Over and above these quantifiable results from international trials, a less easily
measured but equally positive outcome has emerged. A fairly large group of
developed and developing country scientists have enjoyed working together.
New methodology has been developed and standardized. Facilities have been
strengthened. Much still , however, requires to be done , and it is hoped that the
intellectual challenge ofthe problem, and its social relevance, will attract many more
clinical pharmacologists to research on fertility regulating methods.
World Health Organization (1977). Expanded programme of research in human reproduction.
World Health Organization (1979). Special Programme of Research, Development and
Research Training in Human Reproduction. Annual report.
GOSSYPOL ON MALE ANTIFERTILITY
S. Z. QIAN,r. H. HU & L. X. HO
Nanjing Institute ofMateria M ediea, Nanjing,
Teaehing Hospital, Nanjing College ofMedi cine, Nanjing,
People's Hospital ofYangzhou Distriet, Jian gsu,
In 1971, it was found in our laborat ory that the alc oho lic extract ofcotton root bar k, a
C hinese folk med icine for treat ing bron ch itis, was antitussive in mice, the active
princip le being gossypo l. Fro m th e world literature, it was learned th at powdered
cotton root bark was used in western countries as a formal em me nag ogue (Cook &
Mart in, 194 8); no toxic effects were see n when cotto nseed meal , containing
0 .11-0.20% free gossypol , had been fed 60 g dail y for 4. 5 months to human beings
(Ha rper & Smith, 1968); cottonseed meal was incorporated into a food mixture for
feeding children (Bressani , Aguifre & Scrimshaw, 1959 ); person s consuming bread
conta ining 10% cottonseed cake for on e year exhibited no ill react ion s (Bydagyan,
Vladimirev, Levitskii & Shchurov, 1947), and the maximal allowance ofgossypol in
cottonseed food preparations for human use is 450 mg kg-I food in USA and
600 mg kg-I food is recommended by intern ational groups (Berardi & Goldblatt,
1969). All the da ta indicated that the use of food preparations containing gossypol in
man is relatively safe, provided the quantity is within a certain lirnit, and in fact
gossypol itself has been employed clinically in the treatment of cancer with no
seriou s side effects reported (Er ynimov, 1966).
On the basis of the above information, and after ca rrying out aseries of toxicity
experiments and preliminary test-dosing on one of the authors, gossypol was tried
man (Liu, 1957; Hubei, Provincial Epidemie Prevention Station, 1967), certain
criteria of the reproductive system were also inspected in the fertile patients in
addition to routine examination for bronchitis. Results indicated that gossypol was
helpful in controlling the symptoms of chronic bronchitis, but also that, of the five
490 s.Z. QIAN . 1.H. HU. L. X. HO. M. X. SU N. Y. Z. HUAN G & J. H. FANG
not serious. Accordingly, gossypol was put on c1inical trial as a male antifertility
The test-subjects were 20 married volunteers, male, 25-44 years of age, fertile and
apparently healthy, with routine semen indices (count, mobility and percentage of
malformed spermatozoa) in the normal range . They were divided into two groups,
one underwent a high dose gossypol regime and the other, a low dose regime.
Gossypol was given in the above mentioned dose level (60-70 mg daily for 35-42
Routine semen examinations were done before and at different intervals after the
gossypol regime, In all the subjects, necrospermia was achieved one to three weeks
after the regime and thereafter spermatozoa gradually disappeared from the semen
(Table 1). Sperm count and mobility were nearly recovered within 16 weeks after
gossypol, but in a number ofsubjects (group 2, Table I), the recovery was only partial
in this time, however these subjects had received a small maintenance dose of
gossypol or Win. 18446 following the high dose regime . The results indicated that
gossypol had a potent antispermatogenic action in man, which was reversible in the
Table 1 Change in sperm count and mobility after high dose gossypol regime. Group 1patients
(9 patients) had received high dose gossypol only. Group 2 patients had received additionallow
dose maintenance therapy (8 patients) .
