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PREVENTION OF EXPERIMENTAL ALLERGIC ASPERMATOGENESIS BY THYMECTOMY IN ADULT MICE
644
justifiable to accept the provisional conclusion that in this series of patients a difference in dietary habits has influenced the outcome of treatment. Identification of the factor or factors responsible could be of considerable practical importance, and further investigations to this end seems
are
being pursued.
I
am indebted to the Secretary General, East African Common Services Organisation, for permission to publish this communication. My thanks are also due to the headmaster and pupils of the Chopra Public Secondary School, Mwanza, without whose willing cooperation the investigation would not have been possible.
East African Institute for Medical Research,
Mwanza, Tanganyika
M.B.
DION R. BELL Leeds, M.R.C.P., D.T.M. &
H.
Medical Research Officer
metaphase and second meiotic metaphase clearly demonstrated, together with immature spermatids. Thus, there was a complete arrest of spermatogenesis at the early spermatid stage, with azoospermia in the ejaculate. After three injections of 5 mg. of H.P.F.S.H. per week the first spermatozoa were recognised in the centrifuged ejaculate in the 3rd week. During the following weeks the sperm-counts and the proportion of motile spermatozoa increased steadily; in the 1 lth week they reached 41 million per ml., or a total amount of 62 million, and in the 13th week 61 million, or a total of 72 million. The physical properties of the ejaculate were normal. 30 minutes after ejaculation there were 85% of motile spermatozoa, and after 24 hours 15%. 90% of the spermatozoa had a normal morphology.
meiotic prophase and were
DISCUSSION
TREATMENT OF INFERTILITY AFTER PARTIAL HYPOPHYSECTOMY WITH HUMAN PITUITARY GONADOTROPHINS Two partially hypophysectomised patients-a 35-yearold woman and a 38-year-old man-have been treated for infertility with human pituitary gonadotrophins (H.P.F.S.H.). Since operation the woman has been amenorrhoeic, but she conceived after the first induced ovulation, and at full term she gave birth to a normal child. The man had an azoospermia and small testes, and he was impotent; he was treated three times a week and is still under therapy. During the following weeks the number of spermatozoa increased steadily, and by the 13th week of treatment a total amount of 72 million was reached. THE FIRST CASE
The patient had had two abortions in 1957 and 1959. Later in 1959 a chromophobe adenoma was removed, leaving behind a minute amount of pituitary tissue. Postoperatively the tumour bed was irradiated with 6000r. Afterwards she had amenorrhoea and diabetes insipidus, and she was given cortisone and thyroid therapy. After about 2 years she decided to withdraw from all therapy. In 1962 ovulation was induced by daily injections of 5 mg. of H.P.F.S.H. for 10 days, followed 24 hours later by 3000 i.u. of human chorionic gonadotrophin (H.C.G.) for 3 days (5 mg. of H.P.F.S.H. =250-350 H.M.G.-24A units of follicle-stimulatinghormone (F.S.H.) activity in the ovarian augmentation assay plus 100-120 H.C.G. units of luteotrophic-hormone (L.H.) activity in the ventral prostate assay). As expected, ovulation took place on the 12th day of treatment, and she conceived immediately. During the first trimester she had some minor bleeding; otherwise the pregnancy passed uneventfully, and she felt better than she had ever done since 1959. At full term the baby was found in a transverse lie which could not be corrected; but a normal boy, weighing 3-5 kg., was delivered by caesarean section. Postoperatively the blood-pressure showed a tendency to drop, but this was easily controlled by vasopressin intravenously. She was able to breast-feed her baby for only a few days. The boy, now 8 months old, is developing normally. THE SECOND CASE
patient’s wife had a girl in 1954. In 1956, he had a pituitary cyst removed, leaving behind a minute amount of pituitary tissue. After the operation, typical symptoms of a gonadotrophic insufficiency soon developed, with azoospermia, small and soft testes, and impotence. For the first 2 years he had cortisone therapy regularly and androgens irregularly to increase libido and facilitate intercourse. The androgen therapy had no effect on the azoospermia. A testicular biopsy specimen before treatment in 1963 revealed tubules with a decreased diameter but with persistent and active germinal epithelium though the amount was somewhat decreased. No degenerative changes were found in the walls of the seminiferous tubules. The Leydig cells were reduced in number. Using a method of Book and Kjessler (to be described 1), the first The
1.
Bock, J., Kjessler, B. Cytogenetics, 1964 (in the press).
