Androstenedione and progesterone production in vitro by the inner or the outer theca cells in preovulatory follicles of gonadotropin stimulated calves

This study reports some of the steroidogenic characteristics of the interna and externa theca cells taken from young and eCG primed calves. These cells were isolated from large healthy follicles. They were separately cultured for 3 days in absence or in presence of steroid substrates. Androstenedione (A4) and progesterone (P4) were measured by radioimmunoassay (RIA). In control conditions, A4 levels, higher in interna than in externa cells (**P<0.01), decreased during cultures (**P<0.01). In both cell types, A4 increased in presence of 17α-hydroxypregnenolone (17OHP5), pregnenolone (P5) and 22R-hydroxycholesterol (22R-chol) (*P<0.05) but not with P4 or 17α-hydroxyprogesterone (17OHP4) (P>0.05). The most efficient substrate was dehydroepiandrosterone (DHEA) (*P<0.05). In control conditions, P4 levels increased in both cell types. They were higher in externa than in interna cells on day 1, the reverse was observed on day 3. P4 levels increased after addition of 22R-chol and P5 (*P<0.05) but not with 17OHP5, 17OHP4 and DHEA (P>0.05) from day 1 in externa cells and only on day 3 in interna cells. P4 levels measured on day 1 were lower than the quantity of P4 added as a substrate. These results, obtained with theca cells from young calf follicles, indicate: 1/A4 is synthesized by the Δ5 pathway and 17α-hydroxylase activity decreases in vitro, 2/externa and interna cells differ by the quantities of A4 and P4 produced, 3/both lack precursors to produce A4 and P4 but their 3β-hydroxysteroid dehydrogenase activity subsists, 4/P4 could be metabolized during the first 2 days in both cell cultures.     

Transcriptome Comparisons Identify New Cell Markers for Theca Interna and Granulosa Cells from Small and Large Antral Ovarian Follicles

In studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Effect of Actinomycin D and Cycloheximide on Gonadotropin-Induced 17α, 20β-Dihydroxy-4-pregnen-3-one Production by Intact Ovarian Follicles and Granulosa Cells of the Amago Salmon,Oncorhynchus rhodurus*

The effect of actinomycin D and cycloheximide on gonadotropin (partially purified chum salmon gonadotropin, SGA)-induced 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-diOHprog, a maturation-inducing steroid in amago salmon) production was examined in intact ovarian follicles and granulosa cells of postvitellogenic amago salmon, Oncorhynchus rhodurus. Both actinomycin D and cycloheximide blocked gonadotropin-induced 17α, 20β-diOHprog production by intact follicles. In contrast, gonadotropin-induced 17α-hydroxyprogesterone production by intact follicles was not abolished by actinomycin D, but was abolished by cycloheximide, suggesting that postvitellogenic amago salmon ovarian follicles already contain the RNAs necessary for the synthesis of 17α-hydroxyprogesterone. In isolated granulosa cells, chum salmon gonadotropin was able to stimulate 17α, 20β-diOHprog production only when a precursor, 17α-hydroxyprogesterone was provided in the incubation medium, indicating that gonadotropin acts directly on granulosa cells to enhance the activity of 20β-hydroxysteroid dehyrogenase (20β-HSD). Total inhibition of 20β-HSD enhancement in granulosa cells, judged by 17α, 20β-diOHprog production, was achieved when actinomycin D was added between 1 hr before the start of incubation with 17α-hydroxyprogesterone and gonadotropin to 6 hr after. With cycloheximide total inhibition was observed when added in the period of 1 hr before to 9 hr after the start of the incubation. These results suggest that chum salmon gonadotropin acts on granulosa cells to enhance the de novo synthesis of 20β-HSD by a mechanism involving RNA synthesis.     

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.     

Steroid synthesizing cellular sites in the ovaries of Calotes versicolor (Daud.), Hemidactylus flaviviridis (Ruppel) & Chamaeleon calcaratus (Boulenger): histochemical study.

A histochemical study of steroid synthesizing cellular sites in the ovaries of Calotes versicolor (Daud.), Hemidactylus flavivirdes (Ruppel) and Chamaeleon calcaratus (Boulenger) is discussed. THe distribution of delta 5-3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 11beta-hydroxysteroid dehydrogenase, glucose-6phosphate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase and reduced nicotinamide-adenine dinucleotide diaphorase enzyme activities was studied in ovaries of the 3 species of lizards. All the enzyme activities occurred in 1) patches of cells of theca interna; 2) granulosa cells of large preovulatory, postovulatory, and atretic follicles; 3) interstitial cells of the ovarian stroma; and in the 4) ooplasm of the growing oocyte, suggesting their steroidogenic capacity. It was observed that following completion of follicular atresia, the phagocytic granulosa cells degenerate and the remaining cells of theca interna contribute to the formation of interstitial gland cells.     

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.     

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17-hydroxylase/C17–C20 lyase (17-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles. Even in the early stage of atresia, the granulosa cells showed remarkable morphological change characteristic of apoptosis, as visualized by scanning and transmission electron microscopy. Cessation of granulosa cell proliferation may occur earlier than apoptotic change and the degeneration of the granulosa cells becomes rapid once atresia starts.     

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.     

Ultrastructure of the theca interna of ovarian follicles in sheep

Summary The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy.In follicles <3.0mm in diameter, the theca interna consisted of about 8–12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure.In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages.It was concluded that: (1) the theca interna of 2.0–2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.     

17β-hydroxysteroid dehydrogenase from the fungus Cochlioboluslunatus: structural and functional aspects

17β-Hydroxysteroid dehydrogenase (17β-HSD) activity has been described in all filamentous fungi tested, but until now only one 17β-HSD from Cochlioboluslunatus (17β-HSDcl) was sequenced. We examined the evolutionary relationship among 17β-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17β-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17β-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17β-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7α-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17β-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Δ⁴ or 5α, such as: 4-estrene-3,17-dione and 5α-androstane-3,17-dione.     

Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase

The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ⁴-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.     

Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.     

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ–Δ isomerase pathway

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3β-hydroxy-steroid dehydrogenase/Δ⁵–Δ⁴ isomerase (3β-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3β-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3β-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13α-methyl analogue. In contrast to previous studies of fungi with 3β-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one were metabolized solely through the putative 3β-HSD/isomerase pathway, indicating that a 17β-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3β-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.     

Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)