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Molecular Endocrinology, doi:10.1210/me.2001-0320
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Molecular Endocrinology 16 (10): 2297-2309
Copyright © 2002 by The Endocrine Society

The Roles of Circulating High-Density Lipoproteins and Trophic Hormones in the Phenotype of Knockout Mice Lacking the Steroidogenic Acute Regulatory Protein

Tomohiro Ishii1, Tomonobu Hasegawa1, Chin-I Pai, Natalie Yvgi-Ohana, Rina Timberg, Liping Zhao, Gregor Majdic, Bon-chu Chung, Joseph Orly and Keith L. Parker

Department of Internal Medicine (T.I., T.H., L.Z., G.M., K.L.P.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857; Institute of Molecular Biology (C.-I.P., B.-C.C.) , Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China; and Department of Biological Chemistry (N.Y.-O., R.T., J.O.), The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Address all correspondence and requests for reprints to: Dr. Keith L. Parker, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: kparke{at}mednet.swmed.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The steroidogenic acute regulatory protein (StAR) is essential for the regulated production of steroid hormones, mediating the translocation of intracellular cholesterol to the inner mitochondrial membrane where steroidogenesis begins. Steroidogenic cells lacking StAR have impaired steroidogenesis and progressively accumulate lipid, ultimately causing cytopathic changes and deterioration of steroidogenic capacity. Developmental studies of StAR knockout (KO) mice have correlated gonadal lipid deposits with puberty, suggesting that trophic hormones contribute to this lipid accumulation. To delineate the role of gonadotropins in this process, we examined double mutant mice deficient in both StAR and gonadotropins [StAR KO/hpg (hypogonadal)]. Lipid accumulation was ameliorated considerably in StAR KO/hpg mice but was restored by treatment with exogenous gonadotropins, directly linking trophic hormones with gonadal lipid accumulation. To define the relative roles of exogenous vs. endogenous cholesterol in the lipid accumulation, we also examined mice lacking both StAR and apolipoprotein A-I (StAR KO/Apo A-I KO). Steroidogenic tissues of StAR KO/Apo A-I KO mice had markedly decreased lipid deposits, supporting the predominant role of high-density lipoprotein-derived cholesterol in the lipid accumulation caused by StAR deficiency. Finally, we used electron microscopy to compare mitochondrial ultrastructure in StAR KO and cholesterol side-chain cleavage enzyme (Cyp11a1) KO mice; despite comparable lipid accumulation within adrenocortical cells, the effects of StAR deficiency and Cyp11a1 deficiency on mitochondrial ultrastructure were markedly different. These findings extend our understanding of steroidogenic cell dysfunction in StAR KO mice and highlight key roles of trophic hormones and high-density lipoprotein-derived cholesterol in lipid deposits within StAR-deficient steroidogenic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY TROPHIC HORMONES, the predominant regulators of steroid hormone biosynthesis, stimulate steroidogenesis in two temporal phases. The acute phase, which occurs within seconds to minutes, involves enhanced mobilization and delivery of cholesterol to the steroidogenic complex, whereas the chronic phase, which requires hours to days, predominantly involves increased transcription of the genes involved in steroidogenesis (1). The steroidogenic acute regulatory protein (StAR) plays an essential role in the acute phase of steroid hormone synthesis, facilitating cholesterol delivery from the outer mitochondrial membrane to the inner mitochondrial membrane where it is cleaved to pregnenolone by the cholesterol side-chain cleavage enzyme (CYP11A1, Ref. 2, 3, 4, 5). The pivotal role of StAR in steroid hormone synthesis was dramatically revealed when it was shown that StAR mutations in humans cause congenital lipoid adrenal hyperplasia (lipoid CAH; MIM 201710), an autosomal recessive disorder characterized by impaired steroid hormone synthesis and abundant lipid deposits in adrenal glands and gonads (6, 7, 8, 9).

We previously described the phenotype of StAR knockout (KO) mice, which mimicked many features of lipoid CAH (10, 11). Unless rescued with exogenous corticosteroids, StAR KO mice died from adrenal insufficiency shortly after birth, and their adrenal glands exhibited disrupted cellular architecture and abundant lipid deposits. Due to impaired testosterone production by fetal Leydig cells, StAR KO males also exhibited male-to-female sex reversal of their external genitalia. The testes of newborn StAR KO mice contained scattered lipid droplets in presumptive remnants of fetal Leydig cells and later developed diffuse interstitial lipid deposits and Leydig cell hyperplasia at the time of puberty. The ovaries appeared completely normal at birth, but subsequently developed interstitial lipid deposits characteristic of lipoid CAH. Follicular maturation was impaired, and premature ovarian failure was observed in all StAR KO females by 6 months of age. These findings were consistent with a two-hit model for the pathogenesis of lipoid CAH, which proposed that steroidogenic cells in lipoid CAH patients initially retain some capacity for StAR-independent steroidogenesis, but ultimately accumulate cholesterol esters that abrogate steroidogenesis (8). The mechanism(s) that cause cholesterol esters to accumulate in StAR-deficient cells remain unclear, but the temporal correlation of gonadal deposits with the onset of puberty implicates pituitary gonadotropins in their pathogenesis.

