Male infertility.
Explore the source record for details and available documents.
Fertilization in Vitro↗
Biomedical subjects
D M de Kretser
Publications and source records attributed to D M de Kretser.
The Y chromosome and spermatogenesis.
Explore the source record for details and available documents.
Chromosome Deletion↗
Immunosuppressive activities in the seminal plasma of infertile men: relationship to sperm antibodies and autoimmunity.
Semen samples from infertile men were assessed for sperm autoimmunity by direct immunobead assay for immunoglobulin (Ig)A and IgG sperm antibodies and mucus penetration test. Immunosuppressive activity in seminal plasma was measured by an in-vitro bioassay employing dose-dependent inhibition of phytohaemagglutinin-induced activation of rat thymocytes, in the presence or absence of hydroxylamine (0.1 mM), an inhibitor of polyamine oxidation. All seminal plasma samples, regardless of autoimmune status, caused inhibition of T-lymphocyte activation, and hydroxylamine reduced this bioactivity by appproximately 50%. Dialysis (<3500 molecular weight) also significantly reduced seminal plasma bioactivity, both in the presence and absence of hydroxylamine. In the presence of hydroxylamine, there was a negative correlation between IgA, but not IgG, antibody concentrations and lymphosuppressive activity in seminal plasma. Antibody-positive samples displaying impaired sperm function, as indicated by the mucus penetration test, had reduced activity compared with other samples. In contrast, there was no relationship between sperm autoimmunity and lymphosuppressive activity assayed in the absence of hydroxylamine. The data indicate that T-lymphocyte inhibition by human seminal plasma is due to multiple factors, and reduced amounts of these factors may contribute to the development and/or persistence of sperm autoimmunity in infertile men; however, differences in polyamine substrates available for oxidation in semen do not appear to be a major contributing factor.
Animals↗
Evidence that heparin binding autocrine factors modulate testosterone production by the adult rat Leydig cell.
Androgen production by adult rat Leydig cells is stimulated by pituitary LH but can also be modulated in vitro by paracrine stimulatory and inhibitory factors, many of which belong to growth factor families. Their actions are mediated through cell surface or extracellular matrix proteoglycans and the aim of this study was to determine the role of cell surface heparan sulfate proteoglycans in the regulation of testosterone secretion by adult rat Leydig cells. The presence of sodium chlorate (25 mM) and protamine sulfate (10 micrograms/ml) inhibited testosterone production by LH stimulated cells by over 50%, but had no effect on unstimulated cells. The LH responsiveness and testosterone production returned to normal after these agents were removed from the culture media. No significant difference in LH receptor numbers at the end of the culture period was seen between sodium chlorate treated and untreated cells. Testosterone production by dibutryl-cAMP stimulated Leydig cells was also inhibited by sodium chlorate. The addition of heparin inhibited testosterone production by LH stimulated cells in a dose-dependent manner, however, in unstimulated Leydig cells heparin stimulated testosterone production to up to 50% of that seen in LH stimulated cells. These data suggest that cell surface heparan sulfate proteoglycans modulate testosterone production by adult Leydig cells in vitro, and that this may involve the autocrine actions of heparin binding growth factors on the Leydig cells.
Animals↗
Declining sperm counts.
Explore the source record for details and available documents.
Environmental Pollutants↗
Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers.
It is believed that Sertoli cells support a finite number of germ cells and that Sertoli cell number may help determine the spermatogenic capacity of the adult. In the rat, Sertoli cells divide during fetal and early postnatal life, a process controlled in part by FSH. This study examined the effect of recombinant human FSH on postnatal testicular development and the impact of neonatal FSH exposure on the adult animal. Sprague-Dawley rats received FSH (200 IU/kg s.c.) daily from birth (Day 1) until Day 5, 10, 15, and 20. In a second experiment, animals received FSH for the first 10 or 15 days of life and were killed at Day 90. Sertoli and spermatogenic cell numbers were determined by stereological methods (the optical disector technique). FSH treatment significantly (p < 0.001) increased testicular weights (135%, 193%, and 173% of controls at Days 10, 15, and 20, respectively). The absolute volume of the epithelium and interstitium increased significantly as a result of increases in tubule diameter and length. In response to FSH treatment, the number of Sertoli cells increased significantly (p < 0.01) to 168%, 139%, and 151% of control numbers at Days 10, 15, and 20, respectively. After 15 days of FSH treatment, the numbers of spermatogonia and spermatocytes also increased (169% and 220% of control, p < 0.01, p < 0.001, respectively). The labeling index of Sertoli cell nuclei, as determined by bromodeoxyuridine incorporation, was unaffected by FSH treatment, with cessation of Sertoli cell division occurring as normal at Day 15. FSH treatment for the first 10 or 15 days of neonatal life resulted in testicular hypertrophy in adulthood (testis weight 118% and 124% of control, respectively). Similar increases were seen in the absolute volume of seminiferous epithelium and lumen, which were attributed to an increase in tubule length. Sertoli cell numbers increased to 113% and 149% of control after 10 and 15 days, respectively, of FSH exposure, with similar increases in round and elongated spermatid numbers. We conclude that exposure of the neonatal rat to recombinant FSH results in testicular hypertrophy with increases in seminiferous tubule volume and length and proportionate increases in Sertoli and germ cell numbers. This trophic effect of increased perinatal FSH exposure persists into adulthood to augment the spermatogenic potential of the rat.
