Reproductive and Developmental Medicine

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 2  |  Issue : 2  |  Page : 65--73

Gene Expression Pattern of Histone Acetylation Enzymes Changed in the Hypothalamus of Middle-Aged Female Rats: A Putative Mechanism for Female Reproductive Aging


Wen Xu1, Na Zhang2, Li-Sha Li3, Yan Wang3, Lin Wang2, Mei-Rong Du3, Da-Jin Li2, Yan Sun2,  
1 Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai 200011, China
2 Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College; Institute of Obstetrics and Gynecology, The Academy of Integrative Medicine of Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Disease, Shanghai 200011, China
3 Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Disease, Shanghai 200011, China

Correspondence Address:
Yan Sun
Hospital and Institute of Obstetrics and Gynecology, Fudan University, 419 Fangxie Road, Shanghai 200011
China

Abstract

Objective: Female reproductive aging is characterized by reduced responsiveness of the hypothalamus to E2-positive feedback, which can result in alterations of gene expression and luteinizing hormone (LH) surge dysfunction. We hypothesize that age-related changes in E2-responsive gene expression are due to altered histone acetylation by histone deacetylases (HDACs) or estrogen receptor-alpha (ERα) coactivators with histone acetyltransferase (HAT) activity. Methods: In the present study, young and middle-aged female rats were ovariectomized (OVX) and treated with E2 or oil once per day for 2 days. At the time of the expected LH surge, the anterior and posterior hypothalami were dissected, and gene expression of 11 HDACs and 4 ERα coactivators with HAT activity was measured using real-time polymerase chain reaction. Results: In the anterior hypothalamus, age affected the gene expression of 3 HDACs (Hdac3, Hdac5, and Hdac11) and 2 ERα coactivators (Src2 and Crebbp). E2 treatment significantly decreased mRNA levels of 4 HDACs (Hdac4, Hdac5, Hdac10, and Hdac11) and 2 ERα coactivators (Src2 and Crebbp) in young females (3–4 months). However, none of the genes responded to E2 in the middle-aged females (9–11 months), except Hdac10. In the posterior hypothalamus, age influenced Hdac5 and Src1 mRNA expression. E2 treatment increased Hdac4 and Crebbp mRNA levels in the young but not middle-aged females. Conclusions: These data suggest that E2 regulates HDACs and ERα coactivators with HAT activity in an age- and E2-dependent manner, which may contribute to the age-related gene expression changes on the day of LH surge in female reproductive aging.



How to cite this article:
Xu W, Zhang N, Li LS, Wang Y, Wang L, Du MR, Li DJ, Sun Y. Gene Expression Pattern of Histone Acetylation Enzymes Changed in the Hypothalamus of Middle-Aged Female Rats: A Putative Mechanism for Female Reproductive Aging.Reprod Dev Med 2018;2:65-73


How to cite this URL:
Xu W, Zhang N, Li LS, Wang Y, Wang L, Du MR, Li DJ, Sun Y. Gene Expression Pattern of Histone Acetylation Enzymes Changed in the Hypothalamus of Middle-Aged Female Rats: A Putative Mechanism for Female Reproductive Aging. Reprod Dev Med [serial online] 2018 [cited 2019 Dec 13 ];2:65-73
Available from: http://www.repdevmed.org/text.asp?2018/2/2/65/242756


Full Text



 Introduction



Reproductive aging inevitably brings about menopause that regularly occurs at approximately 51 years of age in women and signals the permanent end of fertility. There is no question that changes to the aging ovary, especially the exhaustion of ovarian follicles, are the hallmark of female reproductive aging.[1],[2] However, transplantation of ovaries of old animals to the kidney capsule of young, regularly cycling, but previously ovariectomized female hosts revealed that follicular development and ovulation occurred under the influence of the young neuroendocrine environment.[1],[2] These data are provocative and lend credence to the possibility that initial changes in neuroendocrine axis precede the ovarian reserve loss. Administration of drugs that restore hypothalamic neuropeptidergic activity or electrochemical stimulation of the preoptic area of the hypothalamus of old female rats significantly reinstated luteinizing hormone (LH) surges, estrous cyclicity, and ovulation,[3],[4],[5],[6] highlighted the potential impact of the hypothalamic alteration on female reproductive aging, and supported that manipulation of hypothalamic function would be beneficial to age-related reproductive decline.

A complete understanding of the mechanism underlying neuroendocrine regulation of female reproductive aging is yet to be achieved. Accumulating evidence from a few research groups and ours[7],[8],[9],[10],[11],[12],[13] indicated that the first observable neuroendocrine alteration of female reproductive aging is the delayed and attenuated LH surge under E2-positive feedback condition. A robust LH surge depends on E2 signaling on GnRH neuron excitatory and inhibitory afferent inputs within the hypothalamus, such as Kiss1, GABA, glutamate, and norepinephrine.[7],[8],[9],[10],[11],[12],[13] It is well known that E2 signals responsible for positive feedback events in the hypothalamus are mediated by the estrogen receptor-alpha (ERα).[14],[15],[16],[17] As female rodents reach middle age, there are no age-related changes in ovarian steroid exposure[18] or hypothalamic ERα expression,[19] yet multiple E2-responsive neurons within the hypothalamus become less sensitive to E2 signaling. This leads to changes in gene expression or the release of neurotransmitters that manifest as a delayed and attenuated LH surge.[11],[12],[20],[21],[22] The specific mechanisms underlying reduced responsiveness of the hypothalamus to E2-positive feedback and the associated alterations of gene expression in female reproductive aging rodents remain largely unknown.

Emerging evidence suggests that epigenetic modification, such as histone acetylation, is tightly linked to chromatin organization[23],[24],[25] and ER-mediated gene expression under E2-positive feedback.[26] For example, E2 enhances the formation of a chromatin loop between the promoter and the 3' intergenic region at the Kiss1 locus in the anteroventral periventricular nucleus (AVPV).[26] Histone H3K9/14 acetylation and ERα binding in the AVPV Kiss1 promoter region were induced by E2 and were positively associated with an increase in Kiss1 gene expression in the nucleus,[26] suggesting that histone H3K9/14 acetylation plays a critical role in inducing AVPV Kiss1 gene expression in mice under E2-positive feedback.[26] Typically, histone acetylation status is regulated by a variety of histone deacetylases (HDACs) and histone acetyltransferases (HATs).[27] HDAC2 and HDAC3 were recently shown to be involved in E2-enhanced memory consolidation in the hippocampus through increasing histone H3 acetylation at the Bdnf promoter pII and pIV[28] Specifically, the nuclear receptor coactivators,[29] steroid receptor coactivator-1 (SRC1), SRC2,[30] and the integrator complex p300/CREB-binding protein (CBP)[31] were found to affect ER-mediated transcription as HATs. SRC1 and CBP can also regulate E2-dependent female sex behavior through regulating progestin receptor expression in the hypothalamus.[32] The p300/CBP regulates histone acetylation in the promoters of the LH-induced target genes in mouse granulosa cells undergoing luteinization, following the ovulatory LH surge.[33] These findings support the notion that histone acetylation enzymes are essential for E2-mediated events by modulating histone acetylation and E2 target gene expression; thus, HDAC and HAT expression in the hypothalamus may respond to E2-positive feedback and be involved in E2-induced gene expression on the day of the LH surge.

It is well known that aging impairs ability of E2 to induce gene expression in the hypothalamus at the LH surge.[20],[21],[34] The effects of age on epigenetic modulations of E2 in the hypothalamus, which potently affect chromatin structure and gene expression, have not been investigated. We hypothesize that HDACs and ERα coactivators with HAT activity are involved in the effects of E2-positive feedback on the hypothalamus. On the other hand, age-related alterations of HDACs or ERα coactivators with HAT activity in the hypothalamus may be correlated with reduced responsiveness of E2-positive feedback and altered gene expression in female reproductive aging. The current study determined whether expression of genes encoding 11 HDACs and ERα coactivators with HAT activity in the anterior and posterior hypothalamus is regulated by E2 in young and middle-aged female rats, under E2-positive feedback. We also examined whether there are age-related alterations in the expression of these genes in the anterior and posterior hypothalamus.