Pattern ofchanges in sperm count and mobility.
can be seen from Table 2 that there was a marked decline in sperm mobility after
only two weeks on gossypol, whilst only by the end ofthe gossypol treatment did the
sperm count begin to drop; two weeks after withdrawal of gossypol all the subjects
were necrospermic and four weeks after withdrawal, the majority became
Among the 22 subjects receiving high doses ofgossypol (including the five bronchitic
patients), six had no drug reaction, eight had slight and seven had mild to moderate
drug reactions. Another subject developed gastroenteritis-like symptoms by the end
CLINICAL TRIALOF GOSSYPOLON MALE A NTIFERTILIT Y 491
Table 2 Weekly change in sperm count and mobility during and after high dose gossypol
133,250 129,250 141,570 119,000 26,000 1,13 7 56
±74,550 ±74,040 ± 95,720 ± 55,360 ± 27,280 ± 2,287 ±107
of the gossypol regime , but shortly recovered after symptomatic treatment. The
incidence of different drug reactions was as folIows: decreased appetite (7 cases),
fatiguability (5), dr yness of mouth (5), diarrhoea (4), slight elevation of SGPT (4),
increased appetite (3), upper abdominal discomfort (3), a tendency to sleepiness (3),
dizziness (2), constipation (I), oedema of eyelid (I), testicular pain (I), seem ingly
decreased libido and potency (I), increase in urinary protein from trace to one plus
(I), and depression ofserum potassium level from 4.80 to 3.50 mmol litre-' (I). All the
drug reactions were self-limited or were reversible after cessation of gossypol. A few
requ ired symptomatic treatment. Further investigation will be necessar y to
determine whether these reactions may be entirely ascrib ed to gossypol action.
Besides, ECG , thymol turbidit y and th ymol flocculation tests were examined before
and after gossypol treatment and no significant changes were found . In some subjects,
blood haemoglobin, leucocyte count and differential count, bleed ing and clotting
time , blood pressure, and testicular size and consistency were also examined with no
In order to reduce gossypol side effects, sma ll doses were employed in eight subjects.
In three subjects, gossypol 24-35 mg daily for 51- 55 days induced a drop of sperm
count to 0-4 million ml- 1 (mobility below 15%). In another five subjects who had
received a course of high dose gossypol 10-50 da ys before, a subsequent dosing of
24 mg dail y for 15-40 days dela yed the recovery of sperm for one to three months.
Among these eight subjects, only one had slight fatiguabilit y and oedema of eyelid
(both SGPT and urine routine indices were normal). The results indicated that when
the gossypol dose was decreased and the therapy appropriatel y prolonged, side effects
could be significantly reduced with the antifertil ity effect retained, and that the use of
a small ma intenance do se, after an initial high dose , may dela y sperm reco very.
The health status of all the 25 subjects (including the five bronchitic patients) was
checked approximately one year after gossypol treatment. In all , no significant
changes were found in ECG and SGPT. In most cases , the ph ysical condition was
good , except that two had slight fatiguability and somewhat decreased appetite, and
one had merel y perceptible oedema of eyelid (urine examination of the latter case
revealing 3-6 RBClhigh power field). The significan ce of'these fmdings remains to be
Gossypol was given to 25 fertile men . At a dose of60-70 mg dail y for 35-42 days , the
drug caused a gradual increase of the percentage of immobile sperma tozoa in the
492 s.Z. QIAN,J. H. HU , L. X. HO , M. X. SUN , Y. Z. HUANG & J. H. FANG
ejaculate, followed by oligospermia, necrospermia and azoospermia in all the test
subjects. Recovery could occur around three months after its withdrawal. The side
etTects of the aforesaid dos ing were reversible and generally of mild degree, mainly
including decrease or increase in appetite, fatiguability, dryness of mouth, diarrhoea,
inconsiderable elevation of SGPT and a tendency to sleepiness; individual cases
might have slight oedema of eyelid, seemingly decreased libido and potency, and
insignificant depression of serum potassium level. Gossypol side etTects were much
reduced with its antifertility action retained when the dose was decreased to
24-35 mg daily and duration of therapy appropriately prolonged. The use of a low
maintenance dose of gossypol after a course of high dose could delay the recovery of
sperm. In all subjects, except three who had trivial complaints, the health status was
good as checked one year approximately after gossypol regime. It may be concluded
that gossypol is a quite etTective antispermatogenic agent and as to its side etTects,
although it has not been settled whether these could all be ascribed to gossypol
action, one should not ignore their signiftcance. Further toxicological studies of the
Berardi, L. C. & Goldblatt, L. A. (1969). Gossypo\. In Toxie Const ituents ofPlant Foodstuff ed.
