Acta physiol. scand. 1975. 93. 409-414 F r o m the Institute of Zoophysiology, University of Uppsala, Sweden
Distribution of an Adenohypophysial Constituent in the Body I. Whole-body Autoradiographical Studies in the Mouse BY
JANERIKKIHLSTROM, HAKANHALLand EEVALAKOMAA Received 18 September 1974
Abstract KIHLSTROM, J. E., H. HALLand E. LAKOMAA. Distribution of an adenohypophysial constituent in the body. I. Whole-body autoradiographical studies in the mouse. Acta physiol. scand. 1975. 93. 409-414. A peptide with a molecular weight of about 5000 has previously been shown to affect the output of semen in frogs and probably also in mammals. This sperm-releasing substance is not part of any known gonadotropic hormone. The distribution of this substance has been investigated using whole-body autoradiography. Radioactive material is incorporated into the epididymis, the adenohypophysis and probably also into the ovary.
An extract from the adenohypophysis has been shown to induce the release of spermatozoa in frogs (Kihlstrom and Danninge 1970). The extract probably does not contain any known pituitary hormone (Lakomaa 1974 a, 1974 b). This activity has been demonstrated in extracts from pituitaries from frogs (Kihlstrom and Danninge 1970), perch, rabbits, cats, cattle (Kihlstrom et al. 1971), domestic fowls and humans (Lakomaa and Kihlstrom 1972). The pituitary extract has also been shown to increase the output of semen in rabbits, guinea-pigs and mice (Lakomaa and Kihlstrom 1972). An active constituent (molecular weight approximately 5 OOO) in the extract, called sperm-releasing substance, as suggested by Lakomaa (1974 a), has been highly purified (Lakomaa 1974 b), and used in further studies (Lakomaa 1974 a). In the present work, the distribution of the sperm-releasing substance in the mouse is studied using the whole-body autoradiographical technique.
Material and Methods An acetone powder prepared from bovine adenohypophyses by Biofac A/S (Copenhagen, Denmark) was used when preparing the sperm-releasing substance according to Kihlstrom et al. (1971) and Lakomaa (1974 b), excluding the final isoelectric focusing. The sperm-releasing substance can be lyophilized and stored for a considerable time.
409
410
JAN ERIK KIHLSTROM,
HKKAN
HALL AND EEVA LAKOMAA
As the sperm-releasing substance is a peptide, probably containing at least one tyrosine-residue (Lakomaa, personal communication), it is possible to iodinate the substance with lZ5l (Radiochemical Centre, Amersham, England) according to Greenwood er a / . (1963). 20 jig of the substance in 10 pl of distilled water and 10 o r 50 pg Chloramine T in 10 pl sodium phosphate buffer p H 7.5 were exposed to I mCi Na'"I (carrier free) in about lOpl NaOH-solution (pH 8-1 I ) for 30 o r 60 s respectively. The reaction was interrupted by the addition of 24 or I20 ,ug respectively, Na,S,O, in 50 p1 0.05 M phosphate buffer, pH 7.5. The contents of the reaction vial were transferred to a Sephadex column (0.9 I5 cm. Sephadex G 25 or G 50 eluted with phosphate buffered saline containing 0.1 M EDTA and 0.002 9b NaN,). The column was presaturated by the addition of 0.1 ml 20% Bovine Serum Albumin (BSA), Armour Fraction V, Sigma, and a subsequent addition of 1 mg of a crude preparation of the sperm-releasingsubstance. The reaction vial was washed with 0.1 ml 1 % KI, and this was also transferred to the column. 10 PI of each eluate was diluted and the elution pattern was determined in a gamma counter, type Autowell 11. The sperm-releasing substance was eluted near the void volume, and the free larl near the total volume. The iodinated substance was stored at i - V C , and was used within 10 days (often within 2 days). The mice used in the experiments were albino mice ( N M R I ) of about 2 months of age, obtained from Anticimex, Sollentuna, Sweden. The autoradiographical procedure was similar to the procedure used by Ullberg (1954). About 10-30 pCi of the iodinated sperm-releasing substance in 0 . 1 4 3 ml physiological saline was injected in a tail vein. At different intervals after injection (15 s t o 10 min), the mice were anesthetized with ether for about 30 s, and rapidly frozen in a Dewar vessel containing hexane and dry ice (solid CO,) (about - 70°C). They were then immersed in a solution of carboxymethylcellulose in water, and the whole preparations were frozen in hexane -dry ice for about IS min, and then stored in a freezing-room (-20°C) for 24 h or more. The frozen blocks were sectioned in the freezing room with a microtome, type Leitz, to a thickness of 20-60 /rm and dried at the same temperature. X-ray films (Industrex C, Kodak) were then exposed to the sections for 3-8 weeks and the films were processed in Agfa Gevaert Developer G 150 and Agfa Geavert Fixer G 334.
