Pretranslational Regulation of Sex-dependent Testosterone Hydroxylases by Growth Hormone in Mouse Liver*

The effects of growth hormone on the expression of sex-dependent testosterone 16a- and 15a-hydrox- ylases were studied in growth hormone-deficient Little (litflit) mice at the activity as well as at the mRNA levels. The male isozyme of testosterone 16a-hydrox-ylase (“C”-P-45OIh) was repressed in the liver of male lit/lit mice, and the injection of bovine growth hormone resulted in an increase of the isozyme at both activity and mRNA levels to those seen in control lit/+ male mice. On the other hand, the female isozymes of testosterone 16a- (“I”-P-45Olk) and 15a-hydroxylase (P- 4 5 0 1 ~ ~ ) were increased in livers of both male and female lit/lit mice. The increased I-P-45OlS, and P-45Ola, in lit/lit mice were suppressed by growth hormone but only when it was injected once every 12 h. Thus, the results indicate that growth hormone acts as a masculinizing factor for testosterone hydroxylase activity by activating and inhibiting the expression of male and female isozymes of testosterone hydroxylases in mice, respectively. When growth hormone was infused to simulate a continuous secretion pattern, it showed no significant effect on the expression of hydroxylases in lit/lit mice, suggesting that growth hormone may not be a feminizing factor for testosterone hydroxylase activity in female mice. The changes of specific hydroxylase activities modulated by growth hormone in the mice correlated well with those amounts of hydroxylase mRNAs. The action of exogenous growth hor- mone

The effects of growth hormone on the expression of sex-dependent testosterone 16a-and 15a-hydroxylases were studied in growth hormone-deficient Little (litflit) mice at the activity as well as at the mRNA levels. The male isozyme of testosterone 16a-hydroxylase ("C"-P-45OIh) was repressed in the liver of male lit/lit mice, and the injection of bovine growth hormone resulted in an increase of the isozyme at both activity and mRNA levels to those seen in control lit/+ male mice. On the other hand, the female isozymes of testosterone 16a-("I"-P-45Olk) and 15a-hydroxylase (P-4 5 0 1~~) were increased in livers of both male and female lit/lit mice. The increased I-P-45OlS, and P-45Ola, in lit/lit mice were suppressed by growth hormone but only when it was injected once every 12 h. Thus, the results indicate that growth hormone acts as a masculinizing factor for testosterone hydroxylase activity by activating and inhibiting the expression of male and female isozymes of testosterone hydroxylases in mice, respectively. When growth hormone was infused to simulate a continuous secretion pattern, it showed no significant effect on the expression of hydroxylases in lit/lit mice, suggesting that growth hormone may not be a feminizing factor for testosterone hydroxylase activity in female mice. The changes of specific hydroxylase activities modulated by growth hormone in the mice correlated well with those amounts of hydroxylase mRNAs. The action of exogenous growth hormone to regulate the hydroxylases was so slow that it took 2 days to show a significant effect.
Sexual dimorphism of steroid hydroxylase activities in liver microsomes is a direct reflection of differentiated expression of cytochrome P-450s specific for the activities. Cytochrome P-450 represents a group of terminal oxidases of the membrane-bound monooxygenase system which consists of NADPH-cytochrome P-450 reductase, cytochrome b5, and NADH-cytochrome-b5 reductase.
In inbred mice there are at least two known isozymes ("1"and "c"-P-45016,) of testosterone 16a-hydroxylase whose expressions are sexually differentiated; I-P-45Ol6, is a femalespecific isozyme whereas c-P-45016, is a male-predominant isozyme (1-3). The presence of female-predominant testosterone l5a-hydroxylase activity in mice was also known, and the form of cytochrome P-450 (P-45015,) specific for the 15ahydroxylase activity has been purified and characterized from female mice (4-6). Furthermore, we have recently demon-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. strated by using the hydroxylase cDNAs as hybridization probes that the sex-dependent expression of these hydroxylases is regulated pretranslationally (5, 7).