Aging↗
Microtubule-associated protein-2 in the rat testis: a novel site of expression.
The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.
Animals↗
Discriminative analysis of rat Sertoli and peritubular cells and their proliferation in vitro: evidence for follicle-stimulating hormone-mediated contact inhibition of Sertoli cell mitosis.
A new methodological approach using immunohistochemical markers for Sertoli cells (alpha inhibin), peritubular cells (alpha smooth muscle actin), and S-phase cells (bromodeoxyuridine; BrdU) is presented that allows an accurate and simultaneous analysis of morphogenetic and mitogenic changes occurring in vitro. Sertoli cells and peritubular cells were isolated by sequential enzymatic digestion from 7-day-old rats. Laminin, as a major component of the extracellular matrix of the seminiferous tubule, and FSH, as a hormone stimulating Sertoli cell proliferation, were tested for their ability to influence the morphology or mitotic activity of the cultured cells. After fixation, the coverslips were stained for these antigens with use of specific primary antibodies and horseradish peroxidase- or alkaline phosphatase-labeled secondary antibodies for visualization of the respective antigens with different-colored precipitates. This approach allowed us to distinguish the two cell populations, which could not be done unequivocally without the antibody staining. We scored striking changes in cell densities and cell ratios during the culture period. Peritubular cells showed a consistently higher BrdU-labeling index than Sertoli cells. While Sertoli cells were not labeled until Day 7, peritubular cells proliferated as soon as on Day 3, and their density doubled from Day 3 to Day 7. A linear negative correlation was established for Sertoli cell proliferation in response to their local density on the coverslip, indicating contact inhibition as a signal for cessation of mitosis. At high cell densities, this inhibition was partially overcome in the presence of FSH. The presence of laminin had striking effects on the morphogenetic response but only a minor influence on mitogenesis.
Animals↗
Testosterone withdrawal promotes stage-specific detachment of round spermatids from the rat seminiferous epithelium.
Testosterone (T) has been shown to be essential for the completion of spermiogenesis. Our previous studies showed that when intratesticular T was suppressed for 11 wk, the conversion of round spermatids between stages VII and VIII of the spermatogenic cycle was markedly suppressed and that elongated spermatids were undetectable. The fate of the round spermatids that did not proceed through this conversion was unclear. The current study aimed to investigate this T-dependent loss of round spermatids during stages VII and VIII. Adult male Sprague-Dawley rats received 24-cm T implants for 1 wk to suppress LH while maintaining spermatogenesis. The T24 implants were removed and replaced with 3-cm T plus 0.4-cm estradiol (TE treatment) to suppress intratesticular T and spermatogenesis, and animals were killed at 0 and 4 days and 1, 2, 3, 4, and 6 wk later. The number of homogenization-resistant elongated spermatids in the testis was counted, and round spermatid populations in stages VII and VIII were quantified using stereological techniques. The hourly production rates (HPR) were calculated, and a ratio was made between the HPR of round spermatids in stages VII and VIII to assess the efficiency of their conversion through these stages. Testicular T levels were suppressed to 2-4% of control values by TE treatment. After 2 wk of TE treatment, the number of homogenization-resistant elongated spermatids was significantly suppressed, falling to < 0.5% of the control value by 6 wk. The HPR of round spermatids in stages VII and VIII was not affected by up to 2 wk of TE treatment, nor was the conversion between these stages interrupted. After 3 wk of TE treatment, the HPR of round spermatids in stages VII and VIII was significantly suppressed, as was the conversion between these stages, the ratio falling to 27% of the control value by 6 wk. In rats treated with TE, histological examination of the cauda epididymidis showed occasional round spermatids after 3 wk of treatment, and large numbers after 6 wk. We conclude that the failure of round spermatids to complete spermiogenesis following T withdrawal is due to stage-specific detachment of round spermatids between stages VII and VIII.
