BIOCHEMICAL CHARACTERIZATION OF INHIBIN HORMONE IN THE OVARY OF SHE CAMEL (CAMELUS DROMEDARIES)

Animal Reproduction Research Institute,

AgricultureResearchCenter

Biochemical characterization of inhibin hormone in the ovary of she camel (Camelus dromedaries)

(With One Table and 6 Figures)

By

A.A. Ramadan; A.M. El Behairy*; M.A. Mostafa* and M.A. Al-Gabry

*Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, CairoUniversity

(Received at 9/12/2006)

التعرف على هرمون الانهبين فى مبايض إناث الجمال باستخدام الطريقة الکيميائية الحديثة

أحمد عبد الفضيل رمضان ، عادل محمد أبو الفتوح البحيرى ،

مصطفى عبد الفتاح مصطفى ، محمود عباس ابراهيم الجابرى

هدفت هذه الدراسة الى استخلاص وتنقية ودراسة ترکيب هرمون الانهبين من مبايض الجمال المصرية باستخدام تقنيات حديثة. تم تجميع السائل الموجود داخل حويصلات التبويض الموجودة على مبايض إناث الجمال بغض النظر عن سن الحيوان أو حجم الحويصلات. تم تنقية السائل الذى تم تجميعه من هرمونى الاستروجين والبروجيستيرون باستخدام الفحم النشيط ثم تم استخدام نوعين من الکروماتوجراف السائل لفصل الأنواع المختلفة من البروتينات. النوع الأول من الکروماتوجراف (سيفاکريل س-200) فصل 3 بروتينات بناء على الوزن الجزيئى لها. تم استخدام النوع الثانى من الکروماتوجراف (سيفاديکس ج-100) لفصل البروتينات المکونه للبروتين الثالث المعزول من الکروماتوجراف الأول. تم الحصول على هرمون الانهبين فى صوره نقية من البروتين المستخلص من سيفاديکس ج-100. للتأکد من نقاء الهرمون المعزول من البروتين الثالث، تم استخدام تقنية فصل البروتينات باستخدام التهريب الکهربائى مرتين. فى المرة الأولى تم استخدام مواد تفکک للبروتينات لمکونات صغيره وتم صباغتها بمادة الکوماسى الأزرق اللامع حيث أسفرت عن فصل 5 مرکبات بروتينيه (تراوحت بين 8‚58 -3 ‚ 32 کيلو دالتون). فى المرة الثانية تم حذف المواد التى تفکک البروتينات وتم صباغتها بمادة نترات الفضة حيث أسفرت عن فصل مرکب بروتينى واحد. تم تحليل المرکب المعزول من مبايض إناث الجمال باستخدام الکروماتوجراف السائل عالى الکفائه على عامود سى-18 حيث تم فصل مرکب بروتينى واحد عند الدقيقة 65‚101 مما يدل على نقاء المرکب. تم أيضا استخدام تقنية الکروماتوجراف ذو الألواح الرقيقة عالى الکفائه لفصل الأحماض الأمينيه المکونه للمرکب المعزول، حيث تم الحصول على 7 أحماض أمينيه مختلفة وبترکيزات مختلفة أعلاها ترکيزا الهستيدين.

SUMMARY

Isolation, purification and advanced characterization of hormone inhibin in ovary of female camel are aimed in this study. Pooled follicular fluid was collected from the ovaries of she-camel irrespective to physiological status and age of the animals.  Follicular fluid was subjected to 2 types of gel filtration chromatography; Sephacryl S-200 where three peaks of proteins was obtained.  The suspected peak to contain inhibin (peak III) resolved from S-200 was subjected to Sephadex G-100 where three peaks were obtained, third peak was suspected to contain inhibin in pure form. To verify the purity of the isolated hormone, the lyophilized fraction containing inhibin was subjected to analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both non reducing condition, stained with silver nitrate where a single band was resolved, and under reducing condition, where five bands resolved (ranging between 58.8 to 32.3 KDa). Using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) to fractionate the third peak, subunits of inhibin hormone eluted at different retention times. High Performance-Thin Layer Chromatography (HP-TLC) used to determine the N-terminal amino acids contents of the third peak and  7 amino acids resolved with different concentrations where histidine was the most abundant of the amino acids.

