Control of human luteal steroidogenesis

The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.     

Prolactin stimulates steroidogenesis by human luteal tissue in vitro

Human luteal tissue recovered from varying stages of the luteal phase was minced and incubated for 3 h and the effect of human chorionic gonadotrophin (hCG), prolactin and hCG + prolactin on progesterone and oestradiol production measured. While hCG generally enhanced both progesterone and oestradiol synthesis, prolactin alone at either 20 or 200 micrograms/l had no significant effect on steroidogenesis. When prolactin was added along with hCG in four of six corpora lutea, however, progesterone production significantly increased and in three of six corpora lutea oestradiol production was increased above that induced by hCG alone. It is concluded that prolactin may play some role in the control of steroidogenesis by the human corpus luteum.     

Estrogen regulation of steroidogenesis in rabbit luteal cells

Estradiol is a potent modifier of gonadotropin-stimulated steroidogenesis. By the seventh day after ovulation, estradiol is the only agent required for the stimulation of progesterone synthesis by corpora lutea of superovulated pseudo-pregnant rabbits. To learn which control points in steroidogenesis are susceptible to regulation by estradiol alone, we have studied the production of pregnenolone, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one by corpora lutea of estradiol-stimulated and estradiol-deprived pseudopregnant rabbits. In previous investigations, we learned that estradiol deprivation in vivo, on day 9 of pseudopregnancy, causes an abrupt cessation of progesterone and 20 alpha-hydroxy-4-pregnen-3-one production, which is associated with accumulation of cholesterol and cholesteryl ester in the luteal tissue. We now report that production of pregnenolone, measured as its concentration in serum, also decreases abruptly by 84% within 48 h when the estradiol stimulus is removed on day 9 of pseudopregnancy. In addition, short term incubations of luteal tissue demonstrate that corpora lutea from estradiol-deprived rabbits do not use stores of luteal intracellular cholesterol for production of pregnenolone and progestin. These findings suggest that upon estradiol deprivation, rabbit luteal cells lose their capacity for using stored cholesteryl ester, or cholesterol synthesized de novo, for the production of pregnenolone and progestins. We, therefore, tested the hypothesis that a blockade of steroidogenesis caused by estrogen deprivation occurs at the point of cytochrome P-450 cholesterol side-chain cleavage (P-450scc), a principal rate-limiting step in the conversion of cholesterol to hormonal steroid products. To this end, we assayed the P-450scc activity in mitochondria-rich fractions of corpora lutea from rabbits that were deprived of estradiol for 24 and 48 h beginning on day 9 after induction of superovulation. Surprisingly, withdrawal of the estradiol stimulus did not cause loss of luteal P-450scc activity, measured as the amount of aminoglutethimide-inhibitable conversion of 25-hydroxycholesterol to pregnenolone by mitochondria-rich preparations. From these results, we infer that the luteotropic action of estradiol is probably not effected at P-450scc in the rabbit corpus luteum, but, presumably, occurs at control points that regulate the availability of stored cholesterol and/or its movement to or within the mitochondria for conversion to pregnenolone.     

The effect of melatonin on steroidogenesis by the human ovary in vitro.

Melatonin stimulated steroidogenesis in two compartments of the human ovary in vitro. In a corpus luteum of the menstrual cycle, melatonin increased progesterone synthesis in a dose related manner. Both serotonin and N-acetyl serotonin had no effect on progesterone synthesis. In the ovarian stroma, melatonin stimulated the incorporation of acetate-1-14-C into androstenedione. Binding of radioactive hCG by the corpus luteum was unaffected by melatonin. No specific binding of radioactive melatonin of low specific activity could be detected in homogenates of a human corpus luteum. These observations suggest that melatonin may directly modulate steroidogenesis in the human ovary.     

