Con Mallidis

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

Subfertility in men is a heterogeneous syndrome, its pathophysiology remaining unknown in the majority of affected men. A large number of genes and loci are associated with sterility in experimental animals, but the human homologues of most of these genes have not been characterized. A British study suggested that, in a large proportion of men with idiopathic infertility, the disorder is inherited as an autosomal recessive trait; this provocative hypothesis needs confirmation. Because normal germ cell development requires the temporally and spatially co-ordinated expression of a number of gene products at the hypothalamic, pituitary and testicular levels, it is safe to predict that a large number of autosomal, as well as X- and Y-linked, genes will probably be implicated in different subsets of male subfertility.

Diabetes Mellitus (DM) has been found to have subtle yet profound effects on the metabolic status of the testis, the expression of numerous spermatogenic genes and is associated with increased numbers of sperm with nuclear DNA damage. The precise mechanism causing these detrimental effects remains unknown. The presence of increased levels of the most prominent member (carboxymethyllysine – CML) of the advanced glycation end product adducts and their receptor (RAGE) in the reproductive tract of DM men has provided a new avenue for research. As there are suspicions that the antibiotic (streptozotocin – STZ) employed to induce DM is also capable of causing oxidative stress and DNA damage, we compared CML and RAGE levels in the reproductive tract and sperm nDNA status of STZ mice with the levels in the Ins2Akita mouse to determine which more closely mimics the situation described in the human diabetic. CML was observed in the testes, epididymes and sperm of all animals. Sperm from DM mice showed particularly strong CML immunolocalization in the acrosomal cap, the equatorial region and whenever present, cytoplasmic droplets. Although increased, the level of CML on the sperm of the STZ and Ins2Akita DM mice did not reach statistical significance. RAGE was present on the developing acrosome and epididymal sperm of all animals and in discrete regions of the epididymes of the DM models. Only the epididymal sperm of the Ins2Akita mice were found to have significantly increased (p < 0.0001) nDNA damage. The Ins2Akita mouse therefore appears to more accurately reflect the conditions found in the human and, as such, is a more representative model for the study of diabetes and glycation’s influence on male fertility.

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT–PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant nuclear localization suggests that it may play a role in transcriptional regulation. J. Cell. Physiol. 190: 170–179, 2002. © 2002 Wiley-Liss, Inc.

Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility.

Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s incidence and localization on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. Enzyme-linked immunosorbent assay analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalization was employed to evaluate RAGE&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s precise location on the sperm head. RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001 and P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001, respectively). The presence of RAGE implies that it may play a central role in sperm nDNA damage particularly in diabetic men where the levels are elevated.

The long-held view that diabetes has little effect on male reproductive function has been challenged by findings that the condition influences fertility in numerous previously undetected ways. This retrospective chart review of 3000 couples determined the incidence of couples with a male diabetic seeking assisted reproduction treatment and assessed any relationship between male diabetes and IVF/intracytoplasmic sperm injection (ICSI) outcome. Eight (2.7%) couples were found with a diabetic male partner, of which 18 couples underwent assisted reproduction treatment (five IVF, 12 ICSI, one both), with fertilization rates (IVF 68%, ICSI 62%) similar to non-diabetic patients (IVF 70%, ICSI 71%) and no difference in embryo quality. Two men had retrograde ejaculation and two were azoospermic. Other than reduced sperm motility, the remaining 14 had normal World Health Organization semen parameters. Embryo transfers produced one pregnancy (5% combined IVF/ICSI pregnancy rate/cycle) giving a lower-than-expected rate (28.8%). The pregnancy rate from seven FETs (29%) was comparable to the expected (21.3%). Compared with non-diabetics, approximately three times more couples with diabetic men sought treatment, with a larger percentage having &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;unexplained&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; infertility. Fertilization rates and embryo quality did not differ but pregnancy rates were lower in couples with a diabetic male.

While a multitude of acquired causes can impair spermatogenesis, there is reason to believe that a genetic basis exists in a majority of infertile men (Bhasin et al, 1994; De Kretser et al, 1972; Lamb and Niederberger, 1994; Jaffe and Oates, 1994; Skakkebaek et al, 1994). The occurrence of these genetic defects in infertile men has significant implications for assisted reproductive technologies, particularly intracytoplasmic sperm injection (ICSI) (Bhasin et al, 1994). Because intracytoplasmic sperm injection may allow partners of these infertile men to become pregnant, it is possible that these genetic defects may be transmitted to the male offspring. This raises issues of informed consent and ethical concerns. Similarly, the widespread use of assisted reproductive techniques to induce pregnancy may result in accumulation of genetic defects in the population; these defects would have been otherwise weeded out because of infertility. Substantial prevalence of Y deletions and other known and unknown genetic defects in infertile men and the potential risk of transmitting this genetic disorder to their offspring provide a compelling rationale for genetic screening of infertile men prior to ICSI. The couples undergoing ICSI should be counseled about the potential risk of transmitting this genetic disorder to the offspring. Long term monitoring of ICSI babies for genetic disorders including Y deletions is warranted.