Cholesterol is the obligate precursor of all steroid hormones regardless of tissue of origin (e.g. placenta, testis, ovary, or adrenal gland) or specific physiological role (e.g. glucocorticoid, mineralcorticoid, androgen, estrogen, or progestin). Steroidogenic cells can acquire cholesterol from four different sources: selective uptake of high-density lipoprotein (HDL)-derived cholesterol ester, endocytosis and degradation of low-density lipoprotein (LDL), de novo synthesis from acetate, and hydrolysis of intracellular cholesterol ester. Previous studies suggested that cholesterol substrate for steroid hormone formation is obtained preferentially from circulating lipoproteins (12, 13, 14). Although the relative contributions apparently vary among species or steroidogenic tissues, both HDL and LDL can deliver cholesterol to steroidogenic cells (14, 15, 16). In rodents, cholesterol is provided to steroidogenic cells primarily by the uptake of HDL-derived cholesterol ester through a process that transfers lipids to plasma membrane without the concomitant endocytosis and degradation of the HDL particle (17, 18, 19, 20). This uptake of HDL-derived cholesterol ester is mediated by the scavenger receptor, class B, type I (SR-BI) (21, 22, 23) and also requires apolipoprotein A-I (Apo A-I), which binds SR-BI to facilitate the transfer of HDL-derived cholesterol ester (24, 25). In fact, KO mice lacking SR-BI had reduced cholesterol content of the adrenal glands and ovaries and impaired production of corticosteroids (26). Similarly, Apo A-I knockout (Apo A-I KO) mice had decreased lipid deposits in the adrenal glands and gonads and again showed impaired production of corticosterone in response to ACTH or stress (27). These data suggest that HDL-derived cholesterol in mice is essential for optimal steroidogenesis.

To determine the role of pituitary trophic hormones and the relative contributions of exogenous vs. endogenous cholesterol in lipid deposits in StAR KO mice, we examined the phenotypes of double mutant mice that lacked StAR and either Apo A-I [StAR KO/Apo A-I KO (27)] or gonadotropins [StAR KO/hypogonadal, hpg (28, 29)]. Our results demonstrate an essential role of pituitary gonadotropins in the gonadal lipid deposits of StAR KO mice and implicate HDL-derived cholesterol as the predominant source of lipid deposits in StAR-deficient steroidogenic cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Serum Steroid Hormone Levels at 8 wk of Age
Although StAR KO mice do not require corticosteroid therapy after weaning if they drink normal saline, the StAR KO/Apo A-I KO double mutant mice required ongoing corticosteroid therapy. To keep StAR KO, StAR KO/Apo A-I KO, and StAR KO/hpg mice alive, we supplemented their drinking water with normal saline and administered daily subcutaneous injections of a glucocorticoid/mineralcorticoid cocktail. As reported previously (11), steroid hormones were detectable in StAR KO mice at 8 wk of age, indicating some residual capacity for steroidogenesis (Table 1Go); nonetheless, levels of all steroid hormones except estradiol were significantly lower than those of wild-type mice. Although male and female Apo A-I KO mice were fertile, they also had significantly diminished sex steroid levels. Consistent with previous reports (30), serum testosterone levels of hpg males also were significantly lower than those of wild-type males. In contrast, estradiol levels of hpg females did not differ significantly from those of wild-type females, a finding consistent with the previous observation that serum estradiol levels were comparable in wild-type mice and KO mice lacking the ß-subunit of FSH (31). These findings indicate that female sexual development in mice is so sensitive that normal estradiol levels approach the lower limit of assay sensitivity.


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  		Table 1. Serum Steroid Hormone Levels in 8-wk-Old Mice

 
To determine the roles of Apo A-I and gonadotropins in steroid hormone production in StAR KO mice, we examined the effect of double mutations in StAR and either Apo A-I or GnRH. As shown in Table 1Go, serum estradiol levels of StAR KO/Apo A-I KO females were significantly lower than those of StAR KO females, whereas serum levels of other steroid hormones in StAR KO/Apo A-I KO mice were similar to those of StAR KO mice. Although the serum estradiol levels of StAR KO/Apo A-I KO and Apo A-I KO females did not differ significantly, both were lower than those of wild-type females, supporting an important role of Apo A-I in estradiol production irrespective of the StAR genotype. The serum testosterone levels of StAR KO/hpg males were significantly lower than those of StAR KO males, whereas other serum steroid hormone levels of StAR KO/hpg mice were comparable to those of StAR KO mice. Finally, serum testosterone levels of StAR KO/hpg males were similar to those of hpg males, demonstrating that the hpg mutation impairs testosterone secretion irrespective of the StAR genotype.

HDL Cholesterol Is a Major Source of Adrenocortical Lipid Deposits in StAR KO Mice
The adrenal cortex of newborn StAR KO mice contained prominent cytosolic lipid deposits, as demonstrated by vacuolated regions in hematoxylin-eosin-stained sections and red lipid droplets in oil red O-stained sections (Fig. 1CGo). Lipid deposits were less abundant in adrenocortical cells of newborn StAR KO/Apo A-I KO mice (Fig. 1EGo), suggesting that HDL cholesterol is an important source of the adrenocortical lipid deposits that accumulate in utero in the absence of StAR. In contrast, lipid deposits in the adrenal cortex of newborn StAR KO/hpg mice were comparable to those of StAR KO mice (Fig. 1GGo), consistent with the fact that gonadotropins do not normally regulate adrenocortical steroidogenesis (32).



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  		Figure 1. Oil Red O Staining of Adrenal Glands and Testes from Newborn Mice

A and B, Wild type; C and D, StAR KO; E and F, StAR KO/Apo A-I KO; and G and H, StAR KO/hpg. Sections A, C, E, and G are from adrenal glands (magnification, x200); and B, D, F, and H are those from testes (magnification, x200).

