Chromosomal localization of the human proenkephalin and prodynorphin genes.

DNA probes derived from rat and human proenkephalin and prodynorphin genes have been used to localize these two opiate neuropeptide genes on human chromosomes. Hybridization of probes to Southern blots made with DNAs from a rodent-human somatic-cell hybrid panel indicates localization of proenkephalin to human chromosome 8 and of prodynorphin to human chromosome 20. In situ hybridization to metaphase chromosomes confirms these assignments and indicates regional localizations of proenkephalin to 8q23-q24 and of prodynorphin to 20p12-pter. A human genomic prodynorphin clone reveals a frequent two-allele TaqI polymorphism.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

Immunohistochemical evidence for the presence of peptides derived from proenkephalin,prodynorphin and proopiomelanocortin in the guinea pig pineal gland

Summary By using a plethora of region-specific antisera, this light microscopic immunohistochemical study revealed that derivatives from the three opioid precursors, i.e. proenkephalin, prodynorphin and proopiomelanocortin are differentially distributed in the pineal gland of guinea pig. Various molecular forms of immunoreactive opioid peptides derived from proenkephalin or prodynorphin were prosent in a minority of pinealocytes as well as in nerves. In contrast to this dual distribution pattern of opioid-active peptides, the opioid-inactive derivative from proopiomelanocortin, alpha-melanocyte stimulating hormone, was exclusively present in a large proportion of pinealocytes. A multiple and differential origin and function of opioidergic pineal innervation involving sympathetic, parasympathetic and sensory components is suggested. -MSH is proposed as a pineal hormone which may act in concert with melatonin to regulate pineal rhythms or may function like MSH of pituitary origin.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Deutsche Forschungsgemeinschaft (grant Schr 283/2-1 and We 910/2-1) and by Stifterverband für die Deutsche Wissenschaft, Schering AG and Freunde der Universität Mainz     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.     

Immunohistochemical evidence for the presence of peptides derived from proenkephalin, prodynorphin and proopiomelanocortin in the guinea pig pineal gland

By using a plethora of region-specific antisera, this light microscopic immunohistochemical study revealed that derivatives from the three opioid precursors, i.e. proenkephalin, prodynorphin and proopiomelanocortin are differentially distributed in the pineal gland of guinea pig. Various molecular forms of immunoreactive opioid peptides derived from proenkephalin or prodynorphin were present in a minority of pinealocytes as well as in nerves. In contrast to this dual distribution pattern of opioid-active peptides, the opioid-inactive derivative from proopiomelanocortin, alpha-melanocyte stimulating hormone, was exclusively present in a large proportion of pinealocytes. A multiple and differential origin and function of opioidergic pineal innervation involving sympathetic, parasympathetic and sensory components is suggested. alpha-MSH is proposed as a pineal hormone which may act in concert with melatonin to regulate pineal rhythms or may function like MSH of pituitary origin.     

Effects of testosterone and oestradiol on [3H]-thymidine incorporation by porcine granulosa and theca cells

Experiments were carried out to investigate the effects of varying physiological concentrations (0, 10, 100, and 1000 ng ml⁻¹) of oestradiol or testosterone on [³H]-thymidine incorporation by porcine granulosa and theca cells in vitro. Granulosa cells only were recovered from small (1–3-mm) follicles and both granulosa and theca cells recovered from large (4–8-mm) porcine follicles. Cells were cultured for 72 h in medium containing 10% foetal calf serum, 24 h in serum-free medium, and finally 40 h in serum-free medium containing [³H]-thymidine and appropriate steroid treatment. Although DNA per well was significantly higher (P < 0.05) at the end of culture in the theca cells than in the granulosa cells, neither steroid treatment had a significant (P > 0.1) effect on DNA concentration in either cell type. Overall, cells from small follicles incorporated significantly (P < 0.01) more [³H]-thymidine than those from medium follicles. Both oestradiol and testosterone significantly (P < 0.01) inhibited thymidine incorporation by cells from both follicle size categories, with a significant (P < 0.05) hormone × dose interaction. Finally, there was a highly significant (P < 0.001) interaction between the response of cells to different hormone concentrations and the follicle size from which they were recovered. These results indicate that both oestradiol and testosterone may act in an autocrine/paracrine manner to inhibit proliferation and encourage differentiation in follicular cells and thus are likely regulators of the later stage of antral follicle development in the pig.     

Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca

To evaluate the mechanisms involved in the reduction of estrogen concentrations in porcine follicular fluid during atresia, nonatretic and atretic follicles ranging from 4 to 7 mm in diameter were selected. Follicular fluid estrogen concentrations were 7-16-fold less in the atretic follicles. Isolated granulosa cells from atretic follicles demonstrated a significant reduction in aromatase activity and in follicle-stimulating hormone (FSH)-induced progesterone production in vitro compared to granulosa cells from nonatretic follicles. Isolated theca from atretic follicles also demonstrated a reduction in estrogen production. However, androgen concentrations were equivalent in the follicular fluid of atretic and nonatretic follicles, and theca from atretic follicles maintained testosterone and androstenedione production in vitro. The loss of thecal aromatase activity with atresia is not secondary to a reduction in FSH responsiveness, since FSH did not increase thecal progesterone production in vitro. Cell degeneration also does not account for the reduction in thecal estrogen production, since both androgen output in vitro and follicular fluid androgen concentrations were maintained. These data thus demonstrate that a mechanism other than reduced FSH responsiveness must account for the selective loss of thecal aromatase activity in this stage of atresia.     

