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Impact of Architectural Disruption on Adrenocortical Steroidogenesis In Vitro¹

Departments of Obstetrics and Gynecology (G.A.H., R.A.), and Medicine (R.A.), The University of Alabama at Birmingham, Birmingham, Alabama 35233

Address correspondence and requests for reprints to: Ricardo Azziz, M.D., The University of Alabama at Birmingham, Department of Obstetrics and Gynecology, 549 Old Hillman Building, 618 South 20th Street, Birmingham, Alabama 35233-7333.

	Abstract

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The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis. The impact that disruption of this tissue’s architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear. We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results. To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions. Normal human adrenals (n = 5, three males and two females, age range 17–43 yr) were obtained at the time of organ donation. The three adrenal tissue preparations were incubated in serum-free medium with 10 µM pregnenolone substrate ± 1 µM ACTH. The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay. Initial time course incubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices. In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e. slices less than minces less than suspensions (0.88 vs. 2.1 vs. 3.0 µg/gm tissue, respectively, P < 0.0001). Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs. 37.7 vs. 18.7 ng/gm tissue, P < 0.0028, and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs. 22.2 vs. 31.2 ng/gm tissue, P < 0.297, respectively). It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids. We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.

	Introduction

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ADULT human adrenal glands are functionally and morphologically separated into distinct zones of steroidogenic activity: the zona glomerulosa, fasciculata, and reticularis (1). Overall steroidogenic output is thought to be highly dependent on the complex architectural arrangement of the adrenal, determined in part by the flow of steroid precursors from one zone to another (2), and the proximity of intraadrenal regulatory factors (3). Furthermore, maintenance of cellular integrity is thought to be critical for the optimal and most relevant measures of steroidogenesis (4). However, the in vitro study of adrenocortical steroidogenesis requires that a variable amount of tissue and architectural disruption occur. Among studies of adrenal steroidogenesis in vitro, methods of tissue preparation have varied widely, from primary cell cultures (5, 6), to intact glands, cellular suspensions (7, 8), and tissue slices (9).

In vitro preparations of adrenal tissues result in variable, but definite, amounts of architectural disruption. In turn, this disruption in cell-to-cell contact may alter the same adrenocortical biosynthetic processes that are being examined. We hypothesized that the extent of architectural disruption during tissue preparation would impact the in vitro production of steroids. To test this hypothesis we compared the in vitro production of cortisol (F), androstenedione (A4), dehydroepiandrosterone (DHA), and DHA sulfate (DHS) in fresh tissue preparations from human adrenals with increasing degrees of architectural disruption: tissue slices, tissue minces, and cell suspensions.

	Materials and Methods

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Adrenal tissue preparation

Adrenal glands from five donors were obtained with consent, through the Alabama Organ Center, and were used 1–4 h after procurement. These experiments were approved by the Institutional Review Board. Adherent fat was removed, and adrenals were cross-sectioned with scalpels down the longitudinal axis. Slices with approximately similar proportions of adrenal zonae were used (i.e. extreme head and tail sections omitted).

Intact slices, approximately 2–3 mm thick, were used (average wet weight = 0.12 gm). Similarly, 0.12 gm of adrenal slices were used for the preparation of minces and cell suspensions. Adrenal minces entailed mincing slices with scalpels into approximately 2 mm² cubes. Cell suspensions entailed dispersing the mince of one slice (i.e. 0.12 gm) with 1.0 mg/mL collagenase (type I-A) and 0.1 mg/mL DNAse (Sigma Chemical Co., St. Louis, MO), at 37 C and 5% CO₂ for 1 h; followed by mechanical disruption using plastic transfer pipets. The initial cell suspensions were then centrifuged (500 x g, 3 min, 4 C), the supernatant discarded, and the cellular pellets resuspended in 3 mL serum-free medium (10) in 12-well culture clusters. Similarly, slices and minces were placed in 3 mL serum-free medium in 12-well culture clusters.