Key words: Inhibin hormone, ovary, she camel

Introduction

Inhibins family is present in a wide variety of reproductive and non reproductive tissues, the main source of inhibin in both sexes appears to be the gonads since its concentration is approximately 5-8 folds higher in the ovarian vein than jugular vein and its concentration was undetectable following gonadoectomy (Roser et al., 1994). The ovary is the major source of circulating inhibin and its level was rapidly disappeared from serum following ovarictomy (Robertson et al., 1988). The ovarian tissues contain the mRNA and proteins of inhibin measured in granulosa cell conditioned media and the expression of the subunit mRNAs of α and β subunits of inhibin have changed with follicular development (Findlay et al., 2001). Follicular fluid has shown to be a potent source of inhibin in different species: pigs (Schwartz and Channing, 1977; Channing et al., 1982) cattle (Henderson and Franchimont, 1981) rats (Hermans et al., 1982; Bicsak et al., 1986; Zhang et al., 1987) humans (Channing et al., 1984) and other primates (Noguchi et al., 1987).  Several studies have shown that the amount of inhibin produced by granulosa cells from large follicles was greater than that from small follicles (Channing et al., 1982), and this fact is reflected in the concentrations of inhibin in the follicular fluid (Tsonis et al., 1987). McLachlan et al. (1996) demonstrated that the circulating inhibin levels reflect the mass of active granulosa cells in the ovary, since they correlate significantly with the number of follicles detected by ultrasound. Moreover the expression of βB –subunit of inhibin was greater in the granulosa cells of antral follicles during the luteal-follicular transition, (Hayes et al. 1998), whereas the expression of α- subunit of inhibin appeared relatively constant throughout the follicular development (Drummond et al., 2000 and Findlay et al., 2001). The corpus luteum is also a significant source of inhibin in the luteal phase (McLachlan et al., 1996). Human granulosa cells allowed to be luteinized in culture have the capacity to produce inhibin (Tsonis et al., 1987). In addition the luteal cells of rat and human contained mRNA for the α-subunit of inhibin (Davis et al., 1986, 1987). Indeed, the expression of the βA-subunits of inhibin was highest in the corpus luteum and the dominant follicle (Hayes et al., 1998).

Inhibin was also detected in non-reproductive tissues; the mRNAs that encode inhibin β-subunits have been identified in the heart, skeletal muscle, spinal cord cerebrum, kidney, adrenal, bone marrow, and liver of mid gestational human fetus.

FSH increases inhibin secretion. In primary cultures of rat granulosa cells, crude preparations of FSH stimulated inhibin biosynthesis and its secretion (Woodruff et al., 1987; Turner et al., 1989; and LaPolt et al., 1992). The ovarian steroids may also contribute to the regulation of inhibin subunit gene expression, such as estrogens that can directly increase inhibin mRNAs and its secretion (Turner et al., 1989; Rivier and Vale 1989). The bases of molecular regulation of inhibin subunits synthesis are now clearer than ever. Gonadotropins are potent regulators of inhibin subunit gene expression. Both LH and FSH signal in target cells through G protein-coupled receptors and increase intracellular cAMP levels via activation of adenyl cyclase. Gonadotropins stimulate cAMP response element binding protein (CREB) phosphorylation in granulosa cells and stimulate transcription via CREB-mediated interaction with the CRE in the inhibin tissues specific promoter. This provides an explanation for how the gonadotropins (specifically, FSH) stimulate inhibin (A) expression throughout the majority female reproductive cycle (Mukherjee et al., 1998 and Tanimoto et al., 1996).

Importance of inhibin in the reproductive cycle of many known animals is well established and understood, moreover the isolation and purification of this peptide hormone from these animals has been completed many years ago. Indeed, isolation, purification, and partial identification of this hormone from she camel is aimed in this study for many reasons: first, camel as a domestic animal in Egypt still needs great attention to fully understand its reproductive cycle with all its aspects, second, camel is a unique domestic animal in many physiological, anatomical, pathological, and reproductive aspects, third, increasing the wealth of camel in Egypt needs many efforts to gather information about its reproduction.

