Purification of "inhibin" from human ovarian follicular fluid.

Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 M at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25,000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 microgram/rat.

Inhibition of follicle stimulating hormone binding to granulosa cells in vitro by human follicular fluid.

Evidence for the existence of a low molecular weight (less than 10,000 daltons) factor in human follicular fluid that inhibits binding in vitro of FSH to ovarian granulosa cells is presented.

Inactivation of two hyperactive LH-RH analogues by rat hypothalamic peptidases.

Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are beleived to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself; the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TUB)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptidase with increased biological activity.

Study of testicular feedback in male rats using artificial cryptorchidism as a model.

Plasma LH, FSH and testosterone were measured in testosterone-treated and untreated cryptorchid and castrated male rats. Exogenous testosterone prevented the increase in basal LH but not FSH levels seen in the untreated cryptorchids. Increases in plasma LH and FSH in response to LH-RH were greater in the cryptorchid as compared to the control group and this could not be reversed by exogenous testosterone, suggesting that spermatogenesis-related feedback factors regulate LH as well as FSH at the pituitary level in the intact rat. The results were consistent with a reduced but nevertheless significant secretion of inhibin by the cryptorchid testis. Basal plasma testosterone levels and ventral prostate weights were not significantly different from intact animals.

[Inhibin-like activity in human ovarian follicular fluid (author's transl)]. / Nachweis von Inhibin-Aktivität in der Follikelflüssigkeit menschlicher Ovarien.

A steroid-free proteinaceous substance with inhibin-like activity has been extracted from human ovarian follicular fluid by alcohol precipitation. This substance was capable of inhibiting the ovarian weight increase of hCG-treated immature female rats and also of suppressing the post-castration rise of serum FSH levels in immature male rats. Follicular fluid from small follicls obtained from regularly cycling women was--on the basis of protein- found to be more active than cystic follicular fluid obtained from a premenopausal woman. The possible role of inhibin in the regulation of human ovarian cycle has been discussed.

The effect of local testicular irradiation on testicular histology and plasma hormone levels in the male rat.

Adult male rats were subjected to local testicular irradiation, plasma hormone levels and testicular histology being quantified at intervals up to 52 days thereafter. LH and FSH increased coincidently with spermatid but not with spermatocyte or spermatogonia depletion. Testosterone levels seemed to decrease but this effect was not significant. Oestradiol levels showed no significant changes. From the correlations between the various parameters it was concluded that the lack of inhibin was the main cause of the increase in both LH and FSH and that spermatids provide the signal for production of this non-steroidal inhibitor. The site of inhibin production was not definitively established but the results would be consistent with production of inhibin by the Sertoli cells in association with spermatids.

Some interrelationships between plasma levels of LH, FSH, oestradiol 17beta, androgens and semen analysis data in male infertility patients.

Serum LH, FSH and immunoreactive testosterone-like substances (TLS) have been measured by radioimmunoassay in 130 male infertility patients and oestradiol 17beta in 26 cases. A weak but significant negative correlation was found between FSH and sperm count (rs = -0.19, p less than 0.05) but not LH and sperm count. However, LH and FSH were strongly correlated in the azoospermic (rs = 0.71, p less than 0.01) and oligozoospermic (rs = 0.53, p less than 0.01) groups and levels of both gonadotrophins were significantly elevated in the azoospermic and oligozoospermic as compared to the normozoospermic group. The elevated LH levels in the oligozoo- and azoospermic groups could not be explained by reduced negative feedback of testosterone or oestradiol 17beta since firstly, TLS and oestradiol 17beta levels were similar in all three groups and, secondly, within-group correlations between LH and TLS were either non-significant or positive (azoospermic group r = 0.37, p = 0.06). It is suggested that spermatogenesis-related feedback factor(s) may inhibit LH as well as FSH secretion. No role for oestradiol 17beta as a selective inhibitor of FSH secretion seemed likely as oestradiol 17beta levels were similar in the three groups and were correlated (r = 0.51, p less than 0.01) to TLS levels i.e. to leydig cell rather than spermatogenic function. Seminal fructose was negatively correlated (r = -0.26, p less than 0.01) with sperm count but not significantly with plasma TLS. It would thus seem unlikely that the tendency for seminal fructose levels to increase as sperm count decreases is due to increased androgen production or that seminal fructose can be used as an index of a patient's androgenic status. Patients with varicoceles had hormone levels similar to other patients of similar sperm count. Both sperm morphology and motility were strongly correlated to log sperm count (r = 0.84 and 0.55 respectively) and semen volume was significantly greater in oligozoospermic than normozoospermic patients (p less than 0.01).

