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Effect of castration and testosterone therapy on harderian gland protein patterns of the golden hamster Mesocricetus auratus
Comp. Biochem. Physiol. Vol. 102B, No. 3, pp. 601~503, 1992
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
Printed in Great Britain
EFFECT OF CASTRATION AND TESTOSTERONE THERAPY ON HARDERIAN GLAND PROTEIN PATTERNS OF THE GOLDEN HAMSTER (MESOCRICETUS AURATUS) B. VARRIALE, I. SERINO, S. MINUCCI and G. CHIEFFI Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi", Via Costantinopoli 16, 80138, University of Naples, Italy (Tel.: 3981-5640508; Fax: 3981-5640599) (Received 31 October 1991)
Abstract--1. Sodium dodecyl sulphate 7-12% gradient polyacrylamide gel electrophoresis of male and female hamster Harderian gland whole homogenate shows a clear-cut sexual dimorphism, which consists of the presence of two male-specific glycoproteins (168 and 116 kDa) and two specific female proteins (210 and 190 kDa). 2. In the male, castration causes a significant decrease in the concentration of the two glycoprotein fractions. 3. Replacement therapy with testosterone propionate (T) restores the intact male pattern.
INTRODUCTION The Harderian gland (HG) is a c o m p o u n d tubuloalveolar gland which occupies a large portion of the orbital cavity posterior to the eyeball of animals possessing a nictitating membrane (see review of Sakai, 1981). It is well-developed in rodents where, in addition to producing a lipid secretion, the gland synthesises porphyrins which are stored as solid luminal accretions (Grafflin, 1942). In the golden hamster the gland shows marked sex differences. The male gland has two cell types (Type I and Type II cells) as opposed to one (Type I cell) present in the female gland (Christensen and Dam, 1953). Furthermore, the male gland is larger than that of the female and contains a hundred times fewer porphyrins (Payne et al., 1979). Castration of the male hamster alters the morphology of the male gland towards that of the female and testosterone treatment prevents these changes (Hoffman, 1971). H o h et al. (1984) found significant differences in the electrophoretic patterns of homogenates of the male and female HG. The present investigation was undertaken to determine the effect of castration and testosterone therapy on the protein electrophoretic pattern of the H G of the male hamster.
of the Harderian glands were removed, weighed and homogenized in PBS using a Politron and then centrifuged (6000g) at 4°C for 30 min. Protein content was measured according to the method of Lowry et al. (1951). Equal amounts of proteins (60/~g/sample, analyzed by SDS 7-12% gradient PAGE, were separated on 0.75 mm gel over a distance of 16 cm with 3 cm staking gel according to Laemmli's method (1970). Each sample was heated at 100°C for 5min in loading buffer (10 mM Tris-HCl, 5% (v/v) fl-mercaptoethanol, 2% (w/v) SDS, 20% (v/v) glycerol, 0.1% (w/v) Bromophenol Blue, pH 7.5). The gels were stained with Coomassie Brilliant Blue R (Serva). Prestained standard proteins of 16,000-205,000 mol wt were used. On the same supernatant a SDS 7-12% gradient PAGE was carried out to estimate the electrophoretic pattern of the glycoproteins according to Cowman et al. (1984) with minor modifications. Briefly, 80#g of total proteins for each sample were treated as described before in a buffer deprived of tracking dye, to impair glycoprotein spread along the gel. After the run, gels were washed three times (30 min/wash) in a fixative solution (40% absolute ethanol, 15% acetic acid) and stained (2-3 hr) in 0.5% Alcian Blue (Bio-Rad, Richmond, CA), 2% acetic acid. Gels were destained with several changes of a 2% acetic acid solution. The relative concentration of the proteic fractions was obtained by scanning the electrophoretic plates using a Joyce-Loebl scanner and the results were calculated according to the Svedberg and Pedersen method (1940). Results were statistically analyzed by a one-way analysis of variance (ANOVA) followed by Duncan's test.