Insulin-like growth factor-I stimulates steroidogenesis in rabbit luteal cells

A potential role of insulin-like growth factor I (IGF-I) in the regulation of steroidogenesis in the rabbit corpus luteum was investigated using a primary culture of luteal cells obtained 3 days after ovulation. Dissociated cells were cultured for 1 day in the presence of medium 199 and 10% fetal bovine serum; thereafter, the cells were cultured in medium 199 containing 0.1% BSA, gentamicin (50 micrograms/ml), and hormones or growth factors, and without serum. IGF-I (human recombinant, 100 ng/ml) was as effective as LH (ovine, 10 ng/ml) in maintaining progesterone accumulation through 4 days of culture. Estradiol (10(-8) M), either alone or in combination with LH or IGF-I failed to stimulate progesterone accumulation, which was not surprising since these cells did not possess estrogen receptors. The stimulation of progesterone by IGF-I was not detectable until 24-36 h after introduction of the growth factor to the cultures, whereas stimulation by LH was observed within 2 h. The steroidogenic effect of IGF-I was not attributable to increased cell number, as DNA values or [3H]thymidine incorporation were unchanged by IGF-I. IGF-I increased functional enzymatic activity, observed as increased progesterone accumulation in the presence of 25-hydroxycholesterol used as exogenous substrate. These data indicate that luteal cells have the capacity to respond to IGF-I, raising the possibility that IGF-I has a role in the regulation of steroidogenesis in the rabbit corpus luteum.     

Protein kinase stimulation of steroidogenesis in rat luteal cell mitochondria

The regulatory role of cAMP-dependent protein kinase in steroidogenesis was examined in luteal cell mitochondria prepared from heavily luteinized prepubertal rat ovaries. The cAMP-dependent protein kinase, localized in luteal mitochondria, comprised 5.5% of the total cellular protein kinase activity (cAMP-dependent). Intact mitochondria supported by a suitable electron-donor substrate and inhibited by isoxazole converted cholesterol to a single steroid product, pregnenolone. Neither (Bu)2 cAMP nor a crude preparation of cytosolic protein kinase stimulated pregnenolone production from cholesterol when added to intact luteal cell mitochondria; however, mitochondria treated with 10 mM CaCl2 became responsive to both (Bu)2 cAMP and protein kinase by showing increased pregnenolone production. Likewise, the addition of cytosol protein kinase to incubations of cholesterol and crude cholesterol sidechain cleavage enzyme (cytochrome P-450cscc) isolated from luteal mitochondria, also stimulated pregnenolone production. Cholesterol-poor mitochondria, depleted of endogenous sterol by prolonged preincubation, when subsequently incubated with Ca+2 plus (Bu)2 cAMP and protein kinase showed significantly increased pregnenolone production. Conversely, mitochondria with greatly increased intramitochondrial cholesterol after preincubation with 200 microM cholesterol and a cytochrome P-450cscc inhibitor (aminoglutethimide) synthesized pregnenolone in significantly higher amounts than either normal or cholesterol-poor mitochondria after removal of the aminoglutethimide block. However, addition of (Bu)2cAMP or protein kinase to Ca+2-treated cholesterol-rich mitochondria failed to additionally stimulate pregnenolone synthesis. We conclude from these observations that the mitochondrial membrane normally excludes protein kinase and (Bu)2cAMP from any stimulatory action on cholesterol side-chain cleavage. Disruption of the mitochondrial membrane by high Ca+2 concentrations eliminates this barrier and permits (Bu)2cAMP and protein kinase stimulation of the CSCC enzyme system. The mechanism of stimulation is not clear. It could involve direct action on the CSCC enzyme. Alternatively, an increase in either intramitochondrial transport or binding of cholesterol substrate to the CSCC enzyme could be facilitated by protein kinase action. Direct stimulation of the enzyme by protein kinase seems less likely, since increased enzyme activity was not observed in the presence of high concentrations of intramitochondrial cholesterol substrate.     

