Table Of Contenttm 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
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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
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‘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
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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
