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

Abstract

Estrogen receptors (ESR1 and ESR2) play crucial roles in various processes during spermatogenesis. To elucidate individual roles of ESRs in male fertility, we developed in vivo 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.

Keywords: chromatin remodeling; estrogen receptor; histone modifications; spermatogenesis; spermatozoa.

Figure 1.

Changes in histone modifications in testis. (A) Testicular levels of H3 modifications after PPT and DPN treatment, graphical representation (left panel) and representative Western blot (right panel). Bands representing H3 modifications were observed at 15 kDa and normalized with pan H3 levels. (B) Testicular levels of hyperacetylated H4 graphical representation (left panel) and representative Western blot (right panel). Hyperacetylated H4 bands were observed at 10 kDa and normalised with pan H4 levels. The levels of pan H3 or H4 were unchanged with respect to GAPDH. All values are mean ± SEM. * P value < 0.05; ** P value < 0.01. n = 6.

Figure 2.

Effect of PPT treatment on transcript levels of histone modifying enzymes in the testis. Changes in histone modifiers for (A) H3K4, (B) H3K9, (C) H3K27, and (D) genes involved histone acetylation. All values are mean ± SEM. * P value < 0.05; ** P value < 0.01; *** P value < 0.001. n = 6.

Figure 3.

PPT treatment causes histone retention and Prm1 deficiency in caudal spermatozoa. (A) H3, H4, and H2B histone levels in caudal spermatozoa (left panel) and representative Western blots (right panel). n = 4. (B) Immunofluorescence staining for Prm1 (red) in control (a and d), PPT (b and e), and DPN (c and f). DAPI (blue) used as nuclear stain and graphical representation of mean fluorescence intensity. Arrowheads indicate Prm1-deficient spermatozoa. Inset in (d) indicates negative control and bar in (a) represents 10 μm. (C) Representative flow cytometry histogram for Prm1 staining (right panel) in caudal spermatozoa after control, PPT, and DPN treatments and graphical representation of mean fluorescence intensity (left panel). All values are mean ± SEM. * P value < 0.05; ** P value < 0.01. n = 6.

Figure 4.

Effect of PPT treatment on chromatin remodeling proteins. (A) Transcript levels and (B) protein levels (right panel is a representative Western blot). All values are mean ± SEM. * P value < 0.05. n = 6.

Figure 5.

Changes in histone modifications in spermatozoa after PPT and DPN treatment. Graphical representation (left panel) and representative Western blot (right panel). All bands representing histone modifications were normalized with respect to GAPDH. All values are mean ± SEM. ‘a’ represents P value < 0.05; ‘b’ represents P value < 0.01; ‘c’ represents P value < 0.001. n = 4.

Figure 6.

Proposed mechanism for regulation of epigenetic processes by ESRs during spermatogenesis. Activation of estrogen signaling through ESR1 deregulates several histone modifying enzymes leading to decrease in the activating (H3K4me2/3 and H3ac) and heterochromatin (H3K9me2/3) marks and increase in the repressing marks H3K27me3 and hyperacetylated H4 levels in the testes. The chromatin remodeling proteins Smarce1, Hspa2, Parp1, and Hsp90α were also affected in the testis, leading to increased histone retention and protamine deficiency in spermatozoa. The increased histone retention and aberrant histone modification marks in spermatozoa could contribute to the decreased fertility observed after ESR1 agonist treatment. On the other hand, activation of estrogen signaling through ESR2 decreases the levels of DNA methyltransferases (Dnmts) in the testes, leading to decrease in global and H19 locus methylation. This, along with aberrant histone modification marks in spermatozoa could lead to the decrease in fertility observed after ESR2 agonist treatment. Thus, while ESR1 regulates histone modifications and its machinery, ESR2 regulates DNA methylation during spermatogenesis.