Shao-Yao Ying

Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues.

Publisher Summary This chapter provides an overview of molecular characterization and the distribution and biological activities of fibroblast growth factor (FGF) in various tissues. FGF in the pituitary plays a nonmitogenic role in the maintenance of prolactin and thyrotropin responsiveness, while the same molecule in macrophages may promote wound healing and in the corpus luteum angiogenesis. It is based on the diverse biological activities of somatostatin in brain, hypothalamus, stomach, and pancreas and the wide distribution of numerous neuropeptides in both the central nervous system and the gastrointestinal tract. The concept is equally applicable to FGF and perhaps other growth factors that show a wide distribution in many tissues. The results presented in the chapter suggest that heretofore unidentified biologic activities may be related if not identical to acidic and basic FGFs. These would include ovarian growth factor, macrophage-derived growth factor, cartilage growth factor, tumor angiogenic factor, eye-derived growth factor, retina-derived growth factor, corpus luteum angiogenic peptide, follicular angiogenic factor, adrenal vascularizing factor, and endothelial cell growth factors.

Inhibin and beta type transforming growth factor (TGFβ) have opposite modulating effects on the follicle stimulating hormone (FSH)-induced aromatase activity of cultured rat granulosa cells

We have recently observed that attomolar concentration of exogenously added TGF beta, a molecule structurally related to inhibin, can stimulate the basal secretion of FSH in a pituitary cell culture. Inhibin purified from porcine follicular fluid antagonizes this activity of TGF beta. To understand further the homeostatic regulatory properties of inhibin and TGF beta we have investigated whether the aromatase activity of ovarian granulosa cells is also subject to intra-ovarian modulation by these peptides. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 2 days with androstenedione (10(-7) M) as a substrate, oFSH (2 ng), and different amounts of TGF beta or inhibin. Basal estrogen secretion was negligible and remained unaffected by treatment with purified TGF beta or inhibin (10 ng/ml), whereas treatment with oFSH (2 ng/ml) produced a 100-fold increase in estrogen accumulation. The concurrent application of increasing concentrations (10 pg-10 ng/ml) of TGF beta produced dose-dependent increments in the FSH-stimulated accumulation of estrogen with a ED50 of 0.3 +/- 0.02 ng/ml. On the other hand, concurrent incubation of FSH with inhibin ranging from 10 pg to 10 ng/ml decreases the FSH-mediated estrogen secretion. TGF beta antagonizes the inhibition of inhibin on aromatase activity. These findings suggest that inhibin and TGF beta, two closely related molecules, play novel and opposite roles in modulating the follicular functions.

A homodimer of the β-subunits of inhibin a stimulates the secretion of pituitary follicle stimulating hormone

Summary A 24,000 Dalton protein with follicle stimulating hormone (FSH)-releasing activity, named activin , has been characterized previously from porcine follicular fluid as a heterodimer composed of the β-subunits of inhibins A and B linked by disulfide bond(s) [Ling et al. (1986) Nature, in press]. In this paper we report the isolation of another 24,000 Dalton protein with FSH-releasing activity from porcine follicular fluid, using successive steps of heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and four steps of reversed-phase HPLC, followed by preparative sodium dodecyl-sulfate-polyacrylamide gel electrophoresis chromatography. Based on the molecular weight of the isolated molecule and its deduced NH2-terminal sequence, we propose that this second FSH-releasing substance present in porcine follicular fluid is a homodimeric protein composed of two β-subunits of inhibin A joined together by disulfide bond(s). The name homo-activin-A is proposed for this substance.

Follistatin specifically inhibits pituitary follicle stimulating hormone release invitro

Two forms of purified follistatin, a single-chain polypeptide of mol wt 35,000 (35 Kd) protein, and a related molecule of mol wt 32,000 (32 Kd), which differs from the 35 Kd form in glycosylation or carboxyl terminal truncation, specifically inhibit the release of immunoreactive FSH by primary cultures of rat pituitary cells. Both forms of follistatin and inhibin-A give similar dose-response curves, with identical slopes and maximal effects, suggesting that they may all act through the same mechanism on the pituitary cells. The median effective dose (ED50) of each of the follistatins is 6.2-7.3 ng/ml (1.8 x 10(-10) M), which corresponds to approximately 1/3 of the potency of inhibin. The effect of 35 Kd or 32 Kd follistatin is highly specific for suppressing the release of immunoreactive FSH since there is no demonstrable concomitant effect on the secretion of other pituitary hormones. The effect of follistatins, like that of inhibins, is different from that of the hypothalamic hypophysiotropic factors, requiring greater than or equal to 18 h of incubation in a pituitary monolayer culture system to demonstrate. Coincubation of inhibin and follistatin shows an additive effect in the suppression of FSH release. Pituitary cells exposed to follistatin have significantly less depletion of intracellular FSH (0.01) than those treated with inhibin, indicating that follistatin may act primarily on the suppression of FSH release rather than on both release and synthesis of FSH, as is the case with inhibin.

