Proliferative phase sertoli cells display a developmentally regulated response to activin in vitro

We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using (125)I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.     

Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid

Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.     

An assessment of Leydig cell function after bilateral or unilateral efferent duct ligation: further evidence for local control of Leydig cell function

Leydig cell function was examined in adult male rats at 2 and 4 weeks after bilateral or unilateral efferent duct ligation. Bilateral efferent duct ligation resulted in significantly elevated serum LH levels, an increased size of Leydig cells and an enhanced testosterone response to gonadotropin stimulation in vitro despite a marked loss of LH-hCG receptors. After unilateral ligation of the vasa efferentia, the parameters of Leydig cell function in the ligated testes showed identical changes to those seen after bilateral ligation, whereas such changes were minor or absent in the unligated contralateral testes of the same animals. These results demonstrate that the modifications of Leydig cell function after efferent duct ligation are mainly due to local changes within the testes providing further evidence for an intratesticular control of Leydig cell function.     

Characterization of serum activin-A and follistatin and their relation to virological and histological determinants in chronic viral hepatitis

BACKGROUND/METHODS: Hepatocyte proliferation in viral hepatitis is regulated by a number of growth factors. Activin-A inhibits hepatocyte DNA synthesis while follistatin, a potent activin-A antagonist, promotes liver regeneration. We report the first study of activin-A and follistatin in human viral hepatitis. Sera from 15 normal subjects, 22 hepatitis B and 47 hepatitis C patients were analysed for activin-A and follistatin and correlated with serological and histological markers of liver injury and with specific immunohistochemistry. RESULTS: All groups showed immunoreactivity for activin with hepatocyte localisation. Serum activin-A was significantly increased in viral hepatitis patients compared to controls, was greater in hepatitis B compared to hepatitis C, and correlated with serum aminotransferase and hepatitis B viral replication. A concurrent rise in serum follistatin was not observed in either group, but serum follistatin correlated inversely with hepatitis B DNA levels. Although hepatocyte apoptosis in hepatitis C and proliferation in both groups was significantly elevated compared to controls, there was no correlation with serum activin-A or follistatin. CONCLUSIONS: Activin-A and follistatin are constitutively expressed in human liver and serum concentrations are increased in viral hepatitis. Dysregulation of the activin/follistatin axis may be linked to hepatitis B replication but does not correlate with hepatocyte apoptosis.     

Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men

Testosterone (T) treatment suppresses gonadotropin levels and sperm counts in normal men, but the addition of a progestin may improve the efficacy of hormonal contraception. This study aimed to investigate the speed and extent of suppression of testicular germ cell number induced by T plus or minus progestin treatment and correlate these changes with serum gonadotropins and inhibin B levels, testicular androgens, and sperm output. Thirty normal fertile men (31-46 yr) received either testosterone enanthate (TE, 200 mg im weekly) alone or TE plus depot medroxyprogesterone acetate (DMPA, 300 mg im once) for 2, 6, or 12 wk (n = 5 per group) before vasectomy and testis biopsy. Five men (controls) proceeded directly to surgery. The inclusion of DMPA led to a more rapid fall in serum FSH/LH levels (time to 10% baseline: FSH; 12.6 +/- 2.6 vs. 7.9 +/- 1.4 d; LH, 9.9 +/- 3.4 vs. 3.4 +/- 1.7 d, TE vs. TE+DMPA, respectively, mean +/- SD, both P < 0.0001), yet the mean time to reach a sperm count 10% of baseline was not different (23.7 +/- 7.3 vs. 25.3 +/- 13.9 d, NS). The maximum extent of FSH/LH suppression was identical at 12 wk (mean serum FSH 1.2 and 1.6%, and mean LH 0.3 and 0.2% of baseline: TE vs. TE+ DMPA, respectively) as was sperm count suppression (5 of 5 and 4 of 5 men, respectively, with sperm counts < or =0.1 x 10(6)/ml). Serum inhibin decreased to 55% control at 12 wk in the TE+DMPA group (P < 0.05) but was unchanged by TE treatment (86% control, NS). Testicular T levels declined to approximately 2% of control levels, but testicular dihydrotestosterone and 5alpha-androstane-3alpha,17beta-diol (Adiol) levels were not different to control. Germ cell numbers as determined by stereological methods did not differ between TE and TE+DMPA except at 2 wk when type B spermatogonia and early spermatocytes were significantly lower in the TE+DMPA group (P < 0.05). In all groups, a marked inhibition of Apale-->B spermatogonial maturation was seen along with a striking inhibition of spermiation. We conclude that: 1) the addition of DMPA hastens the onset of FSH/LH suppression, correlating with a more rapid impairment of spermatogonial development, but in the longer term, neither germ cell number nor sperm count differed; 2) testicular dihydrotestosterone and Adiol levels are maintained during FSH/LH suppression despite markedly reduced T levels suggesting up-regulation of testicular 5alpha-reductase activity; and 3) spermatogonial inhibition is a consistent feature, but spermiation inhibition is also striking and is an important determinant of sperm output.     

Quantitative ultrastructural analysis of sperm tails reveals flagellar defects associated with persistent asthenozoospermia.