Contrasting effects of prolactin on luteal and follicular steroidogenesis

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular basis for the inefficacy of 5-FU in human colon carcinoma

Five established human colorectal carcinoma cell lines with distinct phenotypic properties were exposed to different concentrations of 5-FU for varying time intervals. Effects were measured by sequential cell counts and by inhibition of colony formation. Treatment for 1 hr with 1 microgram/ml barely decreased survival of all cell lines as measured by colony formation; at a concentration of 100 microgram/ml, survival was modestly reduced for all cell lines, and for concentrations of 1000 microgram/ml, survival was decreased by greater than 50%. Extending the length of the treatment interval markedly increased the degree of cell kill for all concentrations of 5-FU. Treatment for greater than 24 hrs resulted in almost complete extermination of colony-forming cells, even for relatively resistant cell lines. The effect of 5-FU treatment on cell number was more complex and depended on drug concentration, length of treatment, and type of cell line. In general, decrements in cell numbers were somewhat related to both drug concentration and length of treatment interval, especially if performed 7 days after terminating drug treatment. Earlier cell counts were inconclusive and the same result could be obtained for different drug concentrations or treatment intervals. Furthermore, these results would change on a daily basis. More important, cell count results never correlated with the survival endpoint measured by inhibition of colony formation. Our results suggest that enhancement of the currently poor performance of 5-FU in the treatment of human colon carcinoma could originate from changing the administration modality to long-term infusion.     

Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary

There is increasing evidence that leptin is a physiological link between obesity and infertility. Although leptin receptors have been demonstrated in human ovaries, there is no information regarding the effects of leptin on cells from developing ovarian follicles. To test the direct effects of leptin on human ovarian cells, granulosa cells (GC) and theca cells were isolated from the ovaries of regularly cycling women. Serum was obtained at the time of surgery, and follicular fluid was aspirated from the follicles before isolation of the ovarian cells. Leptin concentrations were similar in follicular fluid and serum. RT-PCR analysis demonstrated that the long, signaling form of the leptin receptor was expressed in both theca and GC. In cultured GC, leptin had no effect on estradiol production, alone or in the presence of FSH, but caused a concentration-related inhibition of the insulin-like growth factor I (IGF-I) augmentation of FSH-stimulated estradiol production. The effect of leptin was specific, because there was no effect on progesterone production. In cultured theca cells, leptin did not alter androstenedione production, alone or in the presence of LH. Leptin caused a concentration-related inhibition of the IGF-I augmentation of LH-stimulated androstenedione production. These data demonstrate that leptin can directly inhibit IGF-I action in ovarian theca and GC at concentrations commonly present in obese women.     

Cellular basis for defective electrolyte transport in inflamed human colon

Electrolyte transport pathways in distal colonic mucosa from patients with noninflammatory and inflammatory (ulcerative colitis, Crohn's colitis) disease of the large bowel were studied in vitro with electrophysiological techniques. Noninflamed tissues exhibited substantial amiloride-sensitive electrogenic sodium transport. In contrast, inflamed but structurally intact tissues exhibited only a modest degree of electrogenic sodium transport, significant increases in total tissue conductance and apical membrane conductance, and a 100% increase in the arachidonic acid content of the cell membrane fraction of mucosal homogenates. Replacement of chloride with gluconate decreased total tissue conductance to a greater extent in inflamed than in noninflamed tissues, and total tissue conductance was higher in inflamed than in noninflamed tissues in the presence of transepithelial potassium and sodium gradients, suggesting enhanced mucosal "leakiness" to anions and cations in acute colitis. Apical addition of nystatin virtually abolished amiloride-sensitive apical sodium uptake in both groups, indicating that the ionophore formed channels in the apical membrane of noninflamed and diseased mucosa. Additional studies showed that mucosal inflammation decreased maximal activity of the basolateral sodium pump by 76%. Thus, defects in the biophysical properties of colonic epithelial cell membranes are likely to be important factors in the pathogenesis of diarrhea in ulcerative and Crohn's colitis.     

Cellular localization of keratin in proliferative epithelial processes and neoplasms of the human ovary

Cytokeratins are one of the intermediate cytoplasmic filaments which contribute to the cytoskeleton. Keratins have recently been demonstrated in normal and neoplastic tissues as well as in human cell lines. It has been suggested that the cellular location of keratin may reflect tissue-specific or epithelial type differentiation. Twenty-three examples of human ovaries containing the full spectrum of epithelial proliferations from inflammatory reactive processes to malignant neoplasia were studied for the cellular distribution of cytokeratin using antisera to human keratin. Nineteen cases contained immunoreactive keratin which was limited to the epithelial cells: 2/2 inflammatory, 8/10 benign tumors, 5/7 borderline tumors, 4/4 carcinomas. There was marked regional heterogeneity in keratin expression such that adjacent morphologically-identical cells could be functionally distinguished by the immunoreactive staining. The predominant cellular localization of keratin varied between histological tumor types in the benign neoplasms: serous = apical, subciliary; endometrioid = apical; mucinous = basal. This pattern was lost in the cytological progression to borderline and malignant tumors. In borderline tumors, the most intense reactivity was noted in areas of cellular atypia and proliferation. In borderline and malignant tumors, keratin was usually present in basal cytoplasmic regions contiguous with stroma.     

