated from the linear trendline equation: y = 0.9545x + 2.1674.
(Fig. 4). SDS–PAGE of the final products under non-reducing condition revealed bands with a MW of 129 and 63 kDa (Fig. 5A and 5B). 3.3. b-Mercaptoethanol reduction, tryptic digestion and immunoreactivity of inhibin The two forms of inhibin, one of high molecular weight (present in fraction no. I, Fig. 1) and another of intermediate molecular weight (present in fraction no. III, Fig. 1), were reduced by b-mercaptoethanol and electrophoresed on SDS–PAGE. The intermediate form of inhibin did not reduce and produced a single band in SDS– PAGE under reducing condition while the high molecular weight form resolved into four components of 63, 43, 29 and 20 kDa (Fig. 6). Out of them, only the 63, 29 and the native 129 kDa were immunoreactive after transferral to PVDF membrane (Fig. 7). A total of five fractions (63, 54, 39, 29 and 25 kDa) were obtained when high molecular weight form was digested by trypsin (Fig. 8A). However, only the 63 and 29 kDa bands were immunoreactive (Fig. 8B). 4. Discussion Inhibin in buFF has not previously been purified and subsequently characterized. In the present investigation we are report-
Fig. 6. SDS–PAGE of high and intermediate molecular weight forms of inhibin isolated from buffalo follicular fluid in the presence of b-mercaptoethanol (reducing conditions). Samples were electrophoresed on 12.5% polyacrylamide gel. M: molecular weight standards; Lanes 1 and 2: high molecular form of inhibin reduced into four components 63, 43, 29, 20 kDa; Lane 3: intermediate molecular weight form did not reduce.
ing different molecular forms of inhibins from the buffalo ovarian FF for the first time. Hyperimmune serum to porcine inhibin detected two major inhibins of different MW in buFF. The major one is a high MW form (129 kDa) and the other one is of interme-
Fig. 5. Molecular weight of purified inhibin from buffalo FF. (A) SDS–PAGE of high molecular weight form of inhibin isolated from buffalo follicular fluid under non-reducing condition. Samples were electrophoresed on 12.5% polyacrylamide gel. Calculated molecular weight of high molecular weight form of inhibin was 129 ± 2.26 kDa. (B) Intermediate form of inhibin showing MW of 63.23 ± 0.81 kDa.
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Fig. 8. Tryptic digestion of high molecular weight form of inhibin. (A) SDS–PAGE (12.5%) of high molecular weight form of inhibin after its tryptic digestion. M: molecular weight standards; Lane 1: high molecular form of inhibin reduced into five components 63, 54, 39, 29, 25 kDa. (B) Western blot of high molecular weight form of inhibin after its tryptic digestion. Out of five components (mentioned in 8A) only 63 and 29 kDa had shown immunoreactivity with antibody against inhibin a(1–32 amino acids).
Fig. 7. Western blot of high molecular weight form of inhibin under reducing conditions. Lanes 1 and 2: showing that out of four components (mentioned in Fig. 6) only 63, 29, and native 129 kDa had shown immunoreactivity with antibody against inhibin a- (1–32 amino acids).
diate MW (63 kDa) with relative OD ratio 1.8:1.4 at 280 nm. The high MW form, upon trypsin digestion produced several fragments; the fragment resembling a MW of 63 kDa did not show similar behaviour as that of a 63 kDa fraction of acetone powder. On reduction and subsequent SDS–PAGE, this intermediate form did not cleave whereas 129 kDa fraction yielded five fractions. Out of these five fractions, 63, 29 kDa and native 129 kDa protein interacted with the antibody in Western blot. This indicates that there is very little proteolytic cleavage/processing inside follicular fluid of the large precursor of inhibin. There is evidence that ovine FF contain a monomeric polypeptide of 65 kDa that resembles albumin in a number of biochemical properties like molecular weight, isoelectric pH and N-terminal amino acid, but differs in bioactivity and CD-spectra. It suggests that ovine FF has a molecule, which acts like inhibin and is not a cleavage product of the high MW form of inhibin (Kumar et al., 1992). van Dijk et al. (1984) have also isolated a 65 kDa non-reducible inhibin from bovine follicular fluid. In bovine follicular fluid, Robertson et al. (1985) found a 58 kDa species as predominant form of inhibin, which was processed to a 31 kDa form when incubated in the presence of serum (McLachlan et al., 1986) or after acid treatment (Robertson et al., 1986). Therefore, it is quite possible that the enzymatic cleavage of native inhibins, produced by granulosa cells in buffalo, takes place during circulation. In bovine follicular fluid, the larger 105 kDa form of inhibin is processed successively to produce the lowest molecular