These results show that, even without gonadotrophic stimulation for more than 3 years, the human ovary can be readily reactivated within a period of 10 days to deliver a normal ovum. This ovum can be fertilised, and it can develop into a normal foetus. A normal pregnancy can then proceed without the presence of the pituitary. It also follows that a special luteotrophic hormone of the pituitary, as in certain animals, is not essential for the function of the pregnant corpus luteum. The mode of action of the gonadotrophins on the human testes has been discussed by many workers. F.S.H. acts on the seminiferous epithelium, but to maintain complete spermatogenesis both F.S.H. and L.H. are necessary.2 The details of the gonadotrophic action on the different stages of spermatogenesis have been more difficult to reveal because of inadequate cytological techniques for the study of nuclear structures in the germ-cells. Schuchardt3 and Tonutti4 claim that F.s.H. is needed for the performance of reduction division and maturation division (meiosis) during spermatogenesis. The fact that all stages of the first meiotic division and the metaphase of the second meiotic division, together with immature spermatids, could be demonstrated in a patient with longstanding gonadotrophic insufficiency, and that after the administration of H.P.F.S.H. the number of normal spermatozoa rapidly increased in the ejaculate, suggest that the meiotic divisions are autonomously performed. The gonadotrophic influence on the germinal epithelium is then mainly restricted to spermiogenesis-i.e., the maturation process of transforming early spermatids into mature
spermatozoa.
ro. --
ro_.
m_.’
CARL GEMZELL M.D.
Department of Obstetrics and Gynæcology, University of Uppsala, Sweden
Stockholm
BERNDT KJESSLER M.D. Goteborg
PREVENTION OF EXPERIMENTAL ALLERGIC ASPERMATOGENESIS BY THYMECTOMY IN ADULT MICE IF the potential danger of autoimmune conditions is due -as suggested by Burnet 5 6-to the persistence in the " body of forbidden clones " capable of reacting under certain conditions against autoantigens, there should be two possible ways of preventing a particular one: (1) by pacifying selectively the particular clone by inducing tolerance to the respective autoantigen; and (2) by removing the thymus as the presumed residence of the forbidden clones. With the model of experimental autoimmune aspermatogenesis (E.A.A.) we were able to 2. 3. 4. 5. 6.
Heller, C. G., Nelson, W. O. Recent Progr. Hormone Res. 1948, 3, 229. Schuchardt, E. Med. Welt, 1961, 21, 1123. Tonutti, E. Med. dans re Monde, 1960, 36, 10. Burnet, F. M. Brit. med. J. 1959, ii, 645, 720. Burnet, F. M. Aust. Ann. Med. 1962, 11, 79.
645 EFFECT OF THYMECTOMY ON SPERMATOGENESIS IN MICE
*Mean of both testes. t
Significantly different from
that of
positive control of similar 7
age.
WITH
E.A.A.
(Student’s t-test;
r<0’01
and 0001
respectively.)
demonstrate the effectiveness of the tolerance approach. Prevention of allergic encephalomyelitis in rats by early thymectomy aseemed to justify the second approach. We thus wondered whether even adult thymus might play a part in autoimmune conditions, and whether thymectomy even in adult life might prevent their induction or interfere with further development of established diseases. In our experiments we used male mice of the inbred strain C57Bl Sn. Three groups, immunised with isologous testicular homogenate in Freund’s adjuvant, were thymectomised at varying stages (see table). They were compared with four non-thymectomised control groupstwo immunised (negatives) and two non-immunised
medians of the experimental and control groups; the null hypothesis was tested by the Wilcoxon criterion.
(positives).
(second positive control). Another finding is a significant increase in the weight of the thymus in mice with a well-developed picture of E.A.A. (first and second negative controls).
Immunisation, quantitative evaluation of spermatogenesis, and statistical analysis were performed as described in an earlier paper." The degrees of spermatogenesis are based on the average frequency of tubules with mature sperms from both testes. The sets of data are characterised by the respective 7. 8.
9. 10.
Vojtíšková, M., Chutná, J., Rychlĩková, M., Pokorná, Z. Folia biol., Prague, 1962, 8, 207. Koprowski, R. in Wistar Institute of Anatomy and Biology, Biennial Research Report 1960-61. Arnason, B. G., Janković, B. D., Waksman, B. H., Wennersten, C. J.exp. Med. 1962, 116, 177. Pokorná, Z., Vojtíšková, M., Rychlĩková, M., Chutná, J. Folia biol., Prague, 1963, 9, 203.