 Methods



Animal care

All animal procedures were approved by the Institutional Animal Care and Use Committee at Fudan University. Young (3–4 months) and middle-aged (9–12 months, retired breeders) female Sprague Dawley rats (Charles River, Beijing, China) were housed in groups and maintained on a 12-h light, 12-h dark cycle (lights on at 0700 h) with free access to chow and water. Rats were handled for 5 min/d for 1 week before monitoring their estrous cyclicity by daily vaginal lavage of sterile saline for 2 weeks. Only rats with at least 2 regular 4–5-day estrous cycles were included in the studies.[22],[35],[36],[37]

Hypothalamic dissection, RNA purification, reverse transcription, and real-time polymerase chain reaction

Twelve young and twelve middle-aged rats were anesthetized with pentobarbital sodium (30 mg/kg, i.p.) and ovariectomized (OVX). At 0900 h on day 7 after OVX, E2-positive feedback conditions were created by giving rats the first of two daily sc injections of 2 μg of E2 benzoate (Hangxiang Inc., Wuhan, China). On the day of the anticipated LH surge, young rats were euthanized by rapid decapitation for gene expression experiments between 1300 h and 1400 h, while middle-aged rats were euthanized between 1500 h and 1600 h.[22],[35],[38] As previously described,[22],[35],[38] the anterior hypothalamus, which includes the preoptic area (POA), and the posterior hypothalamus, which includes the arcuate and ventromedial nucleus of the hypothalamus (VMH), were flash frozen on dry ice and stored at −80°C until quantification of mRNAs.

DNA-free total RNA was purified using the RNeasy lipid minikit (QIAGEN, Valencia, CA, USA), including a deoxyribonuclease step. Reverse transcription (RT)[16] was performed using the high-capacity cDNA RT kit with ribonuclease inhibitor (Applied Biosystems, Foster City, CA, USA). We used conventional gene-by-gene polymerase chain reaction (PCR) for 15 genes of interest in the anterior and posterior hypothalamus: Hdac1, Hdac2, Hdac3, Hdac4, Hdac5, Hdac6, Hdac7, Hdac8, Hdac9, Hdac10, Hdac11, Src1, Src2, Crebbp, p300, and Gapdh [Table 1]. Gene expression was measured by real-time PCR using Lightcycler Roche 480 Absolute Blue quantitative PCR (qPCR) SYBR green master mix (Roche, Indianapolis, IN, USA) and the Applied Biosystems 7700 real-time PCR cycler, according to the manufacturer's instructions. Amplified transcripts were quantified using the comparative threshold cycle method and GAPDH as normalizer. The samples were prepared in duplicate, and the mean cycle threshold (CT) values were used to calculate fold change using ΔΔCT method.[22],[35],[38]{Table 1}

Data analysis

GraphPad Prism 6 software (GraphPad Prism Software Inc., San Diego, CA, USA) was used for statistical analysis. Data are expressed as mean ± standard error of the mean. Two-way ANOVA (age × treatment) was utilized to detect differences in gene expression in the anterior and posterior hypothalamus in young and middle-aged groups. Bonferroni or Turkey's post hoc tests were performed to determine individual group differences following main or interaction ANOVA effects. P < 0.05 was considered statistically significant.

 Results



Effects of E2 and age on histone deacetylase gene expression in the anterior hypothalamus of young and middle-aged rats

