Endocrine Journal 2011, 58 Or i g i n a l Advance Publication doi: 10.1507/endocrj. EJ11-0098

Gonadotropins up-regulate the expression of 2, but not enolase 1, in the rat ovary

Nobuhito Yoshioka1), Noriyuki Takahashi1), Wataru Tarumi1), Masanori T. Itoh2) and Bunpei Ishizuka1)

1)Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan 2)Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa 272-0827, Japan

Abstract. It has been demonstrated that the glycolytic , enolase 1 (ENO1) and (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.

Key words: Enolase, Glycolysis, Gonadotropin, Ovary

Enolase (ENO; 2-phospho-D-glycerate hydro- [2-5]. Recently, Sundblad et al. [5] identified ENO1 lyase; EC 4.2.1.11) is a metalloenzyme that catalyzes as an antigen of antiovarian antibodies. Antibodies the dehydration of 2-phospho-D-glycerate to phospho- directed against ENO1 were found in 19.1% of sera enolpyruvate, the penultimate step of glycolysis [1]. obtained from 110 patients with POF [5]. It has been ENO can also catalyze the reverse reaction, depend- also reported that glycolysis is a major contributor to ing on environmental concentrations of substrates [1]. energy production in ovarian follicles [6-8] and glyco- In vertebrates, there are three subunits of ENO, α, β, lysis in ovarian follicles is required for follicle growth or γ, which can combine to form five different isoen- and estradiol secretion [9]. zymes: αα, αβ, αγ, ββ, and γγ [1]. Three of these isoen- ENO1 mRNA is detected in granulosa cells and theca zymes are commonly found in mammalian cells [1]: cells of preantral and antral follicles in the rat ovary ENO1, the homodimer of α subunits, is found in most [10]. ENO1 protein is also detected in the human ovary tissues, ENO2, the homodimer of γ subunits, is found [5, 11]. On the other hand, ENO2 mRNA is known to in neuron and neuroendocrine tissues, and ENO3, the be expressed in ovaries of neonatal rats [12]. The lev- homodimer of β subunits, is almost exclusively found els of ovarian ENO2 mRNA increases at the primor- in muscle tissues. dial to primary follicle transition [12]. However, the Premature ovarian failure (POF), a syndrome clin- expression of ENO2 and the cellular localization of ically defined by failure of the ovary before the age ENO2 are unknown in the adult ovary. of 40, is frequently associated with autoimmunity In the present study, we examined the expression and cellular localization of ENO2 in the ovary and com- Submitted Jun. 28, 2011; Accepted Aug. 10, 2011 as EJ11-0098 pared with those of ENO1. We found that the levels of Released online in J-STAGE as advance publication Aug. 20, 2011 ovarian ENO2 mRNA, but not ENO1, changed signifi- Correspondence to: Noriyuki Takahashi, Ph.D., Department of Obstetrics and Gynecology, St. Marianna University School of cantly during the estrus cycle. It is known that expo- Medicine, Kawasaki, Kanagawa 216-8511, Japan sure to human CG (hCG) stimulates glycolytic activity E-mail: [email protected] in ovarian follicles [8, 9]. These suggest that gonad-

