H. G. Burger

Isolation of inhibin from bovine follicular fluid

Bovine follicular fluid was used as a source for the isolation of gonadal inhibin, the activity of which was monitored by the dose dependent suppression of the FSH content of cultured pituitary cells. The procedures presented result in over 3000-fold purification of the starting material and the purified inhibin has an apparent molecular weight of 56000. The purified inhibin can be dissociated under reducing conditions into two subunits with molecular weights of 44000 and 14000 daltons.

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.

CHANGES IN THE PITUITARY-TESTICULAR SYSTEM WITH AGE

In order to provide a comprehensive account of pituitary‐testicular function in man, 466 subjects, ranging in age from 2 to 101 years, were studied to examine blood levels of the pituitary gonadotrophins (LH and FSH), the sex steroids testosterone and oestradiol, the binding capacity of the sex hormone binding globulin (SHBG), the free testosterone and oestradiol fractions, and the transfer constant for the peripheral conversion of testosterone to oestradiol. The results were compared with clinical indices of testicular size, sexual function and secondary sex hair distribution. Serum LH and FSH were low before puberty, increased in pubertal adolescents to levels somewhat above those of adults and subsequently increased progressively over the age of 40 years. Testosterone levels fell slowly after the age of 40, while there was a slight rise in plasma oestradiol with increasing age. FSH and testosterone showed small seasonal variations in young adult men, the lowest values being seen in winter. SHBG binding capacity was high in two prepubertal boys, fell in adult men, but increased in old age. Free testosterone and oestradiol levels fell in old age. The metabolic clearance rates (MCR) of testosterone and oestradiol also fell in old age, while the conversion of testosterone to oestradiol was increased. Many correlations were observed between various hormonal and clinical measurements. The evidence is consistent with a primary decrease in testicular function over the age of 40 years.

Testicular control of follicle-stimulating hormone secretion.

Publisher Summary This chapter provides an overview of the testicular control of follicle-stimulating hormone (FSH) secretion. Castration is followed by an increase in circulating levels of FSH than of luteinizing hormone (LH). This suggests the possibility that the mechanisms of gonadotropin secretion either have different thresholds to steroid hormones or a feedback influence associated with the germinal epithelium. The chapter analyzes the factors that control the secretion of gonadotropins in the male and the effect of testicular disorders on the secretion. These disorders are frequently associated with characteristic abnormalities of gonadotropin secretion, observations that are valuable both in diagnosis and treatment of patients with problems of hypogonadism or infertility. All the work reported in the chapter is concerned with the testicular effects on the gonadotropins. The possibility that ovaries can produce a similar or identical substance stems from the fact that postmenopausal FSH levels are raised disproportionately, i.e., more than LH levels and there are many analogies between the functions of the testis and the ovary. There is a significant inverse correlation between the absence of ovarian follicles and the plasma levels of FSH in women with primary or secondary amenorrhea. One compelling stimulus in this field is the possibility of obtaining a substance for contraceptive use that could selectively suppress the secretion of FSH while preserving libido and potency in the male.

The radioimmunoassay of bovine and human follicular fluid and serum inhibin

A radioimmunoassay for inhibin in bovine and human follicular fluid (bFF, hFF) and serum from both species was developed based on an antiserum raised in a rabbit to purified bovine 58 kDa inhibin. Following immunization, parallel changes in plasma FSH and inhibin antibody titre were observed suggesting inhibin neutralization in vivo. The antiserum neutralized bFF, hFF and purified 31 kDa and 58 kDa inhibin activity in an in vitro bioassay system. Purified 58 kDa and 31 kDa inhibin were iodinated using a chloramine-T procedure and the 125I-inhibin purified by elution from Matrex Red A, with the iodinated molecules showing similar physicochemical properties to non-iodinated inhibin. Both tracers were stable in bFF but only 125I-31 kDa inhibin was stable in serum. A second antibody RIA system using either tracer yielded a parallel displacement between purified 31 kDa and 58 kDa inhibin. The cross-reaction in the RIA of inhibin from different species when expressed in terms of their bioactivity was bFF 100%, hFF 30%, ovine FF less than 1% and rat ovarian extract non-detectable. Rat LH and FSH, ovine LH and FSH, hCG, bovine TSH, LHRH, ovalbumin and bovine serum albumin showed less than 0.5% cross-reactivity using either tracer. Similar profiles of both bio- and immunoactive inhibin were observed at each stage of the inhibin purification procedure. The in vitro biological to immunological ratios for a number of purified 31 kDa and 58 kDa inhibin preparations using both tracers in the RIA, ranged from 0.30 to 0.43. The RIA was modified for serum by using 125I-31 kDa inhibin as tracer and an elevated temperature (30 degrees C) to minimize non-specific effects. No detectable activity was determined in steer or human post-menopausal serum whilst bull and human female serum showed parallel dose-response curves to their respective follicular fluid standards with circulating levels of 0.9 and 1.1 ng/ml respectively.

PLASMA INHIBIN LEVELS DURING GONADOTROPIN-INDUCED OVARIAN HYPERSTIMULATION FOR IVF: A NEW INDEX OF FOLLICULAR FUNCTION?

Human plasma inhibin was measured by radioimmunoassay in 26 women undergoing ovarian hyperstimulation for in-vitro fertilisation. Plasma inhibin rose in parallel with oestradiol during gonadotropin therapy (r = 0.79, p less than 0.001). Peak plasma inhibin levels correlated with follicle number as determined by ultrasonography (p less than 0.001) and with the number of oocytes recovered at laparoscopy (p less than 0.05). The increase in production of human ovarian inhibin by exogenous follicle stimulating hormone (FSH) is thus consistent with its proposed role in FSH regulation. Human plasma inhibin may be useful as a circulating protein marker of granulosa-cell function and may have therapeutic implications in human ovulation-induction programmes.

Immuno- and bioactive inhibin and inhibin α-subunit expression in rat Leydig cell cultures

Abstract The rise in serum immunoactive inhibin levels in male rats following hCG stimulation raised the possibility that Leydig cells may produce inhibin. This study therefore evaluated whether Percoll-purified Leydig cells from adult male rats synthesized and secreted inhibin in vitro as measured by Northern blot analysis, radioimmunoassay and in vitro bioassay. Northern blot analysis demonstrated the presence of α-inhibin subunit mRNA in the Leydig cell and inhibin bioactivity was detected in Leydig cell culture media. Levels of immunoactive inhibin increased in culture over 20 h and were directly dependent on the number of Leydig cells in culture. rLH (NIADDK-rLH-I-6) and not rFSH (NIADDK-rFSH-I-6) stimulated immunoactive inhibin levels in a dose-depedent manner. This study demonstrates that Leydig cells express mRNA for the a-subunit of inhibin and produce inhibin which is biologically and immunologically active prompting a re-evaluation of our concepts of testicular inhibin production.

DIAGNOSTIC SIGNIFICANCE OF THYROID MICROSOMAL ANTIBODIES IN RANDOMLY SELECTED POPULATION

A longitudinal study among the population of Busselton, Western Australia, has identified individuals with persistent and transient thyroid microsomal antibodies (TMA). 59 (72%) of 82 subjects with persistent TMA, 18 (72%) of 25 with recently developed TMA, and 12 (23%) of 53 with transient antibody were found to have subclinical hypothyroidism, as indicated by high serum thyroid stimulating hormone concentrations. This study reveals the high specificity, sensitivity, and predictive value of persistent or recently required TMA.

Isolation of a 31 kDa form of inhibin from bovine follicular fluid

The introduction of a pH 4.75 precipitation step to a previously described purification procedure from bovine follicular fluid (bFF) resulted in the isolation of a 31 kDa form of inhibin, in addition to 58 kDa inhibin. The procedure was monitored by an in vitro bioassay based on the suppression of the FSH cell content by pituitary cells in culture. The 31 kDa form was purified 5550-fold with approximately 5% recovery. On SDS-polyacrylamide gel electrophoresis a single band was detected with a molecular weight of 31 000 +/- 1500 (mean +/- SD) which upon reduction gave 2 subunits of 20 200 +/- 300 and 14 800 +/- 600. The biological activity expressed on mg protein basis was similar for both 31 kDa and 58 kDa inhibin although on a molar basis the 58 kDa inhibin was 2-3 times higher. A high degree of cross-reaction was observed between both forms in a radioimmunoassay of bovine inhibin using an antiserum raised against the larger form with either iodinated 31 kDa or 58 kDa inhibin as tracer. Based on the subunit composition of the 31 kDa and 58 kDa inhibin, their similar cross-reaction in a radioimmunoassay system and the apparent generation of the 31 kDa inhibin following a pH precipitation step, it is concluded that 31 kDa inhibin is a smaller form of the 58 kDa inhibin resulting from a shortening of the 43 kDa subunit to a 20 kDa subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

The human placenta: A novel source of inhibin

Human placental extracts contain inhibin bioactivity and immunoactivity giving dose response curves parallel to a human follicular fluid inhibin standard. Inhibin bioactivity in vitro was neutralised by preincubation of extracts with antisera raised to pure bovine inhibin. Umbilical cord blood from term infants contained immunoactivity. Human placental inhibin differs from human ovarian inhibin in terms of its biological: immunological ratio.