Liener, I. E. pp. 211. New York: Academic Press.
Bressani, R., Aguifre, A. & Scrimshaw, N. S. (1959). All vegetableprotein mixture for human
feeding. J. Nutrit., 65, 351-355.
Bydagyan, F. E., Vladimirev, B. D., Levitskii, L. M. & Shchurov, K. A. (1947). Influence of
prolonged consumption of small amount of cottonseed meal on the human organism.
Cook, E. F, & Martin, E, W. (1948). In Remington 's Praetiee ofPharmaey, 9th ed., pp. 798.
Pennsylvania:Mack PublishingCo.
Erynimov, L. C. (1966). Treatment of tumor of urinary bladder with gossypol and ionol in
combination with surgery. Vop. Onko/., 2,29-35.
Harper, G. A. & Smith, K.J. (1968). Status of cottonseed protein. Eeon. Bot., 22,63-72.
Hubei Provincial Epidemie Prevention Station (1967). Collection ofWorks on the Prevention
and Treatment of 'Hanchuan Fever' and 'Xinzhou Paralysis' in Hubei. pp. 18 & 78
Liu, B.-S. (1957). A tentative idea of the use of cooking cottonseed oil for fertility contro\.
Shanghai J. Chinese Med. , 6,43-47.
Division ofBiological and Health Seiences.
Administration ofsynthe tic steroids with biological properties similar to those of the
natu rall y occurring female sex hormones is one of the world's most widely used
They have also been released from vaginal rings (Mishell & Lumkin, 1970) or
intrauterine devices (Ro we, 1977). Experimental rout es of administration include
intra-nasal spray (World Health Organ izat ion , 1979) and release from intracervical
devices (World Health Organization, 1977).
A comprehensive and critical evaluation ofthe literature on contraceptive steroids
in the space avai1able is impossible, so that onl y se1ected top ics of particular interest
The most widely used form of steroidal contraception is that given by the oral
route, ofwhich there are many different pharmaceutical formulations (Briggs, 1977;
Brotherton, 1976). These include fixed-do se and variable-dose combinations of
an oestrogen plus a progestogen, adm inistered for 20 to 24 days per 28-day cycle with
a treatment-free interval. Some use has also been made of dail y progestogen-only
'rnini-pills' which are taken continuously without an interval, especially during the
post partum period, or in women in whom oestrogens are contraindicated (Fotherby,
1977). In most countries the latter products contribute only a small percentage oforal
contraceptive use. The most widely used depot progestogen is medroxyproge sterone
acet ate (MPA) , though there is also significant use of nor ethi sterone (NET)-
Onl y two oestrogens have so far been used in commercial oral contraceptive
products: ethynyloestradiol (EE) and mestranol (MEE) (Figure 1), though some
experimental use has been made of oth er compounds such as quinestrol (QST). In
contrast, a very large range of progestogens is in commercial use. The structures of
fifteen are given in Figure 2. Three main groups of compounds can be recognized in
term s of molecular structure: 17-ethynyl-oestranes, 17-aceto xy-progesterones, and
Figure 1 Oestrogen structu res
Compoun d Form ula R1 Rl RJ R. Rs R. h.
ethynodiol diacetate A O,Ac Me Ac H H H 4
quingestanol A O,CP Me Ac H H H 3,5
norgesterone A* ° Me H H H H 5(10)
chlormadinone acetate B Cl O,Ac Hl 4,6
norethisterone enanthate A ° Me Hp H H H 4
medrox yprogesterone acetate B (I-Me O,Ac Hl 4
megestrol acetate B Me O,Ac Hl 4,6
*Th is compound is a 17-vinyl, rather than a 17-ethynyl, derivative.
Abbreviations: Me = CHl; Et =ClHs; CP = cyclopentyl; Ac =CHlCO; Hp =CHl(CHl)sCO.
Figure 2 Progestogen structures.
Sub-cellular actions of contraceptive steroids
Natural and synthetic sex hormones, including contraceptive steroids, are carried in
blood plasma partially bound to a variety of carrier proteins, including albumin,
corticosteroid-binding globulin, sex hormone-binding globulin (SHBG), and
orosomucoid (Blandford, Wittman, Stroupe & Westphal, 1978; Victor & Johansson,
1977). Binding-site concentrations vary markedly with species (Goldzieher,
Chenault, de la Pena, Dozier & Kraemer, 1978). The SHBG, for example, is the
major carrier for NET and LNG in primate blood, but is absent from plasma ofthe
Protein-bound steroids do not easily pass into cells, except by pinocytosis, but
there is a plasma pool of free steroid in dynamic equilibrium with that associated
with the various proteins. Following diffusion of free steroid through cell
membranes, most actions of sex hormones appear to be media ted by their reaction
with specific receptor-molecules of the cytoplasm. These receptors are proteins and
the interaction involves non-covalent bonding (King & Mainwaring, 1974).
Binding-affmity of these receptors is high (Westphal, 1971) and different cells
contain receptors for different hormones. Specific receptors have been demonstrated
for oestrogens, progestogens, and androgens, as weil as for corticosteroids. Many cells
possess several different receptors for more than one type of steroid hormone. The
major biological effects on target tissues of steroid hormones follow from their
interaction with these specific receptors. The receptor-steroid complex undergoes
translocation into the cell nucleus, where the complex binds to specific affmity-sites
of chromatin (sometimes called 'second receptors'). This alters messenger-RNA
synthesis and changes rates ofprotein synthesis within the target cel!. The net result is
usually increased growth and /or secretion ofthe target-tissue, though in some specific
tissues (for exarnple, hypothalamus and pituitary) sex hormones may be strongly
While several different steroid hormone receptors may be present within the same
cell, specificity is not absolute, and several different classes of hormone (for example,
progestogen and androgen) may compete for binding to one type of receptor (Jänne,
Hemminki, Isomaa, Kokko, Torkkeli, Torkkeli & Vierikko, 1978a).
There are a number of published studies on the relative affmities of natural and
contraceptive steroids for human sex hormone receptors (Briggs, 1975; Illingworth,
Wood, Flinckinger, Mikhail, 1975; Kontula, Jänne , Luukkainen & Vihko, 1973;
Shapiro, Dyer & Colas, 1978; Kasid , Buckshee, Hingorani & Laumas, 1978; Jänne,
Kontula, Vihko, Feil , Bardin, 1978b). The usual method has been to measure
displacement of a tritium-Iabelled hormone from a cytosol receptor preparation
saturated with the label in vitro. Unfortunately, due to differences in techniques and
conditions between laboratories, absolute comparisons are difficult, though some
general conclusions are possible. While the human oestrogen-receptor shows high
affmity for EE, MEE and QST do not bind significantly. Neither oestrogen binds to
the human progestogen-receptor, and neither do many norethisterone derivatives:
NET and LNG bind strongly, while there is a range of binding affmities for the
various pregnanes. Dextronorgestrel (the enantiomer with the t-configuration at
C-13) does not bind, nor do corticosteroids.