©The Japan Endocrine Society

Endocrine Journal Advance Publication 2 Yoshioka et al. otropins may affect ovarian ENO2 expression. Thus, formed on each with ovarian RNA without reverse the effects of gonadotropins on ovarian ENO2 expres- transcription. The PCR products and 100-bp DNA lad- sion were examined. der were electrophoresed in 2% agarose gels contain- ing ethidium bromide and detected by UV irradia- Materials and Methods tion. Intensities of PCR products were quantified using Image J 1.40f program (NIH, http//rsb.Info.nih.gov/ij/). Chemicals The products were subcloned into pT7 blue-T vector All chemicals used in experiments were purchased (Novagen, Madison, WI, USA) for sequencing analy- from Sigma (St Louis, MO, USA) or Wako Pure Chemical sis. Data are expressed as mean ± SEM. Differences Industries (Tokyo, Japan), unless otherwise noted. between the groups were analyzed by a nonparametric analysis of variance (Kruskal-Wallis-H test) followed Animals by Dunn’s post-hoc comparison. Adult (10 to 12 week old) and immature (3 week old) female Wistar-Imamichi rats were used. The In situ hybridization stages of the estrous cycle in adult rats were deter- Ovarian sections were soaked in 0.2M trietha- mined by vaginal cytology. Ovaries and brains were nolamine with gradually added acetic anhydride (final excised immediately after decapitation of the animals 0.5%) for . Then the sections were prehy- (n = 5 per each stage of estrous cycle). In an experi- bridized with 50% formaldehyde in SSC (150 mM ment, oocytes, granulosa cells, and theca and intersti- sodium chloride and 15 mM sodium citrate, pH 7.0) at tial cells were mechanically separated from ovaries of 45C for 30 min. PCR product generated by RT-PCR a 12-week old female rat. Granulosa cells were further was used as a template DNA for further amplifica- purified by density-gradient centrifugation with Ficoll- tion of the region containing site-directed promoter Paque PLUS (GE Healthcare Japan, Tokyo, Japan). sequences in 5’- and 3’-termini. The primer sequences The brain was used as a positive control. ENO2 is are shown in Table 2. Fluorescein-labeled cRNA probes known to be expressed in the neurons [13]. To investi- were obtained using a DIG Northern Starter Kit and gate the effects of gonadotropins on ovarian expression Fluorescein RNA Labeling Mix (Roche, Mannheim, of ENOs, immature rats were subcutaneously injected Germany). Sections were hybridized overnight at 45C with saline, 10 IU eCG or 10 IU hCG (n = 5 per group). with RNA probes dissolved in hybridization solution At 6h after the injection, tissues were dissected and [1× Denhardt’s solution, 10% dextran sulfate, 50% either processed by histological protocols or stored at formaldehyde, 10 mM Tris-HCl (pH 7.6), 600 mM -80 C. For histological analyses, tissues were fixed NaCl, 1 mM EDTA, 0.25% SDS, and 200 μg/mL her- overnight in phosphate-buffered saline pH 7.4 (PBS) ring sperm DNA]. After washing, the sections were containing 4% paraformaldehyde, dehydrated, embed- mounted with ProLong Gold antifade reagent with ded in paraffin, and 6-μm serial sections were prepared. 4´,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, All experimental protocols were approved by the St. CA, USA), and viewed using a Zeiss LSM510 confocal Marianna University School of Medicine Animal Care microscope. The developmental stages of ovarian fol- and Use Committee. licles were determined as described previously [14].

RT-PCRs Western blotting Total RNAs (1 µg) isolated from tissues of each Ovaries and brains were homogenized in ice-cold rat were reverse transcribed, and partial sequences for PBS and then centrifuged. Proteins in the supernatant ENO1, 2 and 3 were amplified using an RNA PCR kit were precipitated by adding trichloroacetic acid (final (TaKaRa, Shiga, Japan). To check the integrity of the 10%). Proteins (20 μg) were resolved by SDS-PAGE RNA from each sample, PCR using a primer set for (12.5% polyacrylamide) and blotted onto a polyvi- β-actin was also performed. The sequences of primer nylidene difluoride membrane using a semi-dry condi- sets and annealing temperatures for each gene were tion. The membrane was incubated overnight at room shown in Table 1. PCR conditions were as follows: each temperature with a rabbit polyclonal antibody raised 30 sec of denaturation at 94C, annealing and extension against ENO2 (Millipore, Billerica, MA, USA) at dilu- at 72C were repeated 30 times. Control PCRs were per- tion of 1:1000 with PBS. The immunoreactive pro-

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Table 1 Sequences of primer sets used in PCR of the present study. Target Anneling Gene Direction Sequence size (bp) temp (C) ENO1 278 55 sense ATCCTACTGCCAGAACTTCAC antisense CCACAACATTCAGTTTCTTGCT ENO2 503 61 sense GGGGCACTCTACCAGGACTTTG antisense GTTCCGGTGTTCAGGCAAGCAG ENO3 509 55 sense GCTACCTAGGCACTCTACTC antisense CGTTGATCACATTGAAGGCA β-actin 166 63 sense GACAACGGCTCCGGCATGTGCA antisense TGAGGATGCCTCTCTTGCTCTG

Table 2 Sequences of primer sets used in in situ hybridization. Target Anneling Gene Direction Sequence size (bp) temp (C) ENO1 224 63 sense TAATACGACTCACTATAGGATCCTACTGCCAGAACTTCA antisense AATTAACCCTCACTAAAGGGTCTTATCATTGTCTCGGAGT ENO2 116 63 sense TAATACGACTCACTATAGGGAGGAGGCTCGCTTCGCGGG antisense AATTAACCCTCACTAAAGGGTTCCGGTGTTCAGGCAAGC