Time course studies and determination of cell viability and protein content

Adrenal tissue from one donor (female, age 17 yr) was used to examine the time course (20 min, 1, 3, 9 hr; n = 2) of steroid production of each tissue preparation. The incubation reactions were initiated by the addition of 10 µM pregnenolone substrate (Steraloids, Wilton, NH) to each tissue preparation and immediately placing these in a humidified culture incubator at 37 C and 5% CO₂. Additional incubations were conducted (0.33, 9 hr; n = 2) to determine cell viability and protein content. For cell viability determinations, slice and mince preparations were reduced to cellular suspensions at the end of each incubation period as described above, and resuspended in the original incubation media. An aliquot of each of these cellular suspensions (corresponding to the original tissue preparation) was mixed with a 0.3% trypan blue solution (11) and 5% FCS; cell viability was determined by using an improved Neubauer hemacytometer. To determine protein content, the tissue preparations at the end of each incubation period were reduced to a conventional subcellular fraction (12) by homogenization in 0.3 M sucrose on ice, filtering through cheesecloth, then centrifugation (8000 x g, 10 min, 4 C). Protein content was estimated using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).

Comparison of steroid production by slices, minces, and cell suspensions

Comparison of steroid production among the three tissue preparations was conducted using adrenals from three males and one female (age range 25–43 yr). Incubations for each donor were performed in triplicate, ± 1 µM ACTH-(1–24) (Organon, Bedford, OH) for 6 h. At the end of each incubation period the media was removed from slice and mince preparations, snap frozen in liquid nitrogen to terminate steroid metabolism, and stored at -70 C. For cell suspension preparations, the entire incubation volume (i.e. cell suspension and incubation media) was removed, and the cellular pellet and incubation media separated as described above. The incubation media (supernatant) was then snap-frozen and stored.

Determination of steroid levels

Stored incubation media was thawed, centrifuged (15, 600 x g, 5 min, 4 C) to remove cellular debris, and the supernatant used to determine steroid levels. DHA and F were determined using antibody-coated tube kits from Diagnostic Products Corporation (Los Angeles, CA). DHS and A4 were determined using liquid phase double antibody and antibody-coated tube kits, respectively, from Diagnostics Systems Laboratories, Inc. (Webster, TX). As these assays are designed for measurement of steroids in human serum, the incubation media samples were diluted or concentrated as necessary to ensure extrapolation of steroid values from the linear portion of the standard curve. Intra- and interassay coefficients of variance for control sample ligands 1, 2, and 3, from Chiron Diagnostics (East Walpole, MA), and pooled human serum were less than 5% and 10%, respectively.

Statistical analysis

The data shown for the time course incubations represent the mean steroid levels from duplicate incubations from a single donor. As such, these results are described in terms of apparent trends without statistical analysis. The data of the comparison between the three different tissue preparations (6-h incubations) represent the mean ± SD steroid levels from all incubations, conducted in triplicate using tissues from four donors. Significant differences in steroid levels among tissue preparations (either with or without ACTH) were determined by ANOVA with Fisher’s protected least significant difference test (PLSD) (P < 0.05).

	Results

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Time course of steroid production

For all steroids (Fig. 1, production either peaked or plateaued at between 1 and 3 h. DHS (Fig. 1A) and DHA (Fig. 1C) production decreased thereafter in some preparations; i.e. DHA and DHS production decreased at 1 h in slices, DHS production decreased at 1 h in minces, and DHA production decreased in cell suspensions after 3 h. No such decrease in production was observed for either F (Fig. 1B) or A4 (Fig. 1D).

View larger version (24K): [in this window] [in a new window]   Figure 1. Time course of: A) dehydroepiandrosterone (DHA); B) cortisol (F); C) DHA-sulfate (DHS); and D) androstenedione (A4) production by slices, minces, or cell suspensions prepared from adrenal tissue from a single donor (female, age 17 yr). Duplicate incubations for each tissue preparation for each time point were performed in serum-free medium in the presence of 10 µM pregnenolone substrate and 1 µM ACTH. The mean of duplicate experiments is expressed as the amount of steroid per gram wet weight of tissue.

Cell viability was similar among slices, minces, and cell suspensions, with 92.1%, 94.6%, and 93.0% mean viability at 20 min, and 90.2%, 92.2%, and 91.9% mean viability at 9 h, respectively. Protein content was generally similar between slices, minces, and cell suspensions, with a mean concentration of 36 mg/dL, 38 mg/dL, and 34 mg/dL at 20 min, and 37 mg/dL, 37 mg/dL, and 35 mg/dL at 9 h.

