The potential of intracytoplasmic sperm injection (ICSI) to transmit genetic defects causing male infertility.
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Cytoplasm↗
Biomedical subjects
D M de Kretser
Publications and source records attributed to D M de Kretser.
Implication of inhibin and related proteins in fetal development.
Initial studies on inhibin, activin and follistatin focussed on their role as regulators of adult gonadal function via feedback regulation of anterior pituitary hormones and via intragonadal control of steroid hormones. The discovery of isoforms of follistatin which are either secreted or retained on the cell surface and which bind activin and, to a lesser extent, inhibin adds a further dimension to the regulation of these peptides. More recently, the cloning of inhibin and activin, and the observation of their close homology to the transforming growth factor-beta family of peptides, has led to an interest in their possible role as growth and differentiation factors. Activin, inhibin and follistatin are expressed in embryonic and fetal tissues, as well as in the placenta. However, although activin is a potent regulator of growth and differentiation in a number of cell types, their role in embryonic and fetal development has yet to be established. High concentrations of inhibin have been observed in the fetal gonads, particularly the testes, and in the fetal adrenals of a number of species and a sex difference in fetal plasma concentrations has also been observed. Although the stimulus for high concentrations of inhibin in the fetus is not know, they are associated with decreased testicular testosterone and a decrease in the concentration of circulating follicle-stimulating hormone (FSH); this suggests that, as in the adult, inhibin may be involved in the regulation of fetal testicular androgen and pituitary FSH secretion during late gestation. The recent reports of elevated concentrations of inhibin and, particularly, activin in amniotic fluid during late gestation and its ability to stimulate the production of prostaglandin E2 by fetal membranes provides yet another potential role for this hormone in the regulation of events leading to parturition.
Activins↗
Acute and short-term actions of serotonin administration on the pituitary-testicular axis in the adult rat.
In this study, adult male rats were injected intraperitoneally with a single dose of serotonin (5-hydroxytryptamine, 5HT; 10 mg kg-1 bodyweight) for 2 h or 18 h, or daily with graded doses of 5HT (0.1-10 mg kg-1) for four days before being killed. Serum and testicular interstitial fluid (IF) concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and immunoreactive-inhibin were measured by radioimmunoassay, and one testis was removed for histological examination. At 2 h after a single injection, 5HT caused a significant inhibition of serum concentrations of LH and inhibin, recovered IF volume and intratesticular testosterone concentrations; testis weight and serum concentrations of testosterone and FSH were unaffected. At 18 h after injection, all parameters had returned to normal, with the exception of intratesticular testosterone concentration which remained lower than normal. The lowest 5HT dose (0.1 mg kg-1) had no effect on any parameter following four daily injections. At a dose of 1.0 mg kg-1 5HT, there was a four-fold increase in the concentration of serum LH, but testis weight, recovered IF volume, testosterone and inhibin concentrations and serum concentrations of FSH were not significantly affected. At the highest dose of 5HT (10 mg kg-1) after four daily injections, testis weight decreased, and IF volume increased nearly three-fold. Testis concentrations of inhibin and serum testosterone were reduced, whereas serum concentrations of both LH and FSH were elevated; intratesticular testosterone concentrations did not differ from controls. Only at the highest dose of 5HT was disruption to the seminiferous epithelium observed, with focal damage ranging in severity from increased degeneration of spermatogenic cell profiles, to complete loss of the germinal epithelium; however, many tubule profiles displayed completely normal spermatogenesis. The acute IF volume reduction and spermatogenic disruption in 5HT-treated rats were consistent with localized ischaemia due to constriction of the testicular arterial supply. The eventual increase in IF volume observed after 5HT treatment appeared to be secondary to the loss of germ cells. Although 5HT also inhibited pituitary LH release and Leydig cell steroidogenesis, these effects appeared to play only a minor role in the induction of spermatogenic damage.
Animals↗
Pregnancies after microsurgical correction of partial epididymal and vasal obstruction.
A group of 16 infertile patients suspected of having a partial epididymal obstruction on the basis of severe oligozoospermia, normal-sized testes and a normal serum follicle-stimulating hormone, underwent scrotal exploration. Evidence of partial obstruction of the epididymis was found in 13 cases and of the vas deferens in one case, and was supported by finding normal spermatogenesis on testicular biopsy. Vasoepididymostomy or vasovasostomy were performed, resulting in a significant improvement of semen analysis in 50% of cases and in six pregnancies in two patients. The diagnosis of partial epididymal obstruction should be considered when the above criteria are met. If pregnancies do not result when intracytoplasmic sperm injection (ICSI) is used with the ejaculated spermatozoa, a testicular biopsy followed by a microsurgical by-pass procedure should be considered whenever normal spermatogenesis is diagnosed. In all cases, the epididymal spermatozoa should be aspirated during the operation and either used immediately for insemination or stored frozen. The remarkable results of the new artificial reproduction technologies and in particular ICSI, question the indication for microsurgical correction in cases of partial epididymal obstruction.
