Adult rat seminiferous tubules secrete a fraction greater than 30 kDa to regulate spermatogenesis

The present study was designed to examine the effects of a >30kDa fraction of medium conditioned for 2 days by adult rat seminiferoustubules on inhibin secretion by cultured tubules, and on spermatogenesisand fertility of male rats. Inhibin secretion was assayed byadding the >30 kDa fraction to 5 cm segments of adult ratseminiferous tubules and measuring inhibin by radioimmunoassayat 2 day intervals. Fertility was assayed by injecting malerats daily for up to 45 days with the >30 kDa fraction andthen mating them with a proestrus female, or by injecting for15 days and mating them with two female rats. The assay usedto evaluate the in-vivo effect of the >30 kDa fraction onthe testis involved an assessment of frequencies of seminiferoustubule stages scored by transillumination on intact tubules.The addition of the >30 kDa fraction to the adult rat seminiferoustubules cultured for 2 days resulted in an inhibition of inhibinsecretion into the medium. This effect was reversed when thefraction was removed and changed with fresh medium and culturedfor a further 4 days. The >30 kDa fraction administered i.p.to adult male rats resulted in a low fertilization rate comparedto control rats (67%) (P < 0.05). The assessment of frequenciesof seminiferous tubule stages scored by transillumination showedan increased frequency of stage VI and decreased frequency ofstages VII and VIII after treatment. The results of the presentstudy provide additional evidence that local regulation of Sertolicell function is mediated by a >30 kDa component or componentssecreted by adult seminiferous tubules which could arrest spermatogenesis.     

Micro- and macro-consequences of ooplasmic injections of early haploid male gametes

This review refers to the evolution of ooplasmic injectionsof round spermatid nuclei ROSNI) or intact round spermatidsROSI). Conclusions from their preliminary application in thehamster, rabbit, mouse and human are discussed. Criteria foridentification of round spermatids and guidelines/quality controlfor application of ROSNI/ROSI techniques are emphasized. Althoughall the animal offspring and the human newborns delivered afterROSNI/ROSI are healthy additional research efforts are necessaryto confirm the safety of these procedures and improve theiroutcome     

The value of quantitative DNA flow cytometry of testicular fine-needle aspirates in assessment of spermatogenesis: a study of 137 previously maldescended human testes

In order to assess the suitability of DNA flow cytometry of fine-needle aspirates for quantiftring spermatogenesis, the results from DNA flow cytometry were compared to histological evaluation of testicular biopsies taken concomitantly from 171 previously maldescended testes. In 137 of 171 cases, sufficient material for flow cytometric as well as histological evaluation was obtained.
Histological analysis of surgical biopsy specimens revealed spermatogenesis including the spermatid stage in 117 of the 137 gonads. In six of the 117 gonads no haploid cells were found using flow cytometry. On the other hand, surgical biopsies failed to reveal spermatogenesis in five cases in which the corresponding aspirates contained haploid cells. Both methods therefore seem equally sensitive in detection of spermatogenesis.
Other types of histological patterns also corresponded to distinct DNA histograms.
Thus, in 11 of 12 cases with Sertoli-cell-only pattern in all tubules, at least 95% of the cells had a diploid DNA content. Furthermore, predominance of tubules with maturation arrest at the primary spermatocyte level corresponded to an increased proportion of tetraploid cells.
When compared to surgical biopsy, DNA flow cytometry of testiclar fine-needle aspirates is a more objective, easy and rapid method, which is more convenient for the patient. This study has indicatedthat DNA flow cytometry is a suitable method of quantitative assessment of spermatogenesis. One of the first target groups might be men with azoospermia. In such men, DNA flow cytometric analysis of fine-needle aspirates and surgical biopsy are apparently of equal sensitivity in detecting gonads with spermatogenesis. We conclude that DNA flow cytometry may become an alternative method for the quantification of spermatogenesis.     

