Porcine Inhibin Research Articles

Circulating Radioimmunoassayable Inhibin during Periods of Transient Follicle-stimulating Hormone Rise: Secondary Surge and Unilateral Ovariectomy1

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.

Effects of passively immunizing ewes against inhibin and oestradiol during the follicular phase of the oestrous cycle

Passive immunization was used to investigate the importance of inhibin and oestradiol in the control of FSH production during the follicular phase of the oestrous cycle in the sheep. Four groups of five mature Scottish Blackface ewes were injected with normal sheep plasma (control), antiserum to the 1-26 alpha peptide fragment of porcine inhibin, antiserum to oestradiol-17 beta, or a combination of the two antisera, 24 h following cloprostenol-induced luteal regression. There was no difference in the concentration of LH in jugular venous plasma between the control and inhibin-immunized groups following the injection of normal sheep plasma or inhibin antiserum, with both groups exhibiting normal LH surges. In both the groups immunized against oestradiol, the basal concentration of LH rose by 25-30% (P less than 0.05) during the 96-h period following injection, while the LH surge and consequent formation of a corpus luteum was inhibited. In all three immunized groups there was a significant (P less than 0.001) rise in the concentration of FSH starting 3.8-4.8 h after the injection of antiserum. The duration of the rise was similar in the groups injected with oestradiol antiserum alone (43.6 +/- 12.8 h) or in combination with inhibin antiserum (40.6 +/- 11.7 h), but was significantly (P less than 0.05) shorter in the group immunized against inhibin alone (17.0 +/- 0.5 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor).

Recently the mesoderm-inducing effects of the transforming growth factor β (TGF-β) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF-β family has mesoderm-inducing ability.

Inhibin, Activin, and Follistatin: Regulation of Follicle-Stimulating Hormone Messenger Ribonucleic Acid Levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.

Passively immunizing ewes against inhibin during the luteal phase of the oestrous cycle raises the plasma concentration of FSH

Passive immunization was used to investigate the importance of inhibin in the negative feedback loop regulating the production of FSH in sheep. An anti-serum raised to the 1-26 peptide fragment of the N-terminus of the alpha-chain of porcine inhibin was first shown to neutralize the suppressive effects of inhibin on the production of FSH by dispersed ovine pituitary cells in vitro. Groups of five mature Scottish Blackface ewes on day 8 of the luteal phase of the oestrous cycle were then injected with either 10 ml plasma from normal ewes (control) or 10 ml ovine inhibin antiserum. On day 10, luteal regression was induced by an i.m. injection of cloprostenol (100 micrograms), and ovulation rate determined 6 days later by laparoscopy. Peripheral plasma samples were collected throughout the experimental period. Following treatment, there was no change in the peripheral plasma concentration of LH in either group. Following injection of the inhibin antiserum, the concentration of FSH rose significantly (P less than 0.001) compared with the control group. The concentration of FSH rose from 1.42 +/- 0.06 to a maximum of 2.58 +/- 0.23 (S.E.M.) micrograms/l by 5.6 +/- 0.9 h, this maximum lasting 9.0 +/- 1.1 h. By 32.8 +/- 6.9 h, the concentration of FSH had returned to pretreatment levels, while the titre of free antibody in the plasma of treated ewes was still high. In the treated ewes, there were one single and four double ovulations compared with three single and two double ovulations in the control group, indicating that the inhibin immunization may have resulted in an increase in ovulation rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay of inhibin in various mammals

A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and 125I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. Sensitivity of the assay was 3.1 ng International Research Standard of porcine inhibin per tube. Parallel inhibition curves were obtained for inhibin preparations from female and male animals of ten species, i.e. cattle, goats, sheep, cats, dogs, monkeys, pigs, horses, rats and man. Inhibin subunits and related proteins cross-reacted minimally with the antiserum used in the study. Plasma concentrations of inhibin in adult male and female rats were measured by the RIA before and at various times after gonadectomy. Inhibin levels in peripheral plasma before gonadectomy were significantly higher in adult female than in adult male rats. Inhibin levels decreased abruptly after gonadectomy in both sexes and they correlated negatively with plasma concentrations of FSH. This inhibin RIA will facilitate studies of the physiology of inhibin in various species of animals.

Immunolocalization and concentrations of inhibin alpha in the ovine testis and excurrent duct system.

