Differences in radiation sensitivity of recovery of spermatogenesis between rat strains.

Previous studies with Lewis/Brown-Norway (BN) F1 hybrid rats indicated that spermatogenesis was much more sensitive to ionizing radiation than in the widely studied outbred Sprague Dawley stock, suggesting that there were genetically based differences; however, the relative sensitivities of various inbred strains had not been established. As a first step to defining the genes responsible for these differences, we compared the sensitivities of seven rat strains to radiation damage of spermatogenesis. Recovery of spermatogenesis was examined 10 weeks after 5-Gy irradiation of seven strains (BN, Lewis, Long-Evans, Wistar Kyoto, spontaneously hypertensive [SHR], Fischer 344, and Sprague Dawley). The percentages of tubules containing differentiated cells and testicular sperm counts showed that BN and Lewis were most sensitive to radiation (< 2% of tubules recovered, < 2 × 10(5) late spermatids per testis), Long-Evans, Wistar Kyoto, Fischer, and SHR were more resistant, and Sprague Dawley was the most resistant (98% of tubules recovered, 2 × 10(7) late spermatids per testis). Although increases in intratesticular testosterone levels and interstitial fluid volume after irradiation had been suggested as factors inhibiting recovery of spermatogenesis, neither appeared to correlate with the radiation sensitivity of spermatogenesis in these strains. In all strains, the atrophic tubules without differentiated germ cells nevertheless showed the presence of type A spermatogonia, indicating that their differentiation was blocked. Thus, we conclude that the differences in radiation sensitivity of recovery of spermatogenesis between rat strains of different genetic backgrounds can be accounted for by differences in the extent of the radiation-induced block of spermatogonial differentiation.

Estrogen-regulated genes in rat testes and their relationship to recovery of spermatogenesis after irradiation.

Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropin-releasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.

Effect of interleukin-1 receptor antagonist gene deletion on male mouse fertility.

Members of the IL-1 family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions in the body. IL-1alpha, IL-1beta, and the IL-1 antagonistic molecule [IL-1 receptor antagonist (IL-1 Ra)] are present in the testis under normal homeostasis, and they further increase upon infection/inflammation. In the present study, we examined the effect of IL-1 Ra gene deletion on male mouse fertility. Male mice [wild type (WT) and IL-1 Ra knockout (KO)] were mated with WT females, and the birth and number of offspring were recorded 21-45 d after mating. Furthermore, the concentration, motility, and morphology of sperm isolated from the cauda of the epididymis were evaluated. The ability of the calcium ionophore (A23187) to induce acrosome reaction (AR) in the sperm of WT and IL-1 Ra KO mice was compared with their ability to fertilize in vitro oocytes from WT females. The direct effect of IL-1alpha and IL-1beta on AR and abnormal morphology in sperm from WT were evaluated. The levels of IL-1alpha and IL-1beta in the testes of WT and IL-1 Ra KO mice were examined by specific ELISA and real-time PCR. Our results show a significant reduction in the capacity of IL-1 Ra KO male mice to fertilize WT females (P < 0.05). Furthermore, the number of offspring in mice fertilized with IL-1 Ra KO male mice was significantly lower than with WT males (P < 0.05). Sperm concentration and the percentage of motile sperm from IL-1 Ra KO and WT were similar; however, the percentage of sperm with abnormal morphology (mainly in the head) and acrosome-reacted sperm cells were significantly higher in the IL-1 Ra KO, compared with that of WT males (P < 0.05). In vitro, the ability of sperm from IL-1 Ra KO male mice to fertilize oocytes from WT females was significantly lower than sperm from WT mice (P < 0.05). In addition, the percentage of reacted sperm from IL-1 Ra KO, spontaneously without ionophore induction, was significantly higher than from WT (P < 0.05). Sperm from WT underwent induction of AR only by ionophore; however, sperm from IL-1 Ra KO were unable to undergo the AR by ionophore, indicating that they are induced and, thus, are inactive in fertilization. Testicular IL-1alpha and IL-1beta levels were significantly higher in IL-1 Ra KO, compared with WT male mice (P < 0.05). The addition of recombinant IL-1alpha or IL-1beta to sperm from a WT mouse induced their AR, and significantly increased abnormal sperm morphology, as compared with controls (P < 0.05). This effect was neutralized by the addition of IL-1 Ra. Our results indicate the involvement of IL-1 in sperm physiology, affecting its morphology and fertilization ability. Higher than homeostatic levels of IL-1 in the testis, as observed in IL-1 Ra KO mice, impaired the ability of sperm to fertilize oocytes. Together, these results may explain some of the male infertility cases with an infection/inflammation background and may hint at the ability to use IL-1 Ra in future therapeutic strategies in these cases.

Lipopolysaccharide increased the expression levels of IL-18, ICE and IL-18 R in murine Leydig cells.

PROBLEM: This study examined the effect of lipopolysaccharide (LPS) on the capacity of Leydig cells to produce and express interleukin-18 (IL-18), IL-18 receptor (IL-18R) and the IL-1beta-converting enzyme (ICE) (IL-18 family), under in vitro conditions. METHOD OF STUDY: Primary Leydig cells (LCs) were isolated from murine testis by the Percoll technique, and cultured both in the presence and absence of LPS (0.1, 1, 5 microg/mL) for 3 and 24 hr. LCs were examined for their capacity to produce and express IL-18 family molecules by using immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA), Western blot and real-time polymerase chain reaction (PCR) analysis. RESULTS: Leydig cells were shown to constitutively express IL-18, as examined by IHC, ELISA, Western blot and real-time PCR analysis. Addition of LPS to LC cultures was shown to significantly increase the basal levels of IL-18, in a dose- and time course-dependent manner, as examined by ELISA, Western blot and real-time PCR analysis. In addition, LPS increased LC cultures to express ICE and IL-18 R, as examined by real-time PCR analysis. CONCLUSION: Our results demonstrate that LPS increased the capacity of murine LCs to produce the IL-18 family molecules. IL-18, in the testis, might be involved in the regulation of physiological and infection/inflammatory processes, and thus, could be a component of the autocrine/paracrine factor net that controls steroidogenesis and male fertility; further studies are needed to confirm this possibility.

In vitro culture of testicular germ cells: regulatory factors and limitations.

Spermatogenesis is regulated mainly by endocrine factors and also by testicular paracrine/autocrine growth factors. These factors are produced by Sertoli cells, germ cells, peritubular cells and interstitial cells, mainly Leydig cells and macrophages. The interactions and the ratio between Sertoli and germ cells in the seminiferous tubules ensure successful spermatogenesis. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers tried to overcome some of the obstacles -- such as the low number of stem cells in the testis, absence of specific markers to identify SSCs -- in addition to difficulties in keeping the SSCs alive in culture. Recently, some growth factors important for the proliferation and differentiation of SSCs were identified, such as glial cell line derived neurotrophic factor (GDNF), stem cell factor (SCF) and leukemia inhibitory factor (LIF); also, markers for SSCs at different stages were reported. Therefore, some groups succeeded in culturing SSCs (under limitations), or more differentiated cells and even were able to produce in vitro germ cells from embryonic stem cells. Thus, success in culturing SSCs is dependent on understanding the molecular mechanisms behind self-renewal and differentiation. Culture of SSCs should be a good tool for discovering new therapeutic avenue for some infertile men or for patients undergoing chemotherapy/radiotherapy (pre-puberty or post-puberty).