Purification of heparin binding oviduct specific proteins and its effect on in vitro embryo development in cattle

Transcription

1 Indian Journal of Experimental Biology Vol. 51, May 2013, pp Purification of heparin binding oviduct specific proteins and its effect on in vitro embryo development in cattle Aditya K Sharma 1, Sushil K Mohapatra 2, A K Mohanty 2 & S K Das 1* 1 Dairy Biotechnology Laboratory, Eastern Regional Station, National Dairy Research Institute, Kalyani , India 2 Animal Biotechnology Centre, National Dairy Research Institute, Karnal , India Received 7 September 2012; revised 27 December 2012 The objective of this study was to see the effect of purified heparin binding oviduct specific proteins (OSP) as media supplement on in vitro embryo developmental competence in cattle. The oviduct specific proteins were isolated from abattoir cattle oviducts and precipitated, dialyzed and at the end purified by high performance liquid chromatography system. The SDS- PAGE profile of eluted heparin binding protein (HBP) fraction showed bands between ~66 - ~97 kda, while heparin unbinding protein (HUBP) fraction showed two bands at ~66 kda and in total protein (TP) bands were ~60 - ~95 kda. Collected all three OSP fractions were used as a media supplement in three different concentrations (0, 5 and 20 µg/ml) for in vitro maturation of immature oocytes, in vitro fertilization and culture of presumptive embryos at 38.5 ºC in 5% CO 2 incubator with maximum humidity. The highest cleavage rate (73.40±2.36%) was observed at 5 µg/ml concentration level and lowest cleavage rate (27.63±1.89%) was obtained in 20 µg/ml total protein (TP) fraction. The highest blastocyst formation (26.47±1.47%) also occurred in 5 µg/ml concentration of total protein (TP) fraction and the lowest blastocyst rate (3.60±1.80%) was achieved at 20 µg/ml HBP fraction. The highest cleavage rate in the control group was 60.45±2.66% in TP fraction and blastocyst formation was 11.66±2.54% in HUBP fraction which was not significantly differ from HBP fraction. These results indicate that at 5 µg/ml of total OSP fraction (TP) and HBP used as media supplement increased the cleavage rate significantly as compared to HUBP fraction, and total OSP fraction (TP) increased blastocyst formation significantly (P<0.05) as compared to HBP & HUBP fraction. Keywords: Cattle, Embryo, In vitro fertilization, Oviduct specific proteins During folliculogenesis and estrus specific physiological and biochemical changes occur in mammalian oviduct which helps to optimize the microenvironment for final maturation of gametes, fertilization, early embryonic development and transport of embryos down the tract to uterus. These events are directly dependent on the oviduct and its secretions which provide a variety of secretory molecules, including mixture of glycoproteins, secreted from non-ciliated epithelial cells of oviduct during the postovulatory phase of the estrous cycle under the control of ovarian steroids 1,2. These proteins associate with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos 3. Oviduct specific proteins enhance sperm binding and oocyte penetration, and may regulate development in early preimplantation embryos 4,5. Other regulatory molecules, protease *Correspondent author Telephone: (O), Cell: Fax: , subratakdas1@gmail.com inhibitors, growth factors, cytokines, enzymes and immunoglobulins have also been identified in the oviductal microenvironment. Goat 5 and cattle 6 oviduct specific proteins have been reported to increase cleavage rate and blastocyst formation rate. Moreover, the secretions of equine oviductal epithelial cells have been reported to increase in vitro fertilization rate 7. Despite the potential importance of oviductal fluid proteins in reproductive processes, there are limited data on fractionated oviductal fluid proteins and their effects on in vitro embryo development. Most of the molecules secreted into the oviductal lumen are protein in nature and they can be fractionated based on their binding property with different ion exchangers. The present communication reports purification of oviduct specific proteins from cattle oviduct and their biological effect on in vitro early embryonic development. Materials and Methods Molecular biology and cell culture grade chemicals and reagents used were from Sigma Chemicals Co. (St. Louis, MO, USA) unless otherwise mentioned.