Observations Radio-activity is found in many organs, as listed below. Lung (Fig. I): No specific pattern of the distribution is found. The activity never exceeds
the activity of the blood. Liver (Fig. I ) : The activity never exceeds the activity of blood. Kidney (Fig. I and 4 ) : The activity of the kidneys is often dominating. Most activity is
found in the cortex. Ureter: In the urine. Bladder: In the urine. Pituitary (Fig. 2): At about the same degree in both the anterior and the posterior part,
though in some cases more activity in the anterior part. Secreting cells (Fig. 2, 4 and 5 ) : Submaxillary gland, epithelial cells of the nose, mouth and small intestine, mucosal cells in the walls of the stomach, and thus also in the contents of the stomach. Thyroid (Fig. 2): Always a great deal of activity. Epididymis (Fig. 3): Especially in the caput epididymidis. The activity seems to be localized to the walls of the tubuli. Testis (Fig. 3): Very weak activity which seems also to be localized to the walls of the tubuli. Ovary (Fig. 4 ) : Follicles and/or corpora lutea. Uterus: Much weaker activity than in the ovary. Slight activity only is found in other male sexual organs (vas deferens, seminal vesicle, prostate, coagulating gland).
AUTORADIOGRAPHICAL STUDIES OF A PITUITARY PEPTIDE
lung
hean
liver
kidney
lung
heart
41 1
liver
small kidney intestine Fig. 1 a. Male injected with 3OpCi. of the iodinated sperm-releasing substance. Interval after injection 15 s, thickness 20 pm. Exposure time 24 days. Activity can be seen in the kidney, lung, liver and is very high in the blood. Fig. 1 b. Male injected with 30 pCi, of the iodinated sperm-releasing substance. Interval after injection 1 min, thickness 20 ,urn. Exposure time 24 days. Activity is found in the kidney, lung, liver, small intestine, stomach and blood. Compare the differences between activity in the blood and in the kidney in this figure with that of Fig. 1 a.
Activity is, at least to some extent, found as soon as after 15 s in all organs mentioned, except in the contents of the stomach where activity is only found after longer incubation times (Fig. 5 ) .
Discussion The activity found in the different organs is not always the result of an incorporation of the iodinated sperm-releasing substance. It is probable, that there is a great deal of free lZ6Iin the sections. This can be due to both decomposition of the iodinated substance during storage, and to a rapid decomposition in the animal during the incubation. Since the distribution is studied after such short times, a great deal of activity still remains in the circulating blood. Therefore the activity found in organs rich in blood (Everett er al. 1956) is not necessarily due to an incorporation of the sperm-releasing substance. In the following list, the probable cause of the activity found in the different organs is discussed.
hypophysis Fig. 2. Male injected with 20 pCi, of the iodinated sperm-releasing substance. Interval after injection 15 s, thickness 40 ,urn. Exposure time 14 days. Activity is found in the submaxillary gland, both parts of the hypophysis and the epitheliar cells of the nose. The activity spots in the brain refer to blood vessels.