weight form of 32 kDa inhibin through the smaller 95 and 55 kDa forms (Sugino et al., 1992). Ireland et al. (1994) demonstrated that amount of most forms of bovine inhibin and a subunits in bovine FF and serum were markedly greater than the 32–34 kDa. It supports that the low molecular weight form, mature form of the bovine inhibin, is a minor by-product of intrafollicular processing of the bovine inhibin precursor. The results of the present investigation also indicate very little proteolytic cleavage/processing of large precursor (129 kDa) of inhibin inside the ovarian follicle. The 29 kDa protein showing positive reaction in Western blot in the present investigation clearly indicates that the native protein from FF may get processed either in FF or during circulation by proteolytic cleavage. The remaining forms isolated and identified by Western blot do not carry much resemblance with the other form of inhibins from different species. However, the 29 kDa fraction, obtained either by tryptic cleavage or b-mercaptoethanol reduction, has definite resemblance to 30–32 kDa forms of inhibin reported in literature (Robertson et al., 1985). In fact, research on the multiple forms of inhibin has appeared mostly in last decade. For instance, Sunderland et al. (1996) identified seven different MW forms of inhibin (>160, 110, 77, 68, 58, 49, 34, and 29 kDa) in individual follicles during all the stages of follicle development. Good et al. (1995) isolated nine different biologically and immunologically active molecular variants of inhibins from bovine follicular fluid (pro alpha C – 29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and >160 kDa). A high proportion of the total activity is present in higher molecular weight forms of inhibin in equine follicular fluid (90, 56, and 32 kDa) than with porcine inhibin (Moore et al., 1994). It now appears that not only among species but also within same species many different forms of inhibin are available from the same source i.e. follicular fluid, because of both biochemical
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and physiological reasons. FF is mainly a transudate of blood plasma; however, it also contains many substances contributed by the granulosa cells. They include inhibins along with growth factors, vitamins, proteoglycans and follicle regulatory proteins etc. (McNatty, 1978). Further, the FF used for isolation of inhibin (s) by different workers has been collected in different ways, at different stages of estrous cycle, at different ages and at different reproductive health of the animals. In most of the cases, the source of isolation or the follicular fluid is a mixture of substances, which are in the state of a dynamic change both due to physiological state and biochemical reactions. If, within a given species, the FF is collected exclusively from a particular size of follicle, which is healthy or non-atretic, at a particular stage of the cycle, perhaps, some uniform results could be obtained. In conclusion, the present study revealed two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements Financial assistance provided to A. Ganguly in the form of Senior Research Fellowship (IVRI) is duly acknowledged. Authors are grateful to Dr. A.K. Mathur, Principal Scientist, Project Directorate on Cattle, Meerut, UP 250001, India for critical reading of the manuscript. References Beard, A.J., Castillo, R.J., McLeod, B.J., Glencross, R.G., Knight, P.J., 1990. Comparison of the effects of crude and highly purified bovine inhibin (Mr 32000 form) on plasma concentration of FSH and LH in chronically ovariectomized prepubertal heifers. Journal of Endocrinology 125, 21–30. Burger, H.G., 1988. Inhibin: definition and nomenclature, including related substances. Journal of Endocrinology 117, 159–160. Burger, H.G., Igarashi, M., 1988. Inhibin: definition and nomenclature including related substances. Journal of Clinical Endocrinology and Metabolism 66, 885– 886. Chandrasekhar, T., Madan, M.L., 1998. Passive immunization against inhibin raises the plasma concentration of FSH in cycling Murrah buffaloes. Indian Journal of Animal Sciences 68 (2), 131–134. de Jong, F.H., Robertson, D.M., 1985. Inhibin: 1985 update on action and purification. Molecular and Cellular Endocrinology 42, 95–103. Findlay, J.K., 1993. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biology of Reproduction 48, 15–23. Findlay, J.K., Xiao, S., Shukovski, L., Michel, U., 1993. Novel peptides in ovarian physiology: inhibin, activin, and follistatin. In: Adashi, E.Y., Leung, P.C.K. (Eds.), The Ovary. Comprehensive Endocrinology, Revised Series, Series Editor, L. Martini. Raven Press, New York, pp. 413–432. Forage, R.G., Ring, J.M., Brown, R.W., Mclnerney, B.V., Cobon, G.S., Gregson, R.P., Robertson, D.M., Morgan, F.J., Hearn, M.T.W., Findlay, J.K., Wettenhall, R.E.H., Burger, H.G., de Kretser, D.M., 1986. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proceeding of the National Academy of Sciences, USA 83, 3091–3095. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K., Matsuo, H., 1986. Isolation of bovine follicular fluid inhibin of about 32-kDa. Molecular and Cellular Endocrinology 44, 55–60. Ghosh, J., Yadav, M.C., Maity, S.K., Meur, S.K., 2005. Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus). Theriogenology 63 (1), 179–189.
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