Animals↗
Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function.
Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.
Animals↗
Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy.
Genes on the long arm of Y (Yq), particularly within interval 6, are believed to play a critical role in human spermatogenesis. Cytogenetically detectable deletions of this region are associated with azoospermia in men, but are relatively uncommon. It has been hypothesized that microdeletions of Yq may account for a significant proportion of men with infertility. The objective of this study was to validate a sequence-tagged site (STS)-mapping strategy for the detection of Yq microdeletions and to use this method to determine the proportion of men with idiopathic azoospermia or severe oligozoospermia who carry microdeletions in Yq. STS mapping of a sufficiently large sample of infertile men should also help further localize the putative gene(s) involved in the pathogenesis of male infertility. Genomic DNA was extracted from peripheral leukocytes of 16 normal fertile men, 7 normal fertile women, 60 infertile men (50 of whom had azoospermia and 10 of whom had severe oligozoospermia with no other recognizable cause of infertility), and 15 patients with the X-linked disorder, ichthyosis. PCR primers were synthesized for 26 STSs that span Yq interval 6. None of the 16 normal men of known fertility had microdeletions. Seven normal fertile women failed to amplify any of the 26 STSs, providing evidence of their Y specificity. No microdeletions were detected in any of the 15 patients with ichthyosis. Of the 60 infertile men typed with 26 STSs, 11 (18%; 10 azoospermic and 1 oligozoospermic) failed to amplify 1 or more STS. Interestingly, 4 of the 11 patients had microdeletions in a region that is outside the Yq region from which the DAZ (deleted in azoospermia gene region) gene was cloned. In an additional 3 patients, microdeletions were present both inside and outside the DAZ region. In 3 subjects, the microdeletions were verified by Southern analysis using labeled PCR products corresponding to the deleted STSs as probes. These data suggest a high prevalence (18%) of Yq microdeletions in men with idiopathic azoospermia/severe oligospermia. The physical locations of these microdeletions provide further support for the concept that a gene(s) on Yq deletion interval 6 plays an important role in spermatogenesis. The presence of deletions that do not overlap with the DAZ region suggests that genes other than the DAZ gene may also be implicated in the pathogenesis of some subsets of male infertility.
Adult↗
Measurement of activin in biological fluids by radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin.
Activin, a dimer of the beta-subunits of inhibin, is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors and has a widespread range of actions in a variety of tissues. The investigation of the physiology of activin action has been facilitated in recent years by the availability of immunoassays in addition to bioassays. Follistatin has been shown to bind to activin with a high affinity and therefore interferes in both radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). In this study we examined the effect of various surfactants and 1.4-dioxane on the measurement of activin in the presence of follistatin by radioimmunoassay. The addition of a combination of sodium deoxycholate. Tween 20 and sodium dodecyl sulphate removed the interference of follistatin in the radioimmunoassay. The measured content of activin in male rat serum, human male serum, human female serum and bovine follicular fluid rose from 3.29 to 4.15, < 0.48 to 2.87, 2.42 to 4.17 and 30.9 to 85.6 ng/ml, respectively, when assayed in the presence of the dissociating reagents. It was unclear whether the altered potencies were due to a dissociation of the follistatin/activin complex rather than the exposure of the epitope on activin recognized by the antiserum. Serum concentrations of activin were lower than those found in testicular cytosols, and after castration no change in serum activin levels was observed, suggesting that the testis does not contribute significantly to circulating activin levels. The use of the dissociating reagents in the radioimmunoassay will enable studies to be carried out that more accurately measure the activin content of various biological fluids, and thus lead to a greater understanding of the physiology of this growth factor.
Activins↗
Expression of activin beta C subunit mRNA in reproductive tissues.
A cDNA encoding the potential activin beta C subunit was produced from human testis RNA using reverse transcription and PCR and then used as a probe in Northern blot analysis of poly(A)+ RNA. A transcript of approximately 1.8 kb was evident in human ovary, placenta and testis samples. A transcript of this size was also detected in adult rat caput and cauda epididymis, and adult and day-15 testis and adult spleen, while adult liver contained a single transcript of approximately 2.1 kb. Poly(A)+ RNA from primary spermatocytes but not from round spermatids contained the 1.8 kb activin beta C mRNA. These findings highlight the need for further studies to determine the physiological role(s) of activin beta C in a wide variety of tissues.
Activins↗
The endocrine regulation of spermatogenesis: independent roles for testosterone and FSH.