As with many liver proteins such as MUP, ap,-globulin, prolactin receptors, and drug oxidases, the sexual differentiation of steroid hydroxylase activity is under multihormonal control. In addition to sex hormones, increasing attention has been given to the role of growth hormone in regulation of sexual dimorphism of steroid hydroxylase activities in liver microsomes and other liver functions (8-10). Norstedt and Palmiter (11) reported that a pulsatile secretion of growth hormone masculinizes expression of MUP and prolactin receptor, while a continuous secretion of growth hormone feminizes their expression in mice. Here we report the use of specific inhibitory antibodies and cDNAs for the mouse hydroxylase isozymes (Iand c-P-45016, and P-45Ol5,) to investigate the regulatory role of growth hormone in the sexdependent expression of these hydroxylases.
Growth hormone-deficient Little mice were used for the present study. Unlike other growth-defected mouse strains such as Snell and Ames or hypophysectomized animals, Little mice have a defect only in growth hormone, with normal levels of other pituitary hormones such as thyroid-stimulating hormone, prolactin, luteinizing hormone, and follicle-stimulating hormone (12), providing the most suitable animal model to investigate the regulation of growth hormone (11,13). The role of growth hormone as a masculinizing but not feminizing factor toward expression of I-and c-P-45016, and P-45015, in mice is discussed in this paper.
Treatments with Hormones-For injection, bovine growth hormone (bGH') was dissolved in 10 mM Tris-HC1 buffer, pH 8.0, containing 0.9% NaCl at a concentration of 250 pg/ml. Two hundred pllmouse of the bGH solution were administered intraperitoneally once every 12 h for 5 consecutive days. The same volume of Tris buffer was injected into control mice. lation kits were obtained from Amersham Corp. ABM and nitrocellulose paper were products from Schleicher and Schuell (Keene, NH). Oligo(dT)-cellulose was purchased from Collaborative Research (Lexington, MA). Somatomedin C was obtained from AM Gen Biochemicals (Thousand Oaks, CA). bGH was a gift from the Pituitary Agency (Baltimore, MD). Minipumps (model 2001) were purchased from Alza Corp. (Palo Alto, CAI. All other enzymes and reagents used in this study were of the highest quality commercially available. Purification of Mouse Testosterone Hydroxylases and Their Antibodies-The two isozymes (C-and I-P-4501,) of testosterone 1601and l5a-hydroxylase (P-45015,) were purified from 129/J mice as described previously (1-2, 4). The antibodies elicited against each purified hydroxylase have been characterized for their specificity for inhibiting the activity due to the homologous antigen (1)(2)(3)(4)(5).
Isolation of Total Liver Poly(A+) RNA and Preparation of Liver Microsomes-Mouse livers for each group of experiments were pooled and minced. The minced livers were divided into two parts, and microsomes and total mRNA were prepared from the separate parts. Liver microsomes were prepared as described previously (1-4) and homogenized in 50 mM potassium phosphate buffer, pH 7.25, containing 20% sucrose, 1 mM EDTA, and 2 pg/ml leupeptin. Total liver RNA was extracted by the guanidine HCl method (14) and subjected to oligo(dT)-cellulose column chromatography to enrich poly(A+) RNA (15).
Northern and Dot Hybridization-Poly(A+) RNA was electrophoresed on 1% agarose gel containing 5 mM methyl mercury hydroxide (16) and transferred to DBM paper. The DBM paper was hybridized with radioactive cDNA probes under the conditions described previously (17). The washed paper was then exposed to x-ray films. One pg of total poly(A+) RNA from mouse livers was spotted on nitrocellulose paper according to the procedure described by Norstedt and Palmiter (11). The paper was hybridized with the radioactive cDNA probes under the conditions reported previously (3). The hybridization was visualized by exposing the paper to x-ray films. The areas hybridized were cut, and their radioactivities were measured by scintillation counting. The spots were duplicated for every poly(A+) RNA sample on the paper, and the radioactivities recovered from the hybridization were averaged for representation of relative amounts of each hydroxylase mRNA. Under these experimental conditions, the amounts of polv(A+) RNA spotted and of hybridization are linearly correlated (3).
Other Anulvtical Methods-Testosterone hvdroxvlase activities were measurei by methods previously reported (1, f). Assay conditions with the antibodies to estimate the specific isozyme-dependent hydroxylase activities were also described previously (3, 4). Total cytochrome P-450 content and protein concentration were determined by the methods described by Omura and Sat0 (18) and Bradford (19), respectively.

RESULTS
Effect of bGH Injection on Specific Testosterone Hydroxylase Activities in Microsomes-"Little" is a mutant mouse strain raised naturally from C57BL/6J which has an autosomal recessive inheritance of growth defect (20). This growth defect is apparently due to a poor transcriptional rate of the growth hormone gene which is regulated by a locus on chromosome 6. The serum growth hormone levels were reported to be 8. 5 and 0.6 ng/ml in males of hetero-(lit/+) and homozygote (lit/ lit), respectively, and these values in lit/+ and lit/lit females were 2.85 and 0.05 ng/ml, respectively (21). Fig. 1 shows the activities catalyzed by each isozyme of testosterone hydroxylase in liver microsomes from lit/+, lit/ lit, and bGH-injected lit/lit mice. Testosterone 16a-hydroxylase activity catalyzed by 1-P-45016, (panel A) was at least 10-fold higher in female than in male lit/+ mice. The specific activity in the lit/lit males was higher than that seen in lit/+ females. I-p-45Ol6,-dependent activity in liver microsomes from lit/lit females was also elevated about 2.5-fold over that in the lit/+ females (Fig. L4). Consequently, the sexual di-  1. Testosterone hydroxylase activities in liver microsomes from control and bGH-injected Little mice. Liver microsomes were prepared from three control and growth hormone-injected lit/+ and lit/lit males and females (see "Experimental Procedures"). I-P-45OI6,-and C-P-45016,-dependent testosterone 16a-hydroxylase activities and P-45OI5,-dependent testosterone 15a-hydroxylase activity were measured by using specific inhibitory antibodies as described in previous reports (1, 2). One-dimensional thin-layer chromatography was used to separate radioactive testosterone metabolites formed by microsomes. Panel A shows I-P-45OI6,-dependent activity; Panel B, C-P-45016,-dependent activity; Panel C, P-45OI5,-dependent activity. The values of hydroxylase activities in this figure were averaged from three separate experiments. The variation of hydroxylase assay was less than 5%.
morphism of the hydroxylase activity due to I-P-45016, was not present in lit/lit mice. To determine whether or not the enhancement of the activity in lit/lit males as well as in lit/ lit females is due to the deficiency of growth hormone in this mouse strain, bGH was injected once every 12 h for 5 consecutive days. As shown in Fig. lA, the injected bGH decreased the activity to a level lower than that seen in the lit/+ females, indicating that the expression of female-predominant I-P-45OI6,-dependent activity in male lit/lit mice is indeed due to growth hormone deficiency. It should be emphasized that the growth hormone deficiency resulted in the induction of female-specific hydroxylases in lit/lit female mice, which implied a complexity of mechanisms by which this testosterone hydroxylase activity is feminized in normal female mice. Testosterone l5a-hydroxylase activity catalyzed by femalepredominant P-45Ol5, was modulated by growth hormone in the same way as was I-P-45OI6,-dependent activity (Fig. IC). The repression by growth hormone, therefore, represents a general regulatory mechanism for other female-specific hydroxylases in male mice.
Contrary to the growth hormone-dependent repression of the female-predominant hydroxylase activities, the expression of C-P-45016,-specific 16a-hydroxylase activity, which is male predominant, was growth hormone dependent in the male mice (Fig. 1B). The specific activities in lit/lit male mice were decreased to the activity levels seen in lit/+ and lit/lit female mice. The bGH injection resulted in an increase of c-P-45016,dependent activity in females as well as in males of lit/lit mice to that found in untreated lit/+ males.
Effects of bGH Injection on Amounts of Testosterone Hydroxylase mRNAs- Fig. 2 shows the hybridization of total A .

8.
