David M. de Kretser

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.

Human Pregnancy by in Vitro Fertilization (IVF) Using Sperm Aspirated from the Epididymis

Spermatozoa were collected by microaspiration from the corpus epididymidis of a 42-year-old man with secondary obstructive azoospermia and used for in vitro fertilization. At insemination 61% of the spermatozoa were motile, with a motility index of 157. One of five eggs was fertilized and this was subsequently transferred to the patients wife at the two-cell stage. Ultrasound examination and changing hormone levels confirmed an ongoing pregnancy, which is currently at 30 weeks of gestation. This technique will provide a useful alternative for the management of some infertile men with obstructive azoospermia.

Follistatin: a multifunctional regulatory protein.

Follistatin was first described in 1987 as a follicle-stimulating hormone inhibiting substance present in ovarian follicular fluid. We now know that this effect of follistatin is only one of its many properties in a number of reproductive and nonreproductive systems. A majority of these functions are facilitated through the affinity of follistatin for activin, where activins effects are neutralized through its binding to follistatin. As such, the interplay between follistatin and activin represents a powerful regulatory mechanism that impinges on a variety of cellular processes within the body. In this review we focus on the biochemical characteristics of follistatin and its interaction with activin and discuss the emerging role of these proteins as potent tissue regulators in the gonad, pituitary gland, pregnancy membranes, vasculature, and liver. Consideration is also given to the larger family of proteins that contain follistatin-like modules, in particular with regard to their functional and structural implications.

The Pituitary-Testicular Axis in Men with Chronic Renal Failure

We studied the effects of uremia on the pituitary-testicular axis in 35 men with creatinine clearances less than 4 ml per minute per 1.7m(2). We found significant elevation (p less than 0.001) of plasma luteinizing hormone and follicle-stimulating hormone (p less than 0.005) and subnormal levels of testosterone (p less than 0.005). Testicular histology revealed severe spermatogenic damage. Human chorionic gonadotropin produced a subnormal testosterone response. The initial response of plasma luteinizing hormone and follicle-stimulating hormone to luteinizing-hormone-releasing hormone was normal, but their subsequent decline was prolonged. The suppression of plasma luteinizing hormone levels by testosterone propionate was normal, but the nadir occurred late; the elevated plasma luteinizing hormone level was due to reduced metabolic clearnace and increased production. Chronic renal failure interferes with testicular steroid production and spermatogenesis.

Men in Australia Telephone Survey (MATeS): a national survey of the reproductive health and concerns of middle-aged and older Australian men

BACKGROUND The Men in Australia Telephone Survey (MATeS) describes the prevalence of self-reported reproductive health disorders as well as related concerns and health behaviours among middle-aged and older Australian men. METHODS A representative sample population (n=5990) of Australian men (>or=40 years) was obtained by contacting a random selection of households with unbiased sampling, stratified by age and state. A 20-min computer-assisted telephone interview was done to assess reproductive health and related knowledge and beliefs, sociodemographic factors, general health, and lifestyle factors. FINDINGS A response rate of 78% (5990/7636) was achieved. 34% (1627/4737) of men surveyed reported one or more reproductive health disorder, all of which were most common in the oldest age group. Age-standardised prevalence of significant lower urinary tract symptoms was 16%, erectile dysfunction was 21%, and prostate disease was 14%. About 50% of participants reported having had a prostate cancer test whereas only 30% (300/1012) of men with erectile dysfunction sought medical help. Willingness to seek medical help for erectile dysfunction was related to age and ethnic origin. Although men aged 40-69 years expressed a moderate or high level of concern about prostate cancer and loss of erectile function, concern about reproductive health was less in the oldest age group (>or=70 years). INTERPRETATION The high prevalence of reproductive health disorders and associated concerns in middle-aged and older Australian men draws attention to the need to develop appropriate services and education strategies specifically directed to improving reproductive health in these men.

Activin A is a critical component of the inflammatory response, and its binding protein, follistatin, reduces mortality in endotoxemia

Activin A is a member of the transforming growth factor-β superfamily, which we have identified as having a role in inflammatory responses. We show that circulating levels of activin increase rapidly after LPS-induced challenge through activation of Toll-like receptor 4 and the key adaptor protein, MyD88. Treatment with the activin-binding protein, follistatin, alters the profiles of TNF, IL-1β, and IL-6 after LPS stimulation, indicating that activin modulates the release of several key proinflammatory cytokines. Further, mice administered one 10-μg dose of follistatin to block activin effects have increased survival after a lethal dose of LPS, and the circulating levels of activin correlate with survival outcome. These findings demonstrate activin As crucial role in the inflammatory response and show that blocking its actions by the use of follistatin has significant therapeutic potential to reduce the severity of inflammatory diseases.

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.

Pituitary Gland Function after Disconnection from Direct Hypothalamic Influences in the Sheep

A surgical procedure is described for isolating the pituitary gland from hypothalamic influences in sheep. The procedure results in total deafferentation of the stalk and median eminence but maintains the blood supply to the pituitary gland. The median eminence, pituitary stalk and anterior face of the pituitary gland were exposed by a transnasal, transphenoidal approach. In early studies section of the pituitary stalk as close as possible to the pituitary gland caused almost total infarction of the gland. Attempts were made to disconnect the pituitary gland from the hypothalamus by section immediately above the lateral inputs of the superior hypophyseal arteries but variable results were obtained, always with infarction of part of the pars distalis. When the pituitary gland was disconnected from the hypothalamus by entering the median eminence above the portal circulation and evacuating the nervous tissue of the tuber cinereum, (hypothalamo-pituitary disconnection; HPD), a small area of infarction was found in the pars distalis of only 1/10 cases. HPD effectively disconnected the pituitary gland from hypothalamic control whilst the pars distalis was not deprived of its blood supply. Complete severance of hypothalamo-pituitary connections also caused atrophy of the pars nervosa and enlargement of the cells of the pars intermedia. Following HPD, plasma LH and FSH concentrations diminished and plasma prolactin concentrations rose. On the day after surgery there were no LH, FSH or prolactin responses to 50 micrograms (i.m.) of oestradiol benzoate indicating the functional isolation of the pituitary gland from the hypothalamus. The isolated pituitaries were capable of responding to gonadotropin releasing hormone by LH release. Disconnection of the pituitary gland from the hypothalamus by subpial deafferentation provides a good in vivo isolated pituitary model in the sheep.