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Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD13436

Low-density lipoprotein receptor affects the fertility of female mice Tao Guo A,B, Liang Zhang B, Dong Cheng C, Tao Liu A,B, Liguo An B, Wei-Ping Li A,D and Cong Zhang A,B,D A

Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200135, China. B Key Laboratory of Animal Resistance Research, College of Life Science, Shandong Normal University, 88 East Wenhua Road, Ji’nan, Shandong, 250014, China. C Shandong Center for Disease Control and Prevention, 16992 Jingshi Road, Ji’nan, Shandong, 250014, China. D Corresponding authors. Email: [email protected]; [email protected]

Abstract. Low-density lipoprotein receptor (LDLR) has been demonstrated to play a central role in lipoprotein metabolism, with Ldlr-deficient (Ldlr–/–) mice developing severe dyslipidemia. In the present study we investigated whether Ldlr knockout could harm female reproduction and explored the mechanisms involved. The results indicate that although the number of litters born to Ldlr–/– mice did not differ significantly from that born to controls, the number of pups per litter was significantly lower in the former group. Interestingly, although Ldlr–/– mice were obese, the weight of their ovaries was lower than that in control mice. Serum cholesterol levels was significantly higher in Ldlr–/– mice than in their wild-type counterparts. In contrast, there were significant decreases in cholesterol, triglyceride and total lipid levels in ovaries of Ldlr–/– mice. Both ovarian lipid deposition, as detected by Oil red O staining, and lipid droplets, as evaluated by transmission electron microscopy, supported decreased lipid levels in ovaries from Ldlr–/– mice. In addition, Ldlr–/– mice had fewer ovarian follicles, more atretic follicles, lower oestrogen levels and spent significantly less time in oestrus than did the controls. Superovulation assays indicated immature Ldlr–/– mice ovulated fewer ova than controls. These results indicate that lack of Ldlr results in dyslipidaemia and poor fertility. Additional keywords: atresia, dyslipidaemia, Ldlr, oestrogen. Received 21 December 2013, accepted 7 May 2014, published online 15 July 2014 Introduction Female reproductive success depends on the development of ovarian follicles, oocyte maturation, ovulation, fertilisation and pregnancy (Dasgupta et al. 2012). The follicle is the functional unit of the ovary that serves to protect and nourish the growing oocyte from a follicle’s formation through to ovulation. In order to mature, follicles need to survive recruitment and selection, during which they develop gonadotropin and steroid receptors to ensure their proper response to the cyclic fluctuation in hormones (Britt et al. 2004a). These steroid hormones have a close relationship with cholesterol metabolism because cholesterol is a precursor for steroid hormones. Thus, it is not surprising that endocrine, especially steroidogenic, tissues and other cell types have evolved multiple pathways to ensure the adequate provision of this crucial lipid, including its synthesis, storage as cholesteryl esters and import from lipoproteins (Rigotti 2003). Current evidence suggests that plasma lipoproteins are the major source of cholesterol for steroid production in the adrenal glands and ovaries (Azhar et al. 2003). Journal compilation Ó CSIRO 2014

The low-density lipoprotein receptor (LDLR) is a cell surface glycoprotein that plays a pivotal role in the homeostatic control of blood cholesterol by mediating the removal of cholesterol-containing lipoprotein particles from the circulation (Jeon and Blacklow 2005). Transcription of the Ldlr gene is regulated by intracellular cholesterol concentration, hormones and growth factors (Singh et al. 2011). At neutral plasma pH, circulating lipoprotein particles bind to the LDLR and enter cells by receptor-mediated endocytosis (Jime´nez et al. 2010). The most important physiological ligand for the receptor is low-density lipoprotein (LDL), which contains a single copy of apolipoprotein B-100 (ApoB-100) and multiple copies of apolipoprotein E (ApoE), b-migrating forms of very low-density lipoprotein (b-VLDL) or certain intermediates, as well as some high-density lipoproteins and chylomicron remnants from the circulation (Rigotti 2003; Jeon and Blacklow 2005). The cholesterol-rich lipoprotein is then degraded intracellularly for cholesterol delivery to the cell interior (Azhar et al. 2003). In addition, VLDL particles are www.publish.csiro.au/journals/rfd

