Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity

Endocrinology ◽  
2020 ◽  
Vol 161 (8) ◽  
Author(s):  
Monica P Goney ◽  
Matthew C J Wilce ◽  
Jacqueline A Wilce ◽  
William A Stocker ◽  
Georgia M Goodchild ◽  
...  
Keyword(s):  
Point Mutation ◽  
Inhibin B ◽  
Targeted Mutagenesis ◽  
Dominant Follicle ◽  
Inhibin A ◽  
Α Subunit ◽  
B Subunit ◽  
Classical Function ◽  
A Subunit ◽  
Synthesis Activity

Abstract Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing β-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of β-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin β A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this β A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin β B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.

scholarly journals The Biological and Immunological Characterization of Inhibin A and B Forms in Human Follicular Fluid and Plasma1

1997 ◽  
Vol 82 (3) ◽  
pp. 889-896 ◽  
Author(s):  
D. M. Robertson ◽  
N. Cahir ◽  
J. K. Findlay ◽  
H. G. Burger ◽  
N. Groome
Keyword(s):  
Molecular Weight ◽  
Inhibin B ◽  
Pituitary Cells ◽  
In Vitro Bioactivity ◽  
Serum Samples ◽  
Human Follicular Fluid ◽  
Inhibin A ◽  
Α Subunit ◽  
Rat Pituitary Cells

Abstract In a previous study (see Ref. 7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin α-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RIA detecting the αN region of the α-subunit, anα -subunit ELISA (Pro-αC) directed to the inhibin forms containing the Pro sequence, and an αC subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r= 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A+B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol wt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-αC immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-αC subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-αC ELISA may provide an appropriate and specific assay for the measurement of free α-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-αC standards were calibrated in terms of their αC subunit content, as determined by anα C subunit immunoassay, with the inhibin B standard containing 60% of the αC subunit content compared with either the inhibin A or Pro-αC standard. After adjustments of the various standards for this difference in αC subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-αC immunoactivity across the hFF and serum chromatograms and compared with levels determined by the α-subunit-directed immunoassays. A high correlation (r = 0.59–0.96) was observed, indicating that the α-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-αC. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33–36K and 50–66K forms of inhibins A and B; and 2) inhibin α-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the α-subunit.

scholarly journals A Spontaneous and Severe Hyperstimulation of the Ovaries Revealing a Gonadotroph Adenoma1

1998 ◽  
Vol 83 (10) ◽  
pp. 3450-3453 ◽  
Author(s):  
Sophie Christin-Maitre ◽  
Catherine Rongières-Bertrand ◽  
Marie-Laure Kottler ◽  
Najiba Lahlou ◽  
René Frydman ◽  
...  
Keyword(s):  
Unusual Case ◽  
Tumor Tissue ◽  
Normal Values ◽  
Inhibin B ◽  
Pituitary Insufficiency ◽  
Rt Pcr ◽  
Inhibin A ◽  
Α Subunit ◽  
Plasma Estradiol ◽  
Dramatic Rise

We report an unusual case of a gonadotroph adenoma in a 34-yr-old woman, revealed by a dramatic rise in the plasma estradiol (E2) concentration (26,800 pmol/L; normal, <370), with nonsuppressed FSH and LH levels (4.9 and 2.4 mIU/mL, respectively). The PRL level was 503 ng/mL. The testosterone and progesterone levels were 7 and 17 nmol/L, respectively. The levels of inhibin α, inhibin A, and inhibin B were increased compared to normal values in both the follicular (fp) and luteal (lp) phases of the menstrual cycle[ inhibin α, 1986 IU/L (fp normal, <700; lp normal, <1650); inhibin A, 254 pg/mL (fp normal, <20; lp normal, <120); inhibin B, 246 pg/mL (fp normal, <150; lp normal, <30 lp)]. Pituitary magnetic resonance imaging revealed a huge pituitary adenoma. After transphenoidal surgery, the patient presented with pituitary insufficiency and diabetes insipidus. RT-PCR of the tumor tissue was positive for LHβ, FSHβ, α-subunit, and PRL. This case is of particular interest because 1) although the E2 level was extremely high, the patient did not present with ascitis, suggesting that chronic elevated E2 does not play a crucial role in the hyperstimulation symptoms; 2) the extreme rise in E2 was related to the cosecretion of FSH and LH, confirming the two-cell two-gonadotropin theory; and 3) the rise in inhibin B is associated with FSH secretion, whereas the rise in inhibin A is probably due to luteinization.

206. Differential regulation of activin and inhibin production by interleukin 1 (IL1), transforming growth factor β1 (TGFβ1) and protein kinase C (PKC) in the Sertoli cell and granulosa cell

2008 ◽  
Vol 20 (9) ◽  
pp. 6 ◽  
Author(s):  
V. Eede ◽  
J. A. Muir ◽  
A. E. O. 'Connor ◽  
W. R. Winnall ◽  
A. E. Drummond ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Granulosa Cells ◽  
Sertoli Cells ◽  
Activin A ◽  
Inhibin B ◽  
Inhibin A ◽  
Α Subunit ◽  
Subunit Mrna ◽  
Subunit Expression