C. Procedures") were electrophoresed on 1% agarose gels containing 5 mM methyl mercury hydroxide and transferred to DBM paper. The electrophoresis was run from top to bottom. The DBM paper was hybridized with nick-translated 32P-cDNA probes (1 X 10' cpm of total radioactivity; R17, p16a-1 or p15a-29), and then the paper was properly washed and exposed to x-ray films. The numbers on the right-hand side of the figure indicate the sizes of P-450 mRNAs. Panel A, hybridization of I-P-45016, with R17; Panel B, hybridization of C-P-45016, with p16a-1; Panel C, hybridization of P-45Ol5, with p15a- 29. liver poly(A+) RNA from various mice with 32P-labeled cDNA probes (R17, p16a-1, and p15a-29). As expected, the amounts 1-P-45016, (Fig. 2 A ) and P-45Ol5, (Fig. 2C) mRNAs were much higher in the females than in the males of C57BL/6J and lit/ + mice, which is consistent with the female-specific expression of hydroxylase activities catalyzed by these isozymes. The amounts of c-P-45016, mRNA were higher in males than in females in these mice (Fig. ZB), which also agreed with the C-P-45OI6,-dependent activities in the mice.
The increased expression of 1-P-45016, and P-45Ol5, mRNA that hybridized with the probes was seen in male and female lit/lit mice, and this was consistent with the increases of the hydroxylase activities in these mice. These increases of mRNA in the lit/lit mice were counteracted by injection of bGH in both males and females.
The content of c-P-45016, mRNA in lit/+ males was decreased to similar to that found in female lit/+ and lit/lit mice. Injection of growth hormone into lit/lit mice enhanced the mRNA in the males and females to those found in male lit/+ and C57BL/6J mice. These modulations of c-P-45016, mRNA by growth hormone were consistent with those of C-P-45016,-dependent hydroxylase activity.
A good correlation of the amount of 1-P-45016, mRNA with the activity levels in the various mice used in the present experiments is demonstrated in Fig. 3. Similar correlations were also obtained in the case of c-P-45016, and P-45Ol5,

TABLE I Effect of infused growth hormone on testosterone hydroxylases in
Little mice Liver microsomes and total liver poly(A+) RNA were prepared from bGH-infused and control lit/lit mice. Two mice were used for each group. Testosterone 16a-hydroxylase activity in microsomes was measured in the presence and absence of anti-I-P-45016, or anti-C-P-45016a antibodies. The isozyme-specific activity was calculated by subtracting the activity with the antibody from the activity without the antibody. The assay conditions were described under "Experimental Procedures." The mRNA contents for the hydroxylases are expressed by the average radioactivity recovered from the two separate dot hybridizations of poly(A+) RNA with the appropriate cDNA probe: R17 for I-P-45016, and pl6a-1 for c-P-45016, (see "Experimental Procedures"). Control bCH infusion (data not shown). Pretranslational regulation is most likely a mechanism of the growth hormone-dependent modulation of these hydroxylases. Effect of bGH Infusion on Expressions of Testosterone Hydroxylases-The activity of Iand c-P-45016, in bGH-infused lit/lit and lit/+ mice and the radioactivity recovered from dot hybridization for the hydroxylase mRNAs are summarized in Table I. The expression of female-specific I-P-45016, and of male-predominant c-P-45016, were not affected either at specific activity and mRNA content by infusing bGH into male and female lit/lit mice. On the other hand, the infusion of bGH in lit/+ male mice resulted in the feminization of these hydroxylases by repressing c-P-45016, and expressing I-P-45016,. Thus, the results suggested that the continuous secretion of bGH itself may not be a feminizing factor, but the destruction of male-specific pulsatile secretion of growth hor-mone happened to feminize the hydroxylase activities in male mice.
During infusion experiments, it was noticed that the implantation of a minipump into Little mice caused stress to the animals and that stress itself was affecting hydroxylase activity. In particular, I-P-4501&-dependent activity was drastically decreased from that in untreated litjlit mice (compared with the activity in Fig. 1).
Time-dependent Decreases of I-P-45OI6, mRNA and Activity by bGH in lit/lit Males-The time-dependent repression of I-P-45016, by bGH in lit/lit males is shown in Fig. 4. A significant decrease of I-p-45O1h was seen 2 days after the first injection, and the maximum bGH affect was obtained 3 days later. This slow effect of bGH is in sharp contrast to the rapid induction of cytochrome P-450 mRNAs by phenobarbital and polycyclic aromatic hydrocarbons. Maximum induction by these inducers was achieved within 20 h after administration (22, 23). Due to the slow effect of growth hormone on the expression of hydroxylases and since somatomedin C has been reported to mediate growth hormone activity in genetically small mice such as Little (24), we tested to see if somatomedin C could mimick the activity of bGH. No effect on the hydroxylases (c-P-45016,, I-p-45016,, and p-45Ol5,) was observed after injection of somatomedin C once every 12 h for 5 consecutive days into lit/lit males (data not shown).