Explore the source record for details and available documents.
Adult↗
The testis is not the major source of circulating follistatin in the ram.
The aims of this study were to determine the plasma concentrations of follistatin in rams and to assess if the testis contributes to circulating follistatin and if there is uptake or production of follistatin by the head in rams. Catheters were inserted in the carotid artery, jugular vein and spermatic vein of intact rams during the non-breeding season (experiment 1; n = 5) and breeding season (experiment 2; n = 4). In experiment 1, blood samples were collected from 5 rams every 10 min for 4 h, commencing 20-60 min after surgery. After 2 h of sampling 1 microgram gonadotrophin-releasing hormone (GnRH) was injected intravenously. In experiment 2, blood samples were collected from 4 of the rams used in experiment 1 by venipuncture 30 and 15 min before surgery and every 15 min throughout surgery. Commencing 1 h after surgery, matched samples were taken from each of the vessels every 10 min for 4 h (1-4 h after surgery), then every hour for 20 h (4-24 h after surgery) and then every 10 min for 4 h (24-28 h after surgery). In both experiments, follistatin secretion was non-pulsatile and there were no significant differences between the concentrations of follistatin in any of the vessels. There was a significant (P < 0.05) increase in the concentrations of follistatin in each of the vessels throughout the 4 h of 10-min sampling in both experiments. In experiment 2 plasma concentrations of follistatin in the jugular vein were significantly (P < 0.05) lower before surgery than at other stages of the experiment. During the non-breeding season (experiment 1) the concentrations of follistatin in all vessels were about 2-fold higher (P < 0.001) than during the breeding season (experiment 2). Concentrations of follistatin were measured in the testicular tissue of the ram, bull, monkey and rat and were found to be 13.6, 2.1, 2.5, 0.8 ng/g testis respectively. In experiment 3, blood samples were collected every 15 min for 4 h from castrated rams (n = 6) in the absence of treatment with testosterone propionate (TP) and after 7 days of treatment with a physiological dose of TP during the breeding and non-breeding seasons. There was no effect of stage of breeding season or TP on the plasma concentrations of follistatin and these concentrations in the castrated rams were similar to the concentrations in the intact rams in experiment 2. In experiment 4, the function of Leydig cells was stimulated by administration of human chorionic gonadotrophin but this had no effect on plasma concentrations of follistatin. These experiments show that the concentrations of follistatin in the plasma of rams are measurable, that the testis is not the major contributor to circulating follistatin and that there is no significant uptake or production of follistatin by the head in rams. It appears that the contribution of the testis to circulating follistatin may vary with the stage of the breeding season, being greater during the non-breeding season than the breeding season. The gonadotrophins and testosterone do not appear to have a direct effect on the secretion of follistatin in rams. The increase in concentrations of circulating follistatin during surgery and more frequent blood sampling suggest a stress-related effect on the production of follistatin.
Animals↗
Follistatin concentrations in male sheep increase following sham castration/castration or injection of interleukin-1 beta.
Plasma follistatin (FS) concentrations were determined after castration (n = 5) or sham castration (n = 4) of mature rams. Both treatments resulted in a prolonged increase in FS between 7 and 19 h after surgery, which returned to pretreatment concentrations by 24 h. Tumour necrosis factor-alpha (TNF-alpha), a sensitive maker of an acute-phase response, was undetectable in plasma, indicating that the FS response was not induced by trauma due to surgery. In a second experiment, injection of castrated rams (n = 4) with ovine recombinant interleukin-1 beta, an acute-phase mediator, resulted in a sustained rise in FS concentrations within 4 h of injection. Plasma TNF-alpha concentrations increased transiently within 1 h of interleukin-1 beta injection, indicating that an acute-phase response had been initiated. Plasma follicle-stimulating hormone (FSH) concentrations were significantly decreased at 8 and 24 h after interleukin-1 beta injection, strongly suggestive of an inhibitory effect of increased FS concentrations on the secretion of FSH. Injection of castrated rams (n = 2) with a control preparation of recombinant interleukin-2 did not induce an acute-phase response, and plasma FS and FSH concentrations were unaffected. These data show that the testis is not a major source of circulating FS, that the increase in circulating FS following sham castration/castration is not due to an acute-phase response, but that conversely FS concentrations are modulated by the acute-phase mediator, interleukin-1 beta.
Acute-Phase Reaction↗
The potential of intracytoplasmic sperm injection (ICSI) to transmit genetic defects causing male infertility.
Explore the source record for details and available documents.
Cytoplasm↗
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↗