214 research outputs found
Immunohistochemical characterization of the anti-Müllerian hormone receptor type 2 (AMHR-2) in human testes
Purpose In males, AMH is secreted by immature Sertoli cells; following exposure to endogenous androgens, Sertoli cells undergo a process of maturation which ultimately inhibits AMH expression to undetectable levels in the serum. However, expression of AMH receptor (AMHR-2) has never been studied in human testes, and high intratubular concentrations of AMH have been reported in recent literature. We therefore assessed expression of AMHR-2 in several testicular tissue samples by immunohistochemistry (IHC). Methods The IHC method was first validated on tissue samples from healthy human testis (n = 2) and from marmoset ovary (n = 1). The same method was then used for assessment on testicular histopathology specimens from patients with mixed atrophy (MA, n = 2), spermatogenetic arrest (SA, n = 2), Sertoli cell-only syndrome (SCO, n = 1), Klinefelter syndrome (KS, n = 1), and nonseminomatous germ cell tumors (NSGCT, n = 1). Tissue samples from two subjects at different pubertal stages (AndroProtect (AP), aged 5 and 14 years) with hematological malignancies were also retrieved. Results In adult men, AMHR-2 was expressed on peritubular mesenchymal cells, with patterns closely mirroring alpha-smooth muscle actin expression. Similar patterns were preserved in almost all conditions; however, in nonseminomatous germ cell tumors the tissue architecture was lost, including AMHR-2 expression. More positive and diffuse staining was observed in tissue samples from prepubertal testes. Conclusions In specimens from both healthy and affected testes, AMHR-2 expression appears weaker in adult than in prepubertal tissue sections. The persistence of AMHR-2 expression seemingly hints at a possible effect of intratesticular AMH on the tubular walls
UVB irradiation as a tool to assess ROS-induced damage in human spermatozoa
One of the consequences of oxygen metabolism is the production of reactive oxygen species (ROS) which in a situation of imbalance
with antioxidants can damage several biomolecules, compromise cell function and even lead to cellular death. The particularities
of the sperm cell make it particularly vulnerable to ROS attack compromising its functionality, mirrored in terms of fertility
outcome and making the study of the origin of sperm ROS, as well as the alterations they cause very important. In the present work,
we used UVB irradiation, an easy experimental approach known as a potent inducer of ROS formation, to better understand the origin
of ROS damage without any confounding effects that usually exist in disease models in which ROS are reported to play a role. To
address these issues we evaluated sperm mitochondrial ROS production using the Mitosox Red Probe, mitochondrial membrane
potential using the JC-1 probe, lipid peroxidation through BODIPY probe and vitality using PI. We observed that UVB irradiation
leads to an increase in sperm mitochondrial ROS production and lipid peroxidation that occur previously to an observable mitochondrial
dysfunction. We concluded that sperm UVB irradiation appears to be a good and easily manipulated in vitro model system to
study mitochondria-induced oxidative stress in spermatozoa and its consequences, which may be relevant in terms of dissecting the
action pathways of many other pathologies, drugs and contaminants, including endocrine disruptors.S.A. is the recipient of a FCT fellowship (SFRH/BPD/
63190/2009). Centre for Neuroscience and Cell Biology (CNC)
funding is supported by FCT (PEst-C/SAU/LA0001/2011)
Serum concentrations of dihydrotestosterone are associated with symptoms of hypogonadism in biochemically eugonadal men
Purpose
Symptoms of hypogonadism are often reported by subjects with normal serum testosterone (T) levels. We aimed to assess the association between clinical symptoms in andrological outpatients and sex steroids levels.
Methods
This is a retrospective cross-sectional cohort study in an Academic clinic and research unit. International Index of Erectile Function (IIEF, EF domain) and Aging Males Symptoms scale (AMS) questionnaires were completed by 635 and 574 men, respectively (mean age: 47.3 ± 13.9 and 47.4 ± 13.8 years, p = 0.829), free of interfering medications with complaints possibly related to hypogonadism.
Results
Serum total/free T as well as dihydro-T (DHT) was associated with IIEF-EF and AMS scores in the overall population using univariate analyses. Multivariate approaches revealed DHT concentrations in subjects with normal T levels (n = 416, Total T > 12 nmol/L) to be significant predictors of AMS scores. A 0.1 nmol/l serum DHT increase within the eugonadal range was associated with a 4.67% decrease in odds of having worse symptoms (p = 0.011). In men with biochemical hypogonadism (Total T < 12 nmol/L), total and free T rather than DHT were associated with AMS results. This association was not found for IIEF-EF scores. Indirect effects of age and BMI were seen for relations with hormone concentrations but not questionnaire scores.
Conclusion
DHT can be associated with symptoms of hypogonadism in biochemically eugonadal men. Serum DHT measurement might be helpful once the diagnosis of hypogonadism has been ruled out but should not be routinely included in the primary diagnostic process
Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of the human FSH receptor promoter.
AIM: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. METHODS: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5'-flanking region fragment of its promoter. RESULTS: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. CONCLUSION: A 1.5kb 5'-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression
Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge
The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood
Male germline stem cells in non-human primates
Over the past few decades, several studies have attempted to decipher the
biology of mammalian germline stem cells (GSCs). These studies provide
evidence that regulatory mechanisms for germ cell specification and migration
are evolutionarily conserved across species. The characteristics and
functions of primate GSCs are highly distinct from rodent species; therefore
the findings from rodent models cannot be extrapolated to primates. Due to
limited availability of human embryonic and testicular samples for research
purposes, two non-human primate models (marmoset and macaque monkeys) are
extensively employed to understand human germline development and
differentiation. This review provides a broader introduction to the in vivo
and in vitro germline stem cell terminology from primordial to
differentiating germ cells. Primordial germ cells (PGCs) are the most
immature germ cells colonizing the gonad prior to sex differentiation into
testes or ovaries. PGC specification and migratory patterns among different
primate species are compared in the review. It also reports the distinctions
and similarities in expression patterns of pluripotency markers (OCT4A,
NANOG, SALL4 and LIN28) during embryonic developmental stages, among
marmosets, macaques and humans. This review presents a comparative summary
with immunohistochemical and molecular evidence of germ cell marker
expression patterns during postnatal developmental stages, among humans and
non-human primates. Furthermore, it reports findings from the recent
literature investigating the plasticity behavior of germ cells and stem cells
in other organs of humans and monkeys. The use of non-human primate models
would enable bridging the knowledge gap in primate GSC research and
understanding the mechanisms involved in germline development. Reported
similarities in regulatory mechanisms and germ cell expression profile in
primates demonstrate the preclinical significance of monkey models for
development of human fertility preservation strategies
Analysis of copy number variation in men with non-obstructive azoospermia
BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridization (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritized the affected genes according to a literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritized genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable
- …