Hormone pretreatment enhances recovery of spermatogenesis in rats after neutron irradiation.

Previous studies showed that a 6-week pretreatment of rats with testosterone plus estradiol enhanced the recovery of spermatogenesis 9 weeks after gamma irradiation, resulting in a dose-modifying factor (DMF) of about 2. To test whether the effect of the hormone treatment was mediated through changes in oxygen tension, thiol levels or DNA repair, we irradiated the testes of rats with neutrons, which depend less on these factors than does low-LET radiation for their cytotoxic action. Control rats and rats treated with testosterone plus estradiol were irradiated with 0.7-2.7 Gy of cyclotron-generated high-energy neutrons. The recovery of spermatogenesis was assessed 9 weeks after irradiation by testis weights, sperm counts and the tubule repopulation indices. Greater recovery of spermatogenesis was observed for all end points, with a DMF of about 2 for rats treated with testosterone plus estradiol compared to the irradiated, cholesterol-treated rats. The equal protection factors for neutrons and gamma rays indicate that oxygen, thiols and repair of DNA damage are unlikely to be involved in the protective effect of the hormone treatment.     

Gene expression profiles of mouse spermatogenesis during recovery from irradiation

Background  

Irradiation or chemotherapy that suspend normal spermatogenesis is commonly used to treat various cancers. Fortunately, spermatogenesis in many cases can be restored after such treatments but knowledge is limited about the re-initiation process. Earlier studies have described the cellular changes that happen during recovery from irradiation by means of histology. We have earlier generated gene expression profiles during induction of spermatogenesis in mouse postnatal developing testes and found a correlation between profiles and the expressing cell types. The aim of the present work was to utilize the link between expression profile and cell types to follow the cellular changes that occur during post-irradiation recovery of spermatogenesis in order to describe recovery by means of gene expression.     

Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation

With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals. Successful depletion of endogenous spermatogenesis was achieved using fractionated ionizing irradiation. A dose of 1.5 Gy followed by a dose of 12 Gy after 24 h reduced the percentage of tubule cross-sections displaying endogenous spermatogenesis to approximately 3% and 10% as evidenced by histologic evaluation of testes at 12 and 21 wk, respectively, after irradiation. At this dose, no apparent harmful side effects were noted in the animals. Upon transplantation, GDNF-overexpressing germ cells were found to be able to repopulate the irradiated testes and to form clusters of spermatogonia-like cells resembling those found in the overexpressing donor mice. The cluster cells in transplanted host testes expressed human GDNF, as had been shown previously for clusters in donor animals, and both were strongly positive for the tyrosine kinase receptor Ret. Thus, we devised an efficient method for depleting the seminiferous epithelium of host mice without appreciable adverse effects. In these host mice, GDNF-overexpressing cells reproduced the aberrant phenotype found in the donor transgenic mice.     

The numbers of Sertoli cells in mature Holstein bulls and their relationship to quantitative aspects of spermatogenesis

Testes from 37 Holstein bulls, 38-99 mo of age, were used to investigate the relationship of Sertoli cell number, Sertoli cell-germ cell ratios and other related factors to daily sperm production (DSP). DSP was assessed by enumeration of spermatids in testicular homogenates, whereas Sertoli cell and germ cell ratios were based on direct counts in 20 round Stage VIII seminiferous tubular cross sections per bull. Numbers of Sertoli cells were calculated as (total homogenization resistant spermatids:spermatid:Sertoli cell ratio)/0.394; the factor of 0.394 adjusted for the presence of homogenization resistant spermatids during only 39.4% of the spermatogenic cycle. Data were subjected to simple linear and second-order regression analyses. Positive linear relationships were observed between DSP and testicular parenchymal weight (p less than 0.005, R = +0.71), DSP per gram (p less than 0.005, R = +0.79), total Sertoli cells (p less than 0.005, R = +0.83), Sertoli cells per gram (p less than 0.01, R = +0.47) and the yield of Step 8 spermatids per Type A spermatogonium (p less than 0.05, R = +0.34). DSP was not related (p greater than 0.10) to the number of germ cells supported per Sertoli cell. Testicular parenchymal weight and DSP per gram were unrelated to each other (p greater than 0.10), but both were related (p less than 0.005) to the total Sertoli cell number (R = +0.61 and +0.62, respectively). Total number of Sertoli cells accounted for more of the variation in DSP between bulls (R2 = 68.2%) than did any other factor examined. It was suggested that total Sertoli cell number may be an important determinant of a bull's spermatogenic potential.     

Ultrastructure of the testes and spermatogenesis in the mite Anystis baccarum

The epithelial lining of testes in Anystis baccarum is glandular and produces a secretory product necessary to form spermatophores. The main stages of spermatogenesis occur in the lumen of the testis in groups of synchronously developing sister cells. Spermatogonia and late spermatids are encircled by glandular cells. Reorganization of developing spermatids is typical of the trombidiform mites and includes formation of the acrosomal complex, cytoplasm elimination, disappearance of the nuclear envelope and formation of invaginations of plasmalemma. The chromatin material condensation is not followed by the entire chromatin body formation. In mature spermatoza, dense chromatin strands (80b nm in diameter) lie along the cell in the peripheral layer of the cytoplasm. Mature spermatozoa lack axonema or any protrusions. A layer of microtubules, visible underneath the outer membrane, may serve for sperm movement in the female genital duct. The acrosomal complex consists of acromal granule, acrosomal filament and subacrosomal substance. This, as well as two aggregates of typical mitochondria, looks plesiomorphic.     

Connexin 43 expression in human and mouse testes with impaired spermatogenesis

Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter's syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter's patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter's patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.     

Differences in spermatogenesis in cryptorchid testes among various strains of mice

The purpose of the study reported here was to define strain differences in spermatogenesis in cryptorchid testes in mice. Mice of strains A/J, BALB/c, CBA/N, C3H/He, C57BL/6 (B6), ddY and ICR were found to be sensitive to heat stress attributable to experimentally induced cryptorchidism. In contrast, mice of strains AKR/N (AKR), MRL/MpJ-+/+ (M+) and MRL/MpJ-lpr/lpr (lpr) were resistant to heat stress. Relative increases of apoptotic cells were detected in the sensitive group, but not in the resistant group. A decrease of proliferating cell nuclear antigen-immunoreactive cells after experimentally induced cryptorchidism was observed only in the sensitive group. These results suggested that heat stress-resistant germ cells were present in MRL and AKR strains, possibly originating from the genetic background.     

Resistance gene analogs in barley and their relationship to rust resistance genes.

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.     

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.