KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

Effect of high intratesticular estrogen on the seminiferous epithelium in adult male rats.

The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiological role of the traditionally female hormone, estrogen, in spermatogenesis. Estrogen receptor alpha knockouts and aromatase knockouts have further accentuated the role of estrogen in germ cell maturation. To delineate the direct action of estrogen in the seminiferous epithelium, we studied the effects of high intratesticular estradiol. The study was based on the fact that administration of exogenous estradiol suppresses the hypothalamus pituitary gonadal axis (HPG) with a dose-dependant concomitant increase in intratesticular estrogen levels. Three doses of 17-beta estradiol, namely 20, 100 and 200 microg/kg/day were administered subcutaneously to different batches of adult male rats for 10 days. The effect of the three doses on serum hormonal profile, intratesticular testosterone (T) and estradiol (E) levels were studied. Twenty micrograms per kilograms per day of 17-beta estradiol affected the hypothalamus-pituitary axis, reducing serum gonadotropins and intratesticular testosterone; however, 100 microg/kg/day of 17-beta estradiol decreased serum FSH and intratesticular testosterone, increased intratesticular estradiol, but had no effect on serum LH. Interestingly, 200 microg/kg/day of 17-beta estradiol decreased serum and intratesticular T without any effect on serum gonadotropins. This could be attributed to the positive feedback effect of estrogens on gonadotropins. In the testis, morphologically two visible effects were seen, namely 'spermiation failure' in all three doses attributed to the suppression of T and FSH and a 'maintenance effect' in the 100 microg/kg/day attributed to E and/or 10% of available intratesticular T. The direct effect of an increase in intratesticular estradiol levels was observed in terms of a decrease in apoptosis in germ cell. The study, therefore, suggests that 100 microg/kg/day of 17-beta estradiol could be used to study the effects of high intratesticular estradiol with a concomitant decrease in intratesticular T and serum FSH levels on spermatogenesis.

Effect of paternal administration of an antiestrogen, tamoxifen on embryo development in rats.

We have earlier reported that oral administration of tamoxifen causes a dose-dependent reduction in the fertility of adult male rats. The decrease in fertility was mainly due to an increase in pre-implantation loss without an effect on fertilizing ability. During the study, an increased incidence of post-implantation loss of conceptuses sired by tamoxifen-treated male rats was observed. A detailed study was undertaken to investigate dose-related changes in pre- and post-implantation loss and the stage(s) of development at which these losses occurred. The present study demonstrates that tamoxifen treatment produced few normal litters as well as significantly increased pre-implantation loss without affecting the rate of fertilization. Also a significant increase in the number of degenerating embryos at the 2-4-cell stage (days 1-2 of gestation), retrieved from the oviduct/uterus of females mated with tamoxifen-treated males was observed. Histology of the resorbed fetuses, in both control and treated groups, showed presence of trophoblast outgrowth indicative of early placenta formation, which normally occurs on days 8-9 of gestation. The present results suggest that pre-implantation loss occurred at the 2-4-cell stage and the post-implantation loss occurred around days 8-9 of gestation, i.e. around midgestation. The possible effects of paternal tamoxifen treatment on embryogenesis may be due to the reduction of androgens or by the blockage of the estrogen receptor by tamoxifen, thereby affecting germ cell maturation during spermatogenesis.

Effect of paternal tamoxifen on the expression of insulin-like growth factor 2 and insulin-like growth factor type 1 receptor in the post-implantation rat embryos.

Nuclear transplantation studies demonstrated the importance of paternal contribution to embryogenesis. Paternal treatment with agents like cyclophosphamide and 5-azacytidine has been shown to cause an increase in pre-implantation loss (PIL) and post-implantation loss (POL). Studies from our laboratory have shown that paternal tamoxifen treatment increases PIL and POL. It was observed that the PIL occurred at day 2 of gestation (embryo at 2-4 cell stage) and the POL occurred around day 9 of gestation (mid-gestation). The insulin-like growth factor (IGF) system represents one of the major growth-controlling system expressed in the embryo. Several studies suggest that in rodents, insulin-like growth factor 2 (Igf2) signaling through the insulin-like growth factor type 1 receptor (Igf1r) modulates embryo growth at around days 9-11 of gestation (mid-gestation). The present study was undertaken to evaluate the expression of Igf2 and Igf1r transcript by RT-PCR in the post-implantation embryos obtained after paternal tamoxifen treatment. It was observed that both the genes were down regulated in resorbed embryos (POL). Since Igf2 is an imprinted gene and the imprint mark is established during spermatogenesis, the present study suggests that paternal tamoxifen treatment may have affected imprinting of the gene during spermatogenesis thereby decreasing its expression and leading to increase in POL. This is to our knowledge the first study correlating the increase in post-implantation embryo loss obtained after paternal drug treatment with the decrease in the expression of Igf2 in these embryos.