Male infertility affects one man in twenty and a genetic basis seems likely in at least 30% of those men. Genetic regulation of fertility involves the inter-related processes of testicular development, spermatogenesis (involving germ cell mitosis, meiosis and spermatid maturation), and their endocrine and paracrine regulation. In regard to spermatogenesis, particular attention has been given to the Yq11 region, where some spermatogenesis genes (&amp;#39;azoospermia factors&amp;#39;) appear to be located. Several candidate genes have been identified but have not been shown to have a defined or essential role in spermatogenesis. Microdeletions of Yq11 are found in approximately 15% of azoospermic or severely oligospermic men. The complexity of the genetic control of male fertility is demonstrated by the evidence for genes involved in spermatogenesis and sexual differentiation on the X chromosome and autosomes. Better understanding of the genetic regulation of normal spermatogenesis will provide new probes for clinical studies; however, at present the majority of spermatogenic failure remains without an identified genetic linkage. The advent of intracytoplasmic sperm injection permits fertility in many previously sterile men and presents the possibility of their transmission of infertility; appropriate counselling is required.

... De Cheung et a/.35). Bardoni et a/.32 localized five commonly used Y-specific probes to the region and O&#x27;Reilly et al.35 correlated the posi-tions of 26 new probes with the original Verg-naud et al.8 map, refining interval 6 in terms of six subintervals. ...

ABSTRACT The introduction of intracytoplasmic sperm injection has led to an unfortunate decrease of interest in male fertility. It is apparent that light microscopy provides limited information and molecular techniques show that DNA abnormalities need to be considered further. Abnormalities include, not only Yq11 deletions, but also DNA strand breaks. Increases in advanced glycation end products in sperm from well-controlled diabetics may provide a mechanism for this damage in nondiabetics. In addition, much publicity is given to decreasing male fertility: this is not confirmed and technical variations and differences in study populations make it difficult to draw conclusions. The generation of stem cell-derived germ cells provides hope for men without germ cells but this is currently only experimental.

Der Diabetes mellitus (DM) bringt zahlreiche systemische Komplikationen mit sich. Im andrologischen Arbeitsgebiet stehen die Erektionsstörung, die retrograde Ejakulation und der Hypogonadismus im Vordergrund. Die Störung der männlichen Infertilität im Zusammenhang mit dem DM ist als solche nicht bekannt. Aufgrund der unzureichenden und teilweise nicht stimmigen Datenlage hinsichtlich der Auswirkungen dieser Erkrankung auf die Spermienqualität erachten nur wenige Fertilitätsspezialisten sie als relevant. Folglich gibt es nur wenige Informationen über ihre Prävalenz bei infertilen Männern. Aufgrund von neuen Studienergebnissen, die zeigten, dass Diabetes minimale molekulare Veränderungen induziert, die für die Spermienfunktion und -qualität wichtig sind, muss diese Einschätzung überdacht werden. Diabetische Männer weisen einen signifikant höheren Anteil von Spermien mit Kern-DNA-Schädigung (nDNA) auf, ein Faktor, der mit einer Einschränkung der Fertilität und erhöhten Fehlgeburtsraten einhergeht. Der Mechanismus, durch den diese diabetogene Spermien-nDNA-Schädigung ausgelöst wird, ist unbekannt. Die Feststellung hoher Spiegel nichtenzymatisch glykosylierter Proteine bzw. Lipide als irreversible Endprodukte („advanced glycation end products“, AGE) und ihres Rezeptors (RAGE) im ganzen männlichen Reproduktionstrakt im Zusammenhang mit Veränderungen der testikulären Metabolitenspiegel und Spermatogenese-Genexpression lassen vermuten, dass die Glykosylierung eine integrale Rolle beim oxidativen Stress spielt, der wiederum eine Spermien-nDNA-Schädigung verursacht. Da die Glykosylierung eine normale Auswirkung des Lebens ist und in die DNA-Fragmentierung verschiedener scheinbar nicht verbundener Bedingungen involviert ist, könnte sie ein allgemeiner Mechanismus für die an Spermien-DNA zu beobachtende Schädigung sein. Whilst diabetes mellitus is known to have many systemic complications, male infertility, beyond impotence, retrograde ejaculation and hypogonadism, has not been widely recognised to be one of them. Due to the paucity of studies and inconsistencies regarding the condition’s impact on semen quality, few fertility specialists consider the condition noteworthy. As a consequence little information exists as to its prevalence amongst infertile men. Recently the prevailing view has been challenged by findings showing that diabetes induces subtle molecular changes that are important for sperm quality and function. Diabetic men have been found to have a significantly higher percentage of sperm with nuclear DNA damage, a factor known to be associated with compromised fertility and increased miscarriage rates. The mechanism by which this diabetes-related sperm nDNA damage occurs remains unknown. The identification of high levels of advanced glycation end products (AGEs) and their receptor (RAGE) throughout the male reproductive tract coupled to changes in testicular metabolite levels and spermatogenic gene expression suggest that glycation may play an integral role in oxidative stress which in turn causes sperm nDNA damage. As glycation is a normal consequence of life and has been implicated in DNA fragmentation in a variety of seemingly unconnected conditions, it may constitute a common mechanism for the damage seen in sperm DNA.