Age-dependent alterations in spermatogenesis in itchy mice

Spermatogenesis is an intricate process in which spermatogonial stem cells divide and differentiate to produce mature sperm. This process strongly depends on protein turnover both in the developing germ cells and the supportive Sertoli cells, and recent evidence has demonstrated the role of the ubiquitin-proteasome system in this protein turnover in the testis. Itch, an E3 ligase important in the immune system, has been implicated in regulating the blood testis barrier. Although the specific role of Itch during spermatogenesis is not yet well understood, its ubiquitous expression and wide array of functional targets suggest multiple and tissue-specific roles. Here the testes of mice that lack Itch protein are evaluated at two developmental time points: peri-pubertal postnatal day (PND) 28 and adult PND 56. Itchy mice demonstrate an increased germ cell apoptotic index compared with wild type C57BL/6J mice at both PND 28 and PND 56. A corresponding 27% reduction in the total number of spermatid heads produced in PND 56 itchy mice was also evident. A histological evaluation of itchy mice revealed a delay in spermatogenesis at PND 28 and disorganization of late stage spermatids at PND 56. An analysis of several apoptotic markers revealed an age-dependent change in cleaved caspase 9, an intrinsic apoptosis mediator. The breeding success of the itchy mice was also significantly decreased, possibly due to a developmental defect. Taken together, these findings indicate that Itch is required for functional spermatogenesis, and that it may play differing cellular roles during development

FasL Gene–Deficient Mice Display a Limited Disruption in Spermatogenesis and Inhibition of Mono-(2-ethylhexyl) Phthalate–Induced Germ Cell Apoptosis

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene–deficient mice (FasL−/−) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL−/− mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 ± 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 ± 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL−/− mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL−/− mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL−/− mice. However, PND 44 FasL−/− mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL−/− mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients

Histological evaluation of the testis of wild type C57BL/6J and <i>Trail^−/−</i> mice at PND 56.

<p>Testicular cross sections (5 µm) from paraffin embedded tissue were examined using PAS-H staining. (A) is from wild type a C57BL/6J mouse, and (B) is from a <i>Trail^−/−</i> mice. At PND 56, <i>Trail^−/−</i> mice show more meiotic figures (B) than wild type C57BL/6J mice (A). (C) Quantification of tubules with meiotic figures in wild type C57BL/6J and <i>Trail^−/−</i> mice at PND 28 and PND 56. Values represent the mean ±SEM with an asterisk identifying a significant difference from control (*p<0.05 and ***p<0.001, Student’s <i>t</i>-test). The bar represents 50 µm.</p

Body weights and testis weights of <i>wild type C57BL/6J and Trail</i>^−/− mice.

<p>Values represent the mean ± SEM with an asterisk identifying a significant difference from control (*p<0.05 and ***p<0.001, Student’s <i>t</i>-test).</p

Spermatid head counts of C57BL/6J and <i>Trail^−/−</i> mice at PND 56.

<p>The total number of mature spermatid heads were counted from 11 C57BL/6J and 8 <i>Trail^−/−</i> mice at PND 56. <i>Trail^−/−</i> mice have significantly lower spermatid head counts than C57BL/6J mice. Values represent the mean ± SEM with asterisks identifying a significant difference from control (**p<0.01, Student’s <i>t</i>-test).</p

Immunohistochemical analysis of intrinsic and extrinsic apoptosis in wild type C57BL/6J and <i>Trail^−/−</i> mice at PND 28 and PND 56.

<p>(A) Cleaved caspase-8 (CC8) and (B) cleaved caspase-9 (CC9) values were calculated as the percentage of tubules containing more than 3 CC8 or CC9 positive cells. <i>Trail^−/−</i> mice have higher intrinsic and extrinsic apoptosis signaling at PND 28 and PND 56 than wild type mice. Values represent the mean ± SEM with an asterisk identifying a significant difference from control (**p<0.01, Student’s <i>t</i>-test).</p

Western blot analysis of FasL expression in wild type C57BL/6J and <i>Trail^−/−</i> mice at PND28 and PND 56.

<p>Total protein from two sets of PND 28 and PND 56 whole testis tissue were analyzed using primary antibodies against FasL. Tubulin was used as a loading control. Total cellular protein expression from whole testis homogenates at PND 28 and PND 56 were detected using primary antibodies against FasL. α-tubulin was the loading control. Values represent the mean ± SEM with an asterisk identifying a significant difference from control (*p<0.05, Student’s <i>t</i>-test).</p