THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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Starting Materi.alF - 94- ?wo possible sourceg of raw material for ovine placental lactogen purification have been examined: (1) ovine placental- cotyledons re¡noved at the time of surgery \4rere kindly provided by Dr. Hanish A. Robertsonf Reproductive Physiology, Animal Research Institute, Agf.iculture Canada, Ottae¡a, Canada; These tissues r¡/ere immediately frozen and stored at. -20 C \^rithout separating maternal and foetal cotyledons. (2) Ovine pLacental cotyLedons (foetal and maternal) a.t mid preqnanci (60 to I20 days of góstation) obtained u,ithin I or z hours after death ìn the s'iaughter house, Auckìañd, New Zeaìand, were inrnediately stored frozen at -20 c. These tissues r,rere sent to ltinnipeg within 30 days after colìection. Arrangements for collection and shipment were coordinated by Drs. Nîcholson and Lapwood, Massey University, New Zea'l and. Hormone Preparations Human growth hormone (NIH''2019G,2.2 IU,/mg) , ovine grohrth hormone (oGH, NIH 0-986f¡, 2.b fv/ng\, monkey growth hormone (mcH, NIH M 9454, 0.9 IU/mg), bovine grolrth hormone (bGH, NIH 8-10034, 2.0 fv/Ítgr, porcine growth hormone (pGH, NIH P-5268, 1.5 IU/mg), canine growth hormone (ccH, NIH D-1004, 1.9 lulmg), rat grov¡th hormone ( rcn, NIH RP-lr 0.6 IU,/ng), human placental lactogen (hPL, NIH, 95t electrophordicaì lJ pure) ¡ ovine prolactin (oPRt, NIH-P-SFIof 26 Tv/mgl , ovine luteinizing hormone (oLH, NIH-LH-Sl8), bovíne prolactin
-95 - (ÞPRL' NIH-P-B3) , and all qther hormone preparat,ions were kindly supplied by the NIA¡4DD of Èhe NIHTUSÀ. Proteih Measurement Protein concentrations of, the fractíons coLl-ected during purification r¡rere estimated by measuring the absorbance aE 278 nm, nraking the assumption that one unit of absorbance was equivaJ-ent to protein concentratíon of I mg/ml. For more accurate determinations, the protein content of the origina3_ crude extract, and of the pools containing opL at. different stages of purification were estimated by the method of lowry (Lowry et al ,l-951) using bovine serum albunin(BSA) as standard. The final purified product was weighed after lyophiJ.izationf and its protein content. per unit weight was determined by spectrofluorometer (Aminco-Botrman), using ovine prolact.in (NIH-P-S-l0) as st,andard¡ excitation was at 278 nm and emission at 350 nm. Concentratioh of volumê by ultrâfiltrat,ion A1I concentration procedures were carried out at 4C in an Amicon Diafto celL of varíous capacity ( 10,60, 4SO, and 2,000 mI). The size of celtused. dependedon the initiat volume required to concentrate and the final volume which was desired. UM-Io membrane fiLters.were used in all concentration steps.
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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
Page 65 and 66: - 43 - syncytiotrophoblast (Tatarin
Page 67 and 68: - 45 - Ib SUBPRTMATE PLACENTAT PR.O
Page 69 and 70: -47 - samples, signif,icant elevati
Page 71 and 72: -49- required for the chorignÍc ao
Page 73 and 74: B. (i) -51 - MONI(ET PLACENTAL I,AC
Page 75 and 76: - 53 - brith 13 minutes for hpl,. T
Page 77 and 78: 55 Pregnarit Marê Sêrum Gonâdót
Page 79 and 80: II b. NON - pRl¡late PT,ACENTAT.
Page 81 and 82: (iv¡ Inmuhologicat Mêthogs: - 59-
Page 83 and 84: _ 6t_ 1974; Fellows et a1, 1974¡ H
Page 85 and 86: 63 Ordêr Rodêhtia (Tat, nicê, ro
Page 87 and 88: Guinea Pi g: 65 Hypophysectomy of t
Page 89 and 90: -67 Orilet Perissodactyla (Horse an
Page 91 and 92: -69 activity in the N-acetyl-lactos
Page 93 and 94: (Íi) coat: -71 The goat is a spont
Page 95 and 96: - 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: - 93- (.TCA) solution were added co
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 and 148: SECTION V : RESULTS (r) LACTOGEN DU
Page 149 and 150: 127 Fractioñâtión of oPL. from c
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'
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¿ o s : t45 Figure 15b. Displaceme
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HoR MoNE coNc€NrRArto ln ß/mt )
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Tissue used for binding ôtudies Li
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Noñ. p..s'ro¡r {N,p.l Pr.eñonr (
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¡( É T 2, \ ¡a.2¡./ir ,..r.¡.
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P 80 \ x.. ¡,azrot¡j .1.¿¡i¡ .
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o c o U o òs -o c2o ao eÀo o 'o .
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F 2 o o o\ ¡¡ig ô 2 Þ 0 (¡ l5e
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! o 't6 l HORMO¡¡ E COr{CENr FAIt
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163 Figure 24 shows the sensltivity
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165 D. STUDIES ON THE SECRETION OF
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E .Dc À o - art & 167 Flgure 26. P
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lJhen blood samples were taken four
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Sheep no. l{6 25í 960 464 5?0 680
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't73 fetal cotylêdons at various d
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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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