2 348 INDIAN J EXP BIOL, MAY 2013 All plasticwares used were from Tarson Products Pvt. Ltd. (Kolkata, India). The disposable syringe filters (0.22 µm) from Axiva Sichem Pvt. Ltd. (Delhi, India) were used. Collection of oviducts and isolation of native oviduct specific proteins (OSP) Fresh cow oviducts were collected from local abattoir and transported to the laboratory within 4 h in normal saline containing 1 mm phenylmethyl sulfonyl fluoride (PMSF) in icepack. After removing fascia and other tissues, oviducts were cut into small pieces and kept in normal saline containing 1 mm PMSF and stored at 4 C. For isolation and purification of native oviduct specific proteins, oviducts were subjected to freezing (-80 C) and thawing (37 C) repeatedly for 4-5 times followed by centrifuged at 16,000 rpm for 30 min at 4 C. The supernatant was collected for purification of different protein fractions. Purification of oviduct specific proteins The collected supernatant was precipitated by adding 60% ammonium sulfate with continuous stirring for 30 min. The sediment was centrifuged at 16,000 rpm for 30 min at 4 C. The precipitate was dissolved in 10 mm phosphate buffer containing 1 mm PMSF, ph 7.0 and dialyzed overnight at 4 C using 10 kda cut off dialysis membrane (Sigma, USA) in same buffer, and any remaining precipitate was removed by centrifugation at 20,000 rpm for 1 h at 4 C. The supernatant (TP) was then filtered through 0.22 µm membrane and stored at 4 C. The sample was then passed through a heparin prepacked column (GE Healthcare, USA) prequilibrated with 10 mm phosphate buffer containing 1 mm EDTA using Acta explorer protein purification system (GE Healthcare, USA). The collected fractions and TP were run on the SDS-PAGE using 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250 to check the presence of oviduct specific proteins based on their molecular size. The concentration of protein was determined by Lowry s method 8. Collection of cattle oocytes and in vitro maturation The ovaries were collected from local abattoir and transported to the laboratory within 4 h in a thermo flask containing saline (30-35 C) supplemented with penicillin (50 IU/mL) and streptomycin (50 µg/ml). Cumulus oocyte complexes (COCs) were collected from 3-5 mm diameter follicles in aspiration medium (Tissue culture media (TCM)- 199 (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) modified) + Dulbecco s phosphate buffered saline (DPBS) % bovine serum albumin (BSA) + 50 µg/ml Gentamycin) using sterile 19 gauge hypodermic needle. The COCs (n=10-12) having more than 3 layers of cumulus cells with homogeneous ooplasm were washed 4-6 times in washing medium (HEPES modified TCM % fetal bovine serum (FBS) mm sodium pyruvate + 50 µg/ml gentamycin) and once in maturation medium (HEPES modified TCM % FBS mm sodium pyruvate + 50 µg/ml gentamycin + 5 µg/ml FSH-P µg/ml L-glutamine + 5% follicular fluid + collected different fractions (TP, HBP and HUBP) of oviduct specific proteins (OSP) in three different concentrations (0, 5 and 20 µg/ml). The treated oocytes were placed in 100 µl droplet of maturation medium covered with mineral oil and incubated at 38.5 C in 5% CO 2 in air with maximum humidity for 24 h. Sperm processing and in vitro fertilization and culture of embryos The spermatozoa used for in vitro fertilization (IVF) throughout the study were from the same batch of one bull. The sperms were prepared for IVF as described 9,10. Briefly, three straws of frozenthawed cattle semen were suspended in 8 ml quenched Brackett Oliphant (BO) medium 11 in 15 ml centrifuge tube and incubated at 38.5 C. After 20 min of incubation progressively motile sperm were collected by taking 5 ml BO medium from the top and centrifuged at 1800 rpm for 5 min. The pellet was dissolved in 2 ml of fertilization-bo (Fert-BO) medium and centrifuged at 1800 rpm for 5 min. Finally the pellet was dissolved in 200 µl of Fert-BO medium. Motile sperm cells ( ) were co-incubated with in vitro matured oocytes (n=10-12) droplets in Fert-BO supplemented with collected OSP (TP, HBP and HUBP) in three different concentrations (0, 5 and 20 µg/ml) for h at 38.5 C in 5% CO 2 in air. The presumptive embryos were washed in TCM-199 supplemented with 10% fetal bovine serum (FBS) and transferred to embryo development medium (EDM: TCM µg/ml sodium pyruvate µg/ml L-glutamine + 50 µg/ml gentamycin + 10 µl/ml essential amino acids + 5 µl/ml non-essential amino acids + 3% BSA) supplemented with collected OSP (TP, HBP and HUBP) in three different concentrations (0, 5 and 20 µg/ml). The cleavage rate was observed after h and embryos were cultured in replacement media (EDM + 10% FCS) supplemented with additional glucose and OSP (0, 5 and 20 µg/ml) for further growth of embryos. The medium (60-70%) was replaced every alternate day.