MaterialS and Methods

Collection of follicular fluid

Large follicles were collected from different abattoirs, including pregnant female camels, irrespective to the physiological status of the follicles, nor the age of the animal. The follicular fluid was aspirated from the ovarian follicles, pooled and transported on ice bags to the laboratory within 30 minutes. Suitable amount of follicular fluid (100 ml) was collected to fulfill the experiments. The follicular fluid was centrifuged at 5000 rpm in cooling centrifuge at 4^o C for 15 minutes; the clear supernatant was carefully aspirated and kept at -20^o C. Protein concentration was estimated using Biuret Reagent as described by (Henary et al., 1968).

Refining follicular fluid from steroids

The follicular fluid was refined from all steroids according to (Welschen et al., 1977) by adding 50 mg of activated charcoal to each    1 ml of follicular fluid while stirred at room temperature for 30 minutes, and centrifuged at 4^o C at 5000 rpm for one hour then the supernatant was carefully aspirated and preserved at -70^o C until subsequent analysis.

Extraction of ovarian peptides

The supernatant of the follicular fluid was added drop-wise to 90% (v/v) acetone; the precipitate was dissolved in 95% acetic acid and stirred overnight at room temperature. The protein was again precipitated by adding ethanol 90% (v/v); the precipitate was dissolved in water, dialyzed, and lyophilized.

Purification of ovarian peptides

1- Purification using Sephacryl S- 200 HR (2.5 x 40 cm) using ECONO System manufactured by Bio-Rad Laboratory USA. The follicular fluid was diluted in 15 ml of 0.05 mol ammonium acetate solution and slowly applied onto the top of the gel. The mobile phase was 0.05M ammonium acetate.  The column effluent was monitored at 280 nm and collected in tubes (1 ml/tube).  Fourteen runs were done, and the number of tubes per each run was ranged between16 to 19 tubes. Protein concentration was estimated using Bradford reagent (1976) and diagrammed to plot the resolved peaks. The tubes representing each peak in every run were pooled together.

2- Purification using Sephadex G-100 column (2.5 x 40 cm) fraction number III produced from the Sephacryl S-200 gel was applied to Sephadex G-100 column and eluted with 25% acetic acid as a mobile phase. One ml fraction size was collected per each tube and protein concentration was estimated and diagrammed to plot the resolved peaks.  The tubes representing each peak in every run were pooled together.

Electrophoresis

The electrophoresis technique was deployed to find out the purity of the suspected protein peak containing inhibin. The technique was done under both non-reducing and reducing conditions using Bio-Rad USA mini gel according to(Laemmli, 1970). Under non-reducing conditions, both SDS and 2- mercaptoethanol (ME-2) were eliminated from the technique and kept under reducing conditions. The molecular weights of protein bands in the gel were estimated in comparison with that of the standard protein markers (Bio-Rad 10,000 to 100,000 Daltons containing sex proteins) using software Gel Pro Analyzer Version 3.1.

Detection of Subunits of inhibin using Reversed Phase High Performance Liquid Chromatography (RP-HPLC) (Moore et al., 1994)

Detection of inhibin subunits was carried out according to the method described by (Moore et al., 1994). An HPLC CBG Englandsupplied with UV detector and a C18 column was used (1cm x 25cm) and the mobile phase consisted of gradient of 0.01% trifluoroacetic acid (TFA) in water and 0.01% TFA in 80% acetonitrile. Total run time was 130 minutes. Flow rate was adjusted to 2 ml/min and the effluent detected at 280 nm using UV detector. The resulted chromatogram was analyzed withWinchrom 3software.

Identification of the N- terminal amino acids contents of the separated peptides using High Performance Thin Layer Chromatography (HP-TLC)

Fraction 3 that contained inhibin was placed in a hydrolysis tube and 200 µL of hydrolysis solution (6 N hydrochloric acid containing 0.1% to 1.0% of phenol)per 500 µg of lyophilized peptide were added.  The sample was hydrolyzed at 110^o C for 24 hours in an inert atmosphere. The hydrolyzed sample then dissolved in 10 µl of 0.2 Msodium bicarbonate and 10 µl 1-dimethylaminonaphthalene-5-sulpfonyl chloride (Dansyl chloride, DNS-CL) and mixed well for labeling of N- terminal amino acids with fluorescent Dansyl chloride. The tube was sealed with parafilm and incubated at 37^o Cfor 1 hour then 10µl of 50% pyridine was added. Silica plates (10X10 cm) were used to separate different amino acids of inhibin. The developing solvent was prepared by adding 5 g EDTA-disodium salt to 50 mL distilled water and the pH was adjusted to 9 using 1 N NaOH, then 10 mL of n-butanol was added and the solution was vortexed well and 35 mL diethylether was added, vortexed and the upper phase was then used as developing solvent. Densitometric evaluation of the amino acids was done by CAMAG TLC Scanner3 using Lab data System and CATS evaluation software. Scanning of the plates was made by fluorescence at 280 nm using mercury lamp, monochromator bandwidth 30 nm; slit dimensions 0.3 x 4 mm. The amino acids were detected in comparison with the standard amino acids solution.