 
To examine developmental effects on the steroidogenic organs, we rescued StAR KO, StAR KO/Apo A-I KO, and StAR KO/hpg mice with exogenous corticosteroids as described in Materials and Methods. As in our previous studies (11), all zones of the adrenal cortex of StAR KO mice at 8 wk of age had considerably increased lipid deposits (Figs. 2DGo and 3DGo). The vacuolization and lipid deposits in the adrenal glands of StAR KO/Apo A-I KO mice at 8 wk of age were substantially less severe than those of StAR KO mice (Figs. 2GGo and 3GGo). This finding indicates that cholesterol derived from circulating HDL is a major source of the lipid that accumulates in StAR-deficient adrenocortical cells. Consistent with the independence of adrenal steroidogenesis from gonadotropins (32), the gross and microscopic appearance of adrenal glands of StAR KO/hpg mice did not differ from those of StAR KO mice (Figs. 2JGo and 3JGo).



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  		Figure 2. Hematoxylin-Eosin Staining of Adrenal Glands, Testes, and Ovaries from 8-wk-Old Mice

A–C, Wild type; D–F, StAR KO; G–I StAR KO/Apo A-I KO; and J–L, StAR KO/hpg. Sections A, D, G, and J are from adrenal glands (magnification, 200x); B, E, H, and K are from testes (magnification, x100); and C, F, I, and L are from ovaries (magnification, x50).

 


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  		Figure 3. Oil Red O Staining of Adrenal Glands, Testes, and Ovaries from 8-wk-Old Mice

A–C, Wild type; D–F, StAR KO; G–I, StAR KO/Apo A–I KO; and J–L, StAR KO/hpg. Sections A, D, G, and J are from adrenal glands (magnification, x200); B, E, H, and K are from testes (magnification, x100); and C, F, I, and L are from ovaries (magnification, x50).

 
Both Apo A-I and Gonadotropins Affect Lipid Deposition in the Testes of StAR KO Mice
The testes of newborn StAR KO mice were indistinguishable from those of wild-type mice in hematoxylin-eosin-stained sections, but oil red O staining revealed scattered foci of interstitial lipid deposits in presumptive remnants of fetal Leydig cells (Fig. 1DGo). Although newborn StAR KO/Apo A-I KO males also had impaired androgen synthesis, as evidenced by complete sex reversal of their external genitalia, the lipid deposits in their fetal Leydig cells were less abundant than those of StAR KO mice (Fig. 1FGo); these studies again support a role of Apo A-I in the acquisition of cholesterol esters by fetal Leydig cells. In contrast, the lipid deposits in newborn StAR KO/hpg mice, which also have impaired virilization, were similar to those in StAR KO mice (Fig. 1HGo), consistent with the observation that pituitary gonadotropins are not major regulators of testosterone production by fetal Leydig cells (33).

At 8 wk of age, the testes of StAR KO mice were comparable in size to those of wild-type mice but remained in the inguinal region rather than descending fully. The Leydig cells exhibited marked hyperplasia and abundant cytoplasmic lipid deposits (Figs. 2EGo and 3EGo). Despite the complete absence of StAR, spermatogenesis in the testes from StAR KO mice was largely preserved, presumably reflecting some residual capacity for StAR-independent synthesis of sex steroids. Similarly, the testes of StAR KO/Apo A-I KO mice were of comparable size to those of wild-type mice, remained in the inguinal region, and exhibited relatively intact germ cell maturation. However, the Leydig cells of StAR KO/Apo A-I KO mice had considerably less lipid deposits than those of StAR KO mice (Figs. 2HGo and 3HGo).

The testes of StAR KO/hpg mice were significantly hypoplastic (mean ± SE, 0.10 ± 0.01 mg/g body weight, n = 10), compared with those of StAR KO males (3.84 ± 0.17, n = 9) (Mann-Whitney U test, P = 0.001), and resided in the inguinal region attached to abundant perigonadal white adipose tissue. The few remaining Leydig cells were completely devoid of lipid deposits in oil red O-stained sections (Figs. 2KGo and 3KGo), whereas the germ cells failed to progress beyond the pachytene spermatocyte stage. Similar histological findings were observed in the testes of hpg mice, but the testes of StAR KO/hpg mice were significantly smaller than those of hpg mice (0.14 ± 0.01, n = 12) (P = 0.009). The seminiferous tubules were less mature in StAR KO/hpg mice; some spermatogonia and Sertoli cell nuclei were located close to the center of the tubules, a finding characteristic of prepubertal testes (34), whereas most spermatogonia and Sertoli cell nuclei in hpg mice were close to the basement membrane of the tubules. These findings suggest that lack of gonadotropin-independent StAR function directly or indirectly affected seminiferous tubule maturation even in postnatal testes.

Both Apo A-I and Gonadotropins Contribute to Ovarian Lipid Deposits in StAR KO Mice
As described previously (10, 11), the ovaries of StAR KO females appeared normal at birth, and the interstitial and theca cells only accumulated lipid after the normal time of puberty (Figs. 2FGo and 3FGo). Consistent with the essential role of StAR in reproduction, follicular maturation was impaired, corpora lutea were never observed, and females were infertile due to impaired ovulation. Despite this, the vaginas of StAR KO females opened by 8 wk of age, and their uteri were indistinguishable from those of wild-type females, consistent with residual capacity for StAR-independent estrogen production. Comparable results were obtained with StAR KO/Apo A-I KO females, which also exhibited vaginal opening by 8 wk of age and had uteri of comparable size to StAR KO and wild-type females. Although the ovaries of StAR KO/Apo A-I KO females at 8 wk of age were smaller than those of StAR KO females, follicular maturation was similarly impaired, and no corpora lutea were observed in any ovarian sections. However, lipid accumulation in the interstitial and theca cells was less severe in StAR KO/Apo A-I KO females than in StAR KO females (Figs. 2IGo and 3IGo), suggesting that HDL cholesterol is a critical source of lipid in the ovaries of StAR KO mice.