3 SHARMA et al: HEPARIN BINDING OVIDUCT SPECIFIC PROTEIN AND EMBRYO DEVELOPMENT 349 Experimental design and statistical analysis In vitro culture media were supplemented with three different concentrations (0, 5 and 20 µg/ml) of TP, HBP and HUBP. The control group was not supplemented with oviduct specific proteins. The data of three independent replicates were statistically analyzed by analysis of variance (ANOVA) with a probability level P<0.05 and expressed as mean ± SE. Results Purification of oviduct specific proteins (OSP) The total protein (TP) after passing through high trap heparin agarose column fractionated into two major fractions representing heparin bound fraction (HBP) and heparin unbound fraction (HUBP). The SDS- PAGE profile of the eluted HBP fraction showed five major protein ranging from ~66 - ~97 kda and one band close to ~110 kda (Fig. 1, lane C). In HUBP fraction four bands between ~45 - ~66 kda (Fig. 1, lane B) were observed. In TP lane all the protein bands which were observed in HBP and HUBP appeared (Fig. 1, lane A). Biological effect of OSP on in vitro embryo development The effects of different OSP fractions in different concentrations on cleavage (Fig. 2A) and blastocyst (Fig. 2B) formation rate have been presented in the Table 1. In brief, total 426 COCs were used for in vitro fertilization and the highest cleavage rate (73.40±2.36%) was observed in total protein (TP) fraction at 5 µg/ml concentration level. The lowest cleavage rate (27.63±1.89%) was obtained when 20 µg/ml concentration of TP fraction was used as a media supplement (Table 1). The highest blastocyst formation occurred (26.47±1.47%) also in total protein (TP) fraction when 5 µg/ml concentration of OSP was used and the lowest blastocyst rate (3.60±1.80%) was Fig. 2 A: Early stages of cattle embryos produced in vitro with the supplementation of cosp in media; B: blastocyst stage of cattle embryos produced in vitro with the supplementation of cosp in media Table 1 Effect of different fractions of cosp on (a) cleavage rate and (b) blastocyst formation rate [Values are means±se] cosp Treatment (concentration of OSP in µg/ml) fractions HBP a 58.21±1.35 a 73.07±2.82 b 30.39±5.16 c b 8.21±1.35 a 19.40±2.01 bab 3.60±1.80 a HUBP a 58.88±4.84 a 68.58±3.90 a 33.11±4.06 b b 11.66±2.54 a 10.68±2.99 ab 7.67±1.42 a Fig. 1 SDS-PAGE profile of purified cosp. Lanes A, B and C represent total protein (TP), heparin unbound OSP (HUBP) and heparin bound OSP (HBP) respectively. TP a 60.45±2.66 a 73.40±2.36 b 27.63±1.89 c b 11.32±0.21 a 26.47±1.47 ba 4.16±2.08 c a) Labeled means within concentrations of a particular protein fractions in a row with superscripts (a,b,c) without a common letter differ significantly, P < b) Labeled means within concentrations of a particular protein fractions in a row with superscripts (a,b,c) without a common letter differ, P < Labeled means within different types of protein fractions of a particular concentration in a column with superscripts (A, B) without a common letter differ, P < 0.05.