teins were chemiluminescently detected using a horse- expressed in the ovary. Brain was used as a positive radish peroxidase-conjugated antibody against rabbit control. Western blot analysis using anti-ENO2 anti- IgG (Millipore) and Supersignal West Dura (Pierce body showed that a single immunopositive band with Biotechnology, Rockford, IL, USA) as a substrate. a molecular mass of 47 kD was detected in both ovary and brain obtained from adult female rat (Fig. 1K). Immunohistochemistry Immnohistochemical analyses using the same antibody Tissue sections were incubated overnight at room revealed that ENO2 protein was detected primarily in temperature with anti-ENO2 antibody at 1:250 dilu- theca cells of preantral and antral follicles (Figs. 1M-O) tion. Immunoreactivities were detected by devel- as well as neurons (Fig. 1P). In addition, ENO2 pro- opment of 3,3’-diaminobenzidine using a Histofine tein was detected in oocytes of primary, preantral and Simple stain rat MAX-PO kit (Nichirei, Tokyo, Japan), antral follicles (Figs. 1L-O). Weak positive signals for followed by counterstaining with Mayer’s hematoxy- ENO2 protein were detected in granulosa cells of pre- lin. No staining was apparent when the primary anti- antral and antral follicles (Figs. 1M-O). bodies were omitted. The ovarian levels of ENO2 mRNA but not ENO1 Results mRNA change in the estrous cycle. We examined the expression of ENO1 and ENO2 The rat ovary expresses not only ENO1 but also ENO2. in adult rat ovaries at 4 stages of the estrous cycle by ENO1 and ENO2 mRNAs were detected in oocytes, RT-PCR analyses. Although no significant difference granulosa cells, and theca and interstitial cells obtained was detected in ovarian ENO1 mRNA levels among the from adult rat ovaries by RT-PCR analyses, however, stages of the estrous cycle (Fig. 2B), ENO2 mRNA lev- ENO3 mRNA was undetectable (Fig. 1A). ENO1 els significantly changed during the estrous cycle (Fig. mRNA was mainly detected in cumulus and mural 2C). Its levels at metestrus were significantly lower granulosa cells of antral follicle (Figs. 1B and C). In than those at estrus. addition, weak positive signals for ENO1 mRNA were detected in oocytes and theca cells. ENO2 mRNA was Gonadotropin injection increases ovarian ENO2 detected in oocytes, granulosa and theca cells of antral expression. follicle (Figs. 1E and F). The effects of gonadotropins on ovarian ENO1 Next, we examined whether ENO2 protein was and ENO2 expression were examined. The injection

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Fig. 1 ENO1 and 2 are expressed in adult rat ovaries. Tissues were obtained from adult (10-12 week old) rats. (A) RT-PCR for ENO1-3 in oocytes (O), granulosa cells (G) and theca and interstitial cells (TI). To check the integrity of the RNA from each sample, PCR using a primer set for β-actin was also performed. (B-G) in situ hybridization of ENO1 (B-D) and ENO2 (E-G) in ovarian sections. (H-J) Negative control using an ENO2 sense probe. Signals for mRNAs of each gene were displayed with green fluorescence (B, E and H) or merged with nuclear staining with Hoechst 33342 (C, F and I). (D, G and J) Differential interference contrast images for morphology. ENO1 mRNA was intensely detected in cumulus and mural granulosa cells, whereas ENO2 was mainly in oocyte (O), granulosa cells (G) and thecal cells. Objective magnification in B-J: x40. (K) Western blotting of ENO2 in ovary (O) and brain (B). In both tissues, a band at 47 kDa corresponding to ENO2 was mainly detected. Immunohistochemistry of ENO2 in adult rat ovary (L-O) and brain (P). ENO2-immunopositive signals were detected as brown. The sections were counterstained by hematoxylin. ENO2 was detected in oocytes, granulosa and theca cells of primary (L), preantral (M), and antral (N and O) follicles. As shown in (P), in brain section, the neuronal cell bodies were immunopositive. Bars in L-P: 100 µm.

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Fig. 2 Expression levels of ovarian ENO2 fluctuate during estrous cycle. Ovaries were obtained from adult (10-12 week old) rats, showing a normal estrous cycle consisting of diestrus, proestrus, estrus and metestrus. (A) Representative data of RT-PCR for ENO1 and ENO2. (B and C) the levels of ovarian ENO2 mRNA, but not ENO1, changed significantly during the estrous cycle. The data obtained from two independent experiments are expressed as the mean ± SEM (n = 5 per group). *p < 0.05 (Dunn’s multiple comparison test).

of hCG, which mimics the effect of LH, into imma- injections of hCG or eCG to immature rats up-regu- ture rats increased significantly ENO2 mRNA levels in lated ovarian expression of ENO2: hCG increased the ovaries (Figs. 3A and B). Similarly, eCG, which ENO2 expression in oocytes and theca cells of prean- shows FSH-like effects, increased ENO2 mRNA lev- tral and antral follicles, and eCG did in theca cells of els. However, hCG and eCG did not affect ovarian these follicles. In contrast, hCG and eCG did not affect ENO1 mRNA levels. the expression of ENO1, which was mainly expressed Immunohistochemistry revealed that hCG mainly in granulosa cells. hCG and eCG are known to mimic increased the expression of ENO2 in oocytes and theca the effects of LH and FSH, respectively. Therefore, cells of preantral and antral follicles (Fig. 3C), while we suggest that endogenous gonadotropins, LH, FSH eCG did in theca cells of these follicles (Fig. 3D). and CG, up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which Discussion would activate glycolysis in these cells. Glycolysis is a major contributor to energy production in ovarian fol- In the present study, we found that adult rat ovary licles [6-8]. Thus, gonadotropin-stimulation of the gly- expressed not only ENO1 but also ENO2. The ovarian colysis in oocytes and theca cells would activate ovar- levels of ENO2 mRNA, but not ENO1 mRNA, changed ian functions such as follicle growth and maturation, significantly in the estrous cycle. Furthermore, single and hormone production. It has been known that expo-

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Fig. 3 Gonadotropins up-regulate ovarian expression of ENO2 but not ENO1. Immature (3 week old) female rats were injected with 10 IU hCG or 10 IU eCG. Ovaries were sampled at 6 h after the injections. (A) Representative data of RT-PCR for ENO1 and ENO2. (B) Ovarian ENO1 and ENO2 mRNA levels. The data obtained from two independent experiments are expressed as the mean ± SEM (n = 5 per group). hCG and eCG up-regulated ovarian expression of ENO2 mRNA but not ENO1 mRNA. *P < 0.05. (C-E) Immunohistochemistry of ENO2 in ovarian sections of animals treated with hCG (C), eCG (D) and saline (E). hCG mainly increased the expression of ENO2 in oocytes and theca cells of preantral and antral follicles, while eCG did in theca cells of these follicles. Bars in C-E: 100 µm.

sure to hCG stimulates glycolytic activity in ovarian ENO1 and ENO2 mRNAs increase at the primordial to follicles [8, 9] and that glycolysis in ovarian follicles is primary follicle transition. required for follicle growth and estradiol secretion [9]. In the present study, we found that ovarian ENO2 In addition, the expression of ENOs varies according mRNA levels decreased at metestrus and that hCG and to the pathophysiological, metabolic or developmental eCG up-regulated ovarian expression of ENO2. In addi- conditions of cells [1, 12], e.g., the levels of ovarian tion, serum gonadotropin levels are low at metestrus [15,

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16]. Thus, endogenous LH and FSH may be involved ral and antral follicles [12, 23-25], demonstrating that in the change of ENO2 mRNA expression during the oocytes metabolize glucose via glycolysis. It has been estrous cycle, although further studies on regulation of suggested that reduced glycolytic activity in the mature ovarian ENO2 expression are necessary. oocyte are indicative of reduced developmental poten- LH/CG and FSH receptors are known to be expressed tial in the resulting embryo [26]. Thus, it is suggested in theca cells [17]. Thus, the present study suggests that that LH and CG stimulates glycolysis in oocytes, which hCG and eCG directly acts on theca cells to up-regulate leads to the activation of meiosis. Alternatively, it has ENO2 expression. However, it is unknown whether been reported that glycolytic activity is low [10, 27] hCG or LH directly acts on oocytes, although oocytes and that glycolysis may not be a major contributor to may express LH/CG receptors [18, 19]. Alternatively, energy production in immature oocytes [25, 27, 28]. It hCG or LH may up-regulate ENO2 expression in is likely that ENO2 up-regulated by hCG in immature oocytes via signaling from surrounding somatic cells: oocytes is utilized for function(s) other than glycolytic LH increases the expression of epidermal growth factor . ENO is known to be a multifunctional pro- (EGF) family proteins amphiregulin (AREG) and epi- tein [1], e.g., ENO functions as a plasminogen binding regulin (EREG) in granulosa cells [20], and then AREG protein, Myc-binding protein, and heat-shock protein. and EREG bind EGF receptor in oocytes, which acti- Further studies will be required to determine the func- vate oocyte functions [21, 22]. In this study, we also tions of ENO2 in the oocyte. found that hCG and eCG displayed different effect, i.e., hCG increased ENO2 expression in oocytes and Acknowledgments theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. The up-regulation of We thank Naomi Hamada for her technical assis- ENO2 expression in oocytes by hCG may occur by the tance. This work was supported by Grants-In-Aid for mechanism involving AREG and EREG. Scientific Research from the Ministry of Education, Glycolytic activity and glycolytic enzymes are Science and Culture of Japan [21592116 to B.I. and detected in mammalian oocytes of primary, preant- 22591839 to M.T.I].

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