StAR KO/hpg females never showed any evidence of postnatal sexual differentiation. Their ovaries and uteri were markedly hypoplastic (ovary, 0.070 ± 0.008 mg/g body weight, n = 8; uterus, 0.27 ± 0.05, n = 3), compared with those of StAR KO females (0.478 ± 0.044, n = 6; 4.68 ± 0.44, n = 3) (P = 0.001; P = 0.03), and the ovaries contained only immature follicles that did not develop beyond the early antral stage (Fig. 2LGo). In striking contrast to StAR KO ovaries, no detectable lipid deposits were present in the interstitial and theca cells of StAR KO/hpg mice (Fig. 3LGo). These findings strongly suggest that gonadotropin stimulation is essential for lipid accumulation in the ovaries of StAR-deficient mice.

Gonadotropin Treatment of StAR KO/hpg Mice Induces Lipid Deposits
To prove that gonadotropins induce lipid accumulation in StAR-deficient gonads, we treated hpg or StAR KO/hpg mice with PMSG, which contains both FSH and LH activities. The weights of testes of StAR KO/hpg mice treated for 10 d (0.56 ± 0.02 mg/g body weight, n = 6) were comparable to those of hpg mice treated with the same protocol (0.53 ± 0.07, n = 6) (P = 0.75). However, the serum testosterone levels of the treated StAR KO/hpg mice (0.053 ± 0.020 ng/ml, n = 3) were significantly lower than those of the treated hpg mice (2.615 ± 0.625, n = 4) (P = 0.049). As shown in Fig. 4Go, C and D, PMSG treatment of hpg males induced luminal expansion of the seminiferous tubules, germ cell maturation to the round spermatid stage, and mild Leydig cell hyperplasia. Treatment for StAR KO/hpg males also induced luminal opening of the tubules and development of a more severe degree of Leydig cell hyperplasia. Moreover, PMSG treatment induced massive lipid deposits within the hyperplastic Leydig cells of StAR KO/hpg mice, whereas their germ cells did not mature beyond the spermatogonial stage (Fig. 4Go, I and J). These effects of the PMSG treatment were progressive, as lipid deposits were more severe in StAR KO/hpg mice treated for 10 d than those for 5 d (data not shown).



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  		Figure 4. Testes of hpg or StAR KO/hpg Mice with or without PMSG Treatment

Where indicated, mice of the indicated genotypes were treated with 5 IU PMSG for 10 d as described in Materials and Methods. A and B, Untreated hpg; C–F, PMSG-treated hpg; G and H, untreated StAR KO/hpg; and I–L, PMSG-treated StAR KO/hpg. Sections A, C, G, and I were stained with hematoxylin-eosin (magnification, x200); and B, D, H, and J were stained with oil red O (magnification, x200). Sections E and K show immunohistochemical analyses with antihuman PCNA antiserum (magnification, x200); and F and L show immunohistochemical analyses with antihuman CREM-1 antiserum (magnification, x200).

 
To evaluate further the effects of the PMSG treatment, we examined the expression of two markers of germ cell maturation: proliferating cell nuclear antigen (PCNA), which is expressed in spermatogonia and early spermatocytes (35), and cAMP-response element modulator-1 (CREM-1), which is expressed predominantly in postmeiotic haploid spermatids in the tubules (36). In PMSG-treated hpg mice, spermatogonia remained adjacent to the basement membrane (Fig. 4EGo), and round spermatids that stained positively with the anti-CREM-1 antiserum were observed in the center of the seminiferous tubules (Fig. 4FGo). In the testes of StAR KO/hpg mice treated with PMSG, spermatogonia were severely diminished in number (Fig. 4KGo), and no germ cells beyond spermatogonia were observed in any tubules, as evidenced by the lack of CREM-1 expression (Fig. 4LGo).

The weights of ovaries and uteri of StAR KO/hpg mice treated sequentially with 5-d PMSG and human chorionic gonadotropin (hCG) (ovary, 0.45 ± 0.06 mg/g body weight, n = 5; uterus, 3.24 ± 0.17, n = 3) did not differ significantly from those of hpg mice treated with the same protocol (0.29 ± 0.04, n = 10; 2.46 ± 0.42, n = 4) (P = 0.05; P = 0.29). In contrast, the serum progesterone and estradiol levels of the treated StAR KO/hpg mice (0.23 ± 0.02 ng/ml and 19 ± 8 pg/ml, n = 3) were significantly lower than those of the treated hpg mice (6.35 ± 1.35 and 90 ± 22, n = 5) (P = 0.03 and P = 0.03). As shown in Fig. 5Go, C and D, PMSG treatment of hpg females induced follicular maturation and lipid accumulation in the interstitial and theca cells. In addition, corpora lutea were observed in the ovaries of hpg females treated sequentially with PMSG and hCG, showing that this regimen of exogenous gonadotropins can induce ovulation in hpg females. Hemorrhagic cysts were observed in the ovaries of PMSG-treated female hpg mice; similar cysts were also seen in the ovaries of StAR KO/hpg mice treated with PMSG, which also exhibited follicular maturation up to preovulatory stage and marked accumulation of lipid deposits (Fig. 5Go, G and H). However, no corpora lutea or ovulated oocytes were identified in the ovaries or oviducts, respectively, even after sequential treatment with PMSG and hCG. As predicted from previous studies of StAR KO mice, gonadotropin-induced lipid deposits were much more severe in the ovaries of StAR KO/hpg females than in hpg females, and these deposits increased progressively with longer PMSG treatment (data not shown). Again, these studies directly link gonadotropin stimulation with gonadal lipid accumulation in StAR KO mice.