Inhibin was localized in the ovine testis, excurrent ducts, and accessory sex glands by using a rabbit antiserum against a synthetic polypeptide representing the first 30 amino acids of porcine inhibin alpha-subunit. Concentrations of inhibin in fluids entering and leaving the epididymis also were determined in a radioimmunoassay using the same antibody. In the testis, immunostaining of inhibin was conspicuous in the seminiferous epithelium. Leydig cells occasionally were stained and the tunica media of blood vessels always was stained. Intense staining was observed in the epithelia lining the rete testis and ductuli efferentes. Staining also was intense in the epithelium of the initial segment and proximal caput epididymidis, and became less intense along the length of the epididymis. These observations were consistent with concentrations of inhibin in rete testis fluid (8.2 pmol/ml) entering the ductuli efferentes and in cauda epididymal plasma (0.67 pmol/ml) leaving the epididymis. Epithelia of ampullary and vesicular glands and of some prostatic acini were positively stained, but bulbourethral glands were never stained. Adrenal cortex, some proximal convoluted tubules in the kidney, and transitional epithelium of the urethra also were stained. Based on radioimmunoassay data and fluid flow rates for the ram, it was concluded that almost all of the 328 pmol inhibin that enters the ductuli efferentes daily is endocytosed in the proximal parts of the excurrent duct system. The physiological role(s) for inhibin, or inhibin-like peptides, in the excurrent duct system remains speculative.

Radioimmunoassay of inhibin in the serum of male monkeys.

A heterologous inhibin radioimmunoassay method to measure inhibin in serum of male cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys has been validated using a specific antibody against bovine 31 kDa inhibin and 125I-labelled 31 kDa inhibin as tracer. A serum pool from male monkeys was used as standard. Serial dilutions of normal monkey serum showed parallel logit-log dose-response curves to purified porcine and bovine inhibin as well as to a female human serum pool. The intra-assay coefficient of variation was 4.2% (n = 10) and the interassay coefficient of variation 5.1% (n = 10). No loss of inhibin immunoactivity was noted after storage at 23 degrees C for 5 days or repeated thawing and freezing of the serum samples. Serum from castrated monkeys showed undetectable levels of inhibin. Treatment with a gonadotrophin-releasing hormone agonist for 15 weeks led to a marked suppression of peripheral serum inhibin to concentrations similar to those after hypophysectomy or pituitary stalk section.

Circulating Inhibin α Concentrations in Infant, Prepubertal, and Adult Male Rhesus Monkeys (Macaca mulatta) and in Juvenile Males during Premature Initiation of Puberty with Pulsatile Gonadotropin-Releasing Hormone Treatment*

Circulating inhibin alpha concentrations were determined in infant, juvenile, and adult male rhesus monkeys with a RIA employing antisera to a synthetic fragment of the alpha-subunit of porcine inhibin. Binding of tracer, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-125I-Tyr, to antibody was inhibited by standard, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-Tyr. and by plasma from adult male monkeys in a parallel fashion. Castration in adults resulted in a 5-fold decline in the levels of immunoreactivity in plasma. Mean (+/- SE) plasma inhibin alpha concentrations in infants and adults (322.9 +/- 51.9 and 460.1 +/- 43.9 pg/ml, respectively) were significantly higher (P less than 0.05) than those in juveniles (191.3 +/- 28.3 pg/ml). Moreover, initiation of puberty in juvenile males, 13-18 months of age, with a chronic (10- to 12-week) intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h) resulted in a progressive rise in circulating inhibin alpha that plateaued, after 5 weeks of pituitary stimulation, at concentrations (343.9 +/- 38.2 pg/ml) comparable to those of infants and adults and twice those observed before initiation of the pulsatile infusion of GnRH. Circulating FSH concentrations increased during the first week of GnRH stimulation from 2.7 +/- 0.1 ng/ml before treatment to 6.0 +/- 1.2 ng/ml, where they remained for the duration of the experiment. Testosterone secretion during the initiation of precocious puberty occurred in discrete episodes that were robustly correlated with GnRH-induced LH discharges. In contrast, changes in circulating inhibin alpha concentrations over the 3-h interval between GnRH pulses were unremarkable. Activation of Sertoli and Leydig cells during initiation of puberty in the juvenile males, as reflected by circulating inhibin alpha and testosterone concentrations, respectively, occurred with similar time courses. At the time of orchidectomy, 10-12 weeks after initiation of GnRH treatment, testicular tissue was prepared for histological examination. In spite of a 2-fold gain in testicular weight and in hypertrophy of Sertoli cells in association with GnRH stimulation, maturation of the germinal epithelium did not progress past prophase I spermatocytes, and the number of these latter cells was meager. These findings indicate that the testis of the infant primate, like that of the adult, secretes significant amounts of inhibin, and that the quiescent Sertoli cell of the juvenile males may be readily provoked by appropriate gonadotropin stimulation into producing inhibin. The results also fail to provide evidence for the view that changes in circulating inhibin concentrations are robustly related, in an inve

Inhibin production by porcine granulosa and luteal cells: development and biological validation of a RIA.