DISCUSSION
Testosterone 16a-hydroxylase activity in mouse liver microsomes is the sum of two sexually modulated isozymes, the female-specific 1-P-45016, and the male-predominant C-P-450160. Testosterone 15a-hydroxylase activity in liver microsomes from female mice is due to the presence of the femalepredominant isozyme called P-45Ol5,. We have demonstrated in this report that when growth hormone is injected once every 12 h for 5 consecutive days to simulate a pulsatile pattern of circulating hormone, it exhibits the repression of 1-P-45016, and P-45015, and the expression of C-P-45ol6, in mice. On the other hand, growth hormone infusion to mimick a continuous secretion pattern has no effect on the modulation of sexually regulated testosterone hydroxylases in mice. Thus, it can be concluded that growth hormone acts as a masculin- izing factor by inhibiting expression of the female isozymes and by activating expression of the male isozyme presumably at the pretranslational levelb) of the hydroxylase in male mice, whereas growth hormone alone may not be a feminizing factor for expression of the hydroxylases in female mice.
According to the report by Norstedt and Palmiter (11), female-predominant expression of prolactin receptor is repressed in lit/lit female and male mice to a level below that in the lit/+ female. The infusion, but not injection, of bovine growth hormone increased the receptor level in lit/lit males to a concentration higher than that seen in lit/+ females. However, an infusion experiment with lit/lit female mice was not described in their report. Nonetheless, the authors concluded that growth hormone, when it is secreted continuously, is a feminizing factor for expression of the receptor in female mice. A similar conclusion has been reached by Gustafsson and co-workers (25) to explain female-specific expression of 5a-androstane-3a,l7P-diol-3,7-disulfate 15P-hydroxylase activity in rat liver microsomes. In their experiments, hypophysectomized rats were used to elucidate the role of growth hormone in feminization of this hydroxylase activity (25). A shortcoming of hypophysectomized animals is that they lose not only growth hormone but a source of all pituitary hormones as well. Therefore, growth hormone may not be the only supplement necessary to reflect what is happening in normal female animals. Our results which show that the levels of female-predominant hydroxylases (I-P-45016, and P-45Ol6,) are higher in lit/lit females than those in lit/+ females suggested that growth hormone has no positive role in the sexdependent expression of 1-P-45016, and p-45ol5, in female mice. This conclusion does not agree with that drawn by other researchers (8-11). The reason for this difference is not certain at the present time. One possibility, however, is that there are growth hormone-dependent and -independent mechanisms regulating sexual dimorphism of liver proteins in female mice.
The male-predominant expression of c-P-45016, in males is regulated by the pulsatile, but not continuous, secretion of growth hormone in mice. This observation is consistent with the previous findings of Norstedt and Palmiter (11) concerning male-predominant MUP expression and of Gustafsson and co-workers (8) concerning the expression of male-predominant steroid 16a-hydroxylase in rats. The feminization of hepatic steroid and drug hydroxylase activities in liver microsomes by a continuous infusion of growth hormone in male rats was originally reported by Kremer and Colby (26) and Mode et al. (27) and recently by Jellinick et al. (28). It is now evident that the repression of male-predominant hydroxylase activities such as testosterone lea-hydroxylase and catechol estrogen formation in growth hormone-infused male rats does not provide direct evidence of a continuous secretion of growth hormone as a feminizing factor, but rather due to the disruption of the pulsatile secretion of growth hormone by the infusion which is a positive regulatory factor for expression of the hydroxylase activity in male animals.
As expression and repression of sex-dependent testosterone hydroxylases (I-and C-P-45Ol6, and P-45Ol6,) are regulated pretranslationally by growth hormone, it is postulated that the hormone modulates sex-dependent transcription of the hydroxylase genes. A transcriptional regulation of a2,-globulin gene by multihormones, including growth hormone, was directly demonstrated by measuring nuclear runoff RNA in vitro (29). The processes by which the pulsatile pattern of growth hormone concentrations in serum transmits its information to hydroxylase genes through the cell membrane and how the genes receive this information are still undescribed.