THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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õ L c o o { c5 I ô zf o dì 't26 Flgure 4. Radi oreceptorassay for grovth hormone or grovrth r,o*on.-rir. activity using'100,000 x g fractions derlved from rabbit Hver. The sensitivity and specificity of binding of the assay is iìlustrated (from Tsushima and Friesen,l973)
127 Fractioñâtión of oPL. from crúde extrác-t by ácidificgtign Although fractìonal precìpitation by lowering the pH of the crude extract was not the most effectlve method( M.Sc. Thesfs,l975), lowering the pH of the crude extract to 6.5 with.,qlacial acetic acid resulted in precipitation of .. some mucous substances whjch Jnterfere with the subsequent anion-exchange chromatography. Uslng thÍs procedure, I couìd eliminate about I5% of the original protein content, but reta.ined about 95% of the original gi^owth hormone activity in the extract. Di ethyani noeth.yl ( DEAE ) -cel I u I ose an i on exchangr__g¡-Ig¡g!9gl9-p¡y. Beforè applyingthe acidified extract to DEAE-ion exchange chromatography, the extract was re-adjusted to a pH of 9.0 with 2N anrnonium hydroxlde. At this pH, nost of the opl was unabsorbed by the column, whereas a considerable amount of protein was absorbed. l{ith lncreasing concentrat.iors of NaCì.in the presence of 0.01 M glycine-OH, pH 9.0, Iittle addìtionaì hormone was eluted as shown in Figure 5. In practice one need not elute with higher salt concentratìon. Carboxylnrethvl (CM)-cel'luìose cation exchanqe chromatography The fractions containing opl after DEAE coìumn chi"omatography were acidified to pH 6.0, centrifuged to remove the prec.ipìtates, and appl ied to a CM-cellulose column for additÍonal purification ag shown in Figure 6. ülhen a stepwise sart gradient ( 0.05M to 0.5M NacL) was enpìoyed to elute the opl fron the coìumn, most of the opl was e'ruted by 0.2M NaCL. Only a smaì.I anount of opl was eluted with 0.5 M NaCL. The O.2M fractions were then further fractionatedon the Cqrboxymethyl -Sephadex column .
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THE UNIVERSITY OF MANITOBA STUDIES
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To My parents and wife: Mn & Mrs. K
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CONTTNT S I. REVIEW 0N PLACENTAL PR
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V. RESULTS E. STUDIES ON THT BIOSYN
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Peptide Hormones: ACTH CG FSH GH LH
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LIST OF FIGURES FIGURE PAGE I Linea
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22 Radioreceptor assay for oPRL us.
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Effect of PGF,. and lactogenic horm
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ABSTRACT STUDIES 0N 0VINE PLACENTAL
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eceptor-b i nd i ng studies show th
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-2- years have seen an increasing i
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-4- secretion of a peptide hor:none
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NHt-Aì a-Pro-Asp-Val -Gl n-Asp-Cys
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6a NH, -Ser-Lys -G'l u.Pro -Leu -Ar
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-8- 197J-; Rayford et al | !9721 .
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-10- sialic acid díd not alter the
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-L2- and Biasucci (1969) denonstrat
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_14_ addition of CAMP (Handwerger e
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-16- conponents I the.faster haying
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_18_ by hcc, as evidenced by increa
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_20- (Mason et a1 , L973i Menon ¡
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21a NH, -Vaì -G1 n-Thr-Val -Pro-Le
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-23- growth-promoting ef,fect, wher
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-25- diffusion teÊt. Josi¡novich
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-27- v¡hereas in the tissue extrac
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-29- found products of molecular we
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-3r- vÍii) S.ecreLion and metabol
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- 33 - metabolic factors may influe
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-35- Although hpI, si¡nuLates the
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-37- C. CIIHER ITUMAN PLACENTAL PEP
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_39_ stimulating activityf is found
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-4t-- (iii) Human Chorionic Cortlco
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- 43 - syncytiotrophoblast (Tatarin
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- 45 - Ib SUBPRTMATE PLACENTAT PR.O
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-47 - samples, signif,icant elevati
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-49- required for the chorignÍc ao
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B. (i) -51 - MONI(ET PLACENTAL I,AC
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- 53 - brith 13 minutes for hpl,. T
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55 Pregnarit Marê Sêrum Gonâdót
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II b. NON - pRl¡late PT,ACENTAT.