Our first objective was to examine whether HDAC gene expression in the hypothalamus was altered by reproductive age and/or E2 bioavailability. Effects of age and hormone treatment on the expression of 11 HDACs (Hdac1–11), in the anterior hypothalamus of young and middle-aged OVX steroid-primed females, were examined using qPCR. All HDAC transcripts, Hdac1–11, were detected in the hypothalamus with differing levels of expression. A significant effect of reproductive age was found for Hdac3 (F = 8.6, P < 0.05), Hdac5 (F = 6.7, P < 0.05), and Hdac11 (F = 10.4, P < 0.01). The mRNA level of Hdac3 was higher, but Hdac5 was lower in OVX middle-aged than young females [Figure 1]a and [Figure 1]c. The Hdac11 mRNA level was higher in E2-treated middle-aged females than young females [Figure 1]e. A significant interaction between reproductive age and E2 was also found for Hdac5 (F = 4.4, P < 0.05) and Hdac11 (F = 7.4, P < 0.05). Although Hdac5 and Hdac11 expressions decreased in young rats following E2 treatment, these effects were absent in middle-aged rats [Figure 1]c and [Figure 1]e. Regardless of age, there was a main effect of E2 on Hdac10 (F = 5.5, P < 0.05) expression. There was an interaction between age and E2 on expression of Hdac4 (F = 7.4, P < 0.05), with lower mRNA levels in E2 than vehicle-treated controls in young females; however, this effect was absent in middle-aged rats [Figure 1]b and [Figure 1]d. Results for the residual genes were not significantly affected in the anterior hypothalamus and are thus not shown.{Figure 1}

E2-regulated expression of genes encoding estrogen receptor-alpha coactivators with histone acetyltransferase activity in the anterior hypothalamus is absent in middle-aged female rats

Cofactor regulation of ER-mediated transcription by modifying chromatin structure through covalent histone modification is an essential estrogen action.[30],[39] We discovered a change in ERα coactivators with HAT activity in the anterior hypothalamus under E2-positive feedback. Gene expression levels of ERα transcriptional coregulators, which are potently able to bring about histone acetylation and ERα-mediated gene expression, were detected. In the anterior hypothalamus, the two Src isoforms, Src1 and Src2, displayed somewhat different patterns of expression. A significant effect of age was found for Src2 (F = 8.6, P < 0.01), with expression lower in middle-aged than young females [Figure 2]a, while Src1 was not affected (data not shown). In addition, there was an interaction between age and E2 on expression of Src2 in the anterior hypothalamus (F = 9.5, P < 0.01). E2 decreased Src2 expression in young but not middle-aged rats.{Figure 2}

The other two homologous ERα cofactors with HAT activity, Crebbp and p300, also showed differential patterns of induction. There was a strong effect of reproductive age on Crebbp (F = 4.7, P < 0.05) but not p300 (data not shown) expression, with a lower mRNA level in middle-aged than young females [Figure 2]b. Crebbp also had a significant effect on hormones (F = 5.5, P < 0.05), with expression reduced in E2 treatment than vehicle controls [P < 0.01, [Figure 2]b]. In addition, there was an interaction between age and E2 (F = 5.4, P < 0.05) on expression of Crebbp in the anterior hypothalamus. Although Crebbp expression decreased in young rats following E2 treatment, this effect was absent in middle-aged rats [Figure 2]b.

Effects of E2 and age on histone deacetylases and estrogen receptor-alpha coactivators with histone acetyltransferase activity expression in the posterior hypothalamus in young and middle-aged rats

To determine whether age or E2 regulates HDACs or ERα coactivators with HAT activity in the posterior hypothalamus, we measured 11 HDACs (Hdac1–11) and 4 ERα coactivators with HAT activity mRNA levels in the posterior hypothalamus. Interestingly, fewer genes were altered by E2 in the posterior compared with the anterior hypothalamus. Regardless of hormone treatment, expression of Hdac5 [F = 5.0, P < 0.05, [Figure 3]b] was lower in middle-aged compared to young animals. There was an interaction between age and E2 on Hdac4 expression [F = 5.4, P < 0.05, [Figure 3]a]. Hdac4 with significant interactions shows upregulation by E2 in young but not in middle-aged females. The Hdac4 mRNA level was higher in E2-treated young than middle-aged females.{Figure 3}

Regardless of hormone treatment, expression of Src1 in the posterior hypothalamus [F = 5.5, P < 0.05, [Figure 4]a] was higher in OVX middle-aged compared to young rats. There was an interaction between age and E2 on Crebbp expression [F = 6.3, P < 0.05, [Figure 4]b]. Crebbp with significant interactions showed upregulation by E2 in young females. The Crebbp mRNA level was lower in OVX young than middle-aged females. Results for residual HDACs and ERα coactivator genes were not significantly affected in the posterior hypothalamus and are thus not provided.{Figure 4}

 Discussion



This study presents novel evidence that exposure to E2-positive feedback induces distinct alterations of histone acetylation enzymes and gene expression in the hypothalamus of young female rats. To our knowledge, this is the first evidence demonstrating that histone acetylation enzymes in the hypothalamus may involve in E2-positive feedback action on the day of LH surge. This is consistent with a recent report indicating that E2 administration led to significant changes in the mRNA expression of several ERα coregulators in the preoptic area of female mice.[39] A previous investigation reported that granulosa cells undergoing luteinization after ovulation induction are coupled with gene expression changes within histone modification enzymes.[33] Our current results support the insights that gene expression of specific HDACs and ERα coactivators with HAT activity is regulated by E2 in the hypothalamus and possibly associated with ERα target gene expression on the day of LH surge. Moreover, our data suggest that histone acetylation enzymes have vastly different expression patterns under E2-positive feedback conditions in an aged hypothalamus. This is supported by the markedly delayed and attenuated E2-dependent LH surges seen in the middle-aged rats, whom have lost sensitivity to the epigenetic response to E2 in their hypothalamus. These data, therefore, strongly suggest that the missed epigenetic response to E2 in the hypothalamus of middle-aged females is an important mechanistic pathway, resulting in insensitivity in E2 target genes and consequently an impaired ERα-mediated gene expression on the day of LH surge.

E2 regulates histone deacetylase expression in the anterior hypothalamus in young females

Recruitment of HDACs to ERα-target genes mechanistically alters the epigenetic equilibrium existing between histone acetylation and deacetylation in breast cancer.[40] Of interest, significant enhanced object recognition memory by E2 is associated with reducing HDAC2 protein levels in the dorsal hippocampus.[28],[41],[42] We therefore hypothesized that HDACs in the hypothalamus would be regulated by E2 in young females. We observed distinct alterations of Hdac4, Hdac5, Hdac10, and Hdac11 expression in the anterior hypothalamus following E2 treatment in young females, with reduced mRNA levels under E2-positive feedback. These data underscore the concept that specific HDACs in the anterior hypothalamus may play critical roles in regulating ERα-mediated gene expression under the E2-positive feedback condition, which may be important for the subsequent GnRH-LH surge. Histone markers, including H3K56ac and H3K14ac, help to control the expression of genes by regulating genome stability.[43] Thus, an important area of future study will be to determine whether the changes in mRNA expression of HDACs are related to histone marker modification and ERα target gene expression. Of note, HDAC mRNAs are extensively distributed throughout the brain.[44] Future studies are needed, focusing on more restricted regions of interest and testing the HDAC protein expression pattern.

E2 modulates ERα coactivators with histone acetyltransferase activity expression in the anterior hypothalamus in young females

Nuclear receptor coactivators with HAT activity are often rate limiting for steroid receptor activation and act as bridging proteins between the receptor and the basal transcriptional machinery.[45] Recent studies found that SRC1, SRC2, and CBP expressed in E2-sensitive regions including the medial preoptic area (MPOA) in the hypothalamus,[46] E2 exposure significantly upregulates mRNA levels of Src1 and Src2 in the VMH of female mice.[39] These observations led us to hypothesize that ERα coactivators with HAT activity in the anterior hypothalamus may be involved in ERα-mediated induction of genes and the E2-dependent LH surge. A surprising find from our present study was the observation that E2 treatment significantly decreased mRNA levels of Src2 and Crebbp in the anterior hypothalamus of young females on the day of LH surge. However, we did not detect a change in Src1 or P300 mRNA expression. Presumably, reduced Src2 and Crebbp gene expression in the anterior hypothalamus under E2-positive feedback is likely to limit inhibitory neurons, correlating with a decreased histone acetylation and gene repression of inhibitory neurotransmitters. SRC2 and ERα were coexpressed in many cells in the MPOA, VMN, and arcuate nucleus (ARC);[47] CBP is coexpressed with progesterone receptors in PR cells in behaviorally relevant brain regions.[46] The identity of SRC2 and CBP cells in the anterior hypothalamus, such as MPOA, will be an important avenue for future investigation.

Middle-aged females are insensitive to epigenetic modifications of E2 in the anterior hypothalamus

Female reproductive aging is characterized by attenuated E2 responsiveness of multiple E2-response neurons within the hypothalamus. In contrast to the young rats, our data showed that gene expression of the four HDACs and nuclear receptor coactivators with HAT activity does not change with E2 in the anterior hypothalamus of middle-aged females. These results demonstrated that middle-aged females are insensitive to the epigenetic modifications of E2 in the anterior hypothalamus. Interestingly, the patterning of histone marks in the genome changes during aging.[48] Histone acetylation depends on a dynamic balance between HDACs and HATs.[27] The absent response of HDACs and the nuclear receptor coactivators with HAT activity to E2 in the middle-aged females may support a missed histone marker modification in the anterior hypothalamus, which probably reflects reduced responsiveness of the hypothalamus to E2 in middle-aged females[13] and age-related alterations of ERα-targeted gene expression.[38],[49] Our previous work identified that E2 induces less Kiss1 mRNA expression in the anterior hypothalamus in middle-aged rats; E2 induces histone acetylation in the Kiss1 gene promoter region in the AVPV under E2-positive feedback.[26] Future studies will need to address the potential age-associated histone acetylation failure in the Kiss1 gene in the AVPV under E2-positive feedback. In addition, the present study found that reproductive age increased expression of Hdac3 but decreased Hdac5, Src1, and Crebbp expression in the anterior hypothalamus in OVX middle-aged rats. However, the physiological significance of these age-related alterations in the anterior hypothalamus is currently unknown.

Differential effects of E2 on histone acetylation enzymes in the posterior hypothalamus in young and middle-aged females

Another important observation from the current study was the revelation that in contrast to reduced expression of Hdac4 and Crebbp in the anterior hypothalamus, E2 increased Hdac4 and Crebbp expression in the posterior hypothalamus in young females. It is possible that upregulation of Hdac4 and Crebbp mRNA in young rats reflects HDAC4 and CBP modulation of excitatory neurotransmitter synthesis in the posterior hypothalamus under E2-positive feedback. In particular, our data demonstrated that there is no change in Hdac4 mRNA under E2-positive feedback in middle-aged females and less Hdac4 mRNA compared with young females, supporting a loss of E2 responsiveness with aging, presented in the posterior hypothalamus as well as the anterior. However, the current data cannot emphasize the significance of increased Crebbp in the posterior hypothalamus of OVX middle-aged rats.

Hypothalamic Hdac5 expression is regulated by nutrient availability and leptin action, implying that HDAC5 is a novel regulator of metabolic homeostasis and an important component of leptin signaling.[50] A high-fat diet resulted in increased expression of HDAC-5 and HDAC-8 in the ventrolateral subdivision of the ventromedial hypothalamus, supporting the concept that HDAC5 may be involved in altering the gene expression profiles in the medial hypothalamus under different metabolic states.[51] Our work demonstrated an age-dependent reduced Hdac5 and increased Src1 in the posterior hypothalamus of reproductive aging females. However, whether the age-related Hdac5 mRNA upregulation in the posterior hypothalamus of middle-aged females is associated with anorexigenic or orexigenic gene expression is currently unknown. Although Src1 expression in the VMH is correlated with E2-regulated sexual behavior in young female rats,[39] the profound increased Src1 expression in the posterior hypothalamus of OVX middle-aged females is necessary for further validation.

In conclusions, our findings provide novel evidence that gene expression of histone acetylation enzymes changes in the hypothalamus of young females on the day of LH surge, under E2-positive feedback conditions. The aged hypothalamus loses its responsiveness to E2. However, there is little evidence that levels of ERα in hypothalamic nuclei differ between young and middle-aged female rats;[19] therefore, it is plausible that the functionality of ERα is influenced by epigenetic modification. Our findings provide key insight into the underlying epigenetic mechanism accountable for the loss of responsiveness to E2-positive feedback regulation of the LH surge, in reproductive aging females.

Financial support and sponsorship

This work was funded by Shanghai Science and Technology Committee (17ZR1403300).

Conflicts of interest

There are no conflicts of interest.

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