MATERIALS AND METHODS
Adult golden hamsters (Mesocricetus auratus) were purchased from Nossan (Italy) and housed under a light: dark cycle of 14:10 hr at 20°C. Animals were housed three per cage and food and water were provided ad libitum. Males (N-- 15) were castrated under anesthesia. Five days after surgical treatment the animals were divided into two groups. The first was used as the castrated animal group; the second was injected three times a week with testosterone (5 mg in 1 ml sesame oil/kg body weight/injection). A third group of intact males (N = 5) served as sham-operated controls, receiving 0.2 ml sesame oil three times a week. At the end of the experiment (4 weeks) animals were killed, the lobes 601
RESULTS AND DISCUSSION The S D S - P A G E gel electrophoresis of the whole homogenate of the male and female hamster Harderian gland shows a definite sexual dimorphism (Fig. 1). In fact, the male gland contains large amounts of two proteic fractions of about 168 and 116 kDa, respectively, which are missing in the female gland. The female gland homogenate reveals two proteic fractions of low concentration, of about 210 and 190 k D a respectively, which are lacking in the male gland.
602
B. VARRIALEet al.
o
o
kDa kDa
205_ 116_
9
205. 116_
84_
84_
47_ 33_ 24_
16_
Fig. I. SDS 7-12% gradient PAGE of Harderian gland proteins. In the male whole gland homogenate the arrows indicate the 116 and 168 kDa fractions. In the female whole gland homogenate the arrows indicate the 210 and 190 kDa fractions. Values on the left line indicate the molecular weight of standard proteins (kDa). Analysis of the glycoproteic pattern (Fig. 2) suggests that the male-specific fractions (168 and 116 kDa) are glycoproteins. The two proteic fractions present only in the female gland (210 and 190 kDa) were both Alcian-negative. As regards the proteic pattern of the experimental groups, castration caused a significant decrease in the concentration of the 168 and l l 6 k D a fractions (Fig. 3). Their relative concentrations were 4.23 + 0.56% for the 168 kDa fraction and 1.49 + 0.23% for the 116 kDa fraction with respect to the relative concentrations of sham-operated males, which were 12.12 + 0.54% (168 kDa) and 2.85_ 0.13% (116 kDa). Replacement therapy, with testosterone, restored the intact male proteic pattern (7.69_ 0.66% and 3.62 + 0.69% respectively) (Table 1). Our observations on the sexual dimorphism of the electrophoretic protein pattern are in agreement with those previously reported by Hoh et al. (1984), where the sex-specific fractions were named HGP1
47 33
Fig. 2. SDS 7-12% gradient PAGE of Harderian gland glycoproteins in male and female whole gland homogenate. Arrows indicate the glycoproteins characteristic of the male pattern which correspond to those shown in Fig. 1. Values on the left line indicate the molecular weight of standard proteins (kDa). (168 kDa) and HGTP1 (116 kDa) in the male, and HGP3 (210 kDa) and HGP4 (190 kDa) in the female. However, an additional fraction (HGP2), isolated by these authors in the male gland only, was found occasionally in the female in our study. Hoh et al. (1984) also carried out electrophoresis on the tubular fraction obtained from sucrose gradients of the original homogenate pellet. This fraction produced two major proteins designated HGTP1 and HGTP2, of 115 and 8 kDa respectively. Two minor proteins with molecular weights of 102 and 95kDa were also identified. In our case, the female gland did not contain any tubular protein. Therefore the two proteic fractions which differentiate the male from the female gland correspond to those previously named HGP1 and HGTP1 by Hoh et al. (1984), the former probably being a structural component and the seco n d a tubular protein component. The presence of membrane-bound clusters of cylindrical tubules in the cytoplasm of the acinar cells is one of the characteristics of the male Harderian gland of the hamster. Bucana and Nadakavukaren (1973) have suggested a possible relationship between the presence of these tubules and the breakdown of porphyrin granules, while Jones and Hoffman (1976) suggested that peritubular complexes may prevent porphyrin biosynthesis by inhibiting the condensation of 6-aminolevulinic acid to porphobilinogen. In fact, in the maturing male, the disappearance of pigment granules coincides with the appearance of
603