-~
thyroid
submaxillary gland
412
JAN ERIK
KIHLSTR~M, HAKAN
corpus epididymidis
caput epididymidis
HALL AND EEVA LAKOMAA
vas deferens
testis
caput epididymidis
cauda epididymidis
testis
Fig. 3 a. Male injected with 20 pCi, of the iodinated sperm-releasing substance. Interval after injection i min. thickness 60 pm. Exposure time 14 days. High activity can he seen in the caput epididymidis. while it is much lower in the other parts of the epididyrnis. No activity is found in the testis. Fig. 3 b. Male injected with 30 pCi, of the iodinated sperm-releasing substance. Interval after injection 3 min, thickness 2 0 p n . Exposure time 24 days. Activity is found primarily in the caput epididymidis, hut also in the other parts of the epididymis and in the vas deferens. The very weak activity in the testis is located to the walls of the tubuli.
Lung: High blood content with remaining activity. Liiwr: High blood content with remaining activity, though also some metabolism of the iodinated sperm-releasing substance (see below). Kidney, ureter and bladder: Excretion of the iodinated sperm-releasing substance and/or free I z 6 I . Pituitary: Free 12al will accumulate in the neurohypophysis, but not in the adenohypophysis (Jentzer 1953). In this study, activity is found in the adenohypophysis. indicating a n incorporation of the constituent. kidney
ovary
contents of the stomach
.~~
kidney
Fig. 4. Female injected with 30 ,ftCi, of the iodinated sperm-releasing substance. Interval after injection I 5 s, thickness 2 0 p ~ 1Exposure . time 24 days. The follicles and/or the corpora lutca of the otarq are incorporated with the constituent. The pattern of the kidney is easily visible. Fig. 5. Male injected with 20 / X i , of the iodinated sperm-releasing substance. Interval after injection 10 min. thickness 60 / t m . Exposure time 7 days. Activity is found almost only in the stomach and i n the kidney.
AUTORADIOGRAPHICAL STUDIES OF A PITUITARY PEPTIDE
413
Secreting cells: Accumulation of free lZ6I(Ullberg and Ewaldsson 1964, Logothetopoulos 1956 a, 1956 b). Thyroid: Accumulation of free lZ6I. Epididymis: Neither blood contamination, nor incorporation of free lZsIseem to be the cause of the high activity found in the epididymis. The blood content of the epididymis does not seem to be higher than that of the testis, when comparing the colours of the two organs in the sections. Radioiodine is not incorporated in the epididymis (Ullberg and Ewaldsson 1964). The picture of the incorporation of the sperm-releasing substance in the epididymis and the testis is very similar to that of FSH (Rajaniemi 1971). From this similarity it can be concluded that this substance like FSH accumulates in the walls of the tubuli of the epididymis and testis. The spermreleasing substance is not identical with FSH or with a fraction of FSH (Lakomaa 1974 a). Ouury: Free lZsIaccumulates in the ovaries (Ullberg and Ewaldsson 1964), but so also does luteinizing hormone (Rajaniemi and Vanha-Perttula 1972, Lee and Ryan 1971). It is unlikely that the sperm-releasing substance is identical with LH, since LH does not accumulate in the epididymis (Rajaniemi 1971). LH, prepared in the same way as the sperm-releasing hormone, does not possess sperm-releasing activity (Lakomaa 1974 a). It cannot be determined from these autoradiograms, whether the accumulation in the ovary is due only to an incorporation of free Y. Uterus: Probably blood contamination.
The increasing amount of activity in the liver and in the kidneys, with increasing intervals after injection, is probably the result of the metabolism of the sperm-releasing substance. The increasing amount of metabolized substance will give free lZsIcirculating in the blood, leading to higher activity in the stomach after longer incubation times (Logothetopoulos 1956 a). Thus, the only organs where the activity scems to be due to an incorporation of the iodinated sperm-releasing substance, are the epididymis, adenohypophysis and possibly the ovary. The incorporation in the adenohypophysis indicates a mechanism of re-uptake, since the substance originates from this organ. The incorporation in the walls of the caput epididymidis cannot at present be explained, since the exact functions of the substance have not yet been investigated. But it is not unexpected to find accumulation in this organ, since the substance probably affects the output of semen also in mammals. A comparison between the distribution of the iodinated sperm-releasing substance and that of LH and FSH can be made. Both LH and FSH accumulate in the testis, but the uptake is not visible in the autoradiograms after such short intervals as have been used in this study with the sperm-releasing substance (Rajaniemi 1971). LH does not incorporate into the epididymis, but FSH on the other hand does (Rajaniemi 1971). Thus the spermreleasing substance has a distribution pattern in the male sexual organs which differs from that of LH, but it does not in this respect differ from that of FSH. This work has been supported by grants from the Helge Axson Johnson Foundation, and from the Swedish Natural Science Research Council to Jan Erik Kihlstrom.
414
JAN ERIK KIHLSTROM, HAKAN HALL AND EEVA LAKOMAA
References BENGTSSON. G., B. EWALDSSON, E. HANSSON and S. ULLBERG, Distribution and fate of lsll in the mammalian ovary. Acra endocr. (Kbh.) 1963. 42. 122-128. N. B., B. SIMMONS and E. P. LASHER,Distribution of blood (Feso)and plasma (PI)volumes of EVERETT, rats determined by liquid nitrogen freezing. Circular. Res. 1956. 4. 419-424. GREENWOOD, F. C., W. M. HUNTERand J. S. GLOVER,The preparation of 1311-labelled Human Growth Hormone of high specific radioactivity. Eiochem. J . 1963. 89. 114-123. JENTZER, A., Relation between the hypophysis and the thyroid gland. Acta endocr. (Kbh.) 1953. 12.264-270. KIHLSTR~M, J. E. and I. DANNINGE, Release of sperm cells in the frog, Rana esculenta L., induced by a pituitary extract, probably without gonadotropic activity. Gen. conrp. Endocr. 1970. 14. 592-593. K I H L S T R ~J. M ,E., E. LAKOMAA and H. HALL,A probably nongonadotropic sperm-releasing activity in the pituitary gland from mammals, amphibians and fishes. Gen. comp. Endocr. 1971. 17. 573-575. LAKOMAA, E., Some properties of a hitherto unknown adenohypophysial substance having sperm-releasing activity. Acra Uniu. upsalien. 1974 a. No. 316. LAKOMAA, E., Purification of a bovine adenohypophysial constituent; which induces the spermiation in the frog, Rana esculenta. 1974 b. In manuscript. M , adenohypophysial extract influencing the output of semen in LAKOMAA, E. and J. E. K I H L S T R ~An rabbits, guineapigs and mice. J. Reprod. Ferril. 1972. 31. 469-472. LEE,C. Y.and R. J. RYAN,The uptake of Human Luteinizing Hormone by slices of luteinized rat ovaries. Endocrinology 197 I . 89. 15 15-1 523. J. H. and N. B. MYANT,Concentration of radioiodide and Ss6-labelled thiocyanate by LOGOTHETOPOULOS, the stomach of the hamster. J. Physiol. (Lond.) 1956 a. 133. 213-219. LOGOTHETOPOULOS, J. H. and N. B. MYANT,Concentration of radioiodide and SS6-labelledthiocyanate by the salivary glands. J. Physiol. (Lond.) 1956 b. 134. 189-194. RAJANIEMI, H., Aivolisikkeen gonadotrofisten hormonien jakautuminen koe-elaimessi ja sitoutuminen kohdekudoksin. Doct. diss. Turku 1971. Specific receptor for LH in the ovary. Evidence by autoradioRAJANIEMI, H. and T. VANHA-PERTTULA, graphy and tissue fractionation. Endocrinology 1972. 90. 1-9. S.,Studies o n the distribution and fate of Ss6-labelled benzylpenicillin in the body. A m radiol. ULLBERG, (Stockh.) 1954. Suppl. 118. ULLBERG,S. and B. EWALDSSON, Distribution of radioiodine studied by whole-body autoradiography. Acta radiol. (Stockh.) 1964. 2. 24-32.
Distribution of an Adenohypophysial Constituent in the Body I. Whole-body Autoradiographical Studies in the Mouse BY
JANERIKKIHLSTROM, HAKANHALLand EEVALAKOMAA Received 18 September 1974
Abstract KIHLSTROM, J. E., H. HALLand E. LAKOMAA. Distribution of an adenohypophysial constituent in the body. I. Whole-body autoradiographical studies in the mouse. Acta physiol. scand. 1975. 93. 409-414. A peptide with a molecular weight of about 5000 has previously been shown to affect the output of semen in frogs and probably also in mammals. This sperm-releasing substance is not part of any known gonadotropic hormone. The distribution of this substance has been investigated using whole-body autoradiography. Radioactive material is incorporated into the epididymis, the adenohypophysis and probably also into the ovary.
An extract from the adenohypophysis has been shown to induce the release of spermatozoa in frogs (Kihlstrom and Danninge 1970). The extract probably does not contain any known pituitary hormone (Lakomaa 1974 a, 1974 b). This activity has been demonstrated in extracts from pituitaries from frogs (Kihlstrom and Danninge 1970), perch, rabbits, cats, cattle (Kihlstrom et al. 1971), domestic fowls and humans (Lakomaa and Kihlstrom 1972). The pituitary extract has also been shown to increase the output of semen in rabbits, guinea-pigs and mice (Lakomaa and Kihlstrom 1972). An active constituent (molecular weight approximately 5 OOO) in the extract, called sperm-releasing substance, as suggested by Lakomaa (1974 a), has been highly purified (Lakomaa 1974 b), and used in further studies (Lakomaa 1974 a). In the present work, the distribution of the sperm-releasing substance in the mouse is studied using the whole-body autoradiographical technique.
Material and Methods An acetone powder prepared from bovine adenohypophyses by Biofac A/S (Copenhagen, Denmark) was used when preparing the sperm-releasing substance according to Kihlstrom et al. (1971) and Lakomaa (1974 b), excluding the final isoelectric focusing. The sperm-releasing substance can be lyophilized and stored for a considerable time.
409
410
JAN ERIK KIHLSTROM,
HKKAN
HALL AND EEVA LAKOMAA
As the sperm-releasing substance is a peptide, probably containing at least one tyrosine-residue (Lakomaa, personal communication), it is possible to iodinate the substance with lZ5l (Radiochemical Centre, Amersham, England) according to Greenwood er a / . (1963). 20 jig of the substance in 10 pl of distilled water and 10 o r 50 pg Chloramine T in 10 pl sodium phosphate buffer p H 7.5 were exposed to I mCi Na'"I (carrier free) in about lOpl NaOH-solution (pH 8-1 I ) for 30 o r 60 s respectively. The reaction was interrupted by the addition of 24 or I20 ,ug respectively, Na,S,O, in 50 p1 0.05 M phosphate buffer, pH 7.5. The contents of the reaction vial were transferred to a Sephadex column (0.9 I5 cm. Sephadex G 25 or G 50 eluted with phosphate buffered saline containing 0.1 M EDTA and 0.002 9b NaN,). The column was presaturated by the addition of 0.1 ml 20% Bovine Serum Albumin (BSA), Armour Fraction V, Sigma, and a subsequent addition of 1 mg of a crude preparation of the sperm-releasingsubstance. The reaction vial was washed with 0.1 ml 1 % KI, and this was also transferred to the column. 10 PI of each eluate was diluted and the elution pattern was determined in a gamma counter, type Autowell 11. The sperm-releasing substance was eluted near the void volume, and the free larl near the total volume. The iodinated substance was stored at i - V C , and was used within 10 days (often within 2 days). The mice used in the experiments were albino mice ( N M R I ) of about 2 months of age, obtained from Anticimex, Sollentuna, Sweden. The autoradiographical procedure was similar to the procedure used by Ullberg (1954). About 10-30 pCi of the iodinated sperm-releasing substance in 0 . 1 4 3 ml physiological saline was injected in a tail vein. At different intervals after injection (15 s t o 10 min), the mice were anesthetized with ether for about 30 s, and rapidly frozen in a Dewar vessel containing hexane and dry ice (solid CO,) (about - 70°C). They were then immersed in a solution of carboxymethylcellulose in water, and the whole preparations were frozen in hexane -dry ice for about IS min, and then stored in a freezing-room (-20°C) for 24 h or more. The frozen blocks were sectioned in the freezing room with a microtome, type Leitz, to a thickness of 20-60 /rm and dried at the same temperature. X-ray films (Industrex C, Kodak) were then exposed to the sections for 3-8 weeks and the films were processed in Agfa Gevaert Developer G 150 and Agfa Geavert Fixer G 334.
Observations Radio-activity is found in many organs, as listed below. Lung (Fig. I): No specific pattern of the distribution is found. The activity never exceeds
the activity of the blood. Liver (Fig. I ) : The activity never exceeds the activity of blood. Kidney (Fig. I and 4 ) : The activity of the kidneys is often dominating. Most activity is
found in the cortex. Ureter: In the urine. Bladder: In the urine. Pituitary (Fig. 2): At about the same degree in both the anterior and the posterior part,
though in some cases more activity in the anterior part. Secreting cells (Fig. 2, 4 and 5 ) : Submaxillary gland, epithelial cells of the nose, mouth and small intestine, mucosal cells in the walls of the stomach, and thus also in the contents of the stomach. Thyroid (Fig. 2): Always a great deal of activity. Epididymis (Fig. 3): Especially in the caput epididymidis. The activity seems to be localized to the walls of the tubuli. Testis (Fig. 3): Very weak activity which seems also to be localized to the walls of the tubuli. Ovary (Fig. 4 ) : Follicles and/or corpora lutea. Uterus: Much weaker activity than in the ovary. Slight activity only is found in other male sexual organs (vas deferens, seminal vesicle, prostate, coagulating gland).
AUTORADIOGRAPHICAL STUDIES OF A PITUITARY PEPTIDE
lung
hean
liver
kidney
lung
heart
41 1
liver
small kidney intestine Fig. 1 a. Male injected with 3OpCi. of the iodinated sperm-releasing substance. Interval after injection 15 s, thickness 20 pm. Exposure time 24 days. Activity can be seen in the kidney, lung, liver and is very high in the blood. Fig. 1 b. Male injected with 30 pCi, of the iodinated sperm-releasing substance. Interval after injection 1 min, thickness 20 ,urn. Exposure time 24 days. Activity is found in the kidney, lung, liver, small intestine, stomach and blood. Compare the differences between activity in the blood and in the kidney in this figure with that of Fig. 1 a.
Activity is, at least to some extent, found as soon as after 15 s in all organs mentioned, except in the contents of the stomach where activity is only found after longer incubation times (Fig. 5 ) .
Discussion The activity found in the different organs is not always the result of an incorporation of the iodinated sperm-releasing substance. It is probable, that there is a great deal of free lZ6Iin the sections. This can be due to both decomposition of the iodinated substance during storage, and to a rapid decomposition in the animal during the incubation. Since the distribution is studied after such short times, a great deal of activity still remains in the circulating blood. Therefore the activity found in organs rich in blood (Everett er al. 1956) is not necessarily due to an incorporation of the sperm-releasing substance. In the following list, the probable cause of the activity found in the different organs is discussed.
hypophysis Fig. 2. Male injected with 20 pCi, of the iodinated sperm-releasing substance. Interval after injection 15 s, thickness 40 ,urn. Exposure time 14 days. Activity is found in the submaxillary gland, both parts of the hypophysis and the epitheliar cells of the nose. The activity spots in the brain refer to blood vessels.
-~
thyroid
submaxillary gland
412
JAN ERIK
KIHLSTR~M, HAKAN
corpus epididymidis
caput epididymidis
HALL AND EEVA LAKOMAA
vas deferens
testis
caput epididymidis
cauda epididymidis
testis
Fig. 3 a. Male injected with 20 pCi, of the iodinated sperm-releasing substance. Interval after injection i min. thickness 60 pm. Exposure time 14 days. High activity can he seen in the caput epididymidis. while it is much lower in the other parts of the epididyrnis. No activity is found in the testis. Fig. 3 b. Male injected with 30 pCi, of the iodinated sperm-releasing substance. Interval after injection 3 min, thickness 2 0 p n . Exposure time 24 days. Activity is found primarily in the caput epididymidis, hut also in the other parts of the epididymis and in the vas deferens. The very weak activity in the testis is located to the walls of the tubuli.
Lung: High blood content with remaining activity. Liiwr: High blood content with remaining activity, though also some metabolism of the iodinated sperm-releasing substance (see below). Kidney, ureter and bladder: Excretion of the iodinated sperm-releasing substance and/or free I z 6 I . Pituitary: Free 12al will accumulate in the neurohypophysis, but not in the adenohypophysis (Jentzer 1953). In this study, activity is found in the adenohypophysis. indicating a n incorporation of the constituent. kidney
ovary
contents of the stomach
.~~
kidney
Fig. 4. Female injected with 30 ,ftCi, of the iodinated sperm-releasing substance. Interval after injection I 5 s, thickness 2 0 p ~ 1Exposure . time 24 days. The follicles and/or the corpora lutca of the otarq are incorporated with the constituent. The pattern of the kidney is easily visible. Fig. 5. Male injected with 20 / X i , of the iodinated sperm-releasing substance. Interval after injection 10 min. thickness 60 / t m . Exposure time 7 days. Activity is found almost only in the stomach and i n the kidney.
AUTORADIOGRAPHICAL STUDIES OF A PITUITARY PEPTIDE
413
Secreting cells: Accumulation of free lZ6I(Ullberg and Ewaldsson 1964, Logothetopoulos 1956 a, 1956 b). Thyroid: Accumulation of free lZ6I. Epididymis: Neither blood contamination, nor incorporation of free lZsIseem to be the cause of the high activity found in the epididymis. The blood content of the epididymis does not seem to be higher than that of the testis, when comparing the colours of the two organs in the sections. Radioiodine is not incorporated in the epididymis (Ullberg and Ewaldsson 1964). The picture of the incorporation of the sperm-releasing substance in the epididymis and the testis is very similar to that of FSH (Rajaniemi 1971). From this similarity it can be concluded that this substance like FSH accumulates in the walls of the tubuli of the epididymis and testis. The spermreleasing substance is not identical with FSH or with a fraction of FSH (Lakomaa 1974 a). Ouury: Free lZsIaccumulates in the ovaries (Ullberg and Ewaldsson 1964), but so also does luteinizing hormone (Rajaniemi and Vanha-Perttula 1972, Lee and Ryan 1971). It is unlikely that the sperm-releasing substance is identical with LH, since LH does not accumulate in the epididymis (Rajaniemi 1971). LH, prepared in the same way as the sperm-releasing hormone, does not possess sperm-releasing activity (Lakomaa 1974 a). It cannot be determined from these autoradiograms, whether the accumulation in the ovary is due only to an incorporation of free Y. Uterus: Probably blood contamination.
The increasing amount of activity in the liver and in the kidneys, with increasing intervals after injection, is probably the result of the metabolism of the sperm-releasing substance. The increasing amount of metabolized substance will give free lZsIcirculating in the blood, leading to higher activity in the stomach after longer incubation times (Logothetopoulos 1956 a). Thus, the only organs where the activity scems to be due to an incorporation of the iodinated sperm-releasing substance, are the epididymis, adenohypophysis and possibly the ovary. The incorporation in the adenohypophysis indicates a mechanism of re-uptake, since the substance originates from this organ. The incorporation in the walls of the caput epididymidis cannot at present be explained, since the exact functions of the substance have not yet been investigated. But it is not unexpected to find accumulation in this organ, since the substance probably affects the output of semen also in mammals. A comparison between the distribution of the iodinated sperm-releasing substance and that of LH and FSH can be made. Both LH and FSH accumulate in the testis, but the uptake is not visible in the autoradiograms after such short intervals as have been used in this study with the sperm-releasing substance (Rajaniemi 1971). LH does not incorporate into the epididymis, but FSH on the other hand does (Rajaniemi 1971). Thus the spermreleasing substance has a distribution pattern in the male sexual organs which differs from that of LH, but it does not in this respect differ from that of FSH. This work has been supported by grants from the Helge Axson Johnson Foundation, and from the Swedish Natural Science Research Council to Jan Erik Kihlstrom.
414
JAN ERIK KIHLSTROM, HAKAN HALL AND EEVA LAKOMAA
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