Purification of Heparin Binding Oviduct Specific Proteins and Its Effect on in Vitro Embryo Development in Cattle

Indian Journal of Experimental Biology Vol. 51, May 2013, pp. 347-351

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

Aditya K Sharma1, Sushil K Mohapatra2, A K Mohanty2 & S K Das1* 1Dairy Biotechnology Laboratory, Eastern Regional Station, National Dairy Research Institute, Kalyani 741 235, India 2Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132 001, 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% CO2 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 inhibitors, growth factors, cytokines, enzymes and physiological and biochemical changes occur in immunoglobulins have also been identified in the mammalian oviduct which helps to optimize the oviductal microenvironment. Goat5 and cattle6 oviduct microenvironment for final maturation of gametes, specific proteins have been reported to increase fertilization, early embryonic development and cleavage rate and blastocyst formation rate. Moreover, transport of embryos down the tract to uterus. These the secretions of equine oviductal epithelial cells have events are directly dependent on the oviduct and its been reported to increase in vitro fertilization rate7. secretions which provide a variety of secretory Despite the potential importance of oviductal fluid molecules, including mixture of glycoproteins, secreted proteins in reproductive processes, there are limited from non-ciliated epithelial cells of oviduct during the data on fractionated oviductal fluid proteins and their postovulatory phase of the estrous cycle under the effects on in vitro embryo development. Most of the control of ovarian steroids1,2. These proteins associate molecules secreted into the oviductal lumen are protein with the zona pellucida, perivitelline space and vitelline in nature and they can be fractionated based on their or blastomere membrane of ovulated eggs and binding property with different ion exchangers. The preimplantation embryos3. Oviduct specific proteins present communication reports purification of oviduct enhance sperm binding and oocyte penetration, and specific proteins from cattle oviduct and their may regulate development in early preimplantation biological effect on in vitro early embryonic embryos4,5. Other regulatory molecules, protease development.

______Materials and Methods *Correspondent author Molecular biology and cell culture grade chemicals Telephone: +91 33 2582 8264 (O), Cell: +91 9433361567 Fax: +91 33 2582 8264, and reagents used were from Sigma Chemicals Co. E-mail: [email protected] (St. Louis, MO, USA) unless otherwise mentioned. 348 INDIAN J EXP BIOL, MAY 2013

All plasticwares used were from Tarson Products Pvt. buffered saline (DPBS) + 0.3 % bovine serum albumin Ltd. (Kolkata, India). The disposable syringe filters (BSA) + 50 µg/mL Gentamycin) using sterile 19 gauge (0.22 µm) from Axiva Sichem Pvt. Ltd. (Delhi, India) hypodermic needle. The COCs (n=10 - 12) having were used. more than 3 layers of cumulus cells with homogeneous Collection of oviducts and isolation of native ooplasm were washed 4-6 times in washing medium oviduct specific proteins (OSP)Fresh cow oviducts (HEPES modified TCM 199 + 10% fetal bovine serum were collected from local abattoir and transported to (FBS) + 0.8 mM sodium pyruvate + 50 µg/ml the laboratory within 4 h in normal saline containing gentamycin) and once in maturation medium (HEPES 1 mM phenylmethyl sulfonyl fluoride (PMSF) in ice- modified TCM 199 + 10% FBS + 0.8 mM sodium pack. After removing fascia and other tissues, pyruvate + 50 µg/mL gentamycin + 5 µg/mL FSH-P + oviducts were cut into small pieces and kept in normal 0.1 µg/mL L-glutamine + 5% follicular fluid + saline containing 1 mM PMSF and stored at 4 °C. For collected different fractions (TP, HBP and HUBP) of isolation and purification of native oviduct specific oviduct specific proteins (OSP) in three different proteins, oviducts were subjected to freezing (-80 °C) concentrations (0, 5 and 20 µg/mL). The treated and thawing (37 °C) repeatedly for 4-5 times followed oocytes were placed in 100 µL droplet of maturation by centrifuged at 16,000 rpm for 30 min at 4 °C. The medium covered with mineral oil and incubated at supernatant was collected for purification of different 38.5 °C in 5% CO2 in air with maximum humidity for 24 h. protein fractions. Sperm processing and in vitro fertilization and

