Porphyrin concentration of the hamster (mesocricetus auratus) harderian gland: Effects of incubation with delta-aminolevulinic acid and various hormones

PORPHYRIN CONCENTRATION OF THE HAMSTER (A4ESOCRICETUS AURATUS) HARDERTAN GLAND: EFFECTS OF INCUBATION WITH DELTA-AMINOLEVULINIC ACID AND VARIOUS HORMONES CARL W. JONES* Department

of Biology,

AND

ROGER A. HOFFMAN

Colgate

University,

Hamilton,

(Recriwd

5 January

1976)

NY 13346, U.S.A.

Porphyrins were measured by fluorescence in 24 hr incubates of hamster Harderian tissue. 2. The addition of delta-aminolevulinic acid (ALA) to incubated Harderian glands caused highly significant increases in both tissue and media porphyrin concentrations, 3. Neither gonadotrophins, estradiol benzoate, testosterone propionate nor melatonin, when added with ALA to the incubation media, had significant effects within 24 hr on tissue or media concentrations of porphyrins. 4. The data support the conclusion that male Harderian tissue is deficient in ALA and that this precursor induces active release of porphyrins from both male and female tissue.

Abstract-l.

INTRODUCTION

1973, 1974); yet the specific regulators have not been identified. For example, although the male gland becomes morphologically female in type, and increases in porphyrin concentrations following castration (Woolley & Worley, 1954; Hoffman, 1971; Wetterberg, 1972; Clabougb & Norvell, 1973), or following estrogen implants in the untreated male (unpublished observation), it is not known whether estrogens enhance, or androgens inhibit, porphyrin biosynthesis in the normal hamster, nor whether the observed changes are due primarily to direct effects of changed gonadotrophin concentrations. Incubation techniques allow in vitro assessment of the direct effects of various invididual substances on the porphyrin concentration of the gland. This report specifically identifies the effects of incubation with delta-aminolevulinic acid (ALA), the presumed precursor to the porphyrins, and with the additional incubation with either gonadotrophins, estrogen, androgen, or melatonin, on the porphyrin concentration of hamster Harderian glands.

In the hamster, the Harderian glands are bilobed compound tubuloalveolar glands located deep within the orbit of each eye. In contrast to rats (Kelenyi & Orban, 1965), mice (Woodhouse & Rhodin, 1963), and the rabbit (Bjorkman et u!., 1960; Kuhnel, 1971) which have no reported ultrastructural sex dimorphisms, the hamster Harderian gland possesses dis-

tinct ultrastructural as well as gross and microscopic dimorphism between males and females (Christensen & Dam, 1953; Woolley & Worley, 1954; Paule et ul., 1955; Hoffman, 1971; Bucana et al., 1972~; Clabough & Norvell, 1973). Harderian glands of males contain no observable pigments, low porphyrin concentrations, and two cell types: Type 2, containing coarse vacuoles characteristically present only in males. and Type 1, seemingly similar to the single cell type in females, containing much finer vacuoles (Hoffman, 1971). The grossly pigmented glands of females contain much greater porphyrin concentrations. Ultrastructurally, Bucana et uI. (1972a) have reported the presence of unusual membrane-bounded clusters of cylindrical tubules in both secretory cell types of only the males, while an extensive cytoplasmic membrane system in the secretory cells of only the females was observed. In the last several years, various in uiuo endocrine manipulations have been performed to elucidate the regulatory mechanisms responsible for these differences in morphology and presumed function (Hoffman, 1971; Wetterberg, 1972; Clabough & Norvell,

MATERIALS AND METHODS Young adult golden hamsters (Me.socricerus auratus), about 67 weeks of age (8GllO g), born and raised in this laboratory on a diurnal light schedule of 14 hr light, 10 hr dark (L:D, 14:10), were anaesthetized and decapitated. Under a plexiglass hood, previously wiped with alcohol, the heads were cleansed with alcohol and the Harderian glands bilaterally removed. Since the smaller superior lobe and region adjacent to the hilum differs histologically from the rest of the gland (Clabough & Norvell. 1973). the posterior portions of only the infehor lobes were taken and divided in half for these experiments. One portion was immediately

* Present address: Department of Anatomy, University of Illinois at the Medical Center. Chicago. IL 60680. U.S.A. 135

CARL W. JONES AND ROGER A. HOFFMAN

136

Table 1. Effects of 24 hr incubation of hamster Harderian glands with delta-aminolevulinic acid (ALA) and ALA plus gonadotrophins (GT) on the porphyrin concentration of tissue and media (mean zt: SEM). See text for statistical analysis. Each group consists of eight cultures