Lee, Kollman, Marsh & Wolff, 1977). High concentrations of NET or LNG inhibit
oestradiol binding to the human uterine oestrogen-receptor (Kasid et al., 1978).
Progestogens in the pregnane series bind to corticosteroid receptors (Di Sorbo,
Rosen , McPartland & Milholland, 1977).
Uniyal, Buckshee , Bhargava, Hingorani & Laumas (1977) have investigated the
binding of(±)-norgestreI (NG) to receptor proteins ofthe human endometrium and
myometrium. Experiments were conducted on receptor proteins from the
endometrial cytosol with sedimentation coefficient 4.9s and 7.8s, and on the 4.3s
receptor protein from myometrial cytosol. It was calculated that the number of
binding sites was 0.34 x 10-12 mol mg-I ofendometrial cytosol protein and 0.30 x 10-12
mol mg-Iofmyometrial cytosol protein. Maximum competition for NG binding sites
A comparison of LNG and progesterone-binding in human uterine cytosol and in
blood plasma has been published by Srivastava, Habib & Stitch (1978). High affinity
binding of LNG was observed in both materials. In the uterine cytosol progesterone
was an inhibitor ofLNG binding, but failed to compete with LNG for binding sites in
plasma. In contrast, dihydrotestosterone, testosterone and oestradiol were strong
competitors for LNG binding in human plasma. Cortisol was a strong competitor for
progesterone binding in plasma, but LNG did not compete at all. Binding proteins for
LNG in uterine cytosol and blood plasma are ditTerent. The binding protein of the
uterine cytosol is the progesterone receptor, whereas the major binding protein of
human plasma is sex hormone-binding globul in. Sex hormone-binding globulin does
not significantly bind progesterone, though the progesterone receptor naturally has
high affmity for this steroid. Progesterone competes with cortisol for sites on
Moguilewsk y & Raynaud (1979) have used uptake of tritiated R5020 to
characterise progesterone receptors in the rat hypothalamus, pituitary and uterus.
Binding by R5020 occurred in all three tissues with the same intrinsic association
constant. NG and norprogesterone competed for the binding sites, while oestradiol,
dexamethasone, aldosterone and testosterone had little etTect. It is concluded that the
progesterone receptor is a similar entity in central and peripheral target tissues.
Using tritiated R5020 , Maclusky & McEwen (1978) have investigated progesterone
receptors in the rat brain. Binding of this synthetic stero id is inhibited by excess
concentration of unlabelled progesterone or by LNG , NET or medroxyprogesterone
acetate (MPA). It is unatTected by dexamethasone, corticosterone, testosterone, or
oestradiol. Two anatomically distinct distributions of progesterone receptors in the
central nervous system ofthe rat are described . One is insensitive to oestrogen, while
the other, like progesterone receptors in non-neural tissues, is increased following
preliminary computer-assisted analysis indicated at least two types of binding sites
for progesterone. Individual Scatchard plots for cortisol and corticosterone binding
were linear with Ks's about 0.4 x 109 M-1• Inhibition analyses and modelling of
competition experiments suggest, however , that the situation is more complex. There
was no binding oflabelled dexamethasone.
Studies on the rhesus monkey uterus by Illingworth, Elsner & Oe la Cruz (1977)
have demonstrated the presence of a specific progesterone-receptor, though the
binding characteristics ditTersomewhat to that of women . This receptor on sucrose
density gradient centrifugation shows a 7.5s peak; a second peak at 4.0s showed only
slight specific binding. The receptor binds progesterone, 5a-dihydroprogesterone,
melengestrol acetate, and NG. Corticosteroids did not compete, while
5ß-dihydroprogesterone and 17a -hydroxyprogesterone showed only weak binding.
Oestrogen treatment ofcastrate animals greatly stimulated the amount ofmyometrial
receptor, but progesterone antagonised this etTect ofoestrogen .