Activin and inhibin are gonadal regulatory proteins comprising an α-subunit and either a βA-subunit or βB-subunit (inhibin A or B), or two βA-subunits (activin A). Synthesis of the α-subunit, and the inhibins, is regulated by FSH via cAMP/protein kinase A. Regulation of the β-subunits in the gonads is less well defined, but the IL1/MAP kinase, TGFβ /Smad and PKC pathways have been implicated. Sertoli cells and granulosa cells were isolated from 18–22 day-old Sprague-Dawley rats under standard conditions and cultured with IL1, TGFβ1 and the PKC agonists, gonadotrophin releasing hormone (GnRH) or phorbol myristate acetate (PMA). Activin A, inhibin A and inhibin B were measured in culture medium (at 48h) by ELISA. Subunit mRNA expression was measured in cell extracts (at 4 h and 8h) using quantitative RT–PCR. IL1 stimulated βA-subunit and activin A production and inhibited α-subunit and βB-subunit expression and inhibin B production in Sertoli cells, but had no effect in granulosa cells. TGFβ1 stimulated activin A in both cell types, as well as the inhibins in granulosa cells. Surprisingly, TGFβ1 had no effect on Sertoli cell α-subunit or βA-subunit mRNA expression, but did cause a slight reduction of βB-subunit expression. GnRH increased activin A and inhibin A, but not inhibin B, production by granulosa cells and had no effect on Sertoli cells, which lack the GnRH receptor. However, direct activation of PKC by PMA stimulated βA-subunit mRNA expression and activin A production and decreased βB-subunit and inhibin B production by Sertoli cells, with marginal effects on inhibin A. These results indicate that activation of the TGFβ or PKC signalling pathways preferentially stimulates βA-subunit expression and/or translation, leading to increased activin A secretion by Sertoli cells and both activin A and inhibin A secretion by granulosa cells. The ability of IL1 to stimulate activin A is confined to the Sertoli cell.

scholarly journals Inhibin A and B in Vitro Bioactivities Are Modified by Their Degree of Glycosylation and Their Affinities to Betaglycan

Endocrinology ◽  
2007 ◽  
Vol 148 (5) ◽  
pp. 2309-2316 ◽  
Author(s):  
Yogeshwar Makanji ◽  
Craig A. Harrison ◽  
Peter G. Stanton ◽  
Radha Krishna ◽  
David M. Robertson
Keyword(s):  
Reversed Phase ◽  
Normal Function ◽  
Inhibin B ◽  
Pituitary Cells ◽  
In Vitro Bioactivity ◽  
Inhibin A ◽  
Α Subunit ◽  
Reproductive Axis ◽  
Rat Pituitary Cells

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn268 or Asn268 and Asn302 in the α-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 ± 0.15 vs. 0.24 ± 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 ± 0.29) than the 34-kDa form (1.08 ± 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC50, 0.68 nm) than the 34-kDa isoform (IC50, 8.2 nm) at displacing [125I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn302 of the α-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

scholarly journals Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro

2005 ◽  
Vol 185 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Y Okuma ◽  
K Saito ◽  
A E O’Connor ◽  
D J Phillips ◽  
D M de Kretser ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Sertoli Cells ◽  
Interleukin 1 ◽  
Activin A ◽  
Inhibin B ◽  
Dibutyryl Camp ◽  
Inhibin A ◽  
Reciprocal Regulation ◽  
Α Subunit ◽  
Subunit Mrna

In several biological systems, the inhibin βA homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1α and IL-1β) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1α or IL-1β, human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of βA-subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin βB-subunit and, to a lesser extent, α-subunit mRNA expression, thereby reducing basal and FSH-stimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.

Vaginally administered estroprogestinic decreases serum inhibin A and inhibin B levels and reduces endometrial thickness

2006 ◽  
Vol 86 (5) ◽  
pp. 1483-1487 ◽  
Author(s):  
S LUISI ◽  
L BORGES ◽  
L LAZZERI ◽  
A DELLANNA ◽  
F SEVERI ◽  
...  
Keyword(s):  
Endometrial Thickness ◽  
Inhibin B ◽  
Inhibin A

scholarly journals Inverse correlation between baseline inhibin B and FSH after stimulation with GnRH: a study of serum levels of inhibin A and B, pro alpha-C and activin A in women with ovulatory disturbances before and after stimulation with GnRH

2000 ◽  
pp. 77-84 ◽  
Author(s):  
FW Casper ◽  
RJ Seufert ◽  
K Pollow
Keyword(s):  
Inverse Correlation ◽  
Strong Relationship ◽  
Serum Levels ◽  
Activin A ◽  
Inhibin B ◽  
Inhibin A ◽  
Before And After ◽  
Pituitary Secretion ◽  
Objective Interest ◽  
A Minor

OBJECTIVE: Interest has focused recently on the influences of the polypeptide factors inhibin and activin on the selective regulation of the pituitary secretion of gonadotropins. DESIGN: Measurement of the concentrations of inhibin-related proteins in relation to the changes in pituitary gonadotropin (FSH, LH) parameters, after GnRH stimulation with a bolus injection of 100 microg gonadorelin, in 19 women with ovulatory disturbances. METHODS: Serum levels of inhibin A and B, activin A, and pro alpha-C were measured using sensitive ELISA kits. RESULTS: Within 60 min after GnRH stimulation, FSH values doubled from 5 to 10 mU/ml (P < 0.001). LH increased 12-fold from 2 to 24 mU/ml (P < 0.001). Activin A showed a significant decrease from 0.47 to 0.36 ng/ml (P < 0.001), whereas pro alpha-C increased from 127 to 156 pg/ml (P = 0.039). The median inhibin A concentration did not show a significant change between baseline and the 60 min value, whereas inhibin B was characterized by a minor, but not significant, increase in the median from 168 to 179 pg/ml (P = 0.408). A significant inverse correlation (P = 0.014) with a mean coefficient of correlation of 0.5516 was found, demonstrating a strong relationship between high inhibin B baseline levels and a small increase of FSH after 60 min. CONCLUSION: Our results show an interesting correlation between the baseline inhibin B and the change in FSH before and after GnRH stimulation. A high baseline inhibin B implies only a minor increase of FSH after 60 min.

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