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  		Figure 5. Ovaries of hpg or StAR KO/hpg Mice with or without PMSG/hCG Treatment

Where indicated, mice of the indicated genotypes shown in this figure were treated with PMSG for 10 d and subsequently received hCG as described in Materials and Methods. A and B, Untreated hpg; C and D, PMSG/hCG-treated hpg; E and F, untreated StAR KO/hpg; and G and H, PMSG/hCG-treated StAR KO/hpg. Sections A, C, E, and G were stained with hematoxylin-eosin (magnification, x100); and B, D, F, and H were stained with oil red O (magnification, x100).

 
Distinct Effects on Mitochondrial Ultrastructure in StAR KO and Cyp11a1 KO Mice
In addition to StAR deficiency, lipoid CAH has been attributed to a deficiency of Cyp11a1 in rabbits (37) and knockout mice (38), and a human subject with apparent haploinsufficiency of CYP11A1 exhibited adrenal insufficiency and male-to-female sex reversal (39). Although both forms of lipoid CAH are associated with profound lipid deposits within steroidogenic cells, the roles and sites of action of StAR and Cyp11a1 suggest that the two variants of lipoid CAH may differ significantly. StAR deficiency should markedly impair the transfer of cholesterol into the mitochondria, whereas Cyp11a1 deficiency should enhance cholesterol loading of the mitochondria. As a first step to assess potential differences in lipid-mediated mitochondrial damage in these two models, we examined the mitochondrial ultrastructure in adrenal glands of newborn wild-type, StAR KO, and Cyp11a1 KO mice. The wild-type adrenal section contained many lipoid droplets of approximately 1-µm diameters (Fig. 6AGo), and the mitochondria cristae had a distinctive vesicular appearance characteristic of the rodent adrenal (Fig. 6BGo) (40). Despite their florid lipid deposits, with diameters of up to 3–5 µm (Fig. 6CGo), the StAR KO adrenal glands maintained the characteristic mitochondrial ultrastructure (Fig. 6DGo). Some cells contained swollen mitochondria of 1- to 2-µm diameters wrapped in multilamellar endoplasmic reticulum or ruptured mitochondria inside lipid-rich structures that may represent compressed multilayered membranes. In the newborn Cyp11a1 KO adrenals, 3- to 4-µm lipid droplets again were observed, as were partially lysed or degenerating mitochondria wrapped in compressed, osmium-stained membranes (Fig. 6EGo); in marked contrast to the StAR KO mitochondria, the Cyp11a1 KO mitochondria exhibited lamellar cristae and the accumulation of abnormal membrane folds within the interior of the mitochondria (Fig. 6FGo). We observed no ultrastructural evidence of the cholesterol crystals in either the cytosol or mitochondrial matrix of StAR KO or Cyp11a1 KO adrenals.



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  		Figure 6. Differences in the Mitochondrial Ultrastructure of Adrenocortical Cells from 3-d-Old StAR KO and Cyp11a1 KO Mice

A and B, Wild type; C and D, StAR KO; and E and F, Cyp11a1 KO. Panels A, C, and E indicate lower magnification views of the zona fasciculata cells, whereas the insets B, D, and F demonstrate high-power views that depict the mitochondrial ultrastructure. L, Lipid droplet; N, nucleus; m, mitochondria. A, Multiple small lipid droplets of approximately 1 µm diameters and typical rounded mitochondria of 0.4–0.6 µm (arrows) in wild-type zona fasciculata cells. The mitochondria of the wild-type cells contain the typical vesicular structures of the inner mitochondrial membranes (arrowheads in inset B). C, Huge lipid droplets of 3–5 µm and swollen mitochondria of 1–2 µm in StAR KO zona fasciculata cells. The mitochondria of the StAR KO cells maintain the vesicular organization of the inner membranes (shown in inset D). Some of the mitochondria are partially lysed (m* in D) or wrapped with multilayered membranes (arrowheads in D). This field also depicts an advanced stage of mitochondrial perturbation, with the mitochondria enfolded within electron-dense material that apparently is compressed osmicated membranes (arrows). E, Large lipid droplets of 3–4 µm and a few large mitochondria in Cyp11a1 KO zona fasciculata cells. Some of the mitochondria are lysed (m*) or wrapped within heavily osmicated compressed lipid multilayers (arrows), as shown in C and D. Inset F depicts a higher magnification of the altered mitochondrial ultrastructure. Note the nondifferentiated mitochondrial cristae that form lamellar or concentric structures (arrowheads in F).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To determine the roles of HDL-derived cholesterol and pituitary gonadotropins in the pathogenesis of endocrine disease associated with StAR deficiency, we examined the phenotype of double mutant mice lacking StAR and either Apo A-I or GnRH. The developmental timing of histological abnormalities in steroidogenic tissues of StAR KO mice previously suggested that gonadotropins play an important role in lipid deposition, consistent with the two-hit model for the pathogenesis of lipoid CAH (11). We now show that the lipid deposits are reduced considerably in StAR KO/hpg mice, establishing a pivotal role of pituitary gonadotropins in lipid accumulation within the gonads of StAR KO mice. We further show that lipid deposits are reduced considerably in adrenal glands and gonads of StAR KO/Apo A-I KO mice, supporting the primary role of HDL-derived cholesterol in the lipid deposits. Finally, we show that there are distinct effects on mitochondrial ultrastructure in two different variants of lipoid CAH: StAR KO and Cyp11a1 KO mice.