Results

Figure (1) represents the fractionation pattern of follicular fluid of she camel. The chromatogram shows 3 protein peaks (I, II, and III) resolved from Sephacryl S-200 HR gel filtration column chromatography.

Fig. 1: Chromatogram of the eluted protein peaks from follicular fluid of she camel on Sephacryl S-200 HR gel filtration column chromatography.

Fig. 2: Represents the fractionation pattern of peak number III resolved from S-200 gel and fractionated on G-100 gel. The chromatogram shows 3 protein peaks.

Fig. 3: Electrophoretic pattern (SDS-PAGE) of isolated and purified inhibin from ovarian follicular fluid of she camel (peak number III from G-100 gel) run under non-reducing conditions and stained using silver nitrate.

Fig. 4: Electrophoretic pattern (SDS-PAGE) of isolated and purified inhibin from ovarian follicular fluid of she camel and run under reducing conditions and stained using Coomassie brilliant blue stain

            Electrophoretic pattern of isolated and purified peak III resolved from G-100 (inhibin) from follicular fluid of she-camel and stained by silver nitrate (Figure 3) clearly shows the presence of only single band of protein (B). The condition of electrophoresis excluded the reducing substances (SDS and ME-2) to allow the sample to run intact without fractionation on the gel. On the other hand running the same sample under reducing condition (in presence of SDS and ME-2) and stained with Coomassie brilliant blue R-250 allowed the fractionation of the isolated inhibin into different subunits according to their molecular weights (Figure 4). There are five protein bands resolved in the gel with different amounts and molecular weights. The following chromatogram shows the fractionation of isolated and purified inhibin on Reversed phase high performance liquid chromatography (RP-HPLC).

Fig. 5: Chromatogram represents different fractions of inhibin with different retention time separated on –RP-HPLC using C-18 column.

Table 1: Showing migration distance (MD), retention factor (Rf) and N terminal of amino acids concentration of she-camel inhibin on HP-TLC

Fig: 6: An HP-TLC Chromatogram represents the concentration of inhibin N-Terminal amino acids (µMol/ml) of she-camel.

Discussion

Inhibin is a peptide hormone secreted mainly from the ovary of all known domestic and experimental animals. It is a member of the transforming growth factor-β (TGF-β) superfamily that is expressed by oocytes, granulosa cells, and theca cells of the developed oocytes.  Inhibin acts as an intraovarian regulatory molecule involved in follicle recruitment, granulosa and theca cell proliferation or atresia, steroidogenesis, oocyte maturation, ovulation, and luteinization. (Knight and Glister, 2003; Kawano et al., 2004; and Ocal et al., 2004). The interactions of peptide and steroid hormone signaling cascades of hormonal eventsin the ovary are critical for follicular growth, ovulation,and luteinization. The pituitary gonadotrophins, follicle-stimulatinghormone (FSH) and luteinizing hormone (LH) play key regulatoryroles and their actions are dependent on other peptides signalingpathways, including insulin-like growthfactor-1 (IGF-1), transforming growth factor-β (TGF-β)family members (e.g., inhibin, activin, growth differentiationfactor-9, and bone morphogenic proteins)(JoAnne et al., 2002). Inhibins family is members of TGF β superfamily of growth and differentiation factors. They were first identified as gonadal-derived regulators of pituitary FSH and were subsequently assigned of multiple actions in a wide range of tissue (Massague, 1998). Isolation and purification of this hormone has been accomplished in many species.  The initial isolation of inhibin was achieved from bovine follicular fluid as a 58 kDa glycoprotein consisting of two disulphide-linked subunits of apparent molecular masses 43 kDa and 15 kDa (Robertson et al., 1985).  Miyamoto et al. (1985) reported that the isolation of a 32-kDa glycoprotein from porcine follicular fluid consisting of two subunits of 20 kDa and 13 kDa, the larger subunit has been termed α, and the smaller was β, these findings were confirmed by Ling et al., 1985 who isolated two forms of inhibin, termed inhibin A and inhibin B and differentiated from each other by the differing NH2 -terminal amino acid sequences of their 13-subunits. In sheep, a 30 kDa form of inhibin with 20 and 16 kDa subunits had been isolated from follicular fluid (Leversha et al., 1987).

