THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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SECTION IV: OBJTCTIVES OF THE PRESENT INVESTIGATIONS They are: 90 The objectives of the present studíes are several fold. l. To establish a method for the purificaffon of ovine placental lactogen (oPL) from ovine placentaì cotyledons in sufficient purity and guantity for chemical and bio'logical studjes. 2. To search for and characterize the receptor sites for oPL in ovine tissues in order to 'locate poss.lbìe sites of action for opL in the sheep during pregnancy. 3. To develop a sensitive radi olmmunoas say for opL that can measure oPL specffically and quantitatively. sheep. 4. To study the secretion of ovine placentat lactogen in the 5. To demonstrate the biosynthesis of opl. 6. To study the bioìogicaì effects of opl in rats.
9t SECTION IV¡ MtTttODs AND IIATERIALS Assay for monitorinq hormoñal iictivltú of opl- Radi oreceptorass ay for growth hormone-l ike activity(RRA-GH). A r adi oreceptoras say for measuring growth hormone or growth hormone-like activity utillzing rabbit liver receptors as described by Tsushlma et al (.t973) wlth sìlght modiflcations was employed to detect the honnonal activlty of ovine placental lactogen during puri fi catlon. Method of isolation of specific receptors: Rabbit liver obtained from mid or late pregnant rabbits was cut into small fragments before being hornogenlied in.S volumes of 0.3 M sucrose solution. Homogenization was carried out at 4 C using Polytron Pt-ìO (Srinkmann) for one minute with the dia.l set at maximum. The homogenate was filtered twice, first with 4 layers and then I layers of cheesec'loth. The filtrate was centrifuged at 7g0 x.g for 20 mln at 4 C, the supernatant was centrlfuged at.15,000 x g for 20 min, and the peìlet was discarded. The supernatant was agaìn centrifuged at .100'000 x g for 45 min to obtain the mlcrosomaì pelìet which contaíns most of the broken cell membrane. 0f the total blnding activity, 70-80 % was recovered in this fraction. The mlcrosomal pellet was suspended in 0.025M Tris-HCl , pH 7.6 containing l0 ml'l MgClr; and kept frozen at -20 C. When requlred for assay the froren receptor was thawed, homogenÌzed in a glass hornogenizer rith an
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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
Page 61 and 62: _39_ stimulating activityf is found
Page 63 and 64: -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: - 89- luteolysis when supesistpessd
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 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
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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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