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primarily cleared from the circulation by the LDLR in the liver (Singh et al. 2011). Many studies have demonstrated that genetic variation at the Ldlr locus is involved in dyslipidaemia, is associated with an increased risk of developing atherosclerosis and determines susceptibility to cardiovascular disease and familial hypercholesterolaemia (FH; Nock and Pillai 2012; Jeon and Blacklow 2005). Dyslipidaemia is characterised by an aggregation of lipoprotein abnormalities, including increased LDL–cholesterol (LDL-C) and high-density lipoprotein–cholesterol (HDL-C; Kwan et al. 2007). The Watanabe heritable hyperlipidaemic (WHHL) rabbit has a defective Ldlr gene and is a model of FH. Dyslipidaemia is associated with polycystic ovary syndrome (Phelan et al. 2010), the most common endocrine disorder among women of reproductive age and a leading cause of infertility (Boomsma et al. 2008). WHHL rabbits are also less fecund than New Zealand white (NZW) rabbits, and the lower fecundity is related to impaired ovarian steroidogenesis due to reduced intracellular availability of cholesterol (Robins et al. 1994). In addition, Ldlr deleted macrophages have reduced basal levels of ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette sub-family G member 1 (ABCG1) and cholesterol efflux (Zhou et al. 2008). These results suggest that LDLR may be an important modulator of cholesterol utilisation. Taking its key roles in plasma lipoprotein and cholesterol metabolism into account, the LDLR may affect steroid production and then impact fertility. The influence of Ldlr deletion on reproduction has not been assessed previously, and we supposed that there is a relationship between dyslipidaemia and hormone synthesis. Previous studies have detected the presence of LDLR in the ovaries of some species, such as mouse, bovine and cutthroat trout (Jong et al. 1999; Argov and Sklan 2004; Luo et al. 2013). Azhar et al. (1999) found that the uptake of selective cholesteryl ester in vitro was upregulated in granulosa cells from ovaries of Ldlr-null (Ldlr–/–) mice; however, little is known about the effects of the LDLR on the fertility of mice. In the present study, we found that lipid metabolism in the plasma and ovaries of Ldlr–/– mice was abnormal. Furthermore, dyslipidaemia influenced oestrogen levels, follicle development and increased follicle atresia, with LDLR deficiency resulting in decreased fertility in mice. Materials and methods Animals Ldlr–/– mice in which the Ldlr gene was knocked out on a C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, ME, USA); wild-type (WT) C57BL/6 mice (8 weeks old) were obtained from the Chinese Center for Disease Control and Prevention (Beijing, China). Breeding colonies were maintained for all mice in the animal facility at Shandong Normal University. The genotypes of the mice were determined by polymerase chain reaction (PCR) analysis. Mice had free access to food and water and were kept on a 12-h light : dark cycle in a pathogen-free mouse room. Mice were manipulated in compliance with the Guidelines of Shandong Normal University for the Care and Use of Laboratory Animals.