Hamster Harderian gland proteins a
b
1. Percentages of total amount of the malespecific proteins loaded on gels, expressed as means + SD Experimental Proteic fractions groups 116 kDa 168 kDa Sham-operated 2.85 + 0.13= 12.12+ 0.54= Castrated 1.49 + 0.23b 4.23 + 0.56b Castrated + T-injected 3.62 + 0.69~ 7.69 + 0.66c
Table
O
kDa 205_ 116_
a vs b: p < 0.01; b VS c: p < 0.01. REFERENCES
84_
47_ 33_ 24_ 16_
Fig. 3. SDS 7-12% gradient PAGE of Harderian gland proteins in the different experimental groups of male golden hamster; (a) sham-operated control; (b) castrated animals; (c) castrated and testosterone-injected animals. Arrows indicate the proteic fractions affected by castration and replacement therapy. Values on the left line indicate the molecular" weight of standard proteins (kDa). tubular clusters (Bucana and Nadakavukaren, 1973), and in the female, testosterone treatment mimics the same phenomenon (Sun and Nadakavukaren, 1980; Spike et al., 1985). The presence of androgen receptors in the Harderian gland of the male hamster is consistent with the testosterone dependence of the gland in the male hamster (Vilkis et al., 1988; Vilkis and Perez-Palacios, 1989). In this report, the testosterone dependence of sex-specific glycoprotein synthesis of the male hamster Harderian gland is conclusively demonstrated. In fact, castration causes a significant decrease of sex-specific protein synthesis, which is prevented by testosterone therapy. Recently, the testosterone dependence of Harderian gland-specific glycoproteins has also been demonstrated in the green frog, Rana esculenta (Chieffi et aL, 1991). Acknowledgements--This work was supported by MURST (40% and 60% grants) and CNR (91.00476.CT04 Grant).
Bucana C. D. and Nadakavukaren M. J, (1973) Ultrastructural investigation of the postnatal development of the hamster Harderian gland. II. Male and female. Z. Zellforsch, mikrosk. Anat. 142, 1-12. Chieffl G., Chieffi Baccari G., Di Matteo L., d'Istria M., Marmorino C., Minucci S. and Varriale B. (1991) Hormonal control of the Harderian gland of Rana esculenta. Belg. J. Zool. 121, 130-132. Christensen F. and Dam H. (1953) A sexual dimorphism of the Harderian glands in hamster. Acta physiol, scand. 23, 333-336. Cowman M. K., Slahetka M. F. Hittner D. M., Kim J., Forino M. and Gadelrab G. (1984) Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosoaminoglycan oligosaccharides. Biochem. J. 221, 707-716. Grafflin A. L. (1942) Histological observations upon porphyrin-excreting Harderian gland of the albino rat. Am. J. Anat. 71, 43~o4. Hoffman R. A. (1971) Influence of some endocrine, hormones and blinding on the histology and porphyrins of the Harderian gland of golden hamster. Am. J. Anat. 132, 463-478. Hoh J. H., Lin W. L. and Nadakavukaren M. J, (1984) Sexual dimorphism in the Harderian gland proteins of the golden hamster. Comp. Biochem. Physiol. 77B, 729-731. Jones C. W. and Hoffman R. (1967) Porphyrin concentration of the hamster Harderian gland. Effect of incubation with 6-ALA and various hormones. Int. J. Biochem. 7, 135-140. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 2 2 7 , 680q585. Lowry O. H., Rosebrough J. N., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Payne A. P., MeGadey J., Moore M. R. and Thompson G. G. (1977) Androgenic control of the Harderian gland in male golden hamster. J. Endocr. 75, 73-82. Sakai T. (1981) The mammalian Harderian gland: morphology, biochemistry, function and phylogeny. Arch. Histol. Jap. 44, 299-333. Spike R. C., Johnston H. S., McGadey Y., Moore M. R., Thompson G. G. and Payne A. P. (1985) Quantitative studies on the effects of hormones on structure and porphyrin biosynthesis in the Harderian gland of the female golden hamster. I. The effects of ovariectomy and androgen administration. J. Anat. 142, 59-72. Sun C. Y. and Nadakavukaren M. J. (1980) Effect of testosterone on the female hamster Harderian gland pigmentation and ultrastructure. Cell Tissue Res. 207, 511-517. Svedberg T. and Pedersen K. O. (1940) The Ultracentrifuge pp. 296-327. Oxford University Press, Oxford. Vilkis F., Hernandez A., Perez A. E. and Perez-Palacios G. (1988) Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster. J. Endocr. 112, 3-8. Vilkis F. and Perez-Palacios G. (1989) Steroid hormone receptors and sexual phenotype of the Harderian gland in hamster. J. Endocr. 121, 149-156.
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
Printed in Great Britain
EFFECT OF CASTRATION AND TESTOSTERONE THERAPY ON HARDERIAN GLAND PROTEIN PATTERNS OF THE GOLDEN HAMSTER (MESOCRICETUS AURATUS) B. VARRIALE, I. SERINO, S. MINUCCI and G. CHIEFFI Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi", Via Costantinopoli 16, 80138, University of Naples, Italy (Tel.: 3981-5640508; Fax: 3981-5640599) (Received 31 October 1991)
Abstract--1. Sodium dodecyl sulphate 7-12% gradient polyacrylamide gel electrophoresis of male and female hamster Harderian gland whole homogenate shows a clear-cut sexual dimorphism, which consists of the presence of two male-specific glycoproteins (168 and 116 kDa) and two specific female proteins (210 and 190 kDa). 2. In the male, castration causes a significant decrease in the concentration of the two glycoprotein fractions. 3. Replacement therapy with testosterone propionate (T) restores the intact male pattern.
INTRODUCTION The Harderian gland (HG) is a c o m p o u n d tubuloalveolar gland which occupies a large portion of the orbital cavity posterior to the eyeball of animals possessing a nictitating membrane (see review of Sakai, 1981). It is well-developed in rodents where, in addition to producing a lipid secretion, the gland synthesises porphyrins which are stored as solid luminal accretions (Grafflin, 1942). In the golden hamster the gland shows marked sex differences. The male gland has two cell types (Type I and Type II cells) as opposed to one (Type I cell) present in the female gland (Christensen and Dam, 1953). Furthermore, the male gland is larger than that of the female and contains a hundred times fewer porphyrins (Payne et al., 1979). Castration of the male hamster alters the morphology of the male gland towards that of the female and testosterone treatment prevents these changes (Hoffman, 1971). H o h et al. (1984) found significant differences in the electrophoretic patterns of homogenates of the male and female HG. The present investigation was undertaken to determine the effect of castration and testosterone therapy on the protein electrophoretic pattern of the H G of the male hamster.
of the Harderian glands were removed, weighed and homogenized in PBS using a Politron and then centrifuged (6000g) at 4°C for 30 min. Protein content was measured according to the method of Lowry et al. (1951). Equal amounts of proteins (60/~g/sample, analyzed by SDS 7-12% gradient PAGE, were separated on 0.75 mm gel over a distance of 16 cm with 3 cm staking gel according to Laemmli's method (1970). Each sample was heated at 100°C for 5min in loading buffer (10 mM Tris-HCl, 5% (v/v) fl-mercaptoethanol, 2% (w/v) SDS, 20% (v/v) glycerol, 0.1% (w/v) Bromophenol Blue, pH 7.5). The gels were stained with Coomassie Brilliant Blue R (Serva). Prestained standard proteins of 16,000-205,000 mol wt were used. On the same supernatant a SDS 7-12% gradient PAGE was carried out to estimate the electrophoretic pattern of the glycoproteins according to Cowman et al. (1984) with minor modifications. Briefly, 80#g of total proteins for each sample were treated as described before in a buffer deprived of tracking dye, to impair glycoprotein spread along the gel. After the run, gels were washed three times (30 min/wash) in a fixative solution (40% absolute ethanol, 15% acetic acid) and stained (2-3 hr) in 0.5% Alcian Blue (Bio-Rad, Richmond, CA), 2% acetic acid. Gels were destained with several changes of a 2% acetic acid solution. The relative concentration of the proteic fractions was obtained by scanning the electrophoretic plates using a Joyce-Loebl scanner and the results were calculated according to the Svedberg and Pedersen method (1940). Results were statistically analyzed by a one-way analysis of variance (ANOVA) followed by Duncan's test.