We describe the development and biological validation of a radioimmunoassay for immuno- and bioactive porcine inhibin. A synthetic 1-32 porcine inhibin peptide was used to raise an antiserum and Tyr-1-32 peptide as tracer. As standard we employed porcine follicular fluid calibrated with the 1-32 alpha-inhibin. Medium obtained from serum-free cultured porcine granulosa cells was chromatographed on Superose S-12 and Mono-Q. Resulting fractions were analysed for inhibin bio- and immunoreactivity. It is shown that granulosa cells produce at least two types of bioactive inhibins, one being also immunoactive in our RIA. We studied secretion of immunoreactive inhibin from porcine ovarian cells under various conditions: Inhibin secretion from mature and immature granulosa cells can be stimulated by FSH, whereas hCG enhances inhibin secretion only from mature granulosa cells. During extended time of culture, the capability of granulosa cells to secrete inhibin is reduced. In contrast, progesterone secretion from these cells increases; this is due to spontaneous functional luteinization. This assumption is supported by the low inhibin secretion of luteal cells in comparison to granulosa cells. Intracellular inhibin content in luteal cells is below detection limit of the RIA, whereas granulosa cells contain readily detectable amounts of this hormone.

Changes in serum concentrations of immunoreactive inhibin during the oestrous cycle of the rat.

Serum and ovarian concentrations of inhibin during the oestrous cycle of rats were determined by a radioimmunoassay (RIA) based on a porcine inhibin RIA. Serum concentrations of LH, FSH, oestradiol-17 beta, progesterone and testosterone were also determined during the cycle. Serum concentrations of inhibin were high (8.47 +/- 0.58 micrograms/l) during the morning of pro-oestrus and then dropped sharply to the lowest level (3.21 +/- 0.38 micrograms/l) at 24.00 h on the day of pro-oestrus after the preovulatory LH and FSH surge. Inhibin concentrations then recovered rapidly, with a peak (7.94 +/- 0.97 micrograms/l) at 15.00 h on the day of oestrus. Inhibin concentrations in serum dropped again to their second lowest value (4.00 +/- 0.12 micrograms/l) at 24.00 h on the day of oestrus. The largest FSH surge observed from midnight of pro-oestrus to the morning of oestrus (the so-called second FSH surge) was shown to be inversely related to the changes in serum concentrations of inhibin, suggesting a close relationship between the serum FSH and inhibin concentrations in cyclic rats.

Changes in the plasma concentrations of inhibin throughout the normal sheep oestrous cycle and after the infusion of FSH

A radioimmunoassay for inhibin was developed using a peptide containing the 1-26 amino acid sequence of the N-terminus of the alpha-chain of 32 kDa porcine inhibin as immunogen, and 125I-labelled tracer. Evaluation of this assay using Sephadex column chromatography, chromatoelectrophoresis and immunoblotting confirmed that it measured all forms of inhibin present in sheep follicular fluid and was suitable for measurement of inhibin in sheep plasma. There was no evidence of the presence of free alpha-subunit in either sheep follicular fluid or ovarian vein plasma. The concentration of inhibin in jugular plasma throughout the follicular and luteal phases of four ewes with ovarian autotransplants was measured. The ovarian secretion of inhibin and oestradiol were also measured simultaneously throughout the follicular phase in a spontaneous cycle and after infusion of NIH-oFSH-S14 at 10 micrograms/h for 48 h following premature luteal regression induced by prostaglandin. The results showed: (1) no change in the peripheral concentration of inhibin throughout the cycle except an increase related to the periovulatory increase in FSH and LH. (2) Following luteal regression, the concentration of FSH fell as the secretion rate of oestradiol increased. During this time there was no significant change in the peripheral concentration of inhibin or ovarian inhibin secretion rate. (3) Following the infusion of FSH there was a marked increase in the concentration of inhibin in both ovarian and peripheral plasma and an increase in ovarian inhibin secretion rate. (4) The calculated metabolic clearance rate of inhibin, 20.3 ml/min, is similar to that of FSH. We conclude that in the ewe the ovarian inhibin secretion rate is stimulated by FSH and, although inhibin may modulate the basal secretion of FSH, a change in its secretion does not account for the fall in FSH which occurs during the follicular phase of the sheep oestrous cycle.

4798885 Compositions of hormonally active human and porcine inhibin containing an alpha chain and 62 chain: Anthony J Mason, Peter H Seeburg assigned to Genentech Inc

4798885 Compositions of hormonally active human and porcine inhibin containing an alpha chain and 62 chain: Anthony J Mason, Peter H Seeburg assigned to Genentech Inc

Changes in serum concentrations of inhibin in cyclic pigs.