Comparison of steroid production by slices, minces, and cell suspensions

In the time course experiment using tissue from one donor, DHS (Fig. 1A) and F (Fig. 1B) production was similar among slices and minces and at least two-fold greater in cell suspensions. DHA production increased steadily over time in mince preparations while decreasing after 1 and 3 h in slice and cell suspension preparations, respectively (Fig. 1C). Similarly, A4 production was greater in minces compared with either slice or cell suspension preparations (Fig. 1D).

In the single time point experiments using tissues from multiple donors, as expected, addition of ACTH to the incubation reaction caused increased production of all steroids compared with the corresponding basal incubations (Fig. 2). DHS production was the most sensitive to changes in tissue preparation: increasing with increasing tissue disruption, i.e. slices less than minces less than suspensions (Fig. 2A, mean values = 0.88 vs. 2.1 vs. 3.0 mg/gm tissue, respectively, P < 0.0001). The production of F (Fig. 2B) and A4 (Fig. 2D) were higher in minces compared to either slices or suspensions (mean values = 25.6 vs. 37.7 vs. 18.7 mg/gm tissue, respectively, P < 0.0028; and 254 vs. 709 vs. 456 ng/gm tissue, P < 0.0042, respectively). The production of DHA was similar among all preparations (Fig. 2C). Age- or gender-specific differences in steroid levels were not evident.

View larger version (24K): [in this window] [in a new window]   Figure 2. Production of: A) dehydroepiandrosterone (DHA); B) cortisol (F); C) DHA-sulfate (DHS); and D) androstenedione (A4) production by slices, minces, or cell suspensions [triplicate incubations for each tissue preparation for each donor, n = 4 (3 males, 1 female, age range 25–43 yr)] in serum-free medium with 10 µM pregnenolone substrate in the presence or absence of 1 µM ACTH. Steroid levels (mean, n = 12 ± SD) are expressed as amount of steroid per gram wet weight of tissue. Data points (among incubations either with or without ACTH) with similar letters are not significantly different.

	Discussion

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Conceivably, the diffusion barriers inherent in each tissue preparation would limit the entry of the substrate (and any other potential regulatory factors including stimulators and inhibitors) into the tissue, and thus limit the enzymatic conversion of the substrate into the measured products. Such diffusion barriers are conceptually the lowest in cellular suspensions, followed by tissue minces, and highest for tissue slices. Thus, the total amount of steroid produced should be inversely proportional to the diffusion barrier. However, this trend was observed only for DHS. While the levels of A4 and F were higher in adrenal minces, compared to slices, the production of these steroids again decreased with further tissue disruption. The bimodal response of A4 and F may reflect the impact of two separate inhibitory effects, namely the decreased diffusion of pregnenolone into the tissue slices and the progressive inhibition of 3ß-HSD activity as cell-to-cell contact is lost.

Cumulative changes in steroid production (i.e. time course incubations), particularly the substantial decrease in DHA at 3 h in slice preparations, may represent one or more mechanisms influencing steroidogenesis. First, a generalized decay in cellular steroidogenesis may be most marked in slices, perhaps secondary to inadequate oxygenation and utilization of nutrients by more centrally located cells. However, this mechanism is unlikely as only DHA and DHS decrease, while A4 and F continue to rise in slice preparations. Secondly, the measured end products may be metabolized to other steroids, which increase after the first hour of incubation. This is also unlikely as there is not a corresponding increase in the concentration of A4 (a DHA product) after the first hour of incubation. Thirdly, an initial rapid release of DHA and DHS from tissue slices, perhaps due to cell death at the periphery, is followed by equilibration. Again, this is unlikely, as similar effects are not seen for A4 and F. Overall it appears that only DHA and DHS production are inhibited over time, with little effect on F or A4. This steroidogenic picture may result from inhibition of the ⁵17,20-lyase activity of P450c17. As reported previously, adrenocortical ⁴17,20-lyase activity (i.e. the direct production of A4 from 17-hydroxy-progesterone) is minimal in the human (13). Furthermore, in vivo studies have shown that acute stress or trauma results in a similar steroidogenic pattern, i.e. the inhibition of DHA and DHS secretion, although F levels continue to rise (14). It is postulated that this alteration in steroidogenesis is the result of altered P450c17 function. Likewise, the adrenal slice, as the most intact of the preparations used, may exhibit this stress response. If so, these data would suggest that maintenance of cell-to-cell contact plays an important role in the regulation of P450c17 activity.