Although our data highlight key roles of gonadotropins and HDL-derived cholesterol in the pathogenesis of lipoid CAH, the precise mechanism by which StAR deficiency impairs cholesterol delivery to the steroidogenic complex remains poorly understood. Further experiments, such as in vitro structure-function analyses with immortalized steroidogenic cell lines derived from StAR KO mice, are required to delineate the mechanisms by which StAR facilitates steroidogenesis. Although the ongoing transfer of HDL-derived cholesterol ester into the cytosol of steroidogenic cells presumably requires an active transfer process, because the solubility of cholesterol ester in the plasma membrane is limited, this transfer system also remains unknown. Further insights into the mechanism of lipid accumulation in StAR KO cells may provide new insights into normal and abnormal cholesterol disposition in steroidogenic cells.

Despite the corticosteroid injections, approximately 40% of the double mutant mice died before 8 wk of age; the data shown here therefore may reflect relatively less severely affected mice. Based on the consistency of our results with the various genotypes, we believe that our conclusions regarding the pathogenesis of lipid deposits are generally applicable to all StAR-deficient mice. We also note that the histological analyses of the adrenal glands in the double mutant mice were potentially affected by the corticosteroid injections that they received. The same corticosteroid replacement regimen, however, did not affect adrenocortical histology in wild-type mice, and we likewise saw no histological differences between corticosteroid-treated StAR KO mice and StAR KO mice kept alive with normal saline after weaning. We therefore believe that histological analyses of adrenocortical structure in these double mutant mice provide valid insights into the pathogenesis of lipoid CAH.

StAR KO mice provide a model system to analyze the relative contributions of different sources of cholesterol to the development of lipid deposits in each steroidogenic tissue. Using in situ hybridization assays, SR-BI gene expression in the adrenal glands and testes of StAR KO mice did not differ significantly from that of wild-type mice (22), suggesting that the compensatory response to StAR deficiency does not include increased SR-BI expression. However, the StAR KO/Apo A-I KO mice had markedly decreased lipid deposits within all of their steroidogenic cells, highlighting the important role of Apo A-I in lipid accumulation within StAR-deficient steroidogenic cells. Apo A-I, a major apolipoprotein in HDL, can directly bind to SR-BI and efficiently mediate the transfer of cholesterol ester from HDL particles into steroidogenic cells without uptake of the apolipoproteins (24, 25). Previous analyses of Apo A-I KO mice and in vitro studies with HDL particles isolated from Apo A-I KO mice demonstrated that Apo A-I deficiency significantly decreases the selective uptake of HDL-derived cholesterol and the intracellular cholesterol ester storage of the steroidogenic cells (27, 41). These findings indicate that HDL-derived cholesterol ester is also a major source of intracellular cholesterol esters in StAR-deficient steroidogenic cells in mice.

The relative roles of different lipoproteins in steroid hormone synthesis in humans are not fully defined. In cell culture analyses, human fetal adrenal glands and corpora lutea predominantly obtained cholesterol for steroidogenesis from LDL, thereby supporting the production of adrenal androgens in late gestation (42) and progesterone during the luteal phase of the menstrual cycle (43). However, accumulating in vivo evidence indicates that HDL-derived cholesterol is also important for human steroidogenesis. First, female patients with abetalipoproteinemia, a hereditary disease resulting in the absence of plasma LDL, had decreased progesterone synthesis during the luteal phase of the menstrual cycle, but generated sufficient estradiol and progesterone to maintain pregnancy (44). Second, human patients with abetalipoproteinemia or familial hypercholesterolemia, a hereditary defect in the LDL receptor, had normal basal cortisol production and exhibited impaired cortisol production only after prolonged stimulation with ACTH (45, 46, 47). Finally, patients with familial hypercholesterolemia treated with cholesterol synthesis inhibitors had normal basal cortisol production and relatively intact cortisol production upon prolonged stimulation with ACTH (48). Collectively, these findings suggest that HDL-derived cholesterol is an important source of cholesterol substrate for steroid hormone synthesis in humans, at least in the adrenal cortex and ovaries. These findings raise the possibility that HDL-derived cholesterol also plays a key role in lipid accumulation in human patients with lipoid CAH.

The temporal link between puberty and the markedly increased gonadal lipid deposits provided correlative evidence for a pivotal role of gonadotropins in lipid accumulation in StAR KO gonads (11). To address this issue directly, we analyzed the gonads of adult StAR KO/hpg mice and found considerably decreased lipid accumulation relative to the abundant lipid deposits in postpubertal StAR KO mice. Moreover, PMSG treatment of StAR KO/hpg mice caused abundant lipid accumulation within the gonads. These results prove that gonadotropins stimulate the deposition of intracellular lipids within gonadal cells lacking StAR.