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Fertility assay For the breeding assay, 8-week-old females were housed with C57BL/6J males of proven fertility for 6 months (two females per male; n ¼ 14 female mice per genotype). Each morning, the vaginal canals were gently checked for copulatory plugs; the parturition dates and number of pups born were recorded and the day pups were born was designated Day 0. Plasma and ovarian cholesterol, triglyceride and total ovarian lipid analysis For plasma analysis, mice were anaesthetised with isofluorane (Baxter Healthcare, Deerfield, IL, USA) on Day 18, 42, 90 and 450 and blood was collected by cardiac puncture (n ¼ 6 per group). Serum lipoproteins were isolated by fast protein liquid chromatography (FPLC; Agilent Technologies, Palo Alto, CA, USA) and separated at a constant flow rate of 50 mL min1 with phosphate-buffered saline (PBS). Plasma concentrations of total cholesterol (TC) and triglycerides (TG) were measured in each group using a colorimetric method with commercially available kits (Sigma Chemical, St Louis, MO, USA). The method for plasma analysis has been described elsewhere in detail (Yesilaltay et al. 2006b; Zhang et al. 2014). For ovarian lipid analysis, 80 mg ovarian tissue was extracted with chloroform : methanol (2 : 1, v/v) in a centrifuge tube and homogenised using a homogeniser and then centrifuged at 2300g for 10 min. For homogenising, the condition used was as follows: at 5000 r.p.m. for 10 s repeated five times. After the supernatant had been collected, the same mixture of chloroform : methanol was added to the precipitate and the sample homogenised again. The two supernatants were pooled together, mixed with 145 mM NaCl, and then centrifuged at 8000 r.p.m. for 5 min at room temperature. The lower chloroform layer, which contained the lipid, was transferred into a weighed Eppendorf tube. Then, the chloroform was evaporated and the tube was weighed again to calculate the total lipid concentration in the ovary. For analysis of ovarian TC and TG concentrations, the methods and kits used were as described above for plasma. Oil red O staining of ovarian lipid deposition Ovaries were frozen in Tissue Tek OCT (Sakura, Torrance, CA, USA). Cryosections (10 mm) were prepared using a Slee Cryostat (Mainz, Germany), fixed in 4% paraformaldehyde (PFA) for 5 min and then washed in alcohol (60%) for 5 min. The sections were then stained with 0.5% Oil red O (O0625; Sigma-Aldrich, St Louis, MO, USA) in 60% isopropanol for 12 min. After two washes in 60% alcohol for 30 s each time, the sections were hydrated for 1 min, counterstained with haematoxylin and then mounted with 50% glycerol. The sections were observed under an Olympus ML2000 microscope (Olympus, Tokyo, Japan) and photographs were taken. Transmission electron microscopy Ovaries isolated from 3-month-old mice were trimmed into small pieces measuring 1  1  1 mm on ice and fixed in 3% glutaraldehyde in 0.2 M phosphate buffer (0.874 g NaH2PO4, 5.158 g Na2HPO4 in 100 mL H2O), post-fixed with osmic acid

LDLR affects the fertility of female mice

for 2 h and processed for transmission electron microscopy (TEM). Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEOL-1011; JEOL, Tokyo, Japan). Lipid drops were counted in three fields of antral follicles per section, and three sections from three different mice were quantified for each mouse genotype. Immunofluorescence analysis Ovaries were snap-frozen in liquid nitrogen, embedded in OCT compound and 10-mm cryosections prepared. Sections were fixed in acetone (208C) for 10 min, then incubated in 0.5% (v/v) Triton X-100 in PBS (pH 7.2) for 20 min. After washing with PBS, the sections were blocked with 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, Santa Clara, CA, USA) for 30 min at 378C. Following blocking, the slides were incubated with LDLR antibody (1 : 200 dilution; SC-11826; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 48C and then incubated with Alexa Fluor 488-conjugated IgG (dilution 1 : 400; Invitrogen-Molecular Probes, Carlsbad, CA, USA) for 1 h. The slides were then incubated with 40 ,60 diamidino-2-phenylindole (DAPI; Roche Applied Science, Mannheim, Germany) for 5 min at room temperature. Sections were then mounted and photographed using an Olympus fluorescence microscope. Histological assessment Ovaries were isolated on postnatal Day 4, 18, 42 and 90 for histological evaluation (n ¼ 6 mice per group). Ovaries were fixed in 4% PFA overnight; after three washes with PBS, ovaries were dehydrated and made transparent using Leica-ASP200S Dehydrator (Leica, Bensheim, Germany). The tissues were embedded in paraffin by the Leica EG1150H Embedding Center. Serial sections (5 mm) were made using a Leica RM2125 microtome and the sections were then stained with haematoxylin and eosin. The study was blinded to prevent bias. In every fifth section, the number of primordial, primary, preantral and antral follicles was counted and the total follicular number was calculated as described previously (Cui et al. 2011). Evaluation of the onset of puberty and oestrous cycles Each morning from the day of weaning pups were evaluated to determine vaginal opening time. Oestrous cycles in 3-month-old female mice were detected over a period of 21 consecutive days by assessing vaginal smears (Thung et al. 1956). The number of oestrus cycles over the 21-day period was recorded. Ovulation response to gonadotropins On Day 21, mice were injected subcutaneously with pregnant mare serum gonadotropin (PMSG; 5 IU), followed 48 h later by human chorionic gonadotropin (hCG; 5 IU) (n ¼ 10 mice per genotype). Mice were killed 16 h after hCG treatment and blood was collected by cardiac puncture to obtain serum for hormone assays. Then, the ovaries and oviducts were dissected free, and the number of ova in the oviduct was determined.