Effects of serum and lipoproteins on steroidogenesis in cultured bovine luteal cells

The effects of serum and lipoproteins on the function of bovine luteal cells in tissue culture were examined. Corpora lutea from regularly cycling dairy cows were dissociated with collagenase and cultured in Ham's F-12 medium with or without serum. The serum-free medium was supplemented with insulin, transferrin and hydrocortisone. Addition of LH to the serum-containing medium did not increase progesterone (P4) production. When the luteal cells were cultured in serum-free medium. LH produced a dose-dependent increase (P less than 0.001) in P4 production during the first 24 h of culture. The responsiveness of the cells to LH then gradually declined, and remained low until days 9-11 of culture, at which time the cells regained their ability to respond to LH. The luteal cells were responsive to dibutyryl cAMP in both serum-containing and serum-free medium. Serum lipoproteins (low and high density) were able to produce a 150-260% increase in progesterone production by the luteal cells cultured in serum-free medium. The results indicate that the presence of serum in the cell culture medium inhibits the responsiveness of luteal cells to LH at a step prior to the increase in cellular cAMP, and that serum lipoproteins can be used to increase progesterone production by cultured bovine luteal cells.     

Interaction of hCG and Lutalyse on steroidogenesis of bovine luteal cells

This study was designed to examine the ability of in vivo administration of human chorionic gonadotropin (hCG, 4000 IU) to alter the effects of Lutalyse (PGF2 alpha, 10 mg) in the cow. hCG significantly increased plasma progesterone concentration in midcycle cows (P less than 0.01), but these elevated levels were not maintained in the presence of Lutalyse (P less than 0.05). Responsiveness of luteal cells in vitro to luteinizing hormone (LH) (100 ng/ml), prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), dibutyryl cyclic AMP (dbcAMP) (10 mM) and PGF2 alpha (1 microgram/ml) + dbcAMP (10 mM) during a 2 h incubation was significantly reduced following in vivo treatment with Lutalyse when compared to in vivo untreated animals. In conclusion, the luteotropic effects of hCG were incapable of preventing Lutalyse-induced regression of the corpus luteum, and treatment of animals with hCG prior to Lutalyse administration could not prevent the significant decrease in responsiveness of luteal cells in vitro.     

Functional differentiation in steroidogenesis of two types of luteal cells isolated from mature human corpora lutea of menstrual cycle

Enriched small and large cell fractions were prepared from mature corpora lutea from 15 women in the midluteal phase by enzymatic dissociation, followed by Percoll gradient centrifugation. The steroidogenic function of each cell type was assessed by measuring the gonadal steroids released into the incubation medium. The large cell fraction was estimated to be 97% pure, with minimal contamination by small cells, whereas the small cell fraction was approximately 68% pure, being contaminated with 10% large cells and 22% nonsteroidogenic cells. In the unstimulated state, large cells were approximately 2-fold more potent in progesterone formation and aromatase activity, but only half as potent in androstenedione and testosterone formation as an equal number of small cells. When stimulated with hCG, the small cells responded with significant increases in progesterone, androstenedione, and testosterone release, but the large cells did not. Both cell types secreted estrone and 17 beta-estradiol in the presence of androgen substrate, but the addition of FSH significantly stimulated aromatization only in large cells. Thus, small and large human luteal cells have steroidogenic properties similar to those exhibited by follicular thecal and granulosa cells, respectively.     