MATERIALS AND METHODS
Adult golden hamsters (Mesocricetus auratus) were purchased from Nossan (Italy) and housed under a light: dark cycle of 14:10 hr at 20°C. Animals were housed three per cage and food and water were provided ad libitum. Males (N-- 15) were castrated under anesthesia. Five days after surgical treatment the animals were divided into two groups. The first was used as the castrated animal group; the second was injected three times a week with testosterone (5 mg in 1 ml sesame oil/kg body weight/injection). A third group of intact males (N = 5) served as sham-operated controls, receiving 0.2 ml sesame oil three times a week. At the end of the experiment (4 weeks) animals were killed, the lobes 601
RESULTS AND DISCUSSION The S D S - P A G E gel electrophoresis of the whole homogenate of the male and female hamster Harderian gland shows a definite sexual dimorphism (Fig. 1). In fact, the male gland contains large amounts of two proteic fractions of about 168 and 116 kDa, respectively, which are missing in the female gland. The female gland homogenate reveals two proteic fractions of low concentration, of about 210 and 190 k D a respectively, which are lacking in the male gland.
602
B. VARRIALEet al.
o
o
kDa kDa
205_ 116_
9
205. 116_
84_
84_
47_ 33_ 24_
16_
Fig. I. SDS 7-12% gradient PAGE of Harderian gland proteins. In the male whole gland homogenate the arrows indicate the 116 and 168 kDa fractions. In the female whole gland homogenate the arrows indicate the 210 and 190 kDa fractions. Values on the left line indicate the molecular weight of standard proteins (kDa). Analysis of the glycoproteic pattern (Fig. 2) suggests that the male-specific fractions (168 and 116 kDa) are glycoproteins. The two proteic fractions present only in the female gland (210 and 190 kDa) were both Alcian-negative. As regards the proteic pattern of the experimental groups, castration caused a significant decrease in the concentration of the 168 and l l 6 k D a fractions (Fig. 3). Their relative concentrations were 4.23 + 0.56% for the 168 kDa fraction and 1.49 + 0.23% for the 116 kDa fraction with respect to the relative concentrations of sham-operated males, which were 12.12 + 0.54% (168 kDa) and 2.85_ 0.13% (116 kDa). Replacement therapy, with testosterone, restored the intact male proteic pattern (7.69_ 0.66% and 3.62 + 0.69% respectively) (Table 1). Our observations on the sexual dimorphism of the electrophoretic protein pattern are in agreement with those previously reported by Hoh et al. (1984), where the sex-specific fractions were named HGP1
47 33
Fig. 2. SDS 7-12% gradient PAGE of Harderian gland glycoproteins in male and female whole gland homogenate. Arrows indicate the glycoproteins characteristic of the male pattern which correspond to those shown in Fig. 1. Values on the left line indicate the molecular weight of standard proteins (kDa). (168 kDa) and HGTP1 (116 kDa) in the male, and HGP3 (210 kDa) and HGP4 (190 kDa) in the female. However, an additional fraction (HGP2), isolated by these authors in the male gland only, was found occasionally in the female in our study. Hoh et al. (1984) also carried out electrophoresis on the tubular fraction obtained from sucrose gradients of the original homogenate pellet. This fraction produced two major proteins designated HGTP1 and HGTP2, of 115 and 8 kDa respectively. Two minor proteins with molecular weights of 102 and 95kDa were also identified. In our case, the female gland did not contain any tubular protein. Therefore the two proteic fractions which differentiate the male from the female gland correspond to those previously named HGP1 and HGTP1 by Hoh et al. (1984), the former probably being a structural component and the seco n d a tubular protein component. The presence of membrane-bound clusters of cylindrical tubules in the cytoplasm of the acinar cells is one of the characteristics of the male Harderian gland of the hamster. Bucana and Nadakavukaren (1973) have suggested a possible relationship between the presence of these tubules and the breakdown of porphyrin granules, while Jones and Hoffman (1976) suggested that peritubular complexes may prevent porphyrin biosynthesis by inhibiting the condensation of 6-aminolevulinic acid to porphobilinogen. In fact, in the maturing male, the disappearance of pigment granules coincides with the appearance of
603
Hamster Harderian gland proteins a
b
1. Percentages of total amount of the malespecific proteins loaded on gels, expressed as means + SD Experimental Proteic fractions groups 116 kDa 168 kDa Sham-operated 2.85 + 0.13= 12.12+ 0.54= Castrated 1.49 + 0.23b 4.23 + 0.56b Castrated + T-injected 3.62 + 0.69~ 7.69 + 0.66c
Table
O
kDa 205_ 116_
a vs b: p < 0.01; b VS c: p < 0.01. REFERENCES
84_
47_ 33_ 24_ 16_
Fig. 3. SDS 7-12% gradient PAGE of Harderian gland proteins in the different experimental groups of male golden hamster; (a) sham-operated control; (b) castrated animals; (c) castrated and testosterone-injected animals. Arrows indicate the proteic fractions affected by castration and replacement therapy. Values on the left line indicate the molecular" weight of standard proteins (kDa). tubular clusters (Bucana and Nadakavukaren, 1973), and in the female, testosterone treatment mimics the same phenomenon (Sun and Nadakavukaren, 1980; Spike et al., 1985). The presence of androgen receptors in the Harderian gland of the male hamster is consistent with the testosterone dependence of the gland in the male hamster (Vilkis et al., 1988; Vilkis and Perez-Palacios, 1989). In this report, the testosterone dependence of sex-specific glycoprotein synthesis of the male hamster Harderian gland is conclusively demonstrated. In fact, castration causes a significant decrease of sex-specific protein synthesis, which is prevented by testosterone therapy. Recently, the testosterone dependence of Harderian gland-specific glycoproteins has also been demonstrated in the green frog, Rana esculenta (Chieffi et aL, 1991). Acknowledgements--This work was supported by MURST (40% and 60% grants) and CNR (91.00476.CT04 Grant).