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Serum oestrogen and progesterone analysis Plasma concentrations of oestrogen were determined with a double-antibody radioimmunoassay system using an iodine [125I] Oestradiol Radioimmunoassay Kit (Jiuding Biological Engineering, Tianjin, China). The primary antibody was rabbit anti-human oestrogen antibody and the secondary antibody was donkey anti-rabbit antibody. Non-specific binding was always ,3%. The inter- and intraassay CV were 8.9% and 7.7%, respectively. The sensitivity of the assay was 2.1 pg mL1. Progesterone concentrations were measured in plasma samples by radioimmunoassay using an iodine [125I] Progesterone Radioimmunoassay Kit (Jiuding Biological Engineering). The primary antibody was rabbit anti-human progesterone antibody and the secondary antibody was donkey anti-rabbit antibody. The sensitivity of the assay was 0.03 ng mL1. The inter- and intraassay CV were 8.9% and 7.2%, respectively. Terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling The terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) assay was performed to detect the number of atretic follicles in the ovary. Cryosections (10 mm) for ovaries were fixed in 4% paraformaldehyde in PBS and subsequently washed three times in PBS for 20 min each time. Sections were stained using the In Situ Cell Death Detection Kit, peroxidase (Roche, Mannheim, Germany) according to the manufacturer’s instructions. For each ovary, two sections were selected at random for staining. The number of atretic follicles and total follicles in the selected sections was counted and the proportion of atretic to total follicles was calculated. Statistical analysis All numerical data are presented as the mean  s.e.m. of at least three separate experiments. All data were analysed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The significance of differences between groups was tested by one-way ANOVA. Values were deemed significantly different if two-tailed P , 0.05. Results Fertility assay The fertility of Ldlr–/– mice was compared with that of ST controls by determining the number of litters, number of pups per litter and the cumulative number of pups per female. All colonies were maintained in the animal facility at Shandong Normal University at the same time under the same conditions. The results showed that Ldlr–/– mice had significantly fewer pups than WT mice over a 6-month period (5.8  1.4 vs 7.1  0.5, respectively; n ¼ 14 in each group; P ¼ 0.0010), even though the number of litters born to Ldlr–/– and WT mice did not differ significantly (Fig. 1a). In addition, the bodyweight of Ldlr–/– female mice was higher than that of WT female mice on Day 90 (20.47  1.32 vs 19.07  1.50 g, respectively; P ¼ 0.0240; n ¼ 10 in each group) and Day 210 (24.75  1.85 vs 22.10  1.12 g, respectively; P ¼ 0.0009; n ¼ 10 in each group;

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Age (days) Fig. 1. Effects of low-density lipoprotein receptor (Ldlr) knockout on the fertility of female Ldlr–/– mice. (a) The cumulative number of pups per female, the number of pups per litter and the number of litters for wild-type and Ldlr–/– mice over a 6-month breeding period. (b) Bodyweight and (c) ovary weight of WT and Ldlr–/– mice. Data are the mean  s.e.m. (n ¼ 14 mice per genotype for (a); n ¼ 10 mice for (b, c)). *P , 0.05, **P , 0.01 compared with control (WT mice).