Adult↗
Human recombinant follistatin-288 suppresses plasma concentrations of follicle-stimulating hormone but is not a significant regulator of luteinizing hormone in castrated rams.
We have tested the hypothesis that treatment of castrated rams (wethers) with human recombinant follistatin-288 (FS-288) suppresses plasma concentrations of FSH but has no effect on plasma concentrations of LH. Wethers were given an i.v. injection of vehicle or 50 micrograms FS-288 followed by a 12-h i.v. infusion of vehicle or 800 micrograms FS-288, respectively. This dose and treatment regimen was identical to that used in a previous study in which wethers were treated with vehicle or human recombinant inhibin A (hr-inhibin). Human recombinant follistatin significantly (p < 0.05) suppressed plasma concentrations of FSH, but vehicle had no effect. The maximal suppression of FSH occurred 12-15 h after the start of treatment, at which time the plasma concentrations were reduced by 20.6%. The reduction in plasma concentrations of FSH caused by FS-288 was about 2.6-fold less than that observed after the same treatment with hr-inhibin. FS-288 had no effect on plasma concentrations of LH; this result was similar to the findings we obtained with hr-inhibin. These data suggest that follistatin is a less potent negative regulator of FSH secretion than inhibin and that follistatin is not a significant regulator of the secretion of LH in rams.
Animals↗
The effects of recombinant follicle-stimulating hormone on the restoration of spermatogenesis in the gonadotropin-releasing hormone-immunized adult rat.
The role of FSH in spermatogenesis is unclear as testosterone alone has been reported to be sufficient in the gonadotropin-deficient rat. This study examined the effects of recombinant FSH on the restoration of spermatogenesis after gonadotropin withdrawal by GnRH immunization. Adult Sprague-Dawley rats received GnRH immunogen (100 micrograms, sc, every 4 weeks) to induce gonadotropin deficiency, with severe spermatogenic regression occurring by 12 weeks. Recombinant human FSH was then given (10 or 50 IU/kg, sc, daily) for 7, 14, and 21 days, with data from both dosages combined in the analyses. Testes were perfusion fixed, and germ cell numbers were quantified by the optical disector technique. After 7 days of FSH, testis weight significantly increased by 43% (P < 0.01), with no further increases at 14 and 21 days. GnRH immunization severely reduced germ cell numbers, which were then significantly (P < 0.05) restored in all cell types, except elongated spermatids, by 7 days of FSH; type A spermatogonia (45%-->61% of control), type B spermatogonia/preleptotene spermatocytes (46%-->65%), leptotene/zygotene spermatocytes (39%-->55%), pachytene spermatocytes in stages I-VIII (11%-->30% control) and IX-XIV (4.3%-->22% control), and round spermatids in stages I-VIII (1.4%-->4.4% control). Prolonged FSH treatment did not further increase type A spermatogonial or pachytene spermatocyte number, whereas round spermatids increased to a peak of 12.8% of the control value. At no stage did FSH increase elongated spermatid numbers above 1% of the control level. The incorporation of bromode-oxyuridine into spermatogonial and early spermatocyte nuclei did not change after GnRH immunization or FSH treatment. Sertoli cell number was not altered by any treatment; however, Sertoli cell nuclear volume was significantly decreased from the control value by GnRH immunization (142 +/- 9 vs. 455 +/- 22 microns 3; P < 0.01) and increased after 7 and 14 days of FSH treatment to 212 +/- 10 and 259 +/- 24 microns 3, respectively. FSH treatment restored serum inhibin levels to normal, but did not increase serum or testicular androgen levels. We conclude that recombinant FSH partially restores spermatogenesis in the gonadotropin-deficient rat by increasing the number of spermatogonia and promoting subsequent maturational steps up to the round spermatid stage. Spermatid elongation was not restored by FSH, indicating the need for an additional factor(s), most likely testosterone.
Animals↗
Isolation and partial characterization of rat sperm tail fibrous sheath proteins and comparison with rabbit and human spermatozoa using a polyclonal antiserum.
Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain. The most intensely staining proteins, using both staining methods, were a double band at 80-87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate 11 of these proteins (116.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Increased numbers of Sertoli and germ cells in adult rat testes induced by synergistic action of transient neonatal hypothyroidism and neonatal hemicastration.
The combined effects of transient neonatal hypothyroidism and neonatal hemicastration were investigated to see whether they were additive. Hypothyroidism was induced in litters of ten male rats for 25 days from the day of birth by administration of 0.1% (w/v) 6-propyl-2-thiouracil in the mother's drinking water; hemicastration was performed on the day of birth. Controls included both normal and sham-operated animals. Numbers of Sertoli cells and round spermatids were quantified at age 135 days using stereological methods. Sham-operation had no effect on testis mass, or numbers of Sertoli or germ cells. Transient neonatal hypothyroidism resulted in an increase in testicular mass of 27% (P < 0.05), whereas neonatal hemicastration resulted in a 33% (P < 0.05) increase over control; the combination of the two procedures resulted in a 62% (P < 0.05) increase. There were corresponding significant increases in the number of Sertoli cells: 82% with hypothyroidism, 18% with hemicastration and 123% with the combination of the two procedures. Numbers of round spermatids showed similar increases: 59% with hypothyroidism, 45% with hemicastration and 95% with the combination of the two procedures. It is concluded that the effects of the combination of transient neonatal hypothyroidism and hemicastration are additive with respect to testicular mass, and numbers of Sertoli and germ cells.
Animals↗
Inhibin and activin in embryonic and fetal development in ruminants.
Inhibin, activin and follistatin are protein hormones with diverse physiological roles. The involvement of inhibin in the regulation of pituitary FSH production and secretion in adult males and non-pregnant females is well established. However, it is unlikely that inhibin plays a similar role in pregnancy in ruminants. Inhibin and activin molecules show a high degree of structural similarity to potent growth and differentiation factors of the transforming growth factor beta (TGF-beta) superfamily of peptides and their localization in a range of embryonic and fetal tissues indicates that they may thus play a role in development. Furthermore, the demonstration that follistatin is also present in a number of embryonic and fetal tissues and fluids has further implications for the actions of activin to which it binds. The role of inhibin, activin and follistatin in early development has yet to be established since gene knockout experiments have so far proved inconclusive. During mid- and late gestation, high concentrations of inhibin are found in the testes and plasma of male fetuses of sheep and cattle. Inhibin may play a role in regulating pituitary FSH release in late pregnancy, but the very high concentrations of this hormone in ovine fetal testes and in male fetal plasma compared with that observed in the fetal ovary and female fetal plasma has yet to be explained. The recent observation of high concentrations of inhibin, activin and follistatin in amniotic fluid surrounding the fetus is intriguing. Excretion via urine or lung liquid is partly responsible for the presence of these proteins in amniotic fluid. The fetal membranes and the placenta are also possible sources. It remains to be established whether these proteins constitute an inactive pool of secreted hormone or whether they have other actions in this fetal compartment.
Activins↗
Multiple factors with steroidogenesis-regulating activity in testicular intertubular fluid from normal and experimentally cryptorchid adult rats.
Steroidogenesis-stimulating activity (SSA) was examined in testicular intertubular fluid from normal, and short-term and long-term (up to 12 months) experimentally cryptorchid rats, using an in vitro Leydig cell bioassay based on testosterone production over 20 h in the presence of a maximum dose of human chorionic gonadotropin. Total fluid volume increased throughout the period of cryptorchidism, while intertubular testosterone concentrations declined. SSA from cryptorchid rats was significantly greater (2- to 3-fold) than normal at all time-points; however, the major increase in activity occurred within the first 4 weeks after treatment. Similar concentrations of lipoproteins were recovered from both untreated and 4-week cryptorchid fluid by density ultracentrifugation, although the bioactivity of the cryptorchid testis lipoprotein fraction was 8-fold higher than the lipoprotein fraction from untreated testes. Moreover, removal of the lipoproteins led to a loss of SSA in the lipoprotein-deficient fraction of the intertubular fluid. Consequently, the in vitro bioassay conditions were modified by addition of a constant level of serum lipoproteins to all assay wells. Employing the lipoprotein-supplemented bioassay, multiple stimulatory and inhibitory activities were resolved by Sephadex G-100 gel filtration in intertubular fluid from both normal and cryptorchid testes: (i) an inhibitory activity eluting in the void volume (> 150 kDa), which decreased after cryptorchidism; (ii) a stimulatory activity (40-80 kDa), which did not appear to be affected by cryptorchidism; (iii) an inhibitory activity (20-40 kDa) which decreased after cryptorchidism, and (iv) a stimulatory activity (12-20 kDa) which increased after cryptorchidism.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Clinical male infertility. I. Prevalence of and progress in understanding male infertility.