Immunohistochemical detection of inhibin in the gonad

Antiserum to inhibin was produced in rabbits by immunization with a synthetic [Tyr30]alpha-chain(1-30)NH2 fragment of porcine inhibin coupled to bovine serum albumin, and the elicited antiserum was used in conjunction with the avidin-biotin immunoperoxidase procedure to localize inhibin-reactive cells in various rat tissue preparations. In the testes, only the Sertoli cells revealed immunoreactivity with the antiserum. Intense staining was also observed in ovarian follicular granulosa cells but not in the theca layer outside the basement membrane. In addition, the luteal cells in the corpus luteum were also stained by the antiserum. The positive staining in the gonadal tissues could be blocked completely by pre-adsorbing the serum with either the synthetic peptide or native inhibin. Immunostaining was not detected in brain, pituitary, thymus, stomach, pancreas, kidney and adrenal section, thus confirming that inhibin is a polypeptide originating only from specific cells of the gonad.

Inhibins and activins

Publisher Summary This chapter discusses the isolation, structure, characterization, and biosynthesis of inhibins and activins. The two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) which regulate the development and maturation of the gonad, are identified. The ability to quantitate FSH and LH in serum revives the inhibin concept, which offers an explanation for the differential regulation of FSH and LH release often observed in humans and experimental animals. Inhibin activity is monitored by an in vitro bioassay using rat anterior pituitary cells in monolayer culture. The message encoding each of the subunits of the inhibins is cloned from porcine and bovine ovarian complementary DNA (cDNA) libraries. DNA sequence analysis shows that the inhibin a subunit is initially synthesized as a large precursor protein with potential N-linked glycosylation sites. The two β subunits are also found to be derived from large polypeptide precursors and cleavage at the processing sites to yield the mature β subunits at the carboxy termini of the precursors. Using the porcine cDNAs as hybridization probes, the human and murine inhibin subunit precursors are also cloned and sequenced.

(DTrp6-Pro9-NEt)-luteinising hormone-releasing factor inhibits follicular development in hypophysectomised rats

THE super-luteinising hormone-releasing factors (super-LRFs) are a group of synthetic analogues at least 100 times more potent than the native peptide in vitro and in vivo1. Several laboratories have demonstrated inhibitory effects of super-LRFs on ovulation, egg implantation, pregnancy, testicular weight and spermatogenesis2–6. Their proposed mechanisms of action involve some type of desensitisation of the pituitary to the highly potent LRF analogue and of the gonads to the gonadotropins released in large quantities by the super-LRf, in both target tissues through receptor down-regulation. Another, seemingly unlikely, explanation for the gonadal inhibitory effects of super-LRFs is that the synthetic peptide acts directly at the level of follicular ovarian tissues. Such a direct effect of super-LRF on the gonads has been suggested by the observation that large doses of LRF prevent cultured granulosa cells from aromatising androgens to oestrogens7. We now report that a super-LRF inhibits follicular development in hypophysectomised rats pretreated with pregnant mare serum gonadotropin.

Purification of Gonadostatin from Bovine Seminal Plasma (BSP)

The concept of a gonadal peptide regulating the secretion of gonadotropins was originally proposed by McCullagh in 1932. This hypothetical material, which he called inhibin, was deduced as being a nonsteroidal substance, probably protein or polypeptidic in nature, that is released into general circulation and acts specifically on the pituitary gland, selectively inhibiting the secretion of FSH, but not that of LH. Such preferential regulation of gonadotropin secretion has generated a great deal of attention during the past decade. Inhibin, or inhibin-like activities, have been reported in extracts of testis (Lee et al., 1976; Keogh et al., 1976; Moodbidri et al., 1976; Nandini et al., 1976; Baker et al., 1976), spermatozoa (Lugaro et al., 1974), rete testis fluid (Setchell and Sirianthsinghji, 1972; Setchell and Jacks, 1974), seminal plasma (Franchimont et al., 1975; Chari et al., 1978), and ovarian follicular fluid (Hopkinson et al., 1977; de Jong and Sharpe, 1976; Welschen et al., 1977; Marder et al., 1977; Schwartz and Channing, 1977).

Gonadocrinins: Peptides in Ovarian Follicular Fluid Stimulating the Secretion of Pituitary Gonadotropins

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two-antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LR, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secret substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second day of diestrus induced secretion of LH comparable to that produced by a 250-ng LRF injection. Gonadocrinin has chemical characteristics different from those of LRF. When purified gonadocrinin or LRF was applied to an identical isocratic high pressure liquid chromatography system, LRF was eluted at a position different from that of gonadocrinin, indicating that, chemically, gonadocrinin is not identical to the hypothalamic decapeptide, LRF.