Disturbance Of Spermatogenesis Research Articles

Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy.

Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time. The non-operated side was used as a control. At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy. Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism. In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis. In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals. In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases. This may be explained by cell arrest before entrance into meiosis. Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases. In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.

Morphological changes in genesial system of men: medico-legal aspects

With the purpose of perfection of examination the character of structural changes in testicles and prostate is investigated; the comparative qualitative and quantitative analysis of cellular reactions of persons with chronic somatopathies (rheumatic disease, idiopathic hypertensia) and chronic household intoxications (alcoholic illness, narcomania) is carried out. The work is carried out on 50 corpses of male persons, 18–35 years old, who died of chronic drunkenness (13 observations), narcomania (20), idiopathic hypertensia (9), and rheumatic disease (8). The suppression of testicles was characterized by disturbances of spermatogenesis at a level of the cell population, characteristic for a zero and first degree of spermatogenesis activity especially expressed in chronic narcotic intoxication. The damage to a prostate was stacked in a picture of a chronic relapsing prostatitis and chronic indurative prostatitis with the outcome of cirrhosis of prostata, being most typical for chronic alcohol intoxication. Morpho-functional investigation of a prostate and testicles affected by chronic somatopathies and chronic household intoxications, both qualitative and quantitative, testify to reduction of function of activity of these organs (hormonal imbalances, mail sterility, sex frustration). Thus, the persons suspected of violent actions of a sexual nature should be examined in order to reveal both erectile dysfunctions and disruption of spermatogenesis.

Subtotal obstruction of the male reproductive tract.

Bilateral obstruction of the male reproductive tract is suspected in men with azoospermia, normal testicular volume and normal FSH. A testicular biopsy is required to differentiate between an obstruction and a testicular insufficiency. Unilateral or subtotal bilateral obstructions and epididymal dysfunction may cause severe oligozoospermia in men with a normal spermatogenesis. However, information on spermatogenesis in oligozoospermic men is lacking, since testicular biopsy is not routinely performed. Men with a sperm concentration of <1 x 10(6) spermatozoa/ml were investigated for possible partial obstruction by performing a testicular biopsy under local anaesthesia. Spermatogenesis was determined by the Johnsen scoring method. A testicular biopsy was performed in 78 men with severe oligozoospermia. The medical history showed male accessory gland infection in 12.8%, previous hernia repair in 14.1% and a history of cryptorchidism in 12.8%. A normal or slightly disturbed spermatogenesis (Johnsen score >8) was present in 39/78 (50%) of the men. Hernia repair occurred more often in men with normal spermatogenesis. A varicocele was predominantly seen in men with a disturbed spermatogenesis. FSH was significantly lower ( P<0.0001) in men with normal spermatogenesis. Subtotal obstruction of the male reproductive tract is a frequent cause of severe oligozoospermia in men with a normal testicular volume and a normal FSH. In other cases, an epididymal dysfunction might explain the oligozoospermia in men with a normal testicular biopsy score.

INSL3 ligand-receptor system in the equine testis.

We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig cells in all types of testes investigated. Quantitative polymerase chain reaction analysis revealed a down-regulation of Insl3 and an up-regulation of the relaxin/INSL3-receptor expression in unilateral cryptorchid versus descended testes. Western blot analysis of protein extracts from adult normal and cryptorchid testes and prepubertal testes showed a single immunoreactive band at 14.5 kDa, which correlates with the predicted size of equine proINSL3. Densitometric analysis of Western blot data of adult normal testes revealed significantly stronger expression of immunoreactive proINSL3 as compared to extracts derived from cryptorchid or prepubertal testes. Thus, decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is transcriptionally regulated. The detection of transcripts for equine Lgr8 in the testis has identified the testis as a potential target of INSL3.

Inhibin B: a novel marker of spermatogenesis

Subfertility affects about 15% of all couples. Assessment of spermatogenesis has a central role in the evaluation of the subfertile couple. Classical markers of spermatogenesis, such as semen analysis, testicular biopsy and endocrine evaluation all have their diagnostic limitations. There is a clear need for accurate additional markers of spermatogenesis. Recently, the serum inhibin B level has emerged as a sensitive endocrine marker of spermatogenesis. This paper summarises the prosand consof different markers of spermatogenesis, with specific focus on serum inhibin B. The serum inhibin B level has been shown to be associated with classical markers of spermatogenesis, particularly testicular histology, and to be the most accurate endocrine marker of spermatogenesis. A subnormal serum inhibin B level clearly reflects disturbed spermatogenesis. Before puberty, when no spermatogenesis takes place, inhibin B is a marker of testicular integrity. Clinical applications of serum inhibin B in childhood and adulthood are given, and a view on future directions and research is presented. The serum inhibin B level has proven to be valuable in the evaluation of spermatogenesis, and holds a promise for further research.

Understanding spermatogenesis is a prerequisite for treatment

Throughout spermatogenesis multiplication, maturation and differentiation of germ cells results in the formation of the male gamete. The understanding of spermatogenesis needs detailed informations about the organization of the germinal epithelium, the structure and function of different types of germ cells, endocrine and paracrine cells and mechanisms, intratesticular and extratesticular regulation of spermatogenesis. Normal germ cells must be discriminated from malformed, apoptotic and degenerating germ cells and tumor cells.Identification of the border line between normal and disturbed spermatogenesis substantiate the diagnosis of impaired male fertility. The profound knowledge of the complicate process of spermatogenesis and all cells or cell systems involved with is the prerequisite to develop concepts for therapy of male infertility or to handle germ cells in the management of assisted reproduction.

Spermatogenese beim Menschen: Grundlagenforschung und Klinik

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.

The influence of environmental factors on male fertility

Potential environmental hazards should be carefully evaluated during clinical assessment of male fertility disorders. Apart from drugs or factors related to lifestyle such as alcohol and tobacco smoke, various environmental and occupational agents, both chemical and physical, may impair male reproductive functions. With regard to the complex development and regulation of the male reproductive system, different compartments and cell types have to be considered as possible targets. Disturbances of spermatogenesis can be routinely identified by detailed semen analysis. However, our knowledge of single exogenous factors and their mechanisms of action in humans is very limited, as extrapolation of results from experimental animal studies remains difficult. For the clinical andrologist, the assessment of relevant exposures to reproductive toxicants by taking detailed histories is still most important. In conjunction with epidemiological studies in occupational and environmental medicine, more data from both exposed and unexposed men have to be collected according to standardized protocols in order to identify or confirm potential reproductive hazards.

Hidden effects of X chromosome introgressions on spermatogenesis in Drosophila simulans x D. mauritiana hybrids unveiled by interactions among minor genetic factors.

One of the most frequent outcomes of interspecific hybridizations in Drosophila is hybrid male sterility. Genetic dissection of this reproductive barrier has revealed that the number of responsible factors is very high and that these factors are frequently engaged in complex epistatic interactions. Traditionally, research strategies have been based on contrasting introgressions of chromosome segments that produce male sterility with those that allow fertility. Few studies have investigated the phenotypes associated with the boundary between fertility and sterility. In this study, we cointrogressed three different X chromosome segments from Drosophila mauritiana into D. simulans. Hybrid males with these three segments are usually fertile, by conventional fertility assays. However, their spermatogenesis shows a significant slowdown, most manifest at lower temperatures. Each of the three introgressed segments retards the arrival of sperm to the seminal vesicles. Other small disturbances in spermatogenesis are evident, which altogether lead to an overall reduction in the amount of motile sperm in their seminal vesicles. These results suggest that a delay in the timing of spermatogenesis, which might be brought about by the cumulative action of many different factors of minor segment, may be the primary cause of hybrid male sterility.

Evidence of disturbed meiosis in a man referred for intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has become the treatment of choice in male infertility due to extreme oligozoospermia. Initially, the technique was limited to the selection and injection of motile spermatozoa with normal morphology; although it has now been shown that spermatozoa with abnormal morphology can also be used successfully.1 We describe a patient referred for ICSI who presented with phenotypically abnormal sperm and we show that in this patient the disturbed spermatogenesis was associated with chromosome abnormalities and abnormal meiosis.

Late effects of ionizing radiations on the testis

Most of the basic data regarding the effect of radiation on the testis are issued from animal studies. They demonstrate the extreme radiosensitivity on the germ cell lineage but little is known about the reversible or definitive aspects of these radiation induced effects. In man, the late non stochastic effects of radiation to the testicule are mainly related to persisting spermatogenesis disturbances or/and hormone related problems. Morphological, physiological, radiobiological specificities of the human testis along with numerous parameters depending on radiation conditions make it difficult to evaluate the late effects and radiation tolerance doses. Evaluation of such effects based on a common scale for therapeutic radiation schedules would improve the present understanding and possibly prevent the occurrence of these delayed effects, for the benefit of patients.

Inhibin B in men with normal and disturbed spermatogenesis.

Inhibin, a dimeric gonadal glycoprotein, inhibits the production and/or secretion of follicle stimulating hormone (FSH). The major species currently recognized are inhibin A (alphabeta A subunit) and inhibin B (alphabeta B subunit). In men, inhibin B seems to be the physiologically important form of inhibin. Therefore we measured serum inhibin B using a new two-site immunoenzymatic assay in 14 men (mean +/- SEM age, 34.5 +/- 0.7 years) with sperm counts >20 x 10(6)/ ml, in 35 men (mean +/- SEM age, 36.4 +/- 1.3 years) with oligozoospermia (sperm count <20 x 10(6)/ml) and in men with azoospermia (three orchidectomized men, three men with Klinefelter's syndrome, 10 men with Kallmann's syndrome). We compared inhibin B concentrations with serum FSH and sperm concentrations. In men with normal sperm concentrations (44.7 +/- 6.4 x 10(6)/ml), the concentration of inhibin was 223 +/- 18 pg/ml and of FSH 5.0 +/- 0.7 IU/l; in patients with low sperm concentrations (3.7 +/- 0.8 x 10(6)/ml), the concentration of inhibin B was 107 +/- 12 pg/ml and of FSH 12.2 +/- 1.5 IU/l. In all patients, except those with hypogonadotrophic hypogonadism, the relationship between inhibin B and FSH concentrations was inverse (r = -0.69, P < 0.0001). In all patients the sperm concentration was positively correlated with inhibin B concentrations (r = 0.70, P < 0.0001) and negatively correlated with FSH concentrations (r = -0.37, P < 0.01). We conclude that inhibin B may be a marker of exocrine testicular function and could offer improved diagnosis and treatment modalities for male infertility.

Prediction of posttreatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay

The hypothesis to be tested was that abnormal sperm chromatin structure is related to disturbed spermatogenesis in patients with testicular cancer. After orchiectomy but before further treatment (“pretreatment”), semen samples from 39 patients with testicular cancer were analyzed for sperm concentration by light microscopy and by the sperm chromatin structure assay (SCSA). In 28 patients assessment of sperm concentration was repeated 12–26 months after orchiectomy (“posttreatment”). The pretreatment SCSA results for the patients were compared to those from 18 healthy semen donors and assessed for correlation with the patients' posttreatment sperm concentration. Twenty-three patients displayed an abnormal chromatin structure in their pretreatment sample. For the nine evaluable patients on the surveillance program, the pretreatment SCSA results were not correlated with the posttreatment concentration. The results from 19 evaluable patients undergoing cytotoxic treatment (radiotherapy, 13; chemotherapy, 6) indicate that posttreatment recovery of spermatogenesis (recovery in 4 of 5 patients) is observed more often in patients with a normal pretreatment chromatin structure than in those with abnormal SCSA values before treatment (recovery in 2 of 14 patients; P = 0.02). The results of SCSA display sperm characteristics beyond those of light microscopically assessed sperm concentration. Pretreatment SCSA results might help clinicians to identify those testicular cancer patients with a high risk of long-lasting posttreatment disturbance of spermatogenesis. Cytometry 30:192–196, 1997. © 1997 Wiley-Liss, Inc.

Prediction of posttreatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay

The hypothesis to be tested was that abnormal sperm chromatin structure is related to disturbed spermatogenesis in patients with testicular cancer. After orchiectomy but before further treatment ("pretreatment"), semen samples from 39 patients with testicular cancer were analyzed for sperm concentration by light microscopy and by the sperm chromatin structure assay (SCSA). In 28 patients assessment of sperm concentration was repeated 12-26 months after orchiectomy ("posttreatment"). The pretreatment SCSA results for the patients were compared to those from 18 healthy semen donors and assessed for correlation with the patients' posttreatment sperm concentration. Twenty-three patients displayed an abnormal chromatin structure in their pretreatment sample. For the nine evaluable patients on the surveillance program, the pretreatment SCSA results were not correlated with the posttreatment concentration. The results from 19 evaluable patients undergoing cytotoxic treatment (radiotherapy, 13; chemotherapy, 6) indicate that posttreatment recovery of spermatogenesis (recovery in 4 of 5 patients) is observed more often in patients with a normal pretreatment chromatin structure than in those with abnormal SCSA values before treatment (recovery in 2 of 14 patients; P = 0.02). The results of SCSA display sperm characteristics beyond those of light microscopically assessed sperm concentration. Pretreatment SCSA results might help clinicians to identify those testicular cancer patients with a high risk of long-lasting posttreatment disturbance of spermatogenesis.

Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis.

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

Endocrine profiles after radiotherapy in stage I seminoma: impact of two different radiation treatment modalities

Background and purpose: In patients with stage I seminoma treated with elective lymph node irradiation, testicular scatter doses are often thought to be responsible for later disturbances in fertility. We studied the influence of radiation field extensions and testicular doses on hormonal function. Materials and methods: FSH (follicle stimulating hormone) and LH (luteinizing hormone) were evaluated before radiotherapy (RT) and by serial analyses after treatment for 4 years. Twenty-three patients were irradiated by hockey stick fields with a mean dose of 31.9 Gy (±4.7 SD) and a mean scatter dose of 54 8 cGy (±16.6 SD). Twenty-one patients received limited RT to the paraaortic nodes with 28.1 Gy (±2.4 SD). The mean testicular dose was only 25 cGy (±7.8 SD). All patients had normal pre-treatment hormonal values. Results: Six months after the end of RT, mean FSH values were significantly elevated in the hockey stick group ( P = 0.032), returning to normal after 3 years. The increase in LH was also significant, but stayed within normal ranges. Limited RT resulted in a minimal, dose-dependent increase of FSH; no changes in LH were noted. Conclusions: In patients with a normal hormonal status after semicastration, FSH is a reliable monitor for transient radiation-induced effects. To avoid treatment-related disturbances in spermatogenesis, scatter doses should be reduced to less than 20 cGy.

Evaluation of testicular toxicity in safety evaluation studies: the appropriate use of spermatogenic staging.

Toxicology of the male reproductive system has received increased interest in recent years partly fuelled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Recently revised regulatory guidelines for the safety assessment of pharmaceuticals and chemicals on reproduction and fertility have emphasized the importance of detailed histopathological examination of the testes as a sensitive method for detecting disturbances in spermatogenesis. Unfortunately this has been accompanied by a general confusion regarding a practical approach to undertaking such a detailed examination, particularly in respect to the use of spermatogenic or tubular staging to identify subtle disturbances in spermatogenesis. The ability to identify tubular stages of the spermatogenic cycle in sections of testis plus a good understanding of the spermatogenic process and its dynamics are essential in order to carry out a sensitive of testicular histopathology and to interpret the changes seen. A rational approach is required initially to detect and subsequently to characterize toxic effects to the male reproductive system. It is important that a distinction is made between these two objectives since different study designs are required and different methodology may be employed to produce the type of information or data required.

Dicentric Y Chromosome Without Evidence of Mosaicism in an Azoospermic Male

A phenotypically male of dicentric Y chromosome with no evidence of sex chromosomal mosaicism is reported. Staining of fluorescence in situ hybridization using Y chromosome centromere-specific DNA probe showed the presence of two bright fluorescent spots in Y chromosome. Testicular histology revealed maturation arrest at the stage of primary spermatocyte. It is suggested that structural anomaly of the Y chromosome is related to the disturbance of spermatogenesis.

Delayed damaging effects of anthracycline antibiotics on the reproductive system of rats

Destructive changes and disturbances of spermatogenesis are found to occur in the testes of Wistar rats 1 and 3 months after a single injection of the anthracycline antibiotic pharmorubicin in the maximum permissible dose. The morphological picture normalizes 6 months postinjection. Disturbances in reproductive function are observed as early as 3 months after treatment. The number of dominant lethal mutations rises one month postinjection.

Effect of oral administration of carbofuran on male reproductive system of rat.

1. Carbofuran was administered orally to adult male rats at dose levels of 0.1, 0.2, 0.4 or 0.8 mg kg-1 body weight, 5 d wk-1 for 60 days. A dose dependent decrease was observed in body weight of rats treated with 0.2-0.8 mg carbofuran kg-1 body weight. 2. A significant decrease in the weight of epididymides, seminal vesicles, ventral prostate and coagulating glands was observed at various test doses of carbofuran except at the lowest dose. 3. Decreased sperm motility, reduced epididymal sperm count along with increased morphological abnormalities in head, neck and tail regions of spermatozoa were observed in rats exposed to 0.2, 0.4, or 0.8 mg carbofuran kg-1 body weight. 4. In addition, significant alterations were observed in the activities of marker testicular enzymes viz. sorbitol dehydrogenase (SDH), glucose-6-P-dehydrogenase (G6PDH) (decreased), lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (gamma-GT) (increased) depending on dose. 5. Histologically, the results indicated the toxicity of carbofuran on testes depending on dose. The changes predominantly consisted of moderate oedema, congestion, damage to Sertoli cells and germ cells, along with the accumulation of cellular debris and presence of giant cells in the lumen of a few seminiferous tubules which showed disturbed spermatogenesis with the higher doses of carbofuran. 6. These observations determined a no effect level dose of 0.1 mg kg-1 body weight of carbofuran on the biochemical and morphological indices studied for male reproductive toxicity assessment in the rat model. The results of the present study provide first hand information on the reproductive toxicity of carbofuran in male rats.