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Animals↗
Stimulatory and inhibitory factors of Leydig cell steroidogenesis are secreted simultaneously by the rat seminiferous tubules and do not affect Leydig cell inhibin production in vitro.
The effect of conditioned medium from rat seminiferous tubules (at Stages VII-VIII and Stages IX-VI) cultured with or without follicle-stimulating hormone (FSH) on the production of testosterone and immunoactive inhibin by Leydig cells was examined. Low doses of conditioned medium from unstimulated tubules at Stages VII-VIII significantly (P < 0.05) increased the mean testosterone production to greater than 31 +/- 11% over that achieved with luteinizing hormone (LH) alone. At the highest dose, the conditioned medium significantly inhibited (P < 0.05) LH-stimulated testosterone production by 13 +/- 7%. Low doses of conditioned medium from unstimulated tubules at Stages IX-VI increased the mean testosterone production to 22 +/- 10%, whereas at higher doses, a significant reversal in the stimulation occurred although not to the same extent as that found with medium from tubules at Stages VII-VIII. Conditioned medium from FSH-stimulated tubules at Stages VII-VIII and Stages IX-VI, significantly increased testosterone production to 39 +/- 7% and 31 +/- 13% respectively. Immunoactive inhibin production by the Leydig cells remained unaffected by exposure to conditioned medium from FSH stimulated and unstimulated tubules at Stages VII-VIII and Stages IX-VI. The data demonstrate that tubule culture medium contains FSH-modulated activities which can specifically stimulate and inhibit testosterone synthesis by adult rat Leydig cells in vitro and therefore explains the contradictory reports in the literature.
Animals↗
Gonadotropin-suppressing activity of human recombinant inhibin in the male rat is age dependent.
We have examined the age-dependent effects of human recombinant inhibin A (hrI) on gonadotropin levels in intact male rats. A single s.c. injection of hrI in the dose range 6.25-100 ng/g BW resulted in a significant and consistent decrease (20-40%) in serum FSH measured 6 h later, at the higher (50 and 100 ng/g) doses, in animals 25-91 days of age. Administration of the same doses in animals < or = 20 days old had no significant effect on serum FSH levels. Similarly, no effect of hrI (100 ng/g) on serum FSH levels was observed in 15-day-old rats when FSH levels were monitored from 1 to 8 h. No effect of hrI was observed on serum LH levels except in 40-day-old rats, which exhibited a consistent and significant reduction at the higher (25-100 ng/g) doses of inhibin. Serum inhibin levels 6 h after the injection were not significantly altered at any dose in young animals (< or = 30 days); however, in older animals (40, 50, and 91 days) there was a significant elevation in circulating inhibin levels at the higher doses, strongly suggesting an age-related change in clearance. Testicular inhibin levels were not affected by administration of the exogenous material except at the highest dose (100 ng/g) in the oldest animals (91 days), when an approximately 30% reduction in both the concentration and the total content of inhibin was observed. The data clearly demonstrate that the effect of hrI on serum gonadotropins is age dependent and that changes in clearance may contribute to the period during which circulating inhibin levels are elevated postinjection.
Aging↗
The isolation of activin from ovine amniotic fluid.
During a study of the levels of inhibin and follistatin in ovine amniotic fluid, we noted that although detectable levels of immunoactive inhibin and follistatin were found throughout gestation, the addition of amniotic fluid to a rat anterior pituitary cell culture resulted in a stimulation, rather than the expected suppression, of FSH concentrations. These data suggested the possibility that activin was present in amniotic fluid. We, therefore, set out to isolate the molecules responsible for this activin-like activity and determine their structure. Amniotic fluid, collected from pregnant sheep between 120-140 days gestation, was used as starting material in the purification and diluted in parallel to a human activin-A standard in the activin RIA employed to monitor the purification. A total pool of 7.4 liters amniotic fluid was processed by dye affinity chromatography, hydrophobic interactive chromatography, gel filtration, and a series of reverse phase HPLC steps. Polyacrylamide gel electrophoresis of fractions from the final HPLC step, which showed both activin immunoactivity and bioactivity, revealed a band with a mol wt of 25.3 kilodaltons (kDa), which reduced to 15.8 kDa, and a minor band of 45 kDa, which reduced to 25 kDa. NH2-terminal amino acid sequences of several active fractions from the same region were identical to the known sequence of ovine activin-A. The identification of immunoactive activin, follistatin, and inhibin in amniotic fluid raises the question of the sites of production of these proteins and their interactions and role in fetal physiology.