Fig. 1b). However, the ovarian weight of Ldlr–/– female mice was lower than that of WT female mice on Day 42 (2.47  0.52 vs 3.13  0.72 mg, respectively; P ¼ 0.0330; n ¼ 10 in each group), Day 90 (4.66  0.75 vs 5.70  0.82 mg, respectively; P ¼ 0.0480; n ¼ 10 in each group) and Day 210 (6.24  0.54 vs 7.82  0.95 mg, respectively; P ¼ 0.0120; n ¼ 10 in each group; Fig. 1c). Plasma and ovarian lipid analysis To explore the effect of Ldlr knockout on lipid metabolism, the different species of plasma lipoproteins were separated and analysed by FPLC. When lipoproteins were analysed using TC kits (Fig. 2a–d), VLDL and LDL were present at lower levels in Fractions 1–31 and HDL (Fractions 32–46) were found to be the

major cholesterol-containing particles in WT mice. In contrast, there was a significant increase in VLDL particles as part of the TC content in Ldlr–/– mice on Day 18, but no difference between Ldlr–/–and WT mice after that time point. Moreover, LDL particles as part of the TC content (Fractions 16–31) were significantly greater in Ldlr–/– mice, whereas HDL particles were decreased. When lipoproteins were subjected to TG assays (Fig. 2e–h), the concentration of VLDL fractions was slightly increased (not statistically significant) in Ldlr–/–compared with WT mice on Days 18, 42 and 90, although there was no difference on Day 450. Plasma and ovary TC and TG levels were measured in Ldlr–/– and WT mice on Day 90. Plasma TC concentrations were markedly increased in Ldlr–/– versus WT mice (326  14 vs 104  5 mg dL1, respectively; P ¼ 0.0008), as were plasma TG levels (260  35 vs 117  5 mg dL1, respectively; P ¼ 0.0210; Fig. 2i, j). In contrast with the plasma profile, TC concentrations in the ovaries of Ldlr–/– mice were significantly lower than those in ovaries of WT mice (7.36  1.35 vs 12.59  2.78 mg g1, respectively; P ¼ 0.043), as were TG levels (20.36  5.45 vs 34.84  7.04 mg g1, respectively; P ¼ 0.048; Fig. 2k, l). Oil red O staining was performed to further validate the condition of lipids in the ovaries of WT and Ldlr–/– mice. The staining was primarily localised in the corpora lutea (CL) and interstitial tissues of both strains of mice, and there were fewer lipids in ovaries from Ldlr–/– versus WT mice (Fig. 3a, d). The total lipid content of ovaries from Ldlr–/– mice was significantly less than that of WT mice (189  32 vs 262  28 mg g1, respectively; Fig. 3g). TEM was used to examine the ultrastructure of ovaries from WT and Ldlr–/– mice and showed that there were fewer lipid droplets in the latter group (Fig. 3b, c, e, f). To further determine the effect of Ldlr knockout on lipid droplets in ovaries, the number of lipid droplets in ovaries from WT and Ldlr–/– mice was counted; the results indicate that there were significantly fewer lipid droplets in ovaries from Ldlr–/– compared with WT mice (3.33  1.15 vs 33.00  8.19, respectively; Fig. 3h). Immunofluorescence analysis of LDLR in ovarian tissue Immunofluorescence analysis was performed to clarify the location of LDLR in ovaries. As indicated in Fig. 4, the LDLR is expressed primarily in the theca, interstitial cells and CL of WT mice. Effects of Ldlr deletion on folliculogenesis Ovarian morphology was examined to investigate whether Ldlr played a role in ovarian follicle development. There was no significant difference in the total number of follicles in ovaries from Ldlr–/– and WT mice on Day 4 (3712  149 vs 3803  257, respectively; P ¼ 0.471), but there were fewer total follicles in ovaries from Ldlr–/– compared with WT mice Day 18 (2955  191 vs 3244  151, respectively; P ¼ 0.0160), Day 42 (2240  181 vs 2522  222, respectively; P ¼ 0.0370) and Day 90 (1196  95 vs 1558  120, respectively; P ¼ 0.0006; Fig. 5a). Similarly, there was no significant difference in the number of primordial follicles from ovaries of Ldlr–/– and WT mice on