In current study, fractionation of follicular fluid of female camel using Sephacryl S-200 gel resolved 3 protein peaks with different molecular weights and protein concentrations. Similar results obtained by Kishiko et al., (1992) who obtained three fractions from bovine follicular fluid. On the other hand, Moore et al., (1994) isolated four fractions from equine follicular fluid and Leversha et al., (1987) found only two fractions in ovine follicular fluid. Using Sephadex G-100 to further fractionate third peak isolated from Sephacryl S-200 also resolved 3 peaks.  Similar findings obtained by Robertson et al., (1992) from bovine follicular fluid, Gordon et al. (1986) from porcine follicular fluid and Moore et al. (1994) from equine follicular fluid. Checking the purity of peak 3 resolved from Sephadex G-100 using electrophoresis, under non-reducing conditions and stained by silver nitrate, revealed only one protein band, indicating purity of the isolated peptide.  Kishiko et al. (1992) reported similar results in bovine and Rivier et al. (1985) in porcine follicular fluids. Running the same protein on electrophoresis under reducing conditions resolved 5 protein bands with the different molecular weights (58, 38, 36, 34 and 32 KDa) which indicate that protein peptide isolated in peak 3 from Sephadex G-100 has multiple subunits or forms. The highest protein concentration detected in the lowest molecular weight band (32 KDa). Miyamoto et al. (1985) mentioned that in porcine follicular fluid inhibin is present in four molecular forms with corresponding MW of 100, 80, 55, and 32 KDa.  More recently, Kishiko, et al. (1992) isolated three forms of inhibin from bovine follicular fluid, the highest molecular weight form (95 and 105 KDa), the intermediate form (55 and 65 KDa) and the low form (32KDa). Also Moore et al. (1994) stated that at least three forms of inhibin were present in equine follicular fluid with different molecular weights, 90, 56 and 32 KDa. It is obvious that the 32 KDa form of inhibin is common in the follicular fluid of most animal species studied so far. The other forms detected with different molecular weights in these animals may be attributed to 1- species differences, 2- different protocols for processing the samples and buffers with different pH values (between acidic and alkaline) used in these studies, and 3- different reproductive and physiological status of ovaries from which follicular fluids were collected. Indeed,Guthrie et al. (1997) reported that follicular production and/or intracellular processing of inhibin dimer and/or inhibin α subunits were changed during different phases of follicular development, supporting the notation of physiological roles for these peptides. Also Mason et al. (1996) concluded that the wide variation in the size of inhibins was due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked sites.

Analyzing peak 3 resolved from Sephadex G-100 using          RP-HPLC, five peaks with different retention times (2.083, 6.117, 68.817, 101.65, and 105.117 minutes) were resolved.  These results are close to results recorded by Moore et al. (1994) who found 5 peaks of peptides in equine follicular fluid with almost same retention times.

Amino acids contents of the she camel follicular fluid inhibin peptide shown to be Arginine, Histidine, Serine, Glycine, Threonine, Aspartic acid and Glutamic acid with concentrations of (0.488, 0.684, 0.449, 0.499, 0.509, 0.506 and 0.503 µmol/ml) respectively. In 1985 Ling et al. found that the sequence analysis of the porcine follicular fluid inhibin (18 and 14 KDa) was Serine, Threonine, Alanine, Proline, Leucine, Tryptophan, Glycine, Glutamic, Aspartic, Asparagine and Arginine. The discrepancy in the results in four amino acids (Alanine, Proline, Leucine, Tryptophan) may be attributed to the method used (we used 6N HCL, and sample heated at 110 C^o for 24 hours) which may caused the destruction of Tryptophan and Leucine and the conversion of Asparagine and Glutamine to Aspartic and Glutamic acids respectively.  The absence of Alanine and Proline from camel inhibin may be attributed to species difference between camel and porcine. Indeed, Rivier et al., (1985) used different technique in studying N- Terminal amino acids of porcine inhibin (32 KDa) and obtained Histidine, Alanine, Serine, Glycine, Leucine, Proline, Threonine and Glutamic acid.  The difference in type of inhibin studied (18 and 14 KDa vs. 32 KDa) and technique adopted in these two studies clearly revealed slight difference in amino acid residues even in the same species; porcine.  Kishiko et al. (1992) found in bovine follicular fluid inhibin (50 KDa) N- Terminal sequence consisting of Cysteine, Histidine, Glycine, Leucine, Glutamic, Aspartic and Arginine. There is a slight difference in only two amino acids, Serine and Threonine found in camel inhibin and Cysteine and Leucine found in bovine inhibin. On the other handLeversha et al. (1987) found that ovine follicular fluid inhibin N- Terminal amino acid contained the sequence of Serine, Proline, Glycine, Leucine, Glutamic, Alanine, Histidine, Valine, Aspartic and Aspargine, where the amino acids Serine, Glycine, Glutamic, Histidine and Aspartic were found in camel inhibin.

In conclusion, in current study inhibin peptide was isolated from the follicular fluid of female camel in our laboratory. Advanced biochemical analyses were adopted to study the structure of this peptide.  The use of 2 types of gel filtration techniques (Sephacryl S-200 and Sephadex G-100) was necessary to obtain inhibin peptide in pure form.  Also the refining follicular fluid from steroids and the extraction of ovarian peptides using chemical techniques has helped in purification process. Inhibin peptide has many forms depending on molecular weights (electrophoretic patterns) and retention times (RP-HPLC). The N-terminal amino acid residues of this peptide also vary according to its form. In our study it is clear that the protein of 32-KDa molecular weight has the greatest protein concentration and when analyzed using HP-TLC technique, it resolved to 7 amino acid residues that have some sharing residues with porcine, bovine, and ovine follicular fluids. Further research is required to clone the genes that control inhibin peptide secretion.  Moreover, we are in the process of raising antibodies against different forms of inhibin isolated and purified in our laboratory. These antibodies will help us in finding out which form has the dominant physiological effect in vivo and also can help us developing therapeutic regime for the reproductive problems originating from some pituitary hormone disturbances.

References

Bicsak, T.A., Tucker, E.M., Cappel, S., Vaughan, J., Rivier, J., Vale, W. and Hsueh, A.J. (1986):Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119, 2711-2719.

Bradford, M.M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Channing, C.P.; Anderson, L.D.; Hoover, D.G.; Kolena, J.; Osteen, K.G.; Pomerntz, S.H. and Tanabe, K. (1982): The role of nonsteoiddal regulators in control of oocyte and follicular maturation. Recet Prog Horm Res 38: 331-340.

Channing, C.P.; Chacom, M.; Tanabe, K.; Gagliano, P. and Tildon, T. (1984):follicular fluid inhibin activity and steroid levels in ovarian tissues obtained at autopsy from human infants from 18 to 200 days of age. Fertil Steril 42: 861-869.

Davis, S.R.; Dench, F.; Nikolaidis, I.; Clements, J.A.; Forage, R.G.; Burger, H.G. and Krozowski, Z. (1986): Inhibin A subunit gene expression in the ovaries of immature female rats stimulated by pregnant mare serum gonadotrophin. Biochem Biophys Res Commun. 138: 1196-1202.

Davis, S.R.; Krozowski, Z.; McLachlan, R.I. and Burger, H.G. (1987):A semineferous tubular factor is not obligatory for regulation of plasma follicle stimulating hormone in the rat. Endocrinology 108: 1035-1039.

Drummond, A.E.; Dyson, M.; Thean, E.; Groome, N.P.; Robertson, D.M. and Findlay, J.K. (2000):Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-nataland immature rat ovaries. J. Endocrinol 166, 339-54.

Findlay, J.K.; Drummond, A.E.; Dyson, M.; Baillie, A.J.; Robertson, D.M. and Ethier, J.F. (2001):Production and actions of inhibin and activin during folliculogenesis in the rat Molecular and Cellular Endocrinology, 180:1-2:139 – 144.

Gordon, W.L.; Liu, W.K. and Ward, D.N. (1986): Inhibin fractionation: a comparison of human and porcine follicular fluid, with particular reference to protease activation. Biol Reprod. 35(1):209-218.

Guthrie, H.D.; Ireland, J.L.; Good, T.E. and Ireland, J.J. (1997):Expression of different molecular mass forms of inhibin in atretic and nonatretic follicles during the early luteal phase and altrenogest-synchronized follicular phase in pigs. Biol. Reprod. 56(4):870-877.

Hayes, F.J.; Hall, J.E.; Boepple, P.A. and Crowley, W. (1998):Differential Control of Gonadotropin Secretion in the Human: Endocrine Role of Inhibin J. Clin. Endocrinol. Metab., June 1, 83 (6): 1835 - 1841.

Henary, R.J.; Cannon, D.C. and Winkleman, J.W. (1968):Clinical Chemistry Principles and Techniques 2^nd Ed Harper and RowN.Y.

Henderson, K.M. and Franchimont, P. (1981):Regulation of inhibin production by bovine ovarian cells in vitro J. Reprod Fertil. Nov; 63 (2):431-42.

Hermans, W.P.; Debets, M.H.M.; Van Leuwen, E.C.M. and de Jong, F.H. (1982): Effects of single injections of bovine follicular fluid on gonadotrophin concentrations throughout the estrus cycle of the rat. J. Endocrinol 92: 425-432.

JoAnne, S. Richards, Darryl L. Russell, Scott Ochsner, Minnie Hsieh, Kari H. Doyle, Allison E. Falender, Yuet K. Lo and Chidananda, S. (2002): Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Progress in Hormone Research 57:195-220.

Kawano, Y.; Fukuda, J.; Nasu, K.; Nishida, M.; Narahara, H. and Miyakawa, I. (2004): Production of macrophage inflammatory protein- in human follicular fluid and culture granulosa cells. Fertile Steril; 82: 1206-1211.

Kishiko, S.; Takanori, N.; Koji, T.; Kaoru, M.; Yoshisha, H.; Masao, I. Kotiti, T. and Hiromu, S. (1992): Purification characterization of high molecular weight forms of inhibin from bovine follicular fluid. Endocrinology 130 (2): 789-896.

Knight, P.G. and Glister, C. (2003): Local roles of TGF-β superfamily members in the control of ovarian follicle development Animal Reproduction Science 78 165–183.

Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

LaPolt, PS.; Piquette, G.N.; Soto, D.; Sincich, C. and Hsueh, A. (1992): Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. Endocrinology 127:820-831.

Leversha, L.J.; Robertson, D.M.; deVos, F.L.; Morgan, F.J.; Hearn, M. T.; Wettenhall, R.E.H.; Findlay, K.J.; Burger, G.H. and de Kretser, M.D. (1987):Isolation of inhibin from ovine follicular fluid. J. Endocrinology 113, 113-221.

Ling, N.; Ying, S.Y.; Ueno, N.; Esch, F.; Denoroy, L. and Guillemin, R. (1985): Isolation and partial characterization of a Mr 32.000 protein with inhibin activity from porcine follicular fluid. Proc-Nat-Acad-Sci 82 (21) 7217-7221.

Mason, A.J.; Farnworth, P.G. and Sullivan, J. (1996): Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A. Mol Endocrinol, 10:1055–1065.

Massague, J. (1998):TGF signal transduction. Annu. Rev. Biochem. 67: 753-791.

McLachlan, R.I.; Robertson, D.M.; Burger, H.G. and de Kretser, D.M. (1996):The radioimmunoassay of bovine and human follicular fluid and serum inhibin. Mol Cell Endocrinol. Jul; 46(2):175-185.

Miyamoto, K.; Hasegawa, Y.; Fukuda, M.; Nomura, M.; Igarashi, M.; Kangawa, K. and Matsuo, H. (1985):Isolation of porcine follicular fluid inhibin of 32K Daltons. Biochem Biophys Res Commun. 14; 129(2): 396-403.

Moore, K.H.; Dunbar, B.S.; Bousfield, G.R. and Ward, D.N. (1994): Initial characterization of equine inhibin. Biol Reprod 51(1):63-71.

Mukherjee, A.; Janice Urban, Paolo Sassone-Corsi and Kelly, E. (1998): Gonadotropins Regulate Inducible Cyclic Adenosine 3',5'-Monophosphate Early Repressor in the Rat Ovary: Implications for Inhibin α Subunit Gene ExpressionMolecular Endocrinology 12 (6): 785-800.