MAC

Media Medn I

Medxa + ALA 10.2) Media + ALA (0.31 Media + ALA (0.4)

1.25 + 0.16 1.26 + 0.14 I52 IO46 I24 * 0.17 1.3Y 2 0.20

0.9 12.0 16.2 13.6 15.3

Media Medza

+ ALA (IX21

I61 i_ I8 17h * 11

202 i 17 237 * 19’

i4lP +61’

2 3 ir 0.6 71 x i 18.2’

204 * IX 309 i 29

Media Medu

+ ALA (0.2) + ALA (0.2)

104 + 0.5 10.8 * 0.5

+X.3’ +90*

29 2 f 3.1 26.3 i 2.3

3Y 6 * 3.4 37.1 It 2x

+ ALA (0.1)

i * k + i

0 I 0.3’ 0.5’ 0.3’ 0.7

-0.4* +107’ + 14.7’ + 12.4’ + 13.9’

0.1 x9.1 105.1 6X.3 88.9

+ + f i I

0.0 2 9’ 3.9” 4 2’ 6.6”

IO 101 1 121.3 x2.4 104.2

* + f + +

0.1 26 4.1 4 3 6.X

Female 2 M2.k 3

2.05 5 0 7.8 1.85 * 0.34

+GT

’ Statistical analysis in first experiment between successive groups only. b The net difference between incubated and unincubated tissue porphyrin concentrations. ’ The combined incubated tissue and media porphyrin concentrations. Not statistically d P < 0.05. r P < 0.01.

assayed for initial porphyrin concentration, the other was placed on a sterile stainless steel screen in a pre-sterilized tissue culture dish containing culture media, incubated at 37 i 2°C in a humidified atmosphere of 95% air, 5% COs, and assayed for porphyrins after 24 hr. Thus each animal served as its own control. Each group consisted of eight cultures. Since there were no differences in the initial porphyrin concentrations between those groups incubated with increasing doses of ALA or ALA plus a gonadotrophin solution (Table 1). and since the addition of ALA to the incubation media always caused highly significant increases in porphyrin concentrations after 24 hr (Difference column, Table l), in all subsequent experiments, half of the Harderian tissue from each gland was incubated with ALA (control group); the other was incubated with ALA plus various hormones (experimental groups; Table 2). One and one-half ml of a sterile stock solution containing 100 ml of Media 199--1X, 5 ml of fetal calf serum, and 2.5 ml of antibiotic-antimycotic solution (Grand island Biol. Co., NY) was added to each incubation dish in addition to varying volumes of ALA stock (2 mg ALA-Sigma Chem. Co., St. Louis, MO-per l.Oml media). In the ex-

analysed.

perimental groups, estradiol benzoate, testosterone propionate, and melatonin (Regis Chem. Co., Morton Grove, IL), dissolved in 950/, ethanol, and a gonadotrophin solution comprised of HCG and FSH dissolved in saline, were added in 0.05 or 0.1 ml vol to the culture dishes in addition to media and ALA. In every case, the same volume of experimental solvent was added to the control cultures. Porphyrins were measured by fluorescence above 470nm as previously described (Hoffman, 1971). Briefly, in both unincubated and incubated samples, Harderian tissue (10-50mg) was homogenized in ethyl acetate:acetic acid (4:l) and diluted with 1.5 N HCi. The diluted homogenate was assayed directly in a Turner fluorometer, Model 110, fitted with appropriate primary and secondary filters (Nos. 110-812; 405, Nos. 110-820; 25) against a tetramethyl coproporphyrin standard (0.05 pg/ml) to determine porphyrin concentrations. The incubation media was also assayed for porphyrin concentration by direct dilution with HCI. In these experiments, no correction factor was applied for saturation of the HCI with ethyl acetate. Because each animal served as its own control, data were statistically analyzed with the matched pairs t test as described in Klugh (1970). Mean concentrations from the dif-

Table 2. Effects of 24 hr incubation of male hamster Harderian glands with ALA (0.2 trill stock) plus various hormones on the porphyrin concentration of tissue and media (means & SEM). Eight cultures/group

Exp. No.