Despite the evidence for some sexual maturation at the normal time of puberty, successful ovulation has not been reported in the StAR-deficient ovaries of mice or women (8, 11, 49, 50). As shown here, the ovaries of StAR KO/hpg females were severely hypoplastic and completely devoid of detectable lipid deposits, suggesting that their steroidogenic cells were spared from any cumulative damage caused by lipid accumulation. Using these mice, we examined whether exogenous gonadotropins could induce ovulation, an issue relevant to assisted reproductive efforts in human patients with lipoid CAH. Although exogenous gonadotropin treatment of StAR KO/hpg mice induced lipid deposition and follicular maturation, the serum estradiol and progesterone levels in StAR/hpg females were considerably lower than those of hpg females, and corpora lutea were never observed in histological sections. We further were unable to reverse the impaired ovulation even when the mice were treated with exogenous progesterone in conjunction with gonadotropins. Thus, our data suggest that the compromised steroid production in the StAR-deficient ovaries precludes ovulation, even if the steroidogenic cells are protected from lipid-induced damage by the absence of gonadotropins until the gonadotropin treatment. To the extent that these results also apply to human patients with lipoid CAH, they suggest that assisted reproduction technologies are unlikely to result in successful oocyte retrieval from these patients.

Electron microscopic analyses of newborn mice revealed that the mitochondria in StAR KO mice generally were less perturbed than those of Cyp11a1 KO mice, despite the fact that lipid droplets accumulated within the cytosol to comparable degrees in both lipoid CAH models. One explanation for these disparate findings is that Cyp11a1 plays a key role in establishing the structure of steroidogenic mitochondria, as suggested by the conversion of mitochondria in 3T3 fibroblasts transfected with an expression plasmid for CYP11A1 from elongated organelles with lamellar cristae to round organelles with vesicular cristae (51). Alternatively, the predicted second hit in the two-hit model for the pathogenesis of lipoid CAH differs in these two forms of lipoid CAH. StAR deficiency, with its florid lipid deposits within the cell and relative sparing of the mitochondria themselves, is associated with some preservation of steroidogenic capacity due to StAR-independent cholesterol transfer. In contrast, the Cyp11a1 KO cells completely lack the enzyme that catalyzes the initial step in the steroidogenic pathway, and their mitochondrial structure is markedly perturbed. Thus, although the molecular basis for the ultrastructural differences in the severity of mitochondrial perturbation between the StAR KO and Cyp11a1 KO mice remains to be defined, these data provide the first insights into potential differences in the pathogenesis of these two forms of lipoid CAH.

In summary, we show that circulating HDL-derived cholesterol is the predominant source of lipid deposits in adrenal glands and gonads of StAR KO mice, we demonstrate that trophic hormone stimulates the deposition of lipids within the steroidogenic cells, and we identify structural differences in the mitochondria of StAR KO and Cyp11a1 KO mice that likely define important differences in the pathogenesis of these two forms of lipoid CAH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
All animal studies were approved by the Institutional Animal Care Research Advisory Committee at University of Texas Southwestern Medical Center (StAR KO, StAR KO/Apo A-I KO, and StAR KO/hpg mice) or by the Institutional Review Committee at the Academia Sinica (Cyp11a1 KO mice). The StAR KO mice were created by gene targeting in embryonic cells as described previously (10). A B6-StAR KO congenic line, produced by backcrossing the StAR KO allele to C57BL/6J inbred mice for 10 generations, was used in this study. The Apo A-I KO mice (strain name B6.129P2-Apoa1tm1Unc, stock no. 002055) and hpg heterozygous mice (strain name HPG/Bm Gnrhhpg/+, stock no. 000804) were purchased from The Jackson Laboratory (Bar Harbor, ME) and introduced into the StAR KO line to produce double mutant mice. The StAR KO, StAR KO/Apo A-I KO, and StAR KO/hpg mice were maintained in the University of Texas Southwestern Animal Resources Center on a 12-h light, 12-h dark cycle, and were given drinking solution (0.9% sodium chloride, Sigma, St. Louis, MO) and standard mouse chow (4% Mouse/Rat Diet 7001, Harlan Teklad, Madison, WI) ad libitum.

The Cyp11a1 KO mice were generated by homologous recombination in embryonic stem cells as described previously (38). They were maintained at the Academia Sinica Animal Resources Center on a 12-h light, 12-h dark cycle.

PCR analyses of genomic DNA were used for genotyping; primer sets included: StAR [common forward primer (5'-GTGCTGGCCATTGGCCAAGA-3'), wild-type allele-specific reverse primer (5'-TCTTACTTAGCACTTCGTCCCCGTTC-3'), and KO allele-specific reverse primer (5'-ATCTCGTCGTGACCCATGGC-3')]; Apo A-I [common forward primer (5'-CCTTCTATCGCCTTCTTGACG-3'), wild-type allele-specific reverse primer (5'-TCTGGTCTTCCTGACAGGTAGG-3'), and KO allele-specific reverse primer (5'-GTTCATCTTGCTGCCATACG-3')]; and GnRH [common forward primer (5'-CACATCTGTAGCCACAGTCC-3'), proximal reverse primer (5'-GCTTGGAGAGCTGTAAGGTC-3'), and distal reverse primer (5'-AGCTCCGAGGCTGTCACTGG-3')]. Genetic sex was determined by PCR analysis for the Sry gene using forward (5'-AAGCGCCCCATGAATGCATT-3') and reverse (5'-CGATGAGGCTGATATTTATA-3') primers. Annealing temperatures were set at 55 C in all PCR. Oligonucleotides and reagents for PCR were purchased from Life Technologies, Inc. (Gaithersburg, MD) and PE Applied Biosystems (Norwalk, CT), respectively.