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)     

Aromatase activity of isolated and recombined hamster granulosa cells and theca

The major synthesis of estrogen by the follicle is postulated to require both theca and granulosa cells. Theca in this scheme provide androgens to the major aromatizing site in the follicle, and the granulosa cell. One aspect of this theory was tested here. We investigated the comparative ability of isolated granulosa and theca, alone and in recombination to aromatize androgen in vitro. We found that the granulosa aromatize [14C]substrate more efficiently than do theca, and compare with the recombined system in their ability to aromatize [14C]androgen. The data therefore substantiates one aspect of the theory regarding the nature of the synergism, i.e., that the granulosa cells, at least in vitro, are the major site of aromatization of the preovulatory follicle.     

Steroid production in vitro by granulosa, theca, and luteal cells from goat ovaries

Basal progesterone (P4) production by isolated goat ovarian cells in vitro was in the order corpus luteum (CL) greater than granulosa (G) greater than theca (TH), while estradiol (E2) production was in the order TH greater than G greater than CL. In G cells, various concentrations (0.01 to 100 micrograms/ml) of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) increased P4 and E2 secretion. Testosterone (T, 10(-9) to 10(-5) M) produced dose-dependent increases in P4 and E2 secretion. Testosterone and LH together had an additive effect on E2 secretion. The combined effect of the lower (less than 10(-6) M) concentrations of T and LH on P4 production was marginally higher than either agent alone, but the increase was statistically insignificant; at higher concentrations of T (10(-6) and 10(-5) M) in combination with LH, P4 secretion was similar to that with LH alone, but was significantly (p less than 0.01 and less than 0.001, respectively) less compared to that with T alone. Follicle-stimulating hormone and T together produced a synergistic effect on E2 and an additive effect on P4 production. In TH cells, a dose-dependent increase in P4 and E2 production was observed with LH and hCG, but the effect of FSH was not significant. Testosterone produced a dose-dependent increase in P4 and E2 secretion. Testosterone and LH together induced higher steroid production than either agent alone. However, the increase was not statistically significant compared to T alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Evidence for interaction between granulosa cells and theca in early progesterone synthesis

The temporal characteristics of steroidogenesis in vitro by hamster preovulatory follicles, were compared to granulosa cells and theca incubated separately. Gonadotropin-stimulated intact follicles or recombined granulosa cells and theca synthesized increased amounts of progesterone by 30-120 minutes of incubation. The granulosa cells and theca, when incubated separately, did not begin to accumulate progesterone until 4 to 6 hours. The relatively rapid rise in follicular progesterone synthesis after in vitro gonadotropin stimulation follows the same time course as the rapid rise in vivo of hamster and rat preovulatory progesterone after the gonadotropin surge. The sharp differences in the temporal characteristics of progesterone synthesis between follicles and separated follicular cell types suggest an interaction between granulosa cells and theca in at least one phase of progesterone synthesis.     

Different action of ovine GH on porcine theca and granulosa cells proliferation and insulin-like growth factors I- and II-stimulated estradiol production

Growth hormone (GH) and insulin-like growth factors (IGFs) are recognized as regulators of ovarian function. This study was designed to compare the effect of GH and IGFs added alone or together on porcine theca interna and granulosa cells proliferation and steroidogenesis. Moreover, the effect of GH on IGF-I secretion was examined. Cells were isolated from medium size follicles and cultured in vitro for 48 h in serum free medium. Estradiol and IGF-I medium concentrations were determined by radioimmunoassays. Proliferation was evaluated by alamar blue assay and by radiolabelled thymidine incorporation. GH increased IGF secretion by granulosa cells while decreased its secretion by theca cells. Proliferation of both cell types was stimulated by IGF-I and IGF-II (30 ng/ml) and modestly inhibited by GH (100 ng/ml). Insulin-like growth factor II increased, in a statistically significant manner, estradiol secretion by both cell types, while IGF-I stimulated estradiol secretion to a greater extent by granulosa then by theca cells. The synergistic action of GH and IGFs on estradiol secretion was stimulatory in theca cells and inhibitory in granulosa cells. These data demonstrate that despite its direct action on estradiol secretion by granulosa and theca cells, GH also modulated estradiol secretion induced by IGFs. Differences in the estradiol production in response to GH alone and the effect of the synergistic action of GH and IGFs suggest that different cellular mechanisms for these hormones are triggered in each cell type.     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro

We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells.