Disturbance Of Spermatogenesis Research Articles

A critical analysis of the biological impacts of plasticizers on wildlife.

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l(-1) to microg l(-1) range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.

Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis

Androgens can induce complete spermatogenesis in immature or prepubertal teleost fish. However, many aspects of the role of androgens in adult teleost spermatogenesis have remained elusive. Since oestrogens inhibit androgen synthesis, we used an oestrogen-induced androgen depletion model to identify androgen-dependent stages during adult zebrafish spermatogenesis. Exposure to 10 nM 17beta-oestradiol (E(2)) in vivo at least halved the mass of differentiating germ cells (from type B spermatogonia to spermatids), while type A spermatogonia accumulated. Studies on the cellular dynamics revealed that a reduction of spermatogonial proliferation together with an inhibition of their differentiation to type B spermatogonia were the basis for the oestrogen-mediated disturbance of spermatogenesis. The capacity of the zebrafish testis to produce 11-ketotestosterone as well as the expression of steroidogenesis-related genes was markedly decreased after in vivo oestrogen exposure. Moreover, the androgen-release response to recombinant zebrafish Lh was lost after oestrogen exposure. We conclude that oestrogen exposure caused a state of androgen insufficiency in adult male zebrafish. Since the downregulation of the steroidogenic system as well as the disturbance of spermatogenesis in testicular explants exposed to E(2) ex vivo was much less severe than after in vivo exposure, the main inhibitory effect appears to be exerted via feedback inhibition of gonadotropin release. This experimental set-up helped to identify spermatogonial proliferation and their differentiation as androgen targets in adult zebrafish spermatogenesis.

Sterility of three brothers in connection with a disturbed carbohydrate metabolism in semen.

Sterile semen of three brothers was investigated biochemically. Most strikingly was the strong similarity of both the amino acid and the carbohydrate composition of the semen. Inside the spermatozoa much more fructose and glucose was present than normally is found. It could be shown that in the seminal plasma peptides and proteins were present all containing more or less beta-alanine and all able to bind glucose and fructose. After incubation with labelled glucose it was evident that inside the spermatozoa enzymes requried for formation of lactic acid were lacking or inactive. Moreover labelled tyrosine and serine was found. Treatment with proviron did not improve carbohydrate metabolism but the amounts of present glucose and fructose inside the spermatozoa had strongly decreased. It is supposed that the sterility of the men is due to a disturbance in spermatogenesis.

Results with Mesterolon in the treatment of disturbances in spermatogenesis

Über die klinischen Erfahrungen bei der Verordnung von Mesterolon (Proviron®) bei 80 Patienten mit Subfertilität wird eingehend berichtet. Es kann gezeigt werden, daß in 63% der behandelten Patienten gute bzw. mäßig gute Resultate im Sinne einer Besserung der Spermiogramme erzielt werden konnten. In 16 Fällen = 20% konnte eine Schwangerschaft eintreten; zu diesem Wert wird ausgeführt, daß die Fertilität der Frauen aus der Gesamtserie in einigen Fällen nicht optimal gewesen ist, so daß auch von dieser Seite Hemmnisse vorhanden waren. Das Fehlen einer Hepatotoxizität und eines Hemmeffektes auf den Hypophysenvorderlappen bei Proviron macht dieses peroral voll wirksame Androgen zu einem wertvollen Medikament für die Behandlungsmöglichkeiten des Andrologen. Literatur auf Anforderung beim Verfasser.

Die Analyse von Spermaproben mit Hilfe impulscytophotometrischer DNS-Bestimmungen*

Semen samples of fertility patients and fertile men were analysed by an improved technique of flow cytophotometry. The DNA-contents of mature spermatozoa were measured with a high level of accuracy. Besides spermatozoa, peaks of spermiogenetic cells and other seminal cells could be demonstrated. Complementary to the results of semen analyses the complex histograms of pulse cytophotometry investigations may give further informations on the nature of the underlying disturbances of spermatogenesis.

Intratesticular testosterone concentration in patients with disturbed fertility.

45 patients with azoospermia or severe oligozoospermia were studied. Testosterone and luteinizing hormone in plasma were in the normal range. Intratesticular testosterone values were higher the greater the degree of tubular atrophy was. Patients with testes of subnormal size (less than 12 ml) had significantly higher values than those with normal sized gonads. 31 out of 37 testes obtained by castration of patients with carcinoma of the prostate showed slight to severe disturbance of spermatogenesis. In this group no positive correlation was observed between the degree of testicular atrophy and intratesticular testosterone concentrations. Intact spermatogenesis was observed in testes with a testosterone content as low as 542 pg/mg tissue.

Bisphenol A Exposure Induces Apoptosis and Upregulation of Fas/FasL and Caspase-3 Expression in the Testes of Mice

There are controversies about adverse effects of bisphenol A (BPA), a ubiquitous xenoestrogen, on reproduction and development of male animals. To understand BPA action and assess its risk more completely, we examined the impact of BPA at high doses on the testes of pubertal male Kunming (China) mice. BPA at 0 (control), 160, 480, and 960 mg/kg/day was given by gavage to mice from postnatal days (PND) 31-44, followed by observation of morphology and detection of apoptosis and expressions of Fas/FasL and active caspase-3 on PND 45, 60, and 90 by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling, immunohistochemistry, and Western blotting. There was no effect of BPA at 160 mg/kg/day, however, at 480 and 960 mg/kg/day there was underdevelopment of testes and disruption of spermatogenesis. There were many apoptotic Leydig and germ cells in the testes with apoptotic indices being significantly increased compared with controls. The expression of Fas and active caspase-3 was localized in the same cell types as apoptosis occurred, and expression levels of Fas, FasL, and active caspase-3 were significantly increased compared with controls. The disturbed spermatogenesis, apoptosis and upregulation of Fas, FasL, and active caspase-3 expression persisted to PND 90. The results suggest that high-dose BPA induces apoptosis of Leydig and germ cells in the mouse testis through the Fas-signaling pathway. Therefore, there is concern about reproductive health for humans occupationally exposed to high levels of BPA.

Semen Characteristics in a Sub-Fertile Arabian Stallion with Idiopathic Teratospermia

A 5-year-old Arabian stallion was managed for breeding with fresh/extended semen during a period of 8 months with a resulting per cycle pregnancy rate of 26.3%. The stallion was in good health and no abnormalities of the reproductive tract were observed. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, that sperm production was normal and that semen quality was extremely poor. The most prevalent sperm defects were abnormal heads and mid-pieces. Most abnormal heads were microcephalic and/or tapered and considerable variation in sperm head dimensions within and among ejaculates was observed. A unique defect characterized by swollen/roughened mid-piece caused by accumulation of cytoplasmic-like material and abnormal mitochondrial sheath was observed. Nuclear vacuoles, acrosome defects, and teratoids were also prevalent and most sperm presented multiple abnormalities. The absence of any clear cause or any signs of testicular degeneration, combined with normal sperm production, and constant abnormal sperm production suggest an inherent, congenital disturbance of spermatogenesis as the cause of teratospermia in this case.

Adjudin Targeting Rabbit Germ Cell Adhesion as a Male Contraceptive: A Pharmacokinetics Study

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

Microdeletions in the Y chromosome as a predictive marker of infertility in males

Results of a molecular-genetics study of microdeletions in the Y chromosome among males with disturbances in spermatogenesis and among patients with cryptorchism are presented. A study of subregions AZFa, AZFb and AZFc with the use of DNA analysis in the STS loci sY84, sY86, sY127, sY134, sY254, sY255, and the gene SRY is performed. Microdeletions in the Y chromosome were found in 13.3% of infertile males studied who exhibited failed spermogram indicators, attesting to the significant information value of the study. The frequency of genetic (cyto- and molecular-genetic) damage among young boys with isolated cryptorchism amounted to 4%, which points to a need for further study of the genetic basis of cryptorchism. Management and optimization of the molecular-genetics study of microdeletions in the Y chromosome are of great importance for medical practice.

Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience

A total of 3179 patients were screened for Y-chromosome microdeletions and 821 patients for partial AZFc deletions. Thirty-nine Y-chromosomal microdeletions were found (2.4% of men with <1 × 10 6/ml spermatozoa): two AZFa, two AZFb, one AZFbc, one partial AZFb, one partial AZFb+c and 32 AZFc ( b2/b4). Partial AZFc deletions were found in 45 patients (5.5%), mostly gr/gr deletions ( n = 28). In patients with AZFc deletion, azoospermia was found in 53.1% and sperm concentrations of mostly <0.1 × 10 6/ml were found in 46.9%. Semen analyses and FSH measurements showed no trend over time. Elongated spermatids were seen in 6/15 AZFc patients and bilateral Sertoli cell-only was found in 4/15. Testicular sperm extraction (TESE) was attempted in 10 patients and spermatozoa were found in six. Compared with infertile men matched by sperm concentration, no differences in hormonal and seminal parameters could be found in patients with AZFc or gr/gr deletions. It is concluded that: (i) frequency of AZF deletions in Germany is much lower than in other countries; (ii) AZFc deletions are associated with severe disturbances of spermatogenesis and TESE is not possible in half of these patients; (iii) AZFc and gr/gr deletions are not associated with any clinical diagnostic parameter; (iv) and no trend is apparent over time.

Mast cells in testicular biopsies of azoospermic men

This work aimed at identifying mast cells in testicular biopsies from 10 normal fertile controls, 20 patients with obstructive azoospermia and 70 patients with nonobstructive azoospermia. The biopsies were stained with haematoxylin and eosin stain for tubular-modified Johnson score and with toluidine blue stain for mast cells. Two populations of mast cells, peritubular and interstitial, were demonstrated in all sections with varied counts. Testicular sections with Sertoli cell only and spermatogenic arrest patterns demonstrated a significant increase in both peritubular and interstitial mast cells compared with other groups, whereas obstructive azoospermia demonstrated a nonsignificant increase compared with the controls. Mast cell count was significantly correlated negatively with Johnson score for both peritubular (P = 0.001) and interstitial (P = 0.001) populations. Whether these results could be a cause or an effect, a special role might be assigned to mast cells in the pathogenesis of disturbed spermatogenesis.

Mutagenicity of Ascaris chymotrypsin inhibitor in germ cells of mice

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI-60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15-21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.

Cellular associations and the differential spermiogram: Making sense of stallion spermatozoal morphology

Morphologic assessment of spermatozoa is one of the most objective measures in a Breeding Soundness Examination of a stallion. There are different systems for morphologic assessment of spermatozoa. The objectives of this article are to review spermatogenesis, describe clinical sample preparation, discuss previous methods of morphologic classification and explain the use of a differential spermiogram. The advantages of the differential spermiogram method of analysis are discussed, along with its use in delineating intrinsic and extrinsic disturbances in spermatogenesis. Case examples of specific cellular associations and their diagnostic relevance are discussed.

Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis

Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN- and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.

Functional changes of mice Sertoli cells induced by Cr(V)

Transport of macromolecules from the interstitial testis tissue to cells at the adlumenal compartment of the seminiferous epithelium occurs naturally through Sertoli cells. In previous studies we have shown that Cr(V) intoxication disturbed spermatogenesis in mice. To test if Sertoli cells are affected by chromium, a well proved carcinogen, the uptake and the horseradish peroxidase transport ability of isolated seminiferous tubules of mice administered with a chromium(V) compound, have been studied. Male CD-R mice were exposed daily for 5 days to [CrV-BT]2- through subcutaneous injection and comparisons were made with groups of vehicle-treated mice. Using an in vitro assay we demonstrated that the seminiferous tubules were able to uptake and transport the tracer, in a much faster way than controls, mainly via intercellular and transcellular pathways, providing evidence that this functional role of Sertoli cells is affected by the Cr(V) compound. These findings might improve the knowledge on the toxicity mechanisms of chromium.

Effects of dibromoacetic acid on murine spermatozoa and testis.

Background and Aims: Bromoacetic acids are a by-product of water ozonation and dibromoacetic acid (DBAA) in particular, which is a by-product of disinfection, inhibits male reproductive functions. In order to understand its effects, the spermatozoa and testes of mice were exposed to DBAA. Methods: Twelve-week-old ICR mice were exposed to 10 p.p.m. DBAA. They were examined in regards to effects on the weights of body, testis and epididymis, the histological changes of tesits and the protein expression in testis. Results: Neither the bodyweight nor the weights of the testis and epididymis of the exposed mice was affected, but approximately 13% of spermatozoa obtained from the cauda epididymis were motile with a drop-shaped head, and structures resembling residual bodies were found in the testis. Moreover, the expression of two testis proteins was changed by exposure to DBAA. Conclusions: It was likely that DBAA inhibited male reproductive functions by disturbance of spermatogenesis via change of protein expression. (Reprod Med Biol 2004; 3: 85-93).

Genomic imprinting in disruptive spermatogenesis

The possibility of imprinting disease transmission by assisted reproductive technologies has been raised after births of children with Angelman's and Beckwith-Wiedemann's syndromes. To investigate whether imprinting defects were associated with disturbed spermatogenesis, we studied two oppositely imprinted genes in spermatozoan DNA from normozoospermic and oligozoospermic patients. In the mesodermal specific transcript gene (MEST), bisulphite genomic sequencing showed that maternal imprinting was correctly erased in all 123 patients. However, methylation of the H19 gene did not change in any of 27 normozoospermic individuals (0%, 95% CI 0-13%), compared with methylation changes in eight moderate (17%, 8-31%, p=0.026) and 15 severe (30%, 18-45%, p=0.002) oligozoospermic patients. Our data suggest an association between abnormal genomic imprinting and hypospermatogenesis, and that spermatozoa from oligozoospermic patients carry a raised risk of transmitting imprinting errors.

Detection of structural and numerical chromosomal abnormalities by ACM-FISH analysis in sperm of oligozoospermic infertility patients.

Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients. We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region. There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy. Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

Previously uncharacterized roles of platelet-activating factor acetylhydrolase 1b complex in mouse spermatogenesis.

Platelet-activating factor (PAF) has been shown to affect sperm motility and acrosomal function, thereby altering fertility. PAF acetylhydrolase 1b (PAFAH1B) hydrolyzes PAF and is composed of three subunits [the lissencephaly (LIS1) protein and alpha1 and alpha2 subunits] and structurally resembles a GTP-hydrolyzing protein. Besides the brain, transcripts for Lis1, alpha1, and alpha2 are localized to meiotic and early haploid germ cells. Here, we report disruptions of the alpha2 (Pafah1b2) and alpha1 (Pafah1b3) genes in mice. Male mice homozygous null for alpha2(alpha2-/-) are infertile, and spermatogenesis is disrupted at mid- or late pachytene stages of meiosis or early spermiogenesis. Whereas mice homozygous mutant for alpha1(alpha1-/-) have normal fertility and normal spermatogenesis, those with disruptions of both alpha1 and alpha2 (alpha1-/-alpha2-/-) manifest an earlier disturbance of spermatogenesis with an onset at preleptotene or leptotene stages of meiosis. Testicular Lis1 protein levels are up-regulated in the alpha2-/- and alpha1-/-alpha2-/- mice. Lowering Lis1 levels by inactivating one allele of Lis1 in alpha2 null or alpha1/alpha 2 null genetic backgrounds (i.e., alpha2-/-Lis1+/- or alpha1-/-alpha2-/-Lis1+/- mice) restored spermatogenesis and male fertility. Our data provide evidence for unique roles of the PAFAH1B complex and, particularly, the lissencephaly protein Lis1 in spermatogenesis.