4 350 INDIAN J EXP BIOL, MAY 2013 achieved when 20 µg/ml HBP fractions was used as a media supplement (Table 1). No significant difference in the rate of cleavage and blastocyst formation in both TP and HBP suggests that overall effect on cleavage and blastocyst formation is primarily imparted by HBP. The highest cleavage rate in the control group was 60.45±2.66% in TP fraction and blastocyst formation was 11.66±2.54% in HUBP fraction which was not significantly differ (P<0.05) from HBP fraction (Table 1). Discussion Results of the present study showed that addition of TP as a culture media supplement increased the rate of cleavage and blastocyst formation in some fractions at specific concentrations. At the concentration of 5 µg/ml comparatively pronounced effect was observed. The highest cleavage rate was in TP fraction at 5 µg/ml concentration level, though it was not significantly different from HBP fraction (P<0.05). An increased cleavage rate (73.40±2.36 %) obtained in the treatment group was higher than the average cleavage rate (60.45±2.66 %) achieved in the control group. These results are in agreement with that of Martus et al. 12 in bovine where presence of bovine oviduct glycoprotein (OGP) during h incubation period significantly (P < 0.05) increased fertilization rates as compared to control group (62.00 vs 31.20%). An increased blastocyst formation (26.47±1.47%) at 5 µg/ml concentration was comparable to the average blastocyst formation (11.32 ± 0.21%) in the control group. In a similar study a significant increase (P < 0.05) in blastocyst number was found by Kouba et al. 13 when porcine OSP (posp) was included during pre-incubation/ivf compared to the control group. However, no additional effect of posp added during IVC was detected on the number of obtained blastocysts and high concentration of posp tended to decrease the observed effect on development. Pradeep et al. 5 reported cleavage and blastocyst rate as and 24.50% respectively in goat when goat oviductin was used at 10 µg/ml as a media supplement. McCauley et al. 14 checked the effect of OSP on cleavage rate and embryo development and observed that both transient and continuous exposure of posp (10 µg/ml) significantly enhanced cleavage (57.0±3.1 and 57.7±3.3% respectively) and blastocyst formation rate (35.6±1.8 and 36.6±1.8% respectively) as compared with the control (P < 0.01). When we compared the rate of cleavage among TP, HBP and HUBP fractions used at a concentration of 5 µg/ml, the increased rate of cleavage in TP and HBP as compared to HUBP was observed (73.40±2.36%and 73.07±2.82% respectively as compared to 68.58±3.90% in HUBP- Table 1). Regarding blastocyst formation rate among TP, HBP and HUBP, there was increase rate in TP (26.47±1.47%) as compared to HBP and HUBP (19.40±2.01% and 10.68±2.99% respectively, Table 1). It is clear from the observation that a concentration of 5 µg/ml OSP in media is a concentration for in vitro embryo development. The increase of cleavage and blastocyst formation may be due to presence of proteins which promote both cleavage and blastocyst formation. The cleavage and blastocyst rate tended to decrease gradually as the concentrations of OSP increased above 5 µg/ml which may be due to inhibitory effect of higher concentration. Question arises as to why similar observation without any significant variation was observed in TP and HBP, while the rate of cleavage and blastocyst formation was less in HUBP fraction. This observation indicates that HBPs primarily play a major role in fertilization and at the same time HUBP may have either no role or an inhibitory role in fertilization. In conclusion, total oviduct specific protein (TP) and heparin-binding protein (HBP) fractions increased the cleavage rate as compared to HUBP fraction, and TP alone increased blastocyst formation rate as compared to HBP & HUBP fractions at a concentration of 5 µg/ml. Hence, total oviduct specific proteins can be used as a media supplement at a concentration of 5 µg/ml for in vitro embryo development. 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