HtrA2 is up-regulated in the rat testis after experimental cryptorchidism

AIM: The aim of the present study was to elucidate the role of high temperature requirement A2 (HtrA2) in germ cell loss in the heat-stressed testis. METHODS: We examined the expression of HtrA2, caspase-9 activity and proteolytic activity of HtrA2 in the rat testis, and their in vivo responses to experimental cryptorchid treatment. RESULTS: Northern analysis revealed the expression of HtrA2 mRNA peaked at days 1 and 7 after cryptorchid treatment. While expression of HtrA2 mRNA was seen in the spermatogonium, spermatocytes and some spermatids in normal adult rat testis, experimental cryptorchidism treatment resulted in a marked increase in its signal intensity in spermatocytes and some spermatids, and the layers of spermatogonium and early primary spermatocytes became negative at days 1 and 7 after the treatment. However, the spermatogonium, Sertoli cells and interstitial cells appeared to have strong intensities at days 14, 28 and 56 after the treatment. Western analysis revealed the expression of HtrA2 protein peaked at day 2 coinciding with the increase of positive spermatogonium, the appearance of protein-positive interstitial cells, and day 28 coinciding with the reappearance of protein-positive interstitial cells. Caspase-9 activity peaked at day 2 and HtrA2 proteolytic activity peaked at day 28. Consequently, the first peak of HtrA2 mRNA expression was followed by the peak of caspase-9 activity and the second peak was followed by the peak of proteolytic activity; however, the second peak of mRNA expression had considerable chronological difference from that of the protein. CONCLUSION: These findings suggest the probabilities that the heat stress results in germ cell death by a caspase-independent manner with the elevation of HtrA2 proteolytic activity, as well as a caspase-dependent manner with the elevation of caspase-9 activity.     

Prolonged suppression of spermatogenesis by oestrogen does not preserve the seminiferous epithelium in procarbazine-treated rats

We examined the hypothesis that induction of reversible testicular atrophy, subsequent to withdrawal of gonadotrophin support, would alleviate the testicular toxicity of the anti-cancer drug procarbazine. In rats, severe but reversible testicular atrophy and suppression of spermatogenesis were induced 56 days after the subcutaneous insertion of a silastic implant containing oestradiol-17 beta. The effect of this treatment upon the testicular toxicity of four weekly doses of procarbazine (200 mg kg-1) was examined 56 days after the termination of procarbazine/oestrogen treatment. At this time the testicular endocrine and spermatogenic functions were close to normal in rats which has received only oestradiol-17 beta. Procarbazine produced severe testicular atrophy which was associated with azoospermia and destruction of the germinal epithelium. Serum LH and FSH concentrations were raised and were associated with low serum concentrations of both testosterone and androgen-binding protein. The combination of procarbazine with the oestrogen treatment did not change any of the testicular toxicity and in some cases it appeared to be exacerbated. In contrast to these experiments other studies have indicated that the testis can be protected if spermatogenesis is reversibly suppressed by other agents which are also active via the pituitary endocrine system. The data would therefore suggest that protection is achieved either by some testicular change other than withdrawal of pituitary gonadotrophin support or that oestradiol-17 beta has additional activity which is permissive for the development of the testicular toxicity of procarbazine.     

Efficacy of the steroidal fraction of fenugreek seed extract on fertility of male albino rats

T. foenum-graecum seed extract (100 mg/day/rat) was fed orally to male albino rats for 60 days. The sperm count and motility of cauda spermatozoa declined significantly leading to negative fertility tests. The organ weights as well as androgen dependent parameters (protein, sialic acid and fructose) were lower, revealing the reduction in circulating androgen. Cholesterol is a precursor of androgen. The increase in testicular and serum cholesterol may be co-related with its nonutilization by the system, leading to a fall in circulating androgen and hence the altered histoarchitecture observed. Therefore T. foenum-graecum extract exerts both antifertility and antiandrogenic activities.     

Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.     

Mutation screening and CAG repeat length analysis of the androgen receptor gene in Klinefelter's syndrome patients with and without spermatogenesis

BACKGROUND: Mutations of the androgen receptor (AR) gene give rise to a wide array of phenotypic abnormalities. A systematic analysis of the AR gene in patients with 47,XXY has not previously been performed. METHODS: Mutations of the AR gene and expansion of the CAG repeats in exon 1 of the AR gene were studied in 13 patients with Klinefelter's syndrome either with (n = 1) or without (n = 12) spermatogenesis. RESULTS: No abnormalities in the AR gene were detected by single strand conformational polymorphism analysis. The CAG lengths ranged from 17 to 27 (mean +/- SD 22.8 +/- 3.3, median 23) for Klinefelter patients or from 17 to 28 (mean +/- SD 23.2 +/- 2.6, median 23) for control subjects. X-inactivation analysis for the methylation status of the AR gene was performed in seven patients who were heterozygous for CAG repeats of different length, showing that the longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two. CONCLUSIONS: An AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome.