The C18-4 cell line, a mouse spermatogonial stem cell line, was established by Dr. Martin Dym’s team at Georgetown University . In our tests, C18-4 cells were cultured in Germ Cell Media containing 0, 25, 50, 75, 100 mM Curcumin respectively. After treatment for 0 h, 24 h, 48 h, 72 h, the proliferation rate of each group was indirectly measured by MTS Assay. As shown in Figure 1, at successive time point, the relative cell number in the 25-mM Curcumin group was similar to that in the control group (0 mM Curcumin) (p.0.05). However, in the 50-mM Curcumin group, cell reduction appeared by 48 h and became apparent at 72 h (p,0.05); in the 75-mM and 100-mM groups, the negative effect by Curcumin was manifest since 48 h (p,0.05). Combining these series of results, we found Curcumin treatment led to a loss of cell viability in a dose-dependent manner. In that context, we applied a moderate treatment of 50 mM Curcumin in vitro in the following tests.Curcumin-treated groups (Figure 3. A). However, if we examined merely on the haploid spermatids, there was an obvious increasing of apoptosis in the treated ones (Figure 3. B). Using a 48 h assay, increased apoptosis was observed in the treated testicular cells (Figure 3. C). It seemed that, haploid spermatids were more susceptible to Curcumin treatment than other testicular cell types (Figure 3, Figure S2), implying the regulation of acetylation has much importance in spermatids.
The Acetylated Histone Level in Spermatids was Downregulated after Curcumin Treatment
In normal testis, there was a constitutive expression of AcH4 among Step 1? spermatids, followed by a peak of hyperacetylation in elongating spermatids. After 50 mM Curcumin treatment, we detected the expression of AcH4 in the FACS-sorted spermatids. The developmental steps of spermatids were classified according to the published standards, which based on the morphological features, especially the shape of acrosome . We counted at least 30 spermatids at each step. In a 3 h test, no changes were observed for the AcH4 pattern by immunofluorescence staining (data not shown). By contrast, in a 48 h test, the signal of AcH4 retained in Step 1? spermatids, but completely disappeared after Step 8, distinct from that in the control group (Figure 4.A, Table S3). This result was further confirmed by Western blot assay. As we expected, after 50 mM Curcumin treatment for 48 h, the signal of AcH4 was drastically weakened in the experimental group (Figure 4. B). However, there was also a noticeable decrease in the expression level of the internal control protein b-actin. One feasible explanation was that, we loaded protein samples from the same amount of cells onto the each lane of SDS-PAGE gel, but not at the consistent protein concentration. Therefore, a relative longterm Curcumin treatment might negatively affect the global protein levels, involving a rather complex mechanism. As a result, the abnormal acetylation level would lead to a failure of histone replacement and later nuclear condensation. Eventually, the spermatogenesis would be hampered.
Apoptosis was Induced in Primary Haploid Spermatids by Curcumin Treatment
The primary testicular cells were prepared and treated by 50 mM Curcumin for 3 h or 48 h. The apoptosis analysis was carried out immediately after the treatment. Otherwise, the haploid spermatids were sorted by FACS before the apoptosis analysis. In this study, the purity of haploid spermatids we obtained was stably .95%, certificating the reliability of the following tests (Figure 2). In a 3 h assay for total testicular cells, there was no significant difference between the control and the
Figure 2. One representative assay of haploid spermatids aggregating by FACS. (A). Hoechst33342 stained primary testicular cells were analyzed in SORP FACSAria II FACScan flow cytometer. P4 indicated the haploid cell population. (B). The gathered haploid spermatids were confirmed by DNA content analysis (lower) with the total testicular cells used as a control (upper).
Curcumin Influenced the mRNA Expression of Histone Acetylases and Deacetylases
After Curcumin treatment, we found an abrupt absence of AcH4 signal after Step 8. We assumed that, there was HAT specifically responsible for the hyperacetylation wave in Step 9?2 spermatids, becoming the direct or indirect target of Curcumin treatment. Aim to confirm the inferred HATs, we detected the mRNA levels of several critical enzymes in spermatids by quantitative real-time PCR (qPCR). The products of Cdyl, Cbp and Myst4 are HATs, while Hdac1 and Hdac4 encode HDACs belonging to different families. Among these genes, Cdyl [11,12] and Myst4  were considered testis-specific. Our results showed that, in a 3 h test, the mRNA expression of HAT genes Cdyl and Cbp seemed to decline in the treated group (p.0.05). In contrast to Cdyl and Cbp, the mRNA level of Hdac4 tended to go up (p.0.05), but actually raised for Hdac1 (p,0.05). For Myst4, there was no meaningful changes observed (p.0.05) (Figure 5. A). Surprisingly, in a 48 h test, all the detected genes were downregulated (p,0.05), except Hdac4 (Figure 5. B). Our results indicated, these detected genes were truly transcripted in spermatids, and responsed to the Curcumin treatment independently. Thus, the products of Cdyl, Cbp and Myst4 might be involved in the normal histone hyperacetylation process.
The Dynamics of Types of Chromatin Associated Factors were Disturbed by Curcumin Treatment
In that sense, we were going to examine the dynamics of vital CAFs after Curcumin treatment. By immunofluorescence tests, we found the basal transcription factors TBP and TAF1 disappeared earlier from the spermatids nuclei (Figure 6.A, Table 1), which was coordinated with the fact of disrupted transcription. Then we identified the same pattern to the transcription regulator AP2a, remodeling factor TOPOIIb, and to epigenetic markers H3K4Me3 and H4K20Me3, all of which were cleared before transcription silence (Figure 6.A, Table 1). These phenomena implied the crosstalk between different epigenetic modifications. At the same time, signals of GC sequence and TP2 remained in elongated spermatids, indiscriminating to those in normal spermiogenesis (figure not shown, see Table 1). Considering the anti-GC antibody recognized the euchromatin compartment, it was shown that, our results were not derived from a premature chromatin condensation, or the limitation of our protocols. For those spermatids in which histones had been replaced by transition proteins, the binding of TP2 seemed unaffected.