Bucana C. D. and Nadakavukaren M. J, (1973) Ultrastructural investigation of the postnatal development of the hamster Harderian gland. II. Male and female. Z. Zellforsch, mikrosk. Anat. 142, 1-12. Chieffl G., Chieffi Baccari G., Di Matteo L., d'Istria M., Marmorino C., Minucci S. and Varriale B. (1991) Hormonal control of the Harderian gland of Rana esculenta. Belg. J. Zool. 121, 130-132. Christensen F. and Dam H. (1953) A sexual dimorphism of the Harderian glands in hamster. Acta physiol, scand. 23, 333-336. Cowman M. K., Slahetka M. F. Hittner D. M., Kim J., Forino M. and Gadelrab G. (1984) Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosoaminoglycan oligosaccharides. Biochem. J. 221, 707-716. Grafflin A. L. (1942) Histological observations upon porphyrin-excreting Harderian gland of the albino rat. Am. J. Anat. 71, 43~o4. Hoffman R. A. (1971) Influence of some endocrine, hormones and blinding on the histology and porphyrins of the Harderian gland of golden hamster. Am. J. Anat. 132, 463-478. Hoh J. H., Lin W. L. and Nadakavukaren M. J, (1984) Sexual dimorphism in the Harderian gland proteins of the golden hamster. Comp. Biochem. Physiol. 77B, 729-731. Jones C. W. and Hoffman R. (1967) Porphyrin concentration of the hamster Harderian gland. Effect of incubation with 6-ALA and various hormones. Int. J. Biochem. 7, 135-140. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 2 2 7 , 680q585. Lowry O. H., Rosebrough J. N., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Payne A. P., MeGadey J., Moore M. R. and Thompson G. G. (1977) Androgenic control of the Harderian gland in male golden hamster. J. Endocr. 75, 73-82. Sakai T. (1981) The mammalian Harderian gland: morphology, biochemistry, function and phylogeny. Arch. Histol. Jap. 44, 299-333. Spike R. C., Johnston H. S., McGadey Y., Moore M. R., Thompson G. G. and Payne A. P. (1985) Quantitative studies on the effects of hormones on structure and porphyrin biosynthesis in the Harderian gland of the female golden hamster. I. The effects of ovariectomy and androgen administration. J. Anat. 142, 59-72. Sun C. Y. and Nadakavukaren M. J. (1980) Effect of testosterone on the female hamster Harderian gland pigmentation and ultrastructure. Cell Tissue Res. 207, 511-517. Svedberg T. and Pedersen K. O. (1940) The Ultracentrifuge pp. 296-327. Oxford University Press, Oxford. Vilkis F., Hernandez A., Perez A. E. and Perez-Palacios G. (1988) Hormone regulation of the rodent Harderian gland: binding properties of the androgen receptor in the male golden hamster. J. Endocr. 112, 3-8. Vilkis F. and Perez-Palacios G. (1989) Steroid hormone receptors and sexual phenotype of the Harderian gland in hamster. J. Endocr. 121, 149-156.