Purification of oviduct specific proteinsThe culture of embryosThe spermatozoa used for in vitro collected supernatant was precipitated by adding 60% fertilization (IVF) throughout the study were from the ammonium sulfate with continuous stirring for same batch of one bull. The sperms were prepared for 30 min. The sediment was centrifuged at 16,000 rpm IVF as described9,10. Briefly, three straws of frozen- for 30 min at 4 °C. The precipitate was dissolved in thawed cattle semen were suspended in 8 mL quenched 10 mM phosphate buffer containing 1 mM PMSF, pH Brackett Oliphant (BO) medium11 in 15 mL centrifuge 7.0 and dialyzed overnight at 4 °C using 10 kDa cut tube and incubated at 38.5 °C. After 20 min of off dialysis membrane (Sigma, USA) in same buffer, incubation progressively motile sperm were collected and any remaining precipitate was removed by by taking 5 mL BO medium from the top and centrifugation at 20,000 rpm for 1 h at 4 °C. The centrifuged at 1800 rpm for 5 min. The pellet was supernatant (TP) was then filtered through 0.22 µm dissolved in 2 mL of fertilization-BO (Fert-BO) membrane and stored at 4 °C. The sample was then medium and centrifuged at 1800 rpm for 5 min. Finally passed through a heparin prepacked column (GE the pellet was dissolved in 200 µL of Fert-BO medium. Healthcare, USA) prequilibrated with 10 mM Motile sperm cells (2-4 × 106) were co-incubated with phosphate buffer containing 1 mM EDTA using Acta in vitro matured oocytes (n=10 - 12) droplets in Fert-BO explorer protein purification system (GE Healthcare, supplemented with collected OSP (TP, HBP and HUBP) USA). The collected fractions and TP were run on the in three different concentrations (0, 5 and 20 µg/mL) for

SDS-PAGE using 12% polyacrylamide gel and 16-18 h at 38.5 °C in 5% CO2 in air. The presumptive stained with Coomassie Brilliant Blue R-250 to check embryos were washed in TCM-199 supplemented with the presence of oviduct specific proteins based on 10% fetal bovine serum (FBS) and transferred to their molecular size. The concentration of protein was embryo development medium (EDM: TCM-199 + 30 8 determined by Lowry’s method . µg/mL sodium pyruvate + 100 µg/mL L-glutamine +

Collection of cattle oocytes and in vitro 50 µg/mL gentamycin + 10 µL/mL essential amino maturationThe ovaries were collected from local acids + 5 µL/mL non-essential amino acids + 3% BSA) abattoir and transported to the laboratory within 4 h in supplemented with collected OSP (TP, HBP and a thermo flask containing saline (30-35 °C) HUBP) in three different concentrations (0, 5 and supplemented with penicillin (50 IU/mL) and 20 µg/mL). The cleavage rate was observed after 40-42 streptomycin (50 µg/mL). Cumulus oocyte complexes h and embryos were cultured in replacement media (COCs) were collected from 3-5 mm diameter follicles (EDM + 10% FCS) supplemented with additional in aspiration medium (Tissue culture media (TCM)- glucose and OSP (0, 5 and 20 µg/mL) for further 199 (4-(2-hydroxyethyl)-1-piperazineethane sulfonic growth of embryos. The medium (60-70%) was acid (HEPES) modified) + Dulbecco’s phosphate replaced every alternate day. SHARMA et al: HEPARIN BINDING OVIDUCT SPECIFIC PROTEIN AND EMBRYO DEVELOPMENT 349

