Developmental Regulation of Histone H3 Methylation at Lysine 4 in the Porcine Ovary

REPRODUCTIONRESEARCH Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary Marcelo M Seneda2, Maren Godmann, Bruce D Murphy1, Sarah Kimmins and Vilceu Bordignon Department of Animal Science, McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Que´bec, H9X 3V9 Canada, 1Centre de Recherche en Reproduction Animale (CRRA), Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montreál, Saint-Hyacinthe, Que´bec, J2S 7C6 Canada and 2Departamento de Clıńicas Veterina´rias, Universidade Estadual de Londrina, Londrina, Parana´, 86051–990 Brasil Correspondence should be addressed to S Kimmins; Email: [email protected] V Bordignon; Email: [email protected] Abstract Follicular growth and oogenesis involve highly dynamic changes in morphogenesis, chromatin structure, and gene transcription. The tight coordination of these events leads to ovulation of a mature oocyte and formation of the luteal tissue necessary to regulate embryo implantation and development. This entire process is regulated by numerous endocrine and in situ mechanisms. The role of epigenetic mechanisms in folliculogenesis, such as the biochemical modification of the DNA packaging proteins, the histones, is not well understood. Our objective was to determine the cellular and follicular stage-specific patterns of histone H3 methylation at lysine 4 (K4) in porcine preovulatory follicles and during luteinization in pig ovaries. Ovary tissues were collected from slaughtered prepubertal and cyclic gilts at various stages of the estrous cycle, pregnancy, and from ovaries recovered from gonatropin-treated gilts at 0, 24, and 38 h post human chorionic gonadotropin (hCG) injection. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin and sectioned using standard histological protocols. Immunofluorescent staining was performed on 3 mm thick sections. The immunostaining pattern of mono-, di-, and tri-methylated histone H3-K4 and lysine-specific demethylase 1 (LSD1, also known as KDM1 or AOF1) was assessed. Interestingly, H3-K4 mono-, di-, and tri-methylation in follicles of prepubertal gilts was specifically distributed and developmentally regulated. While granulosa cells of primary, secondary, and early antral follicles were negative for H3-K4 methylation those from large antral follicles showed a striking upregulation in the cells located in the proximity to the oocyte. Specifically, the cumulus oophorus displayed intense staining for H3-K4 methylation and signals were strongest in the granulosa cells in the inner two cell layers of the follicular wall. Although all oocytes from primary to large antral stage follicles were positive for H3-K4 mono-, di-, and tri-methylation, the patterns of distribution were altered through oocyte follicle development. H3-K4 methylation in granulosa cells was dramatically reduced as time to ovulation approached and was low to undetected at 38 h post hCG treatment. H3-K4 mono-, di-, and tri-methylation in large luteal cells increased as differentiation evolved but remained low in small luteal cells. Strikingly, LSD1 (KDM1) expression was found to be restricted to the corpus luteum. In summary, this study provides new information on histone H3-K4 methylation patterns in the oocyte and follicle during folliculogenesis, which suggests that these epigenetic markers serve an essential regulatory role during folliculogenesis. Reproduction (2008) 135 829–838 Introduction Pepin et al. 2007). Despite the fact that in vivo and in vitro experiments and transgenic mouse models have The ovary is responsible for the generation of oocytes revealed key mechanisms that regulate folliculogenesis capable of undergoing fertilization and for the pro- (Pangas & Matzuk 2004, Skinner 2005), our under- duction of steroid hormones required to prepare for standing of the involvement of epigenetic processes is fertilization and pregnancy. The follicle is the primary just emerging (LaVoie 2005). Follicular development in structure of the ovary and is comprised of an oocyte mammalian species is driven by endocrine signals, encompassed by one or more layers of somatic cells. including estrogens and the gonadotropin and intra- Recent advances in the study of folliculogenesis in ovarian regulators such as growth and differentiation mammals demonstrate a tightly regulated program of factor 9 (GDF9; Su et al. 2004, Knight & Glister 2006). genomic, epigenetic, and hormonal events that direct Progression from the primordial to preantral stage is, in survival, proliferation, and differentiation of somatic part, regulated by oocyte-derived factors that include cells (DeManno et al. 1999, Ruiz-Cortes et al. 2005, GDF9 and bone morphogenic protein (BMP15), and is q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0448 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 03:15:26AM via free access 830 M M Seneda and others not dependent on gonadotropin support (Matzuk et al. referred to as germinal vesicle 0 (GV0) and consists of a 2002). As the follicle develops to the early antral stage, it nucleolus with diffuse filamentous chromatin distributed becomes responsive to gonadotropin and its develop- over the nuclear area, which is seen in most of the ment is also influenced by intra-ovarian regulators (Craig oocytes present in follicles smaller than 1 mm (Sun et al. et al. 2007). Signaling between the oocyte and granulosa 2004). With the progression of growth and differen- cells has emerged as a key regulator of the primary- tiation, oocytes undergo a dramatic change in nuclear to-secondary-to-antral follicle transition, and members organization in which chromatin becomes progressively of the transforming growth factor-b (TGF-b) family are condensed, forming a heterochromatin rim surrounding required for these transitions (Pangas & Matzuk 2004). the nucleolus, a configuration termed surrounded Estrogen and follicle-stimulating hormone (FSH) are nucleolus (SN; Debey et al. 1993), or GV1–4 (depending critical to the formation of antral follicles (Britt et al. on the condensation state of the chromatin) in the pig 2002, 2004),whichfailtoforminFSHreceptor (Sun et al. 2004). Accompanying these alterations in knockout mice (Dierich et al. 1998). chromatin structure are changes in transcription levels; Eukaryotic DNA is packaged into chromatin formed by oocytes showing the NSN configuration undergo high nucleosomes composed of a histone octamer (H2A, H2B, rates of transcription whereas those in the SN state H3, and H4) wrapped by 147 bps of DNA. Epigenetic display only a low level (Bouniol-Baly et al. 1999, mechanisms regulate chromatin higher order and the Maddox-Hyttel et al. 2005, De La Fuente 2006). access of gene regulatory factors to the DNA (Lietal. 2007). In the porcine ovary following the preovulatory LH DNA methylation and histone modifications are important surge, the luteinization process begins with hyperanemia epigenetic mechanisms regulating gene expression, and enlargement, followed by expulsion of the ovum development, and are deregulated in some cancers (Murphy et al.2001). Following ovulation, under the (reviewed in Goldberg et al. 2007). Post-translational influence of the gonadotropins, prolactin and insulin, modification of the amino terminus of histones acts to granulosa and thecal cells differentiate. This process is control access of the transcriptional machinery to the DNA accompanied by alterations in steroidogenesis whereby through recruitment of effector molecules, or can trigger the thecal and granulosa cell participate in the synthesis gene silencing through recruitment of transcriptional and secretion of progesterone (Murphy et al. 2001). At this repressors (Kouzarides 2007). Histone modifications point, the roles of histone modification in the control of associated with gene activation include histone H4 gene transcription are just beginning to be elucidated. For acetylation and histone H3 methylation at lysine 4 example, acetylation of histone H3 at lysine 9 has been (H3-K4; Barski et al. 2007, Kouzarides 2007). Genome revealed to increase transcription of Niemann-Pick C1 in wide studies in eukaryotes localize H3-K4 methylation to porcine granulosa cells (Gevry et al.2003)andpro- gene regulatory regions (Santos-Rosa et al. 2002, Ng et al. gesterone synthesis capacity is facilitated by upregulation 2003, Sims et al.2003). More recently, in high-resolution of cholesterol synthesis for which STAR is a key gene profiling of the human genome, H3-K4 methylation was whose expression appears to be regulated in part by correlated with gene activation (Barski et al. 2007). epigenetic mechanisms (Lazzaro et al. 2006). During Prior to birth in the mammalian ovary, oocytes are luteinization, granulosa cells undergo acetylation of arrested at prophase I of meiosis. Once the ovarian histone H3 and consequent transcriptional activation of follicle undergoes activation, oocytes are maintained the STAR gene (Christenson et al. 2001). Given the under meiotic arrest at the diplotene stage during importance of epigenetic modifications in the control of postnatal development. At puberty and from each gene expression and chromatin organization, the present estrous cycle on, the gonadotropin surge of luteinizing study was undertaken to determine the cell and develop- hormone (LH) promotes meiotic resumption (Eppig mental-specific patterns of histone H3-K4 mono (m1)-, di 2001). Epigenetic imprints in the form of DNA (m2)-,

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