Estrogen, through estrogen receptor 1, regulates histone modifications and chromatin remodeling during spermatogenesis in adult rats

<p>Estrogen receptors (ESR1 and ESR2) play crucial roles in various processes during spermatogenesis. To elucidate individual roles of ESRs in male fertility, we developed <i>in vivo</i> selective ESR agonist administration models. Adult male rats treated with ESR1 and ESR2 agonist for 60 days show spermatogenic defects leading to reduced sperm counts and fertility. While studying epigenetic changes in the male germ line that could have affected fertility, we earlier observed a decrease in DNA methylation and its machinery upon ESR2 agonist treatment. Here, we explored the effects on histone modifications, which could contribute to decreased male fertility upon ESR agonist administration. ESR1 agonist treatment affected testicular levels of histone modifications associated with active and repressed chromatin states, along with heterochromatin marks. This was concomitant with deregulation of corresponding histone modifying enzymes in the testis. In addition, there was increased retention of histones along with protamine deficiency in the caudal spermatozoa after ESR1 agonist treatment. This could be due to the observed decrease in several chromatin remodeling proteins implicated in mediating histone-to-protamine exchange during spermiogenesis. The activating and repressing histone marks in spermatozoa, which play a critical role in early embryo development, were deregulated after both the ESR agonist treatments. Together, these epigenetic defects in the male germ line could affect the spermatozoa quality and lead to the observed decrease in fertility. Our results thus highlight the importance of ESRs in regulating different epigenetic processes during spermatogenesis, which are crucial for male fertility.</p

Estrogen signaling, through estrogen receptor β, regulates DNA methylation and its machinery in male germ line in adult rats

<p>Estrogen, through its receptors, regulates various aspects of spermatogenesis and male fertility. To understand the roles of estrogen receptors (ERα and ERβ) in male fertility, we have developed <i>in vivo</i> selective ER agonist administration models. Treatment of adult male rats with ERα or ERβ agonist for 60 d decreases fertility and litter size mainly due to increased pre- and post-implantation embryo loss. Since epigenetic mechanisms like DNA methylation play a crucial role in male fertility, we investigated the effects of the ER agonists on DNA methylation in spermatozoa. Treatment with ERβ agonist causes a significant decrease in DNA methylation both at the global level and at the <i>H19</i> differentially methylated region (DMR). This could be due to decrease in DNA methyltransferases in the testis upon ERβ agonist treatment. The hypomethylation observed at the <i>H19</i> DMR corroborates with aberrant expression of <i>Igf2</i> and <i>H19</i> imprinted genes in the resorbed embryos sired by ERβ agonist-treated males. Thus, our study demonstrates that ERβ regulates DNA methylation and methylating enzymes during adult rat spermatogenesis. Activation of estrogen signaling through ERβ could therefore cause DNA methylation defects leading to impaired male fertility. These results define a role for estrogen in epigenetic regulation of male germ line, suggesting that epigenetic insults by exposure to environmental estrogens could potentially affect male fertility.</p

Additional file 1: of Comparative analysis of syndromic and PCR-based diagnostic assay reveals misdiagnosis/ overtreatment for trichomoniasis based on subjective judgment in symptomatic patients

Multilingual abstract in the six official working languages of the United Nations. (PDF 421 kb

Summary of Parasitemia Data in Experiment #1

<p>‘Day 1^st parasitemia’ is the day after sporozoite challenge when parasites were first seen on malaria smear. ‘Day >1% parasitemia’ is the day of drug treatment (‘unknown’ means that monkeys were drug treated before reaching 1% parasitemia). Priming with 3 doses of DNA in PBS gave better protection than priming with 1 dose of DNA in PBS or 3 doses of DNA on PLG (<b>*</b>p = 0.04 Fisher's Exact test).</p

Immune response to PkCSP antigen at time of challenge for individual animals by day to first parasite seen in blood.

<p>Data from Experiments #1, 2, and 3 are plotted. Data from Control animals are not included. Panel A shows PkCSP endpoint ELISA titers where there is no significant correlation. Panel B shows PkCSP interferon-γ ELIspot titers with a linear regression line included. There is a significant negative correlation (p = 0.04), animals with lower ELIspot responses having longer times to the appearance of first parasite in the blood.</p

Panel A. Interferon-γ ELIspot was tested for only two antigens: PkCSP and PkMSP1.

<p>Spots in medium controls were subtracted from antigen test wells, and the results averaged. Priming with DNA on PLG gave a stronger interferon-γ response than DNA in PBS but this was not significant (p = 0.58). Panel B. Geometric mean antibody titers by ELISA at time of sporozoite challenge for each of the four malaria vaccine antigens. Priming with 3 doses of DNA gave higher antibody levels than did a single DNA priming dose for PkCSP, PkMSP1 and PkAMA1 antigens (p<0.05). Priming with 3 doses of DNA in PBS produced higher serum antibody titers than did priming with 3 doses of DNA on PLG for PkCSP, PkSSP2, and PkAMA1 antigens (p<0.05).</p

Panels A–D show the % parasitemia for individual monkeys by vaccine group according to the day after sporozoite challenge for Experiment #1.

<p>Data shows the first day parasites were detected and continues until the animal was drug treated when parasitemia exceeded 1% ( 3 animals inadvertently treated at lower parasitemias have open symbols). For comparison, in each panel the grey line shows the mean parasitemia for the Control group. In panel C, one animal never became parasitemic as indicated by x-x. Panel E shows the geometric mean parasitemias for vaccine groups for all days in which at least three animals had not been drug treated. (The monkey from the DNA Pk4×3/COPAK group which did not become infected was excluded from the average).</p

Immunization schedule for Experiment #2

<p>Five monkeys in each of five vaccine groups were immunized and challenged with Pk malaria sporozoites. The four antigen vaccine given Group 1 was identical to that given to Group 1 in Experiment 1. Group 2 received only the PkCSP components of the vaccine given to Group 1. Group 3 received a ten-fold larger dose of the PkCSP DNA than Group 2 and the same dose of PkCSP COPAK. Group 4 received only the PkCSP and PkSSP2 components of the vaccine given to Group 1. Group 5, the Control group, received a mock vaccine consisting of plasmid DNA without malaria antigen inserts followed by parental COPAK without malaria inserts. (See text for more details).</p

Panels A–C show the % parasitemias for individual monkeys by vaccine group according to the day after sporozoite challenge for Experiment #3.

<p>Data shows the first day parasites were detected and continues until the animal was treated with anti-malaria drugs. For comparison, in each panel the grey line shows the mean parasitemia for the Control group. Panel D shows the geometric mean parasitemias for all the vaccine groups for all days in which at least three animals had not been drug treated. For each day 8–11 the mean parasitemia for the group receiving the booster dose at the 21 week interval was lower than the other groups (p<0.05, Student's T test).</p

Summary of parasitemia data in Experiment #2

<p>‘Day 1^st parasite’ is the day after sporozite challenge when a parasite was first seen on malaria smear. ‘Day >2% parasitemia’ is the time of drug treatment. The group receiving the four antigen vaccine and the single antigen PkCSP vaccine had a delay in the appearance of parasites in the blood (<b>*</b>p = 0.03 vs Control group, Fisher's Exact Test). The group receiving high doses of PkCSP DNA priming was not as well protected. Co-immunization with PkSSP2 and PkCSP was worse than immunization with PkCSP alone (p = 0.03). Only the monkeys receiving all 4 antigens were protected against high parasitemias (<b>**</b>p<0.001 vs Control group).</p