A sensitive and specific radioimmunoassay (RIA) system for porcine ovarian inhibin has been developed. Antisera to porcine inhibin of molecular weight 32,000 (32 kDa inhibin) were raised in male chickens. The recognition site of the antiserum used in the present study was the N-terminal region of the alpha-subunit of 32 kDa inhibin. The antiserum could recognize higher molecular weight forms of inhibin present in porcine follicular fluid as well as the 32 kDa form. The average effective dose and the least detectable amount of inhibin in this RIA were 643 and 30.7 pg/tube respectively. Non-specific effects of serum on the RIA could be overcome by including 100 microliter serum from a castrated pig in the standards and by incubating at 30 degrees C. Serum concentrations of inhibin fluctuated between 0.6 and 2.5 micrograms/l during the oestrous cycles of the pigs. The amount of serum inhibin gradually increased from the late luteal phase to the early follicular phase and reached a maximum of 2.48 micrograms/l at day -4 (day 0 = day of ovulation). Concentrations then decreased rapidly to reach a minimum of 0.6 micrograms/l. Two small peaks were also observed during the luteal phase, although the concentration was relatively low during this phase. Changes in serum concentrations of oestradiol-17 beta did not parallel those of inhibin, especially during the luteal phase when serum concentrations of oestradiol-17 beta remained quite low. Serum concentrations of FSH were inversely related to those of inhibin rather than to those of oestradiol-17 beta, suggesting that the secretion of FSH during the oestrous cycles of pigs is mainly controlled by ovarian inhibin.

Age-dependent changes in physiological action, content, and immunostaining of inhibin in male rats.

We examined the role of endogenous inhibin in modulating FSH secretion in male rats during the infantile (days 10 to 21), juvenile (days 22 to 35), and pubertal (days 36 to 90) periods by 1) neutralization of endogenous inhibin using specific antibodies, 2) measurement of plasma and testicular levels of immunoreactive inhibin, and 3) immunohistochemical detection of testicular inhibin. In all studies, we used an antiserum against the first 26 N-terminal amino acids of the alpha-chain of porcine inhibin (anti-alpha-inhibin). Plasma immunoreactive inhibin levels were highest in young rats (8-15 days old), then decreased steadily with age. In addition, iv injection of the inhibin-alpha antiserum caused significant (P less than or equal to 0.01 or P less than or equal to 0.05) elevations in plasma FSH levels in male rats aged 10-24 days, whereas no significant (P greater than 0.05) changes occurred in older animals. In the testes, immunoreactive inhibin levels, expressed as femtomoles per mg wet weight, also declined with age. Inhibin immunostaining was most prominent in the Sertoli cells, with the greatest staining in the testes of 8 to 15-day-old rats. These results suggest that endogenous inhibin plays a physiological role in suppressing FSH secretion in infantile male rats, at an age when Sertoli cells contain the largest amount of this protein.

Inhibin: studies of stored and secreted forms by biosynthetic labeling and immunodetection in cultured rat granulosa cells.

The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the alpha-chain of porcine inhibin (pI alpha 1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and delta 4-androstenedione (10(-7) M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,000), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI alpha 1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin beta-A chain (pI beta A81-113) indicated that the 11,000 Mr peptide was the inhibin beta-A chain. Extracts of cells treated with FSH contained only a high Mr alpha-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin alpha antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10(-5) M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin alpha-chain precursor with 49,000 Mr. Although some of the high Mr alpha-form was secreted, the majority of the alpha-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr beta-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin alpha- and beta-gene expression as well as subunit dimerization and secretion.

Complementary Deoxyribonucleic Acid (cDNA) Cloning and DNA Sequence Analysis of Rat Ovarian Inhibins*

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.

Purification and Characterization of an FSH Releasing Protein from Porcine Ovarian Follicular Fluid

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species1–5 and characterized as a heterodimeric protein consisting of α- and β-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine6,7 and human7 inhibin α-subunits and two distinct porcine inhibin β-subunits (βA and βB)6 have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH8–14. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells3. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin βA-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50)∼25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.

Purification and characterization of Erythroid Differentiation Factor(EDF) isolated from human leukemia cell line THP-1

We isolated a protein, from a cell line of human origin, which exhibits extensive differentiation inducing activity toward Friend leukemia cells. The protein, called Erythroid Differentiation Factor(EDF), was found in a 4 day culture of THP-1 cells performed in the presence of 4 β -phorbol 12-myristate 13-acetate(PMA). EDF is a homodimer of a molecular weight of 25,000, with an NH 2-terminal sequence identical to that of the β A-chain of porcine Inhibin. It was suggested that a single protein species is responsible for the activities of both EDF and FRP, a FSH releasing protein isolated from porcine ovarian follicular fluid.

Hormonal regulation of inhibin production by cultured Sertoli cells

The hormonal regulation of inhibin production by cultured rat Sertoli cells was examined using a specific radioimmunoassay (RIA) which detects the N-terminal portion of the porcine inhibin alpha chain. FSH, but not hCG or prolactin caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathway to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system.