.I

6

Media Media Med,a Media Medh

+ + + + +

ALA ALA + E.B. ALA ALA i T.P. ALA

Media+ ALA + MEL

101 iI0 10.2 * 0.7 l, i * 0.6 132”07 11.0 2 07

35.5 31)x 681 62 9 545

it.1 + 0.6

60.3i 5.1

ALA. delta-aminolevulinic acid; E.B., estradiol benzoate; MEL. melatonin. u The combined tissue and media porphyrin concentrations.

T.P.,

It 4.9 + 60 li_60 I 4.6 i- 40

testosterone

45.6 50.0 792 76.1 65.5

i + + 2 i

5.8 64 6.1 5.0 44

71.4i 5.0

propionate;

Porphyrin concentration ferent groups in the first experiment were compared with the student’s t test. RESULTS

While tissue from males decreased in porphyrin concentration after incubation in media alone for 24 hr (P < 0.05), there was a highly significant increase (P < 0.01) in tissue incubated with 0.1 ml ALA stock (Experiment 1, Table 1); the female incubates, however, increased in porphyrin concentration regardless of the presence or absence of ALA, even though a greater increase occurred (P < 0.01) with the addition of ALA (Experiment 2, Table 1). Incubation of tissue from both males and females with ALA also caused a significant increase (P < 0.01) in media porphyrin concentration (Table 1). Since previous experiments demonstrated that male Harderian tissue incubated with ALA contained about one-tenth as much porphyrin as the female, larger amounts of ALA stock (0.2ZO.4ml) were utilized to determine the optimal concentration of this substrate, and to ascertain whether higher doses could induce porphyrin concentrations typical of normal females. Although differences were found in both tissue and media porphyrin concentrations between groups incubated with successively larger amounts of ALA, except in tissue porphyrin concentration between the 0.3 and 0.4 ml ALA groups (Experiment 1, Table l), the addition of 0.14.4 ml ALA stock to the male incubates produced grossly similar increases. The addition of 0.2 ml ALA caused the highest tissue and media porphyrin concentrations, and was used in all further experiments (Experiments 36, Tables 1 and 2). Apparently larger doses of delta-aminolevulinic acid will not induce porphyrin concentrations typical of the normal female hamster when added to male incubates. Because previous reports suggested that increased gonadotrophin secretion might cause both the morphological changes from male to female type of gland following castration (Hoffman, 1971; Clabough & Norvell, 1973), and result in much higher porphyrin concentrations, male Harderian tissue was incubated with either ALA or ALA plus a gonadotrophin solution (50 iu. HCG + 1 mgFSH/culture). After 24 hr, there were no differences between groups in either incubated tissue or media porphyrin concentrations, and the total porphyrin concentrations were still far less than those of normal females (Experiment 3, Table 1). Possibly, estrogens enhance porphyrin synthesis. Consequently, male Harderian tissues were incubated with ALA or ALA plus estradiol benzoate (5 pg/culture). Again the experimental treatment had no effect over the 24 hr incubation period; no differences in either incubated tissue or media porphyrin concentrations were evident (Experiment 4, Table 2). Castration causes a gradual loss of Type 2 cells in the male hamster, and the Harderian glands

of the hamster

137

become morphologically female in type with concurrent increases in porphyrin concentrations (Hoffman, 1971; Wetterberg, 1972; Clabough et al., 1973). Thus it is possible that testosterone suppresses the synthesis of porphyrin in the male gland. Therefore, Harderian tissue was incubated with ALA or ALA plus testosterone propionate (500 pg/culture). No difference between groups was found in incubated tissue or media porphyrin concentrations (Experiment 5, Table 2). Clabough and Norvell (1973, 1974) have suggested that heightened pineal activity may cause a decrease in the formation or storage of porphyrins in the hamster gland. Consequently, one might expect a decreased porphyrin production in Harderian tissue incubated with ALA plus melatonin, a pineal constituent. There were no differences in either incubated tissue or media porphyrin concentrations between the ALA-incubated control, and tissue incubated with both ALA and melatonin (1 pg/culture; Experiment 6, Table 2). Apparently this dose of melatonin has no effect on porphyrin synthesis in the male hamster Harderian gland over the 24 hr incubation period. DISCUSSION

