2. Medizinische Klinik und
Tierversuchsanlage Germany
der Universität Düsseldorf,
ENDOCRINE TESTICULAR FUNCTION IN MINK DURING THE FIRST YEAR OF LIFE
By E.
Nieschlag
and H. Bieniek
ABSTRACT The endocrine testicular function in mink (Mustela vison) was investigated during the first year of life encompassing puberty, the first mating season and the phase of regression thereafter. The mink, relatively easily accessible as a semi-domesticated animal, was chosen as an example of a mammalian seasonal breeder. In plasma samples from 7 to 17 animals collected on 10 occasions between July and April testosterone and \g=D\4\x=req-\ androstenedione were determined by simultaneous radioimmunoassay. A steady increase of both androgens from November to early March, when the mating season occurs, and a rapid decline to pre-pubertal levels thereafter was observed. The ratio of testosterone to \g=D\4-androstenedione was 1:1 from November to April. These findings parallel the known morphological transitions of the testes.
The mink (Mustela vison) appears to be well suited for the study of reproduc¬ tive functions in mammalian seasonal breeders since, as a semi-domesticated animal, it is more easily accessible and available in larger numbers than other seasonal breeders previously investigated like the roe deer (Stieve 1950; Short 8c Mann 1966), the bat (Glover 1973) or the rock hyrax (Neaves 1973). Al¬ though seasonal changes in the testicular morphology of the mink are well
Supported by the Deutsche Forschungsgemeinschaft. Requests for reprints should be addressed to: Dr. E. Nieschlag, D 4 Düsseldorf, Moorenstr. 5, Germany.
2. Medizin. Univ.
Klin.,
by the studies of Onsted (1967), Hemmigsen (1967) and Venge (1973), nothing is known about the endocrine testicular function accompanying the morphological alterations. For this reason we were prompted to investigate androgens in peripheral blood of the male mink during its first year of life which encompasses puberty, the first mating season and the phase of regression documented
thereafter.
During the first year of life testicular development in the mink is charac¬ terized by a relatively slow growth rate of 0.2 g per month from birth in May to onset of puberty in November. This slow growth is followed by a steep weight increase of approximately 1 g per month for the next 4 months, until a maximal weight of 5-6 g is reached in March, when the mating season, ex¬ tending over 3-4 months, occurs. Thereafter the testes undergo rapid involution so that by May their weight returns to almost pre-pubertal dimensions (Onsted 1967; Hemmigsen 1967; Venge 1973).
METHODS Seven male Standard mink originating from three litters born in the first week of May were followed through the first year of life. Plasma samples from each animal were obtained on 10 occasions over a period beginning in July of the first year and ex¬ tending to April of the following year. Plasma was also collected on the dates indi¬ cated in Fig. 1 from 3 to 10 additional mink born in the same week as the previous 7, but originating from different litters. All animals were kept under identical con¬ ditions in an open air farm (natural light conditions). In all plasma samples testosterone and zH-androstenedione were estimated by a simultaneous radioimmunoassay after separation by thin layer chromatography (Nieschlag et al. 1975). A maximum of 22 samples were analysed together so that at least all samples from one date were estimated simultaneously. Intra- and interassay precision are 8 and 11% respectively in the testosterone assay, and 9 and 12% in the d4-androstenedione assay.
RESULTS
Results
are shown in Fig. 1. The testosterone levels remain fairly constant the first 5 months of life, increase slowly through January and then during rise rapidly towards the mating season in March. Thereafter a rapid decline occurs, so that by April pre-pubertal levels are reached. ¿)4-Androstenedione follows the pattern of testosterone very closely. How¬ ever, while zl4-androstenedione is always lower than testosterone until October, showing a ratio of approximately 1:3, d4-androstenedione and testosterone levels are almost identical during the following months. After the return to pre-pubertal levels in April, the ratio of the two steroids remains the same.
Plasma testosterone(·—»land androstenedione (o—o) in mink through the first year of live (
mean
values i
se.)
1500
500
July
Aug
Sept
Oct
Dec
(solid line) through the first
Plasma testosterone
Febr
Jan
12
10
March 16
10
Fig. 1. and zH-androstenedione (broken year of life (mean values + se).
April 17
line)
in mink
DISCUSSION
Since seasonal variations of the reproductive functions in mink of the northern hemisphere occur almost simultaneously (Venge 1973), it would appear justified to compare our endocrine results with morphological findings obtained from animals raised in Scandinavia. The pattern of androgen levels in peripheral plasma of the mink throughout its first year of life is almost identical with the curves for testicular weight and tubular diameter presented by Ousted (1967) and Venge (1973), thus indicating good correlation between morpho¬ logical transitions and the endocrine activity of the testes. However, the fall in plasma androgens after the mating season appears to precede the involution of the testicular tissue slightly. There is also a good correlation between histochemical and endocrine findings. Ousted (1967) observed that the lipid content of the mink Leydig cell increases from the fourth month
on
and attains
a
maximum in the seventh
month of life and decreases thereafter. Hence the depletion of lipid droplets occurs at the onset of the steep increase of circulating androgens. This is con¬ sistent with results found by Clegg (1966) showing that in the pubertal rat Leydig cells contain lipids only until the early phase of maximal growth of the accessory reproductive glands is achieved. Furthermore, Aoki (1970) demon¬ strated a drastic lipid depletion of Leydig cells in immature mice following administration of HCG. It could be concluded that the lipid accumulation in the Leydig cells represents a reservoir of precursors which are converted to androgens under the influence of pituitary stimulation at the onset of puberty. Since it is not easy to obtain blood samples from mink we were unable to take into consideration diurnal or short-term fluctuations of plasma testoste¬ rone, now well established in bulls (Katangole et al. 1971), rams (Purvis et al 1974) and men (Nieschlag 1974). Such fluctuations are also likely to exist in the mink and may explain the relatively high scatter of values found on any one date and reflected in the sem (Fig. 1). The highest single testosterone value obtained during the breeding season amounted to 3170 ng per 100 ml plasma. Such exceedingly high values have never been found under physiolo¬ gical conditions in continuous breeders like men, but are reported to occur during the mating period in other seasonal breeders like the rock hyrax (Neaves 1973) or during muths of elephants (Jainudeen et al. 1972). The rise in plasma testosterone during sexual development has been described in other species like the rat (Knorr et al. 1970; Miyachi et al. 1973), the guinea pig (Resko 1970), the pig (Elsaesser et al 1973) and men (August et al 1972). Resko (1970) and Frasier et al (1969) measured testosterone together with zP-androstenedione during puberty of guinea pigs and boys, respectively, and described a shift in the testosterone/zP-androstenedione ratio, while zP-androstenedione levels remain relatively constant, as a characteristic of puberty. We do not find a similar shift in the ratio of these androgens in the mink but rather /P-androstenedione levels paralleling testosterone levels and reaching the same high concentrations as testosterone. The biological significance of the high /P-androstenedione levels remains to be elucidated. Since it was suggested that in men peripheral zP-androstenedione levels may arise to a substantial amount from steroid metabolism in the hair follicles, where zP-androstenedione is the most prominent androgen meta¬ bolite (Sansone-Bazzano et al 1972), the idea that the high /P-androstenedione levels in the mink could be associated with the quality of the fur is intriguing,
yet highly speculative.
ACKNOWLEDGMENT We
appreciate
the skilful technical assistance of Ms. M. Pier and Ms. G. Müller.
REFERENCES Aoki .: Protoplasma 71 (1970) 209. August G. P., Grumbach M. M. 8- Kaplan E. L.: J. clin. Endocr. 34 (1972) 319. Clegg E. ].: J. Anat. (Lond.) 100 (1966) 369. Elsaesser F., Pomerantz D. K., Ellendorff f, Kreikenbaum K. Sc König .: Acta endocr.
(Kbh.) Suppl.
173
(1973)
148.
Frasier S. D.. Gafford F. Se Horton R.: J. clin. Endocr. 29 (1969) 1404. Glover T. D. In: G. Raspé, Ed. Schering Workshop on Contraception: The Masculine Gender. Pergamon Press/Vieweg, Braunschweig (1973) p. 235. Hemmigsen B.: Nord. Vet.-Med. 19 (1967) 71. Jainudeen M. R., Katangole C. B. Se Short R. V'..· J. Reprod. Fértil. 29 (1972) 99. Katangole C. B., Naftolin F. Se Short R. V'.: J. Endocr. 50 (1971) 457. Knorr D. W'., Vanha-Perttula T. 8e Lipsett M. B.: Endocrinology 86 (1970) 1298. Miyachi Y., Nieschlag E. Se Lipsett M. B.: Endocrinology 92 (1973) 1. Neuves W. B.: Biol. Reprod. S (1973) 541. Nieschlag E. In: Aschoff J. and Halberg F., Eds. Chronobiological Aspects in Endo¬ crinology. Schattauer Verlag, Stuttgart (1974). Nieschlag £., Mauss J., Coert A. Sc Kicovic P.: Acta endocr. (Kbh.) 79 (1975) 366. Onsted O.: Acta endocr. (Kbh.) Suppl. 117 (1967) 1. Purvis K., lllius A. W. Sc Haynes N. B.: J. Endocr. 61 (1974) 241. Resko J. .: Endocrinology 86 (1970) 1444. Sansone-Bazzano G., Reisner R. M. Se Bazzano G.: J. clin. Endocr. 34 (1972) 512Short R. V. 8c Mann T.: J. Reprod. Fértil. 12 (1966) 337. Stieve H.: Z. mikr.-anat. Forsch. 55 (1950) 427. Venge O.: Kgl. Vet.- og Landbohejsk. Ârsskr. (Kbh.) (1973) 95. Received
on
August 27th,
1974.
Tierversuchsanlage Germany
der Universität Düsseldorf,
ENDOCRINE TESTICULAR FUNCTION IN MINK DURING THE FIRST YEAR OF LIFE
By E.
Nieschlag
and H. Bieniek
ABSTRACT The endocrine testicular function in mink (Mustela vison) was investigated during the first year of life encompassing puberty, the first mating season and the phase of regression thereafter. The mink, relatively easily accessible as a semi-domesticated animal, was chosen as an example of a mammalian seasonal breeder. In plasma samples from 7 to 17 animals collected on 10 occasions between July and April testosterone and \g=D\4\x=req-\ androstenedione were determined by simultaneous radioimmunoassay. A steady increase of both androgens from November to early March, when the mating season occurs, and a rapid decline to pre-pubertal levels thereafter was observed. The ratio of testosterone to \g=D\4-androstenedione was 1:1 from November to April. These findings parallel the known morphological transitions of the testes.
The mink (Mustela vison) appears to be well suited for the study of reproduc¬ tive functions in mammalian seasonal breeders since, as a semi-domesticated animal, it is more easily accessible and available in larger numbers than other seasonal breeders previously investigated like the roe deer (Stieve 1950; Short 8c Mann 1966), the bat (Glover 1973) or the rock hyrax (Neaves 1973). Al¬ though seasonal changes in the testicular morphology of the mink are well
Supported by the Deutsche Forschungsgemeinschaft. Requests for reprints should be addressed to: Dr. E. Nieschlag, D 4 Düsseldorf, Moorenstr. 5, Germany.
2. Medizin. Univ.
Klin.,
by the studies of Onsted (1967), Hemmigsen (1967) and Venge (1973), nothing is known about the endocrine testicular function accompanying the morphological alterations. For this reason we were prompted to investigate androgens in peripheral blood of the male mink during its first year of life which encompasses puberty, the first mating season and the phase of regression documented
thereafter.
