tm i n i m m mb a m ium * fo r tm ABB AX OF CBORXOHXC OOlAPOfROFSIS By Amb&al la r lla l Min Thesis auhmittsd to the Faculty of the arsdusts School of the University of Maryland In partial fu lfilliasn t of tbs requiremanta for the degree of Boo tor of fhiloaophy 1051 UMI Number: DP70257 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI Dissertation Publishing UMI DP70257 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code uest ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Wm author M i aiaoore gratltado to Dr* Cm Mm Chapman for I I I valmabla a&vlea, cFtfcteia© and auggoationa during t&* oourao of tola study* to# A* w* SloteM a for him aaaiaiooao in a ta tla tle a l evaluation of tlaa ramXta* and H as Ooorglaum S* OittiniEjer for Oar &oi& and onoouFaj^jsont • fo Ayarst, I d i m & lorriooo Ltd* Montroal* Canada* H0 ftitlop aoknowlo&gos* vltfci thank* tha parg»iy of oiiorlonie gonadotrophin nsod in l l l i study* 162823 tmm q& c o a fs s ts I'-’TROT3 JCTI0#,* * * * * * * » • * * • « • • • • * # * * • • # * • » * • • • • * * * • * « • . . # * * * 1 X • C:OB ltd O t,T@ph.% WtB * » 1*' IX , PhyaiolGCT o f fr o g TC produotlwo aysiom ****♦* 3 XXX* M oihodu o f asway*#*•**#*••«*•#**#***•####*•*•>*#♦ ♦ 0 XT* l^.X© ffe g w ih o d o f aoitoy*** •«•##»* * * •••« * » * •« ••* 331 II XPBf? 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Seasonal varl&tieft**************** *****##*•«**##* 33 S* uti 50 swd h value «•»* * * *•* * * ***** # *♦ # *#*• ** * ****** 54 ?« le e u r se y o f w t& ed s o f e s le o la t lm * ****** • * • * • • • » 33 S* /& vanta@ ss and. aeon raey o f fr o g m tteod**** • * •* * • • 33 Svl’t* ABX# ******************* *******•*♦***•****#*******»#••* 33 5lli1 - X DXB AT-'trr* * * * * * • * #*•#»*♦###**♦♦•*♦*•##*•#***#****#•*•#* 30 IITdQIWfXOlf ‘Hie Q3 0s| of chorionic gonado trephine u#od Tor a I laical purposes ooraKudLng and oap&uaive * km thod general** Xy used lor tm purpose is oaeod cm the *vaginal aoioarrr reae«* tlon using ligature fe&isle rats, as rcooiaaicnded Gy tho darmanent Commission on Biological 11 mid ar&ifcat I on of the iuisitn ^rgeni*e» i: I on of the League of Nations (IB) * ih© present investigation was -undertaken to develop an saeuruto, cheep and r&gla ivathocl of using the GalIi~»l&lninl {61} reaction on mala frogs, and to compare results of several assays op fci*e proposed male frog toohniguo with the potency as determined by ta§ rat vaginal amesr method# I. GOB ADOTROi’iU MB $ GernAotrophic hOTOOiies may be divided into two im ln groups, one of which is derived from pituitary ond. the other from living chorion!e tisane* The former includes those obtained directly from the blood and urine of normal rmn m £ women fit renc-pet**©» The s© ©end --re op of gonodo trephine o one I eta of active material of the nrine or bleed of women diaring pregnancy rod of the blood, ef pre &nant r are a C1S4} • interior fltu itergp fonadotropMng; In the $ rrly work of he^e? (4) on the effacts of hypophys act ©my In doge* atrophy of genital ormans was seen* Iftas and his - oworters (45) show** ad enlargement of ovaries and excessive Xu te Ini t, at .ion in normal rats treated with pituitary extracts, and later the pa Inveatlge* tors indicated the chemical die tine tlon of the gonadotrophic factor from ton growth promoting factor in tm extract* Urn m a t advances i n oar kno«l©&$© of th© gonadotrophins have a#on stimulated by t&e development of taalmi^uaa for hypophysestosfgr toy various- worker a using various animals# In Xfl? L • £* hx&th, (II?) aancunced that rats could to conveniently nypophyso©tomis- ed ana that this operation caused stro p s of fcha gcnade* Xbeae effects could &o reversed. by Luo implantation of rat pifcuitarles# fu el of tuo recent work is based oa this* toreat progress has boon made in lac ira c t1©nation of anterior pituitary extracts# ihe separation of too hormone& "am been achieved toy fractio n al p recip itatio n with organic solvents and inorganic salts# and toy adjustment of the ph# A detailed account of tala uork oas oooa ^ivon toy -ui and &vans (8S) • /t le a st threo gonadotrophic joori&ane* have bean isolated ircm crude p itu itary extracts# Ibey are the fo llic le stim ulating hormone CiSH) # tii© lute in i sing hormone (hi*) sometimes referred to as the interstiiiaX -celi-stim uX stoX ag hormone (iCotf) and lute© trophic hormone {prolactin# lactogenic imrrnQm) (IBij# in# iso latio n of f-h. in ©1©ctropnore tlo aliy homogenous state from sheep p it a lta r is a am b##n achieved only recently toy hi efc al# (of)# its chief function in to© female is to induce development of tine ovarian fo llic le a up to the point of ovula­ tion# ana in the mule the development of sem iniferous tubles ia i maintains**©© of apsrrtfatGgensais# if* pure i t uoco not cause iu t© in is a tlon directly# an in tact aniuala Xu. t# lula a t i on may occur eventually cat to in© lib eratio n of .hi from the anim als1 own pituitary# i t was one# believco that *“f.h cause a the release of estrogen* from the ovaries# tout there is ©via©moo that pure & preparation© do not do this im hypophyaeetomlsod rots sod that a small amount of LH mist bo pm m nt for th is o ffset to occur* Pure FSB