is apparent that substantial variation in porphyrin concentrations exists both within and between experiments of this study. Undoubtedly the true porphyrin content of the hamster Harderian gland varies greatly between animals of the same age and sex. In these experiments, young adult male hamsters varied as much as 2.9 pg/lOO mg tissue, while littermate females varied more than 100 pg/lOOmg tissue. In spite of this, the addition of delta-aminolevulinic acid to the incubation media consistently induced increases in the porphyrin concentration of cultured male tissue; without ALA, the porphyrin concentration decreased. Furthermore. female tissue increased in porphyrin concentration upon incubation regardless of the presence or absence of ALA in the media. This suggests that one of the fundamental differences between male and female hamster Harderian glands, the relatively low porphyrin concentration in the male, may be caused, in part at least, by a lack of available ALA in this tissue. Assuming that the increase in porphyrin concentration of these incubated tissues is the result of rle no00 synthesis due to the addition of ALA, then the production of delta-aminolevulinic acid might be normally repressed in the male gland. In the mouse Harderian gland, the rate-limiting enzyme in porphyrin synthesis is believed to be ALA synthetase (Margolis, 1971); a decreased production of this enzyme, or its rapid inactivation in the male hamster could account for the characteristically lower porphyrin concentrations, In either case, the addition of increasingly larger doses of ALA should result in progressively greater porphyrin concentrations, a result not observed in these experiments at the concentrations It

138

CARL

W.

JONES AND

utilized. Furthermore, the total porphyrin concentrations are much less than those of normal females. Perhaps ALA can only be produced and utilized for porphyrin synthesis in certain cells. Recently, Clabough et al. (1973) reported a marked increase in Type 2 cells in the Harderian glands of simultaneously blinded~astra~d male hamsters without the appearance of porphyrin, and moderate to heavy porphyrins in blind + castrated + pinealectomized hamsters which contained Type 1 cells exclusively. These data are suggestive of a correlation between Type 1 cell concentration and porphyrins. If ALA were produced and utilized only in Type 1 cells, which comprise half the cells present in male tissue (Clabough et al., 1973, 1974), one might expect the porphyrin concentrations to increase to about half those of female tissue, which is comprised only of this cell type. The total porphyrin concentrations of male, ALA-supplemented incubates do approximate these values, and thus we cannot yet exclude this possibility. Presently we know of no reports of possible differences in secretory product between the two cell types. A correlation between the appearance of microtubular clusters and the disappearance of pigment granules (porphyrins) in the maturing male hamster, and the absence of these tubules in females of any age, has led Bucana et ul. (1973) to suggest that these tubules contain those enzymes necessary for breakdown of pigment molecules. Since the difference in porphyrin concentration between unincubated tissue, and tissue incubated with ALA suggests that ALA is normally inactivated or degraded in the male hamster Harderian gland, these membrane-bounded tubular clusters might also have some function in limiting the amount of ALA available for porphyrin production. The observations that these tubules are often found in the vicinity of mitochondria (Bucana et al., 1973), and that ALA is synthesized within. while porphobilinogen (PBG) is synthesized outside the mitochondrion (Tomio & Grinstein, 1968) would suggest that these tubules might inhibit the condensation of ALA to PBG. This would account for low porphyrin concentrations independent of cell type, and might also help explain the variability in porphyrin concentrations observed while incubating with increasing amounts of ALA in the first experiment. Furthermore, incubation of female tissue with ALA could be expected to cause relatively greater increases in porphyrin concentrations than in males, a result observed in the present studies. Porphyrins present in the incubation media could represent tissue secretion or a “leeching” effect. Significant amounts of porphyrins are present in the media only when tissue had been incubated with deltaaminolevulinic acid; tissue incubated in media alone released extremely small amounts. Furthermore, even though female Harderian tissue increased in porphyrin concentration after 24 hr incubation without ALA. there was little porphyrin present in the media;

ROGERA. HOFFMAN media in which female tissue was incubated with ALA significantly increased in concentration of porphyrin. If the porphyrin present in the media were due to a “leeching” effect, then much more porphyrin would be expected in the media of female tissue incubated without ALA. Thus the porphyrin present in the media probably represents active tissue secretion. Evidently ALA promotes active secretion in both sexes. The reason for this remains obscure. We have not answered the question of whether hormones directly affect porphyrin biosynthesis in the Harderian gland of the male golden hamster, or whether they modify cellular constituents, which themselves are responsible. These experiments do indicate only that the hormones studied have no measurable effect on porphyrin synthesis induced by the addition of ALA within the 24 hr incubation period. A longer incubation period, or more appropriate dose levels of these hormones may be required to demonstrate unequivocal effects directly on Harderian tissue. This culture procedure could, however, be utilized to determine early morphological changes in male hamster Harderian tissue associated with increased porphyrin production, Acknvtute~~rr?~~nrs--Partial support for these studies has been furnished by the McGrorty Foundation and by the Colgate Research Council.

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