During the first year of life testicular development in the mink is charac¬ terized by a relatively slow growth rate of 0.2 g per month from birth in May to onset of puberty in November. This slow growth is followed by a steep weight increase of approximately 1 g per month for the next 4 months, until a maximal weight of 5-6 g is reached in March, when the mating season, ex¬ tending over 3-4 months, occurs. Thereafter the testes undergo rapid involution so that by May their weight returns to almost pre-pubertal dimensions (Onsted 1967; Hemmigsen 1967; Venge 1973).
METHODS Seven male Standard mink originating from three litters born in the first week of May were followed through the first year of life. Plasma samples from each animal were obtained on 10 occasions over a period beginning in July of the first year and ex¬ tending to April of the following year. Plasma was also collected on the dates indi¬ cated in Fig. 1 from 3 to 10 additional mink born in the same week as the previous 7, but originating from different litters. All animals were kept under identical con¬ ditions in an open air farm (natural light conditions). In all plasma samples testosterone and zH-androstenedione were estimated by a simultaneous radioimmunoassay after separation by thin layer chromatography (Nieschlag et al. 1975). A maximum of 22 samples were analysed together so that at least all samples from one date were estimated simultaneously. Intra- and interassay precision are 8 and 11% respectively in the testosterone assay, and 9 and 12% in the d4-androstenedione assay.
RESULTS
Results
are shown in Fig. 1. The testosterone levels remain fairly constant the first 5 months of life, increase slowly through January and then during rise rapidly towards the mating season in March. Thereafter a rapid decline occurs, so that by April pre-pubertal levels are reached. ¿)4-Androstenedione follows the pattern of testosterone very closely. How¬ ever, while zl4-androstenedione is always lower than testosterone until October, showing a ratio of approximately 1:3, d4-androstenedione and testosterone levels are almost identical during the following months. After the return to pre-pubertal levels in April, the ratio of the two steroids remains the same.
Plasma testosterone(·—»land androstenedione (o—o) in mink through the first year of live (
mean
values i
se.)
1500
500
July
Aug
Sept
Oct
Dec
(solid line) through the first
Plasma testosterone
Febr
Jan
12
10
March 16
10
Fig. 1. and zH-androstenedione (broken year of life (mean values + se).
April 17
line)
in mink
DISCUSSION
Since seasonal variations of the reproductive functions in mink of the northern hemisphere occur almost simultaneously (Venge 1973), it would appear justified to compare our endocrine results with morphological findings obtained from animals raised in Scandinavia. The pattern of androgen levels in peripheral plasma of the mink throughout its first year of life is almost identical with the curves for testicular weight and tubular diameter presented by Ousted (1967) and Venge (1973), thus indicating good correlation between morpho¬ logical transitions and the endocrine activity of the testes. However, the fall in plasma androgens after the mating season appears to precede the involution of the testicular tissue slightly. There is also a good correlation between histochemical and endocrine findings. Ousted (1967) observed that the lipid content of the mink Leydig cell increases from the fourth month
on
and attains
a
maximum in the seventh