New Inventions IMAGE INVERTER FOR THE ZEISS ’ TUTOR ’ EYEPIECE THE use of a microscope as an aid to surgery of small structures carries with it the disadvantage that the student or assistant is denied a chance of seeing and taking part in what is being done. The Zeiss binocular operating microscope is provided with a secondary ocular on a short tube at right angles to the surgeon’s line of vision; swivelling on that axis, the tube can be set in convenient positions for a second viewer. Though it provides a good view of the surgeon’s field, the usefulness of the eyepiece is limited because the view is as seen through a mirror-left and right reversed-hence it needs some mental gymnastics to relate the image to the object, and the assistant is virtually unable to perform some of the finer
The results show that marked protection can be afforded by thymectomy performed not only before immunisation (at age 5 weeks) but also between the two immunising doses (age 8 weeks) and even 3 weeks after them. In this third case damage to the testes at the time of thymectomy (age 12 weeks) must have been considerable (as in the first negative control): absence of the thymus obviously accelerated recovery, for 4 weeks later the damage was significantly less than in the non-thymectomised (second negative) controls, the state of spermatogenesis being similar to that in the non-immunised mice
Full details of this work together with the morphology of the thymus of mice with E.A.A. will be published elsewhere." MARTA VOJTÍŠKOVÁ M.D. Prague Institute of Experimental Biology and Genetics, ZORA POKORNÁ Na cvičišti 2, Prague, PH.D. Prague Czechoslovakia 11. Vojtíšková, M., Viklicky, J. ibid. 1964, 10, (in the press).
SPECIAL SHOWER FOR INCONTINENT AMBULANT PATIENTS INCONTINENT ambulant patients are usually cleaned in standard baths in hospitals for subnormal and geriatric cases-a slow procedure which entails thorough cleansing of the bath
manipulations. I have devised an optical train which reverts the image and be fitted into any ZeissTutor ’eyepiece. It does not interfere with the surgeon’s optical system; it does not introduce any further light loss; nor does it change the external form of the instrument. The view is exactly that seen by the surgeon and is correctly oriented so that fine manipulations are easy. can
I am indebted to Dr. Brian Stanford for improving the design and perîecting the optics of the prototype. It is manufactured by Optec Reactors Ltd., 54, Upper Montagu Street, London, W.1.
Royal Hospital, Sheffield
J. D. GRAY M.B.
Sheff.,
F.R.C.S.
Special shower bath for incontinent patients.
justifiable to accept the provisional conclusion that in this series of patients a difference in dietary habits has influenced the outcome of treatment. Identification of the factor or factors responsible could be of considerable practical importance, and further investigations to this end seems
are
being pursued.
I
am indebted to the Secretary General, East African Common Services Organisation, for permission to publish this communication. My thanks are also due to the headmaster and pupils of the Chopra Public Secondary School, Mwanza, without whose willing cooperation the investigation would not have been possible.
East African Institute for Medical Research,
Mwanza, Tanganyika
M.B.
DION R. BELL Leeds, M.R.C.P., D.T.M. &
H.
Medical Research Officer
metaphase and second meiotic metaphase clearly demonstrated, together with immature spermatids. Thus, there was a complete arrest of spermatogenesis at the early spermatid stage, with azoospermia in the ejaculate. After three injections of 5 mg. of H.P.F.S.H. per week the first spermatozoa were recognised in the centrifuged ejaculate in the 3rd week. During the following weeks the sperm-counts and the proportion of motile spermatozoa increased steadily; in the 1 lth week they reached 41 million per ml., or a total amount of 62 million, and in the 13th week 61 million, or a total of 72 million. The physical properties of the ejaculate were normal. 30 minutes after ejaculation there were 85% of motile spermatozoa, and after 24 hours 15%. 90% of the spermatozoa had a normal morphology.