Effects of prolactin on steroidogenesis and cAMP accumulation in rat luteal cell cultures

The effects of prolactin (PRL), alone and as a modulator of human chorionic gonadotropin (hCG) action, on steroidogenesis and cAMP accumulation in rat luteal cell cultures were examined. Cultured rat luteal cells were prepared from immature rats primed with pregnant mare serum gonadotropin and hCG. In vitro treatment was performed with 0.1 and 0.2 IU/ml hCG and 1-100 ng/ml PRL. Cultures were incubated for 48 h for evaluation of progesterone (P4) secretion and for 1 h for measurement of cAMP accumulation. The same doses of hormones and incubation periods were also used in preovulatory rat granulosa cell cultures and found to cause a significant, dose-dependent inhibition in estradiol, P4 and cAMP accumulation. In luteal cell cultures, on the other hand, P4 secretion was significantly elevated, in a dose-dependent manner, by PRL. Moreover, identical doses of PRL caused a significant, dose-dependent stimulation of cAMP accumulation. Basal levels of P4 were also significantly elevated by PRL alone, but no such stimulation by PRL was detected in basal levels of cAMP. Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, increased the stimulation of P4 and cAMP by hCG + PRL in a manner dependent on PRL concentrations. The overall data therefore demonstrate divergent effects of PRL on cAMP accumulation and steroidogenesis in the ovary: inhibitory in the preovulatory and stimulatory in the postovulatory state. Moreover, these findings suggest a possible common mechanism linking the effects of PRL before and after ovulation: inhibition of cAMP accumulation via enhanced breakdown of the nucleotide.     

Androgen modulation of follicle-stimulating hormone-induced granulosa cell steroidogenesis in the primate ovary

The role of androgen in regulating FSH-induced steroidogenesis in primates was investigated in granulosa cell cultures from reproductively suppressed (acyclic) marmoset monkeys. Progesterone accumulation and induction of aromatase activity were measured during a 48-h culture of granulosa cells (isolated from 0.5-1.0 mm diameter follicles) in medium 199 containing human (h) FSH and/or various sex steroids. Steroidogenesis in control cultures was minimal, but the presence of hFSH (0.3-100 ng/ml) caused dose-dependent stimulation. Maximal responses (mean +/- SE) were observed with 30 ng/ml of hFSH (aromatase, 1.0 +/- 0.2 pmol estradiol/10(3) cells X 3 h; progesterone, 4.5 +/- 0.8 pmol/10(3) cells X 48 h) and were 100 times basal values. The presence of testosterone (10(-6)M) during the 48-h culture enhanced the responses to hFSH two- to six-fold over the range 0.3-3.0 ng hFSH/ml. In the presence of a submaximal stimulatory dose of hFSH (3 ng/ml), the effects of testosterone on granulosa cell steroidogenesis were dose-related. Maximum responses were obtained with doses of testosterone between 10(-8) and 10(-7)M. Similar dose-dependent effects were found with 5 alpha-dihydrotestosterone (a non-aromatizable androgen), but not with estradiol, suggesting specific androgen synergism with FSH. Maximal aromatase activity induced after in vitro treatment with hFSH approached that in granulosa cells freshly isolated from a preovulatory follicle of a cyclic animal. These results demonstrate steroid modulation in vitro of FSH-responsive function, similar to that observed in rodent granulosa cells. Therefore, androgen may play a local role in the regulation of FSH-stimulated granulosa cell function during follicular development in primates.     

Immunohistochemical localization of pituitary gonadotrophins and gonadal steroids confirms the 'two-cell, two-gonadotrophin' hypothesis of steroidogenesis in the human ovary

Ovaries from 37 women with normal menstrual cycles were analysed for localization of pituitary gonadotrophins and gonadal steroids using an immunohistochemical method. In the follicular phase, FSH and oestradiol-17 beta localized in the granulosa layer, and LH, progesterone and testosterone localized in the internal thecal layer. In the luteal phase, gonadotrophins and steroids localized in luteal cells. Particularly in the early luteal phase, FSH and oestradiol-17 beta localized in large luteal cells, and LH, progesterone and testosterone localized in small luteal cells. The results of the present immunohistochemical analysis confirm the two-cell, two-gonadotrophin hypothesis of steroidogenesis in the human ovary.