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Fig. 2. Lipoprotein profiles of wild-type (WT) and low-density lipoprotein receptor-knockout (Ldlr–/–) mice. (a–h) The contribution of different lipoprotein fractions to plasma total cholesterol (a–d) and triglyceride (e–h) concentrations in female WT and Ldlr/ mice on Days 18, 42, 90 and 450, as determined by fast protein liquid chromatography. The approximate positions of the different lipoprotein fractions are as follows: very low-density lipoprotein–cholesterol, Fractions 1–16; low-density lipoprotein–cholesterol, Fractions 17–31; high-density lipoprotein–cholesterol, Fractions 32–46. (i–l) Total cholesterol and triglyceride concentrations in the plasma (i, j) and ovarian tissues (k, l) of WT and Ldlr/ mice on Day 90. Data are the mean  s.e.m. *P , 0.05, **P , 0.01 compared with control (WT mice).

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Fig. 3. Oil red O staining and detection of lipid deposition by transmission electron microscopy (TEM) of ovaries from 3-month-old wild-type and low-density lipoprotein receptor-knockout (Ldlr–/–) mice. (a) Typical Oil red O staining of lipids and counterstaining with haematoxylin showing lipid deposition (red) in ovaries from WT females. (b, c) Electron micrographs of ovaries from WT mice; the inset in (c) shows a higher magnification view of a lipid droplet. White arrows indicate lipid droplets. (d) Oil red O staining of the ovaries from Ldlr–/– mice. (e, f ) Electron micrographs of ovaries from Ldlr–/– mice; the inset in (f ) shows a higher magnification view of a lipid droplet. o, oocyte; g, granulosa cells. (g) Concentration of total lipid in ovaries of WT and Ldlr–/– mice. (h) The number of lipid droplets in ovaries from WT and Ldlr–/– mice. Data are the mean  s.e.m. *P , 0.05, **P , 0.01 compared with control (WT mice).

Day 4 (3260  155 vs 3323  285, respectively; P ¼ 0.647) and Day 18 (1169  82 vs 1231  65, respectively; P ¼ 0.180), but there were fewer primordial follicles in ovaries from Ldlr–/– compared with WT mice on Day 42 (873  96 vs 1002  98, respectively; P ¼ 0.0450) and on Day 90 (301  26 vs 343  27, respectively; P ¼ 0.0220; Fig. 5b). Again, there were no significant differences in the number of primary follicles in ovaries from Ldlr–/– and WT mice on Day 4 (452  47 vs 480  34, respectively; P ¼ 0.260) and Day 18 (657  47 vs 700  41, respectively; P ¼ 0.121). However, on Day 42, there were significantly more primary follicles in ovaries from Ldlr–/– than WT mice (847  64 vs 752  61, respectively; P ¼ 0.0250). On Day 90, there were significantly fewer primordial follicles in ovaries from

Ldlr–/– than WT mice (305  30 vs 405  30, respectively; P ¼ 0.0004; Fig. 5c). Although there was no significant difference in the number of preantral follicles in ovaries from Ldlr–/– and WT mice on Day 42 (290  37 vs 318  44, respectively; P ¼ 0.255), there were significantly fewer preantral follicles in ovaries from Ldlr–/– than WT mice on Day 18 (950  53 vs 1093  102, respectively; P ¼ 0.0120) and Day 90 (355  31 vs 485  40, respectively; P ¼ 0.0008; Fig. 5d ). There were significantly fewer antral follicles in ovaries from Ldlr–/– than WT mice on Day 18 (179  31 vs 220  32, respectively; P ¼ 0.0480), Day 42 (230  34 vs 440  43, respectively; P ¼ 0.0003) and Day 90 (235  19 vs 326  28, respectively; P ¼ 0.0006; Fig. 5e).