Noguchi, K.; Keeping, H.S.; Winter, S.J.; Sarto, H.; Oshima, H. and Troen, P. (1987): Identification of inhibin secreted by cynomolgus monkey sertoli cell cultures J. clin Endocrinol Metab 64: 783- 788.

Ocal, P.; Aydin, S.; Cepni, I.; Idil, S.; Idil, M.; Uzun, H. and Benian, A. (2004): Follicular fluid concentrations of vascular endothelial growth factor, inhibin A and inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol Reprod Biol; 115: 194-199.

Rivier, J. and Vale, W. (1989): Immunoreactive inhibin secretion by the hypophysectomized female rat: demonstration of the modulating effect of gonadotropin-releasing hormone and estrogen through a direct ovarian site of action. Endocrinology 124195-198.

Rivier, J.; Spiess, J.; McClintock, R.; Vaughan, J. and Vale, W. (1985): Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun. 1985 Nov 27;133(1):120-127.

Robertson, D.M.; Foulds, L.M.; Leversha, L.; Morgan, F.J., Hearn, M.T.; Burger, H.G.; Wettenhall, R.E. and de Kretser, D.M. (1985): Isolation of inhibin from bovine follicular fluid. Biochem Biophys Res Commun.16;126 (1):220-226.

Robertson, D.M.; Foulds, L.M.; Prisk, M. and Hedger, M.P. (1992): Inhibin/Activin β subunit monomer: Isolation and Characterization. Endocrinology 130: 1680-167.

Robertson, D.M.; Tsonis, C.G., McLachlan, R.I.; Handelsman, D.J.; Leask, R.; Baird, D.T.; McNeilly, A.S.; Hayward, S.; Healy, D.L. and Findlay, J.K. (1988): Comparison of inhibin immunological and in vitro biological activities in human serum. Journal of Clinical Endocrinology & Metabolism, Vol 67: 438-443.

Roser, J.F.; McCue, P.M. and Hoye, Y. (1994): Inhibin activity in the mare and stallion. Domest Anim Endocrinol 11:87–100.

Schwartz, N.B. and Channing, C.P. (1977): Evidence for ovarian “inhibin” suppression of the secondary rise in serum follicle stimulating hormone levels in proestrus rats by injection of porcine follicular fluid. Proc Natl Acad Sci 74: 5721-5724.

Tanimoto, K.; Eisaku Yoshida, Shunji Mita, Yutaka Nibu, Kazuo MurakamiandAkiyoshi F. (1996): Human Activin β A Gene J Biological Chemistry. 271,( 51) 20: 32760-32769.

Tsonis, C.G.; Messinis, I.E.; Templeton, A.A.; McNeilly, A.S., and Baird, D.T. (1987):Gonadotropic stimulation of inhibin secretion by the human ovary during the follicular and earlyluteal phase of the cycle J. Clin. Endocrinol. Metab. 66: 915-921.

Turner, I.M., Saunders, P.T.K.; Shimasaki, S. and Hillier, S.G. (1989): Regulation of inhibin subunit gene expression .by FSH and estradiol in cultured rat granulosa cells. Endocrinology 125: 2790-2798.

Welschen, R.; Hermans, W.P.; Dullaart, J. and de Jong, F.H. (1977):  Effect of an inhibin- like factor present in bovine and porcine follicular fluid on gonadotrophin levels in ovarictomized rats. J Reprod. Fert. 50: 129- 131.

Woodruff, T.K.; Meunier, H.; Jones, P.B.C.; Hsueh, A.J. and Mayo, K. (1987): Rat inhibin: (Y- and P-subunit complementary deoxyribonucleic acids and expression in the ovary. Mol Endocrinol (1):561-568.

Zhang, Z.; Carson, R.S.; Herrington, A.C.; Lee, V.W.K. and Burger, H.G., (1987): Follicle stimulating hormone and somatotidin C stimulate inhibin production by rat granulosa cells in vitro. Endocrinology 120: 1633-1638.

Fig. 1: Chromatogram of the eluted protein peaks from follicular fluid of she camel on Sephacryl S-200 HR gel filtration column chromatography.

Fig. 2: represents the fractionation pattern of peak number III resolved from S-200 gel and fractionated on G-100 gel. The chromatogram shows 3 protein peaks.