Concentration of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle.

Changes in the concentrations of ovarian steroids and pituitary gonadotrophins were measured by radioimmunoassay in the jugular plasma of six Clun Forest ewes throughout the oestrous cycle. The concentration of oestradiol began to rise 12-14 h before the onset of oestrus from values of 11-2 +/- 0-36 (S.E.M.) pg/ml during the luteal phase to 21-1 +/- 2-01 pg/ml at -8 to 0 h (oestrus). There was no distinct increase during the luteal phase. Circulating progesterone varied in a cyclic manner with the highest values at the mid-luteal phase (3-70 +/- 0-28 ng/ml; n = 28). In five out of six ewes the concentration was still quite high (1-86 +/- 0-43 ng/ml) at 35 h before the onset of oestrus. The concentration declined rapidly thereafter, reaching minimum values about 12 h before oestrus coincident with the increase in oestradiol concentration. Plasma LH increased from very low values of 2-59 +/- 0-09 ng/ml during the luteal phase to 75-3 +/- 7-4 ng/ml about 9 h after the onset of oestrus. Two peaks of plasma FSH concentration were detected after the onset of oestrus. The first peak (171-0 +/- 35-5 ng/ml) coincided with the LH peak and the second (133-0 +/- 10-7 ng/ml) occurred about 24 h later at a time when LH values were low. The mean FSH concentration at other times during the cycle was 61-9 +/- 2-8 ng/ml.

Testosterone undecanoate: a new orally active androgen.

Oral testosterone undecanoate (TU) in arachis oil has been evaluated with a view to its possible use as a means of androgen replacement therapy. A single 100 mg dose was found to elevate plasma androgen levels and urinary 17-ketosteroid excretion in 6 normal men. Ninety mg/day and 60 mg/day doses taken by a hypogonadal man resulted in sustained levels of androgen which appeared physiological when measured by radioimmunoassay without chromatography. However, upon separation of the steroids by chromatography it was found that much of the androgen present was in fact dihydrotestosterone not testosterone. Both TU and dihydrotestosterone undecanoate were detected in plasma by gas chromatography and it is suggested that the ester is absorbed as such from the intestine and the unesterified steroid subsequently released by hydrolysis. The convenience of oral administration, the resulting prolonged elevated plasma androgen levels and the probable lack of deleterious effects on the liver may render oral TU of value where androgen replacement therapy is indicated.

Further studies on enzymic inactivation of luteinizing hormone- releasing hormone (LH-RH) by peptidases in the rat hypothalamus.

Luteinizing hormone-releasing hormone (LH-RH) is known to be inactivated by peptidases in the rat hypothalamus with consequent loss of LH-releasing ability. To make a further study of the peptidases' action on the decapeptide, synthetic LH-RH and its [1-9NH2] analogue were incubated with the supernatant hypothalamic fraction containing the enzyme activity. Using an assay system measuring gonadotrophin release in ovariectomized/steroid-primed rats, both LH-RH and the [1-9NH2] analogue were found to be inactivated to different extents after incubation with the fraction, the analogue completely losing both LH- and FSH-releasing activity, and the releasing hormone almost completely losing its LH- and totally losing its FSH-releasing activity. The findings extend the initial studies by showing that the peptidases can remove both the decapeptide's intrinsic LH- and FSH-releasing activity and that these enzymes act on LH-RH at a site other than the C-terminal glycinamide, since they are able to inactivate the [1-9NH2] analogue lacking this residue.