has thus no o ffset on the uterus# the gonadotrophins in the urine in female castrates* or at the menopause or 1m the male w in e are probably mainly FBI mixed with a lit t le LH (64)* The luteinizing hoimmm (LB or XCSR) has been obtained in pure form m m single homogenous protein from pig’s pltuitsrtea by Chow et si* (§4) m d from sheep’s pltnit& ries by Li, et si* {©€)» Tim hommm® from the two sources* identical in their biological actions * are d istin ct substances which d iffer tenunologlcally end In their chemical properties such as molecu­ lar weight, isoelectric point, and amine acid composition* It stim ulates the in te r stitia l colls in the gonads of either sex of rat* In the male this causes the release of androgens with secondary effects upon the prostate and the seminal resid e* In the female the effect depends ifsry much on the presence of fo llic le s In the ovary* In young hypophyseefccsaieed female rats there are no fo llic le s and* though the effect on the In terstitia l c e lls can be detected histologically* there is lit t le or no increase In the weight of the ovary and no release of estrogens* If such rats are fir s t treated with FSB to form fo llicles* the injection of LB causes ovulation* luteIni&atien* and the re­ lease of estrogens* This is called sometimes the synergistic effect (64)* Luteotrephin (lactogenic) Hormone* Corpora lu tes formed under the action of FSB and LB do not secrete progesterone un­ t i l stimulated to do so by a third substance present in the extracts of the anterior pituitary* This substance is called 4 Xutaotrophln, but It seems to be identical with lactogenic hmmmm (A) * Human Chorionic Conadotrobhin (03 b the fir s t known naturally ©©©urrliig gon©dotrophln was that found 1b th© blood and urla© of pregnant w«wii| and originally nanaO. Prolan by Aseheist ana Zondek (3)# Its pm m w m was da tooted by ita ab ility to produce precocious maturity (eatrous* fo llicu la r dewelopmsmt* ovulation and lute ini* at Ion) in immature rodents* rats and mie© within a period of 94 hours# That i t d iffers from gonadotroph!© hormone© of anterior pituitary origin was demonstrated by Ewans ©t a l. (46) (4?)# Hlstoehamioal studies of wislockl and Bennett (13B) indicated that eytotrophobast Is responsible for Its secretion, lwiden©© from tissue culture is in agreement with other ©widen©© in showing that ©horloiiicepltbeXia* produces the hormone (63) (?8)# Chorionic gonadotrophin* or Cd is now the generally accepted appelation for the honson©# Glee a son ©t si# (Et) claim to haw© isolated a pure* crystalline* e lec trophore tic ally homogenous product, II ataman et el# (325 haw© described a simple method for the extraction of 00 from pregnancy urine* and practical In obtaining © large amount of hormn© for further biological and chemical inweatlga** tlons# Their procedure is b riefly as follows!** Urine obtained during the fir s t half of the pregnancy is ©hilled filtered* and acidified to pM 3*6 with glacial acetic acid* after filtra tio n the clear filtr a te is percolated through a m lw m containing Permit! t# Adsorption is complete when 10 litr e s of urine per hour are passed through a column hawing a 0 % G M IN* f0t I G ft m ** m %h (a m» & •S3 fIt *ft S3* t r O l i n m s u H 0 la Hm 3 f8f I t *0 & l s o m n a « £ «♦ 1 0 H *3 o # i ** $4k ft r £ ft ft i * S0? S£T ft n t S' f ft 0 e m r r I s H H t s C3s & & ft i $ t ®Q §X1 * i a m N* 0 l p* 3 N* * I ! I m & 0 » 0» e e p*?* a P » I l # la # 1 f* # 0 H* N* s f w i t * h sr I I » l *-* i b t * 1 e r a & i ti m o &1 ft n nj ft s 09 28 »i £ o f % s s a n *3 ft i d *0 ft m r $ o g * e n s ) 6 Seminal vesicles mm strongly stlmalated in the hypophgrsectemie* od male and sperms tesoa formation may occur (48)# 4 pro para** tlon contalnlixs 12*000 to 13*600 I*U* per mg* .has teen mat© (98) • i l l the four gonadotrophic bonNM i (1CSH* FSH* fms t md CG) mm prepared la different degrees of parity# All are glye#** proteins* la addition to the amino ©old constituent®* the aute at fine© a ©on tain ha acorn© and texoa amine* Destruction of e ertehy** drat# residua by a nay®# a @«us«« laaetlvatlom of the hormones# Similarly* modffieatlon of protein group© deereases the gonad©** trophic activity* thus carbohydrate as ©all as protein parts are ©SMntlal for the physiological action* Presence of pros- thetle groups in the so hormones are not Indicated* The complete composition of the gonadotrophins has not yet been revealed (&?)• ix # pHXsioioay of maim m oo m m o m cfxm sxstm ; The anatom and histology of the reproductive system of male frog Hans pinions, have teen thoroughly studied by if ugh (106)* but i t seems necessary to describe here some of the physiology of the male reproductive system in order to under** stand the mmmmr of action of the gonadotrophins on the male frog* The breeding season of the Sorth American frog (Ream pinions)* is usually from the .latter part of February to April# The maturing of the spermatosoa and the appearance of sexual in stin cts of male frog* is correlated with an increased development of the base of the Inner d ig it of the fore leg and an enlargement of certain muscles which are concerned in the clasping reflex - in