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administration in both groups. However, Ldlr–/– mice shed fewer ova compared with age-matched WT controls (41.00  8.91 vs 50.86  9.75, respectively; P ¼ 0.036; Fig. 7e). Discussion

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Fig. 4. Ovarian tissues from wild-type (WT) mice subjected to fluorescent immunohistochemistry to localise the low-density lipoprotein receptor (LDLR) protein. (a, b) Immunofluorescence staining of the LDLR. (c, d ) 40 ,60 -Diamidino-2-phenylindole (DAPI) staining of nuclei. (e, f ) Merged image of LDLR and DAPI; the inset shows a higher magnification of a follicular wall with visible granulosa and theca cells. NC, negative control.

Apoptosis in ovaries The TUNEL assay revealed that there were more TUNELpositive follicles in ovaries from Ldlr–/– than WT mice (Fig. 6a–c). To further determine the effect of Ldlr knockout on follicles in the ovary, the number of atretic follicles and total follicles in eight sections from four WT and four Ldlr–/– mice was determined. The ratio of atretic : total follicles revealed that there was a significantly higher percentage of atretic follicles in ovaries from Ldlr–/– than WT mice (40.80% vs 28.33%, respectively; P ¼ 0.009; ig. 6d ). Effects of Ldlr deletion on plasma hormones and reproductive physiology Hormone analyses revealed no significant differences in serum progesterone levels between WT and Ldlr–/– mice, but there was a significant decrease in serum oestrogen concentrations in Ldlr–/– mice (Fig. 7a, b). Vaginal opening was similar in Ldlr–/– and WT (28.00  1.15 vs 30.33  5.61 days (D), respectively; P ¼ 0.215; Fig. 7c). However, the days in oestrous over a 21-day period was less in Ldlr–/– compared with controls (Fig. 7d). Immature mice on Day 21 that were given PMSG ovulated in response to hCG

The present study demonstrates that the dyslipidaemia caused by Ldlr gene deletion has a significant effect on the fertility of female mice. The Ldlr deletion significantly decreased fertility, oestrogen levels and oestrus length, disturbed plasma and ovarian lipid metabolism and restrained ovarian follicular development by promoting follicle atresia. Moreover, Ldlr–/ – prepubertal mice ovulated fewer oocytes in response to a superovulatory regimen of PMSG þ hCG. In conclusion, Ldlr knockout harms female reproduction. Female Ldlr–/– mice produced fewer pups than WT mice over a 6-months breeding period, although the number of litters born to Ldlr–/– mice was not significantly different from that of controls. A previous study on the infertility of HDL receptor-negative (Scarb1–/–) female mice also suggested a link between female fertility and abnormal lipoprotein metabolism (Yesilaltay et al. 2006a). In the present study, FPLC analysis of lipoproteins indicated that the increased total plasma cholesterol in Ldlr–/– mice was a consequence of a marked increase in LDL particles. The LDLR is capable of binding lipoproteins containing ApoE or ApoB100 and plays a major role in removing from the plasma remnant products of VLDL metabolism, including the ApoB-100and/or ApoE-containing VLDL remnants and the ApoB-100containing LDL particles, mainly in the liver (Heath et al. 2001; Singh et al. 2011). The bulk of intermediate density lipoprotein (IDL) particles are also cleared in the liver via the LDLR. When Ldlr is defective, as in FH homozygotes or WHHL rabbits, the IDL particles remain in the circulation, where they are converted to LDL (Ishibashi et al. 1994). In addition, a large proportion of the ApoB-100- and/or ApoE-containing VLDL remnants are converted to LDL-C (Meddings and Dietschy 1986; Spady et al. 1987). This may explain the situation in Ldlr–/– mice: because of a lack of the LDLR, the VLDL particle content of TC on Day 18 and LDL content on Days 18, 42, 90 and 450 in the plasma may not have been cleared by liver, so that VLDL-C levels on Day 18 and the LDL-C levels were increased compared with levels in WT mice on Days 18, 42, 90 and 450. These phenomena support the conclusion that the LDLR is important for controlling circulating plasma LDL-C concentrations (Osono et al. 1995). In addition, we found that the HDL fractions in cholesterol decreased. The reduced HDL levels may also have been due to dysfunction of the LDL pathway, because the deletion of Ldlr reduces basal levels of ABCA1 (Zhou et al. 2008). ABCA1 plays a key role in hepatic cholesterol efflux, induces pathways that modulate cholesterol homeostasis in the liver and establishes the liver as a major source of plasma HDL-C (Basso et al. 2003), and low ABCA1 levels contribute significantly to low plasma HDL-C levels (Haghpassand et al. 2001; Oram and Lawn 2001; Cohen et al. 2004). In addition, in the face of a lack of LDLR function, other pathways involved in the uptake of cholesterol for peripheral tissues may be enhanced. For example, it has been reported that scavenger receptor class B type 1

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(SCARB1), which mediates the selective uptake of HDL-C into cells, is upregulated in Ldlr–/– mice (Azhar et al. 1999). This could explain the low HDL-C levels in plasma in Ldlr–/– mice. Tissues synthesise cholesterol de novo and take up LDL and/or HDL from the plasma for steroidogenesis, with plasma lipoproteins being the major source of cholesterol for steroid production in ovaries (Goldstein and Brown 1985; Azhar et al. 2003; Connelly and Williams 2004). We observed that Ldlr knockout led to increased bodyweight in mice, but decreased ovary weight compared with WT mice, observations that are consistent with those of a previous report (Repas 2011). Histochemical analysis of ovaries from Ldlr–/– mice showed reduced Oil red O staining of lipids compared with WT mice. This suggests there was reduced cholesteryl ester storage, as reported previously in ovaries from Scarb1-knockout mice (Trigatti et al. 1999). Moreover, TEM studies revealed few lipid droplets in ovarian cells from Ldlr–/– mice. The total lipid content of ovaries from Ldlr–/– mice, as well as TC and TG levels, were significantly decreased compared with WT mice. All these observations indicate that LDLR pathway deficiency affects the uptake

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of TC from the plasma to the ovary, and lower TG levels may be associated with fewer lipid droplets in ovaries from Ldlr–/– mice. As a precursor for the conversion to steroid hormones, a constant supply of cholesterol is required within ovarian cells. In the mouse, the LDLR is located in theca and interstitial cells, as well as in the CL. The diverse sites of Ldlr expression may indicate multiple roles in ovarian metabolism. In the present study, hormone analyses revealed no significant differences between the two genotypes in terms of serum progesterone concentrations, but oestrogen concentrations were significantly lower in Ldlr–/– compared with WT mice. In the ovary, progesterone is synthesised mainly in the CL. In mice, an important source of cholesterol for steroidogenesis in the CL is HDL imported via SCARB1 (Jime´nez et al. 2010). It was reported that cholesterol imported via SCARB1 for progesterone synthesis was enhanced in Ldlr–/– mice (Azhar et al. 1999); furthermore, the de novo synthesis of cholesterol may be augmented to compensate the dysfunction of the LDL pathway by increasing the expression of 3-hydroxy-3-methylglutaryl CoA reductase, a rate-limiting enzyme in the cholesterol pathway

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Fig. 5. Effects of low-density lipoprotein receptor (Ldlr) deletion on the number of ovarian follicles in wild-type (WT) and Ldlr–/– mice at various ages. The number of (a) total follicles, (b) primordial follicles, (c) primary follicles, (d) preantral follicles and (e) antral follicles per ovary is shown. Data are the mean  s.e.m. of six mice for each genotype and age. *P , 0.05, **P , 0.01 compared with control (WT mice).

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Fig. 6. Terminal deoxyribonucleotidyl transferase-mediated dUTP