meiotic prophase and were
DISCUSSION
TREATMENT OF INFERTILITY AFTER PARTIAL HYPOPHYSECTOMY WITH HUMAN PITUITARY GONADOTROPHINS Two partially hypophysectomised patients-a 35-yearold woman and a 38-year-old man-have been treated for infertility with human pituitary gonadotrophins (H.P.F.S.H.). Since operation the woman has been amenorrhoeic, but she conceived after the first induced ovulation, and at full term she gave birth to a normal child. The man had an azoospermia and small testes, and he was impotent; he was treated three times a week and is still under therapy. During the following weeks the number of spermatozoa increased steadily, and by the 13th week of treatment a total amount of 72 million was reached. THE FIRST CASE
The patient had had two abortions in 1957 and 1959. Later in 1959 a chromophobe adenoma was removed, leaving behind a minute amount of pituitary tissue. Postoperatively the tumour bed was irradiated with 6000r. Afterwards she had amenorrhoea and diabetes insipidus, and she was given cortisone and thyroid therapy. After about 2 years she decided to withdraw from all therapy. In 1962 ovulation was induced by daily injections of 5 mg. of H.P.F.S.H. for 10 days, followed 24 hours later by 3000 i.u. of human chorionic gonadotrophin (H.C.G.) for 3 days (5 mg. of H.P.F.S.H. =250-350 H.M.G.-24A units of follicle-stimulatinghormone (F.S.H.) activity in the ovarian augmentation assay plus 100-120 H.C.G. units of luteotrophic-hormone (L.H.) activity in the ventral prostate assay). As expected, ovulation took place on the 12th day of treatment, and she conceived immediately. During the first trimester she had some minor bleeding; otherwise the pregnancy passed uneventfully, and she felt better than she had ever done since 1959. At full term the baby was found in a transverse lie which could not be corrected; but a normal boy, weighing 3-5 kg., was delivered by caesarean section. Postoperatively the blood-pressure showed a tendency to drop, but this was easily controlled by vasopressin intravenously. She was able to breast-feed her baby for only a few days. The boy, now 8 months old, is developing normally. THE SECOND CASE
patient’s wife had a girl in 1954. In 1956, he had a pituitary cyst removed, leaving behind a minute amount of pituitary tissue. After the operation, typical symptoms of a gonadotrophic insufficiency soon developed, with azoospermia, small and soft testes, and impotence. For the first 2 years he had cortisone therapy regularly and androgens irregularly to increase libido and facilitate intercourse. The androgen therapy had no effect on the azoospermia. A testicular biopsy specimen before treatment in 1963 revealed tubules with a decreased diameter but with persistent and active germinal epithelium though the amount was somewhat decreased. No degenerative changes were found in the walls of the seminiferous tubules. The Leydig cells were reduced in number. Using a method of Book and Kjessler (to be described 1), the first The
1.
Bock, J., Kjessler, B. Cytogenetics, 1964 (in the press).
These results show that, even without gonadotrophic stimulation for more than 3 years, the human ovary can be readily reactivated within a period of 10 days to deliver a normal ovum. This ovum can be fertilised, and it can develop into a normal foetus. A normal pregnancy can then proceed without the presence of the pituitary. It also follows that a special luteotrophic hormone of the pituitary, as in certain animals, is not essential for the function of the pregnant corpus luteum. The mode of action of the gonadotrophins on the human testes has been discussed by many workers. F.S.H. acts on the seminiferous epithelium, but to maintain complete spermatogenesis both F.S.H. and L.H. are necessary.2 The details of the gonadotrophic action on the different stages of spermatogenesis have been more difficult to reveal because of inadequate cytological techniques for the study of nuclear structures in the germ-cells. Schuchardt3 and Tonutti4 claim that F.s.H. is needed for the performance of reduction division and maturation division (meiosis) during spermatogenesis. The fact that all stages of the first meiotic division and the metaphase of the second meiotic division, together with immature spermatids, could be demonstrated in a patient with longstanding gonadotrophic insufficiency, and that after the administration of H.P.F.S.H. the number of normal spermatozoa rapidly increased in the ejaculate, suggest that the meiotic divisions are autonomously performed. The gonadotrophic influence on the germinal epithelium is then mainly restricted to spermiogenesis-i.e., the maturation process of transforming early spermatids into mature
spermatozoa.
ro. --
ro_.
m_.’
CARL GEMZELL M.D.
Department of Obstetrics and Gynæcology, University of Uppsala, Sweden
Stockholm
BERNDT KJESSLER M.D. Goteborg
PREVENTION OF EXPERIMENTAL ALLERGIC ASPERMATOGENESIS BY THYMECTOMY IN ADULT MICE IF the potential danger of autoimmune conditions is due -as suggested by Burnet 5 6-to the persistence in the " body of forbidden clones " capable of reacting under certain conditions against autoantigens, there should be two possible ways of preventing a particular one: (1) by pacifying selectively the particular clone by inducing tolerance to the respective autoantigen; and (2) by removing the thymus as the presumed residence of the forbidden clones. With the model of experimental autoimmune aspermatogenesis (E.A.A.) we were able to 2. 3. 4. 5. 6.