The changes in adrenocortical steroidogenesis due to tissue disruption in vitro might suggest a role for altered cell-to-cell signaling in some of the physiopathologic alterations noted in vivo, for example, the decline in adrenal androgens noted with age (15). This hypothesis is supported by two findings. During human aging the boundary between the outer zona fasciculata and the inner zona reticularis becomes more diffuse as columns of the opposing zonal cell types begin to interdigitate across the zonal boundary (1, 16), thus suggesting a change in adrenocortical architecture. In addition, subjectively there is a dramatic age-related decrease in the overall consistency (texture) of the adrenal cortex, suggesting a loss in intercellular matrix (personal observation), although quantification of this process remains to be performed. Hence, physiologic changes in adrenal architecture could potentially represent a regulatory mechanism affecting adrenal steroid production.

Aside from the potential physicochemical discussion of the differences in steroid production, we must consider how these findings affect our knowledge of adrenocortical steroidogenesis. The majority of investigations of adrenocortical steroidogenesis have been accomplished using well-defined systems for monolayer cultures of either primary adrenal cell suspensions (5, 10) or adrenal carcinoma cell lines (17, 18). It is unclear to what extent these experiments reflect the actual steroidogenic processes in vivo. Although in situ adrenal studies likely provide more physiologically-relevant results, the results of in vitro studies do maintain some degree of cellular organization necessary for steroidogenesis (4) and do reflect some degree of steroidogenesis in vivo (19). Furthermore, the use of freshly prepared adrenal tissues is suggested to better reflect in vivo conditions compared to cell cultures (20). As noted previously, the functional activity of the human adrenal cortex is thought to depend upon the flow of steroids from one zone of the adrenocortical cells to the next and upon intraadrenal regulatory factors (2, 3). Obviously, the complex morphological architecture of the human adrenal cortex is completely lost under conditions of cell culture, acellular preparations, or dispersed cells. Hence, our data would suggest that, given sufficient donor adrenal tissue, the use of a tissue preparation that maintains some degree of overall tissue integrity, such as a tissue mince or slice, should be preferred.

We conclude that adrenocortical steroidogenesis is altered by changing the degree of architectural disruption of the adrenal tissue. Similarly, in an extensive review of glomerulosa function in rat adrenals (21), increasing tissue disruption had differential effects on aldosterone and corticosterone production in vitro. This mechanical disruption, which is necessary to study steroidogenesis in vitro, may impact the many physiological interactions within the adrenal gland (e.g. innervation, immune and endothelial cells, peptide growth factors, gap junctions) that are involved in the regulation of adrenocortical steroidogenesis (3). Nonetheless, at the gross tissue level, increasing tissue disruption appears to favor sulfotransferase activity in vitro and, depending on the time of incubation, either increases or decreases 3ß-HSD and ⁵17,20-lyase activities: although this remains to be confirmed molecularly. Moreover, the use of in vitro systems for the study of adrenocortical steroidogenesis that result in a significant loss of normal architecture may preclude observing differences in steroidogenesis that are the result of physiopathological alterations in cell-to-cell signaling. These results underscore the need for carefully considering technical aspects of in vitro adrenocortical studies, particularly when comparing results between reports using different modes of tissue preparation.

	Acknowledgments

 
We would like to thank the organ procurement and preservation staff of the Alabama Organ Center (University of Alabama Health Service Foundation and University Hospital, Department of Surgery, Division of Transplantation) for their assistance in providing the adrenal glands.

	Footnotes

 
¹ Supported in part by Grant RO1-HD29364 from the National Institutes of Health, Bethesda, Maryland (R.A.).

Received July 17, 1998.

Revised December 14, 1998.

Accepted January 5, 1999.

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