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(iv¡ Inmuhologicat Mêthogs: - 59-
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_ 6t_ 1974; Fellows et a1, 1974¡ H
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63 Ordêr Rodêhtia (Tat, nicê, ro
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Guinea Pi g: 65 Hypophysectomy of t
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-67 Orilet Perissodactyla (Horse an
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-69 activity in the N-acetyl-lactos
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(Íi) coat: -71 The goat is a spont
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- 73- When serum from a pregnant go
Page 97 and 98: -75 Gusdon et al (1920)using . haer
Page 99 and 100: 77 (c) Imnunologicaì Properties of
Page 101 and 102: -79 - SECTION II : OYINE REpROpucTI
Page 103 and 104: BI which contain' as lvell as the o
Page 105 and 106: 83 ce11s usually contaln either num
Page 107 and 108: 85 non-pregnant values did not diff
Page 109 and 110: 8rl Leaf blank to corect nr.rmberin
Page 111 and 112: - 89- luteolysis when supesistpessd
Page 113 and 114: 9t SECTION IV¡ MtTttODs AND IIATER
Page 115 and 116: - 93- (.TCA) solution were added co
Page 117 and 118: -95 - (ÞPRL' NIH-P-B3) , and all q
Page 119 and 120: 97 buffer, the eluates were pooled,
Page 121 and 122: Char4ctêf ization Pfocedures l. Ch
Page 123 and 124: t0t (c) À.nalvticaL .{rod.iu¡n do
Page 125 and 126: 103 B : MFTÍÍOD OF DETECTION AND
Page 127 and 128: AFsa)¡- ProceiiureS 105 Specific b
Page 129 and 130: 107 ', C : IIEBI{OÐ OF DEVELOPMENT
Page 131 and 132: 109 D. RTNG PREGNôXCY (l) Maternâ
Page 133 and 134: l1ì (wtlvo'). After the hornogenat
Page 135 and 136: (4) Tissúe distributiori df radloi
Page 137 and 138: l'1 5 (2) Procg.ssjnq, gf. incuÞ¡
Page 139 and 140: 1.l7 for 30 min. Then the precipita
Page 141 and 142: (2) Assav of ìuteo.trgpic effec.t
Page 143 and 144: EXPERIMENT # TI (7 days after HCG)
Page 145 and 146: 123 to progesterone (l/300 djlution
Page 147: SECTION V : RESULTS (r) LACTOGEN DU
Page 151 and 152: 129 Figure 6. carborymethyì(cM,)-c
Page 153 and 154: t 3't óo0 ,oo 800 900 FRACIION r,t
Page 155 and 156: E o ø ô¡ t- z ao É, o '133 .--.
Page 157 and 158: TABLE I TABLE OF PURIFICATION OF OV
Page 159 and 160: 137 Figure lì. A, Poìyacrylamide
Page 161 and 162: Leaf blank to correct nr.rmbering
Page 163 and 164: 14r Anal.vtical Sodi um Dodecyl Sql
Page 165 and 166: o 9 x F- -)- 9 rU I æ -t f () LT'
Page 167 and 168: ¿ o s : t45 Figure 15b. Displaceme
Page 169 and 170: HoR MoNE coNc€NrRArto ln ß/mt )
Page 171 and 172: Tissue used for binding ôtudies Li
Page 173 and 174: Noñ. p..s'ro¡r {N,p.l Pr.eñonr (
Page 175 and 176: ¡( É T 2, \ ¡a.2¡./ir ,..r.¡.
Page 177 and 178: P 80 \ x.. ¡,azrot¡j .1.¿¡i¡ .
Page 179 and 180: o c o U o òs -o c2o ao eÀo o 'o .
Page 181 and 182: F 2 o o o\ ¡¡ig ô 2 Þ 0 (¡ l5e
Page 183 and 184: ! o 't6 l HORMO¡¡ E COr{CENr FAIt
Page 185 and 186: 163 Figure 24 shows the sensltivity
Page 187 and 188: 165 D. STUDIES ON THE SECRETION OF
Page 189 and 190: E .Dc À o - art & 167 Flgure 26. P
Page 191 and 192: lJhen blood samples were taken four
Page 193 and 194: Sheep no. l{6 25í 960 464 5?0 680
Page 195 and 196: 't73 fetal cotylêdons at various d
Page 197 and 198: TABLE VI OPI CONCENTRATTON IN FETAL
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l. Súrglcat Method 't77 Figure 3l
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= >€ ,3 ; I 179 rwt 76-0450 o----
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dUrlng PrêOnancy in thè Ewes tBr
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! ñ' l83 NoRMON! C oNCENTßArlO þ
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l ol
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I I I "+ _t rl \l :- 'i rl -l l87 a
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l89 Flgure 37. Percentage ot l2sl-o
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19t column is shown in Figure 3BA.
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- 193 3H -couNdro _________SPECtFtC
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TABLE IX /- BIOASSAY OF qROIItIfH p
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z o 197 Figure 40, Radioreceptor as
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5A L lNl Pgtzq cô-tlzt Pgfl6 + cl
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SFCT.IoN II : pl.scussr0N 4d ã 1È
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Ch aracteri zati on 203 Analytical
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205 size, corresponding to the mole
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207 the specific binding of oPL to
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209 than with prolactin-like effect
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2t1 the jncrease in oPRL binding si
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213 The resuìts obtained indicate
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215 on the role af PL in the fetus.
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Surgi ca1 method- 217 Our studies o
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219 5.Relationships amonq opRL. oGH
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221 The nature of thelarge nolecula
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223 data argue for a close interact
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]. TABLE OF CHEMICAL PROPERTIES GUI
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cor,t coAT (bPL) (cpl) 3. TABLE OF
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À) E 229 Figure 43. The secretion
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231 H. POSSIBLE ROLT OF OVINE PLACE
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233 Our studies on the blnding of o
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235 through its binding to cotpus l
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237 Furthermore, oPL neasurement in
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239 circulationand alìantoic f'lul
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SECTICN IX PUBLICATIONS A) Manuscr
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SECTION X: BIBLIOGRAPHY Abramovich,
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Beas,F., Saìinas, 4., GonzalezrF.,
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247 Brody,S,, and Carlstrom,G.,J.Cl
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249 Crosignanì ,P.G. , Nencioni ,
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25t Friesen,H.G., Endocrinoìogy 76
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253 Grinwich,D.L., M. Hichens, and
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25s Hum, V.G., Knipfel , J.G., and
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?57 Knopp,R,H,, H,J. Ruder,E. Herra
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259 Manku,M.S., and D.F. Horrobin,
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261 , :iir .,.-;;r-,:, Morgan, F.J.
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263 Pierce,G.B., Midþley,A.R. and
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?65 Shepard, T.H.., Gen. Comp. Endo
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267 Tsuruhara,T., Dufau,M.L., Hickm
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