Activins↗
Partial characterization of inhibin, activin, and follistatin in the term human placenta.
To determine whether the human term placenta contains inhibin, activin, and follistatin, placental homogenates from normal placentae were subjected to several fractionation procedures: 1) dye affinity chromatography; 2) hydrophobic interaction chromatography using phenyl sepharose; 3) gel filtration under acid conditions; 4) reversed phase-high pressure liquid chromatography (RP-HPLC); and 5) preparative polyacrylamide gel electrophoresis and electroelution. Purification was followed by RIA for inhibin, activin, and follistatin as well as in vitro bioassay based on the FSH cell content of rat anterior pituitary cells in culture. Two peaks of immunoactive and bioactive inhibin with differing elution patterns on RP-HPLC were shown to have molecular weights of 33K and 32K on polyacrylamide gel electrophoresis. These peaks may represent inhibin A and B. Three peaks of immunoactive activin on RP-HPLC had molecular weights of 26.5K, 25.5K, and 27K and may represent activin A, AB, and B. Immunoactive follistatin eluted on RP-HPLC as a broad peak, which on polyacrylamide gel electrophoresis consisted of three main activities consistent with molecular weights of 31K, 35K, and 38K. These studies demonstrate that the term placenta contains inhibin, activin, and follistatin, which have been partially characterized. The presence of these substances within the placenta raises the possibility of their function in local paracrine interactions.
Activins↗
Clinical review 64: Pathophysiology and natural history of male infertility.
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Animals↗
Inhibin and follistatin concentrations in fetal tissues and fluids during gestation in sheep: evidence for activin in amniotic fluid.
The concentrations of inhibin and follistatin in amniotic fluid and in tissue extracts from the placenta, gonads and adrenals of fetal sheep were measured using radioimmunoassays. These tissue extracts were from whole fetuses from days 16 to 45 and from the individual organs from day 46 to 145 (term) and were assayed at multiple dilutions. The capacity of these extracts to alter FSH production of rat anterior pituitary cells in culture was also assessed at multiple dilutions. Immunoactive inhibin concentrations in amniotic fluid from both sexes increased during gestation and levels were significantly greater in males than females. Peak concentrations of immunoreactive inhibin of 11.2 +/- 1.9 ng/ml were found in males at 116-125 days of gestation. Follistatin concentrations did not change throughout gestation and no significant difference was noted between sexes. Mean follistatin levels throughout gestation were 3.0 +/- 0.9 ng/ml for males and 3.7 +/- 0.9 ng/ml for females. Despite the potential for FSH inhibition by inhibin and follistatin, amniotic fluid from both sexes at all stages of gestation stimulated FSH secretion in the pituitary cell bioassays, suggesting the presence of activin which was confirmed by the measurement of immunoactive activin (13.3 +/- 2.5 ng/ml) in a specific radioimmunoassay. Maximum concentrations of immunoactive and bioactive inhibin in placental extracts were observed in late gestation (2.2 +/- 0.6 and 3.8 +/- 1.6 ng/g respectively) and there was no significant difference between sexes. Follistatin concentrations in placental cotyledons ranged from 11.5 to 27.1 ng/g with no significant difference between sexes. In view of the higher follistatin concentrations compared with inhibin, it is likely that the capacity of placental extracts to suppress FSH production by pituitary cells in culture is due predominantly to follistatin. Immunoactive inhibin was observed in high concentrations in the fetal testis throughout gestation; with concentrations increasing to a maximum of 1993.0 +/- 519.7 ng/g at 126-135 days of gestation with a ratio of bioactive: immunoactive inhibin of 1:20. Although bioactive and immunoactive inhibin was also observed in fetal ovaries and adrenals from both male and female fetuses, concentrations were lower than those observed in fetal testes. Follistatin concentrations in the fetal testis were elevated between 70 and 95 days (97.6 ng/g) and then declined. Similar concentrations were found in the adrenal glands of both sexes (males 83.5-103.3 ng/g: females 55.3-95.8 ng/g).(ABSTRACT TRUNCATED AT 400 WORDS)
Activins↗