Heller, C. G., Nelson, W. O. Recent Progr. Hormone Res. 1948, 3, 229. Schuchardt, E. Med. Welt, 1961, 21, 1123. Tonutti, E. Med. dans re Monde, 1960, 36, 10. Burnet, F. M. Brit. med. J. 1959, ii, 645, 720. Burnet, F. M. Aust. Ann. Med. 1962, 11, 79.
645 EFFECT OF THYMECTOMY ON SPERMATOGENESIS IN MICE
*Mean of both testes. t
Significantly different from
that of
positive control of similar 7
age.
WITH
E.A.A.
(Student’s t-test;
r<0’01
and 0001
respectively.)
demonstrate the effectiveness of the tolerance approach. Prevention of allergic encephalomyelitis in rats by early thymectomy aseemed to justify the second approach. We thus wondered whether even adult thymus might play a part in autoimmune conditions, and whether thymectomy even in adult life might prevent their induction or interfere with further development of established diseases. In our experiments we used male mice of the inbred strain C57Bl Sn. Three groups, immunised with isologous testicular homogenate in Freund’s adjuvant, were thymectomised at varying stages (see table). They were compared with four non-thymectomised control groupstwo immunised (negatives) and two non-immunised
medians of the experimental and control groups; the null hypothesis was tested by the Wilcoxon criterion.
(positives).
(second positive control). Another finding is a significant increase in the weight of the thymus in mice with a well-developed picture of E.A.A. (first and second negative controls).
Immunisation, quantitative evaluation of spermatogenesis, and statistical analysis were performed as described in an earlier paper." The degrees of spermatogenesis are based on the average frequency of tubules with mature sperms from both testes. The sets of data are characterised by the respective 7. 8.
9. 10.
Vojtíšková, M., Chutná, J., Rychlĩková, M., Pokorná, Z. Folia biol., Prague, 1962, 8, 207. Koprowski, R. in Wistar Institute of Anatomy and Biology, Biennial Research Report 1960-61. Arnason, B. G., Janković, B. D., Waksman, B. H., Wennersten, C. J.exp. Med. 1962, 116, 177. Pokorná, Z., Vojtíšková, M., Rychlĩková, M., Chutná, J. Folia biol., Prague, 1963, 9, 203.
New Inventions IMAGE INVERTER FOR THE ZEISS ’ TUTOR ’ EYEPIECE THE use of a microscope as an aid to surgery of small structures carries with it the disadvantage that the student or assistant is denied a chance of seeing and taking part in what is being done. The Zeiss binocular operating microscope is provided with a secondary ocular on a short tube at right angles to the surgeon’s line of vision; swivelling on that axis, the tube can be set in convenient positions for a second viewer. Though it provides a good view of the surgeon’s field, the usefulness of the eyepiece is limited because the view is as seen through a mirror-left and right reversed-hence it needs some mental gymnastics to relate the image to the object, and the assistant is virtually unable to perform some of the finer
The results show that marked protection can be afforded by thymectomy performed not only before immunisation (at age 5 weeks) but also between the two immunising doses (age 8 weeks) and even 3 weeks after them. In this third case damage to the testes at the time of thymectomy (age 12 weeks) must have been considerable (as in the first negative control): absence of the thymus obviously accelerated recovery, for 4 weeks later the damage was significantly less than in the non-thymectomised (second negative) controls, the state of spermatogenesis being similar to that in the non-immunised mice
Full details of this work together with the morphology of the thymus of mice with E.A.A. will be published elsewhere." MARTA VOJTÍŠKOVÁ M.D. Prague Institute of Experimental Biology and Genetics, ZORA POKORNÁ Na cvičišti 2, Prague, PH.D. Prague Czechoslovakia 11. Vojtíšková, M., Viklicky, J. ibid. 1964, 10, (in the press).
SPECIAL SHOWER FOR INCONTINENT AMBULANT PATIENTS INCONTINENT ambulant patients are usually cleaned in standard baths in hospitals for subnormal and geriatric cases-a slow procedure which entails thorough cleansing of the bath
manipulations. I have devised an optical train which reverts the image and be fitted into any ZeissTutor ’eyepiece. It does not interfere with the surgeon’s optical system; it does not introduce any further light loss; nor does it change the external form of the instrument. The view is exactly that seen by the surgeon and is correctly oriented so that fine manipulations are easy. can
I am indebted to Dr. Brian Stanford for improving the design and perîecting the optics of the prototype. It is manufactured by Optec Reactors Ltd., 54, Upper Montagu Street, London, W.1.
Royal Hospital, Sheffield
J. D. GRAY M.B.
Sheff.,
F.R.C.S.
Special shower bath for incontinent patients.