A brief summary is given by MacLusky, Krey, Lieberburg & McEwen (1978) on
progestogen receptors in the Bonnet monkey (M'radiata) and in the rat. Progesterone
receptors in the monkey and rat were compared using radio-labelled R5020 . The
receptors were identified by Sephadex gel filtration and density gradient centri-
fugations. Examinations were conducted on cytosol fractions of the hypothalamus,
preoptic area, pituitary and uterus. In 10% glycerol the receptor sedimented at 6-7s
and had a high affinity for R5020. Binding ofthis progestogen was inhibited by excess
concentrations of progesterone, NG, NET or 5 a-dihydroprogesterone. It was
amygdala, midbrain, brain stern or cerebellum. Alterations in the distribution and
concentration of progesterone-receptors of the brain were detected in oestrogen
on brain and pituitary function may be quite different.
Taking the progesterone receptor as an exarnple, most studies are in agreement
that the binding characteristics are similar, irrespective oftissue examined. There are
however, species differences. A comparison (Briggs, 1980) of the relative affmities of
synthetic progestogens for the progesterone receptors of dog myometrium and
mammary gland suggests that the two tissues contain very similar receptors.
However, the canine receptor differs markedly from the progesterone receptor ofthe
human myometrium. Particularly striking is the relatively low affmity of NG and
NET for the receptor of dog tissues, as compared to man. This may explain the
observation (Nelson, Botta & Weikel, 1973) that NET is only a weak progestogen in
the dog but is potent in women . A comparison mayaiso be made with the guinea pig
uterine proge sterone-receptors, which show high affmity for NET (Atger, Baulieu &
Milgrorn, 1974) but low affmity, unlike both the dog and human receptors, for MPA
and CMA (Feil , Miljkovic & Bardin, 1975).
An interesting discussion on molecular conformation and protein binding affmity
for progestogens by the progesterone receptors is given by Duax, Cody, Griffin,
Rohrer & Weeks (1977). They present results on an analysis ofX-ray crystallographic
da ta concerning the molecular conformation and interactions of277 steroids. These
are a wide range ofcompounds in the oestrane, androstane and pregnane series. Ofl9
compounds observed to have equal or greater binding affinity than progesterone for
the progesterone receptor of either rabbit or human uterus, all were b,4-3-one
conjugates as measured by crystallographic torsion angles of the 0-3-C-3-C-4-C-5
configuration . It appears that a specific conformation is required for interaction with
progesterone receptors, and that intramolecular control of directionality hydrogen
Changes in binding-site concentrations undoubtedly occur following sex hormone
treatment, but are difficult to interpret. Administration of a 'pulse' ofsex hormone is
followed by migration of the steroid-receptor cornplex into the nucleus, with a
corresponding depletion ofreceptor in the cytosol. Some hormones, however, appear
to change the concentration of receptors to wh ich they do not bind. Oestrogens, for
exarnple, increase progesterone-receptor concentration while progesterone itself may
have an opposite action (Jänne, Kontula, Luukkainen & Vihko, 1975).
There appear to be marked differences between tissues in changes in receptor
concentrations following sex hormone treatment (King & Mainwaring, 1974;
but has no effect on the amount ofthese receptors in the cerebral cortex (MacLuskey
& McEwan, 1978). Strikingly different distribution patterns ofprogesterone receptors
in the brains ofrats and monkeys have been reported by MacLuskey et al. (1978).
The concentration of unoccupied progesterone receptors and oestrogen receptors
Kalra & Spellacy (1978). Progesterone receptor concentrations were measured using
tritiated R5020 while oestrogen receptors were measured with tritiated oestradiol.
Concentrations ofoestradiol receptors ranged from 0-500 fmol mg protein:' , whereas
ovulation the distribution profile ofboth receptors became very steep with more than
a tenfold difTerence in receptor levels being found between the fundus and the cervix
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