Experimental design and statistical analysis µg/mL concentration of TP fraction was used as a In vitro culture media were supplemented with three media supplement (Table 1). The highest blastocyst different concentrations (0, 5 and 20 µg/mL) of TP, formation occurred (26.47±1.47%) also in total protein HBP and HUBP. The control group was not (TP) fraction when 5 µg/mL concentration of OSP was supplemented with oviduct specific proteins. The data used and the lowest blastocyst rate (3.60±1.80%) was 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 Fig. 2A: Early stages of cattle embryos produced in vitro with in vitro fertilization and the highest cleavage rate the supplementation of cOSP in media; B: blastocyst stage of (73.40±2.36%) was observed in total protein (TP) cattle embryos produced in vitro with the supplementation of fraction at 5 µg/mL concentration level. The lowest cOSP in media cleavage rate (27.63±1.89%) was obtained when 20 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 0 5 20 HBP a 58.21±1.35a 73.07±2.82b 30.39±5.16c b 8.21±1.35a 19.40±2.01bAB 3.60±1.80a

HUBP a 58.88±4.84a 68.58±3.90a 33.11±4.06b b 11.66±2.54a 10.68±2.99aB 7.67±1.42a

TP a 60.45±2.66a 73.40±2.36b 27.63±1.89c b 11.32±0.21a 26.47±1.47bA 4.16±2.08c 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 < 0.05. b) Labeled means within concentrations of a particular protein fractions in a row with superscripts (a,b,c) without a common letter differ, P < 0.05. Labeled means within Fig. 1SDS-PAGE profile of purified cOSP. Lanes A, B and C different types of protein fractions of a particular concentration in a represent total protein (TP), heparin unbound OSP (HUBP) and column with superscripts (A, B) without a common letter differ, heparin bound OSP (HBP) respectively. P < 0.05. 350 INDIAN J EXP BIOL, MAY 2013

achieved when 20 µg/mL HBP fractions was used as used at a concentration of 5 µg/mL, the increased rate a media supplement (Table 1). No significant of cleavage in TP and HBP as compared to HUBP difference in the rate of cleavage and blastocyst was observed (73.40±2.36%and 73.07±2.82% formation in both TP and HBP suggests that overall respectively as compared to 68.58±3.90% in HUBP- effect on cleavage and blastocyst formation is Table 1). Regarding blastocyst formation rate among primarily imparted by HBP. The highest cleavage rate TP, HBP and HUBP, there was increase rate in TP in the control group was 60.45±2.66% in TP fraction (26.47±1.47%) as compared to HBP and HUBP and blastocyst formation was 11.66±2.54% in HUBP (19.40±2.01% and 10.68±2.99% respectively, Table 1). fraction which was not significantly differ (P<0.05) It is clear from the observation that a concentration from HBP fraction (Table 1). of 5 µg/mL OSP in media is a concentration for in vitro embryo development. The increase of Discussion cleavage and blastocyst formation may be due to Results of the present study showed that addition of presence of proteins which promote both cleavage and TP as a culture media supplement increased the rate blastocyst formation. The cleavage and blastocyst rate of cleavage and blastocyst formation in some tended to decrease gradually as the concentrations of fractions at specific concentrations. At the OSP increased above 5 µg/mL which may be due to concentration of 5 µg/mL comparatively pronounced inhibitory effect of higher concentration. Question effect was observed. The highest cleavage rate was in arises as to why similar observation without any TP fraction at 5 µg/mL concentration level, though it significant variation was observed in TP and HBP, was not significantly different from HBP fraction while the rate of cleavage and blastocyst formation (P<0.05). An increased cleavage rate (73.40±2.36 %) was less in HUBP fraction. This observation indicates obtained in the treatment group was higher than the that HBPs primarily play a major role in fertilization average cleavage rate (60.45±2.66 %) achieved in the and at the same time HUBP may have either no role control group. These results are in agreement with 12 or an inhibitory role in fertilization. that of Martus et al. in bovine where presence of In conclusion, total oviduct specific protein (TP) and bovine oviduct glycoprotein (OGP) during 16-18 h heparin-binding protein (HBP) fractions increased the incubation period significantly (P < 0.05) increased cleavage rate as compared to HUBP fraction, and TP fertilization rates as compared to control group (62.00 alone increased blastocyst formation rate as compared vs 31.20%). An increased blastocyst formation to HBP & HUBP fractions at a concentration of (26.47±1.47%) at 5 µg/mL concentration was 5 µg/mL. 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