Sperm tail morphology was examined in 10 infertile asthenozoospermic men to determine whether poor sperm motility was caused by ultrastructural defects of the flagellum. In this quantitative analysis, the numbers of outer doublet and central pair microtubules, outer and inner dynein arms and radial spokes were counted in transverse sections of 75 axonemes from each patient and compared with similar data previously collected from 10 men with normal semen characteristics. Four patients had axonemal defects: two had severe microtubule abnormalities and two had more subtle but statistically significant deficiencies of dynein arms. These abnormalities would not have been detected by more commonly used qualitative examination. Three patients had no detectable ultrastructural abnormalities of the sperm tail, possibly indicating a metabolic deficiency. A further three patients had mid-piece abnormalities. Two had few, if any, flagellar mitochondria and the third patient had irregular and disorganized mitochondria. Quantitative ultrastructural analysis has revealed axonemal abnormalities in seven of 10 patients with previously unexplained asthenozoospermia.     

Serum testosterone response to single injection of hCG ovine-LH and LHRH in male rats

A biphasic pattern of testosterone secretion in response to a single injection of 100 IU hCG has been observed in the rat. Serum testosterone increased from basal levels of 8.7 pL 3.1 ng/ml (mean pL SEM) to 23.0 pL 1.4 ng/ml within 2 h of hCG-stimulation and returned to control levels by 2 days. A second, delayed, but significant increase in serum testosterone occurred, reaching a peak of 24.6 pL 4.0 ng/ml at 3 days and declining to basal values at 5 days. To study this response further, lower doses of hCG were tried. Administration of 10 IU hCG produced a single peak of testosterone, which did not occur until 24 h. Differences in the serum testosterone response were related to the concentration of hCG measured in the serum after injection, as injection of 1 IU, which failed to increase serum hCG levels above detection, was also inadequate to increase serum testosterone. The response after stimulatin with 500 μg ovine-LH or 0.1–10.0 μg LHRH was also evaluated. Injection of 500 μg ovine-LH produced a significant rise in serum testosterone reaching a peak at 2 h of 25.2 pL 2.6 ng/ml and subsequently declining over the next 48 h to control levels where it remained for 5 days. Stimulation with doses of 0.1–10.0 μg LHRH produced rapid and short increases in serum LH concentration which induced peaks of testosterone up to 48.8 pL 14.1 ng/ml 1 h post injection. No secondary peak of testosterone followed. Failure of ovine-LH and LHRH to produce a second testosterone peak suggests that this response may be due to a re-stimulation of the Leydig cell by elevated levels of hCG which persist until the fourth day after injection.     

Studies on seminiferous tubule fluid production in the adult rat: effect of hypophysectomy and treatment with FSH, LH and testosterone

Seminiferous tubule fluid production in adult rats was studied using the technique of unilateral efferent duct ligation (EDL), the production rate representing the difference in testis weight over the time since ligation. Following EDL, fluid production increases linearly for 6 h and linearly at a slightly slower rate for a further 18 h with a sharp decrease thereafter. No differences in fluid production were noted for rats aged between 90–310 days. Forty-eight hours after hypophysectomy there was a significant fall (26%) in fluid production prior to any significant decrease in testis weight. Fluid production continued to decline with time after hypophysectomy eventually reaching a plateau 16–44 days later at levels approximately 15% of those found in control rats. Treatment of rats hypophysectomized 4 days earlier with ovine FSH for 3 days did not restore fluid production, but treatment with ovine LH, testosterone propionate (TP) or FSH together with TP for a similar duration all restored fluid production to normal. On the other hand, treatment of intact adult rats with ovine LH significantly increased fluid production but the effect of treatment with testosterone alone did not reach significance. The results indicate that in the adult rat, seminiferous tubule fluid production is controlled principally by testosterone secreted by the Leydig cell in response to LH stimulation.     

Factors affecting the variability of semen analysis results in infertile men

Infertile men who had 3 or more semen analyses performed in one laboratory were placed in 2 groups (I) oligozoospermic group (n = 106), mean sperm concentration between 1 and 20 million/ml (II) asthenozoospermic group (n = 71), mean sperm concentration greater than 20 million/ml, and mean motility less than 60%. With increasing durations of abstinece from ejaculation before the tests there were significant increases in semen volume and sperm concentration. Semen volume increased over the first 4 days to a similar extent in both groups. Sperm concentrations increased over 15 days, but the effect of abstinence was much greater in the asthenozoospermic group than in the oligozoospermic group (14% compared with 1.4% of the within subject variation). Significant changes in results accompanied repeated testing, notably rises in sperm concentration and motility. Sperm motility was lower in winter and higher in summer in both groups and also, but to a lesser extent, in artificial insemination donors who collected semen in the laboratory.     

The Mechanical Fragility of the Red Cell in Patients with Lead Poisoning

Observations on the mechanical fragility of the red cell in 68 workers having had exposure to lead in varying degrees, and on blood to which lead had been added in vitro in concentrations up to 5μg./ml., showed that the mechanical fragility index did not vary significantly from that of a control group.

Furthermore, the use of various anticoagulants produced no significant alterations in the mechanical fragility index. It was found, however, that in subjects suffering from anaemia of varying types, the mechanical fragility index was always greatly increased if the haemoglobin was below 80%.