StAR KO mice, irrespective of their Apo A-I or hpg genotypes, were rescued with injections of a glucocorticoid/mineralcorticoid cocktail as previously described (11). In brief, all newborn pups were injected sc daily with 20 µg hydrocortisone (Sigma), 0.025 µg fludrocortisone acetate (Sigma), and 0.025 µg dexamethasone 21-phosphate (Sigma) until the StAR genotype was determined by PCR analysis, and corticosteroid injections were continued in all mice homozygous for the StAR KO allele. In addition, 0.9% sodium chloride (Sigma) was provided ad libitum as drinking solution.

Hormone Assays
Serum steroid hormone concentrations were determined by RIA. Mice (8 wk of age) were anesthetized with 2.5 mg ketamine (Fort Dodge Animal Health, Fort Dodge, IA), 0.25 mg xylazine (Phoenix Scientific, Inc., St. Joseph, MO), and 0.05 mg acepromazine malate (Burns Veterinary Supply, Rockville Centre, NY), and blood was collected by cardiac puncture between 1200 h and 1600 h. Steroid hormones analyzed included corticosterone [limit of detection, 4.5 pg/ml; intraassay percentage coefficient of variation (CV), 3.2%; % percentage recovery, 89.8], progesterone (limit of detection, 4.8 pg/ml; intraassay percentage CV, 4.6%; percentage recovery, 75.7), testosterone (limit of detection, 4.6 pg/ml; intraassay percentage CV, 6.3%; percentage recovery, 75.9), and estradiol (limit of detection, 1.0 pg/ml; intraassay percentage CV, 9.7%; percentage recovery, 62.5). Statistical significance in differences between different groups was determined by the Mann-Whitney U test, and differences with P < 0.05 were considered significant.

Histological Analyses
Tissues were harvested from anesthetized mice as described above. Morphological analyses used Bouin’s fixed tissues stained with hematoxylin (Richard-Allan Scientific, Kalamazoo, MI) and eosin (Richard-Allan Scientific) or frozen tissues stained with oil red O (Sigma) and counterstained with hematoxylin. The Bouin’s fixative was purchased from Polysciences, Inc. (Warrington, PA). The Bouin’s fixed tissues were embedded in double of purified paraffin and plastic polymers of regulated molecular weights (Oxford Labware, St. Louis, MO) and cut in 7-µm sections with a Rotary Microtome HM 330 (Microm GmbH, Heidelberg, Germany). The frozen tissues were embedded in optical cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA) and sectioned at 10 µm with a Cryostat CM1900 (Leica Corp. Instruments GmbH, Nussloch, Germany).

Immunohistochemical analyses were performed with 5-µm sections from Bouin’s fixed tissues, rabbit polyclonal antibodies, and the Vectastain ABC kit H (Vector Laboratories, Inc., Burlingame, CA) including biotinylated goat secondary antibodies against rabbit IgG, avidin DH, and biotinylated horseradish peroxidase H. The rabbit polyclonal antibodies against the full length of human PCNA or CREM-1 and normal rabbit IgG for control reactions were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Visualization was performed with diaminobenzidine (Vector Laboratories, Inc.) and hydrogen peroxide (Vector Laboratories, Inc.) followed by counterstaining with hematoxylin.

For electron microscopic analyses, adrenal glands were trimmed free of fat, fixed in 0.1 M sodium phosphate (pH 7.4) containing 1% paraformaldehyde [Electron Microscopy Sciences (EMS), Fort Washington, PA] and 2.5% glutaraldehyde (EMS), prestained (osmication) by use of 1% osmium tetroxide in the above buffer, embedded in epoxy resin (EMS), and cut in 70-nm sections with an Ultratome 3 (LKB, Stockholm, Sweden). Sections were examined by transmission electron microscopy (Technai 12 workstation, Philips, Eindhoven, The Netherlands) equipped with a MegaView II camera (Soft Imaging System GmbH, Münster, Germany).

PMSG Treatment
Hpg or StAR KO/hpg mice at 8 wk of age were injected at approximately 1200 h with 5 IU PMSG ip (Sigma) daily for either 5 or 10 consecutive days. To induce ovulation, the females treated with PMSG were subsequently treated with hCG (5 IU ip, Sigma) at approximately 1800 h on the last day of the PMSG injection. Relevant tissues were harvested at about 1200 h on the day after the last injection.


   ACKNOWLEDGMENTS
 
We thank Dr. David Hess (Oregon Health Sciences University, Beaverton, OR) for measurements of circulating steroid hormone concentrations.


   FOOTNOTES
 
This work was supported by NIH Grants DK-54028 and DK-54480 (K.L.P.), by Grant NSC 90-2311-B-001-110 from National Science Council (B.-C.C.), by Academia Sinica, Republic of China (B.-C.C.), and by Grant 1999-315-2 from The United States-Israel Binational Foundation (J.O.).

1 T.I. and T.H. contributed equally to this study. Back

Abbreviations: Apo A-I, Apolipoprotein A-I; CAH, congenital adrenal hyperplasia; CREM, cAMP response element modulator; CV, coefficient of variation; hCG, human chorionic gonadotropin; HDL, high-density lipoprotein; hpg, hypogonadal; KO, knockout; LDL, low-density lipoprotein; PCNA, proliferating cell nuclear antigen; SR-BI, scavenger receptor, class B, type I; StAR, steroidogenic acute regulatory protein.

Received for publication November 26, 2001. Accepted for publication June 14, 2002.


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