month of life and decreases thereafter. Hence the depletion of lipid droplets occurs at the onset of the steep increase of circulating androgens. This is con¬ sistent with results found by Clegg (1966) showing that in the pubertal rat Leydig cells contain lipids only until the early phase of maximal growth of the accessory reproductive glands is achieved. Furthermore, Aoki (1970) demon¬ strated a drastic lipid depletion of Leydig cells in immature mice following administration of HCG. It could be concluded that the lipid accumulation in the Leydig cells represents a reservoir of precursors which are converted to androgens under the influence of pituitary stimulation at the onset of puberty. Since it is not easy to obtain blood samples from mink we were unable to take into consideration diurnal or short-term fluctuations of plasma testoste¬ rone, now well established in bulls (Katangole et al. 1971), rams (Purvis et al 1974) and men (Nieschlag 1974). Such fluctuations are also likely to exist in the mink and may explain the relatively high scatter of values found on any one date and reflected in the sem (Fig. 1). The highest single testosterone value obtained during the breeding season amounted to 3170 ng per 100 ml plasma. Such exceedingly high values have never been found under physiolo¬ gical conditions in continuous breeders like men, but are reported to occur during the mating period in other seasonal breeders like the rock hyrax (Neaves 1973) or during muths of elephants (Jainudeen et al. 1972). The rise in plasma testosterone during sexual development has been described in other species like the rat (Knorr et al. 1970; Miyachi et al. 1973), the guinea pig (Resko 1970), the pig (Elsaesser et al 1973) and men (August et al 1972). Resko (1970) and Frasier et al (1969) measured testosterone together with zP-androstenedione during puberty of guinea pigs and boys, respectively, and described a shift in the testosterone/zP-androstenedione ratio, while zP-androstenedione levels remain relatively constant, as a characteristic of puberty. We do not find a similar shift in the ratio of these androgens in the mink but rather /P-androstenedione levels paralleling testosterone levels and reaching the same high concentrations as testosterone. The biological significance of the high /P-androstenedione levels remains to be elucidated. Since it was suggested that in men peripheral zP-androstenedione levels may arise to a substantial amount from steroid metabolism in the hair follicles, where zP-androstenedione is the most prominent androgen meta¬ bolite (Sansone-Bazzano et al 1972), the idea that the high /P-androstenedione levels in the mink could be associated with the quality of the fur is intriguing,
yet highly speculative.
ACKNOWLEDGMENT We
appreciate
the skilful technical assistance of Ms. M. Pier and Ms. G. Müller.
REFERENCES Aoki .: Protoplasma 71 (1970) 209. August G. P., Grumbach M. M. 8- Kaplan E. L.: J. clin. Endocr. 34 (1972) 319. Clegg E. ].: J. Anat. (Lond.) 100 (1966) 369. Elsaesser F., Pomerantz D. K., Ellendorff f, Kreikenbaum K. Sc König .: Acta endocr.
(Kbh.) Suppl.
173
(1973)
148.
Frasier S. D.. Gafford F. Se Horton R.: J. clin. Endocr. 29 (1969) 1404. Glover T. D. In: G. Raspé, Ed. Schering Workshop on Contraception: The Masculine Gender. Pergamon Press/Vieweg, Braunschweig (1973) p. 235. Hemmigsen B.: Nord. Vet.-Med. 19 (1967) 71. Jainudeen M. R., Katangole C. B. Se Short R. V'..· J. Reprod. Fértil. 29 (1972) 99. Katangole C. B., Naftolin F. Se Short R. V'.: J. Endocr. 50 (1971) 457. Knorr D. W'., Vanha-Perttula T. 8e Lipsett M. B.: Endocrinology 86 (1970) 1298. Miyachi Y., Nieschlag E. Se Lipsett M. B.: Endocrinology 92 (1973) 1. Neuves W. B.: Biol. Reprod. S (1973) 541. Nieschlag E. In: Aschoff J. and Halberg F., Eds. Chronobiological Aspects in Endo¬ crinology. Schattauer Verlag, Stuttgart (1974). Nieschlag £., Mauss J., Coert A. Sc Kicovic P.: Acta endocr. (Kbh.) 79 (1975) 366. Onsted O.: Acta endocr. (Kbh.) Suppl. 117 (1967) 1. Purvis K., lllius A. W. Sc Haynes N. B.: J. Endocr. 61 (1974) 241. Resko J. .: Endocrinology 86 (1970) 1444. Sansone-Bazzano G., Reisner R. M. Se Bazzano G.: J. clin. Endocr. 34 (1972) 512Short R. V. 8c Mann T.: J. Reprod. Fértil. 12 (1966) 337. Stieve H.: Z. mikr.-anat. Forsch. 55 (1950) 427. Venge O.: Kgl. Vet.- og Landbohejsk. Ârsskr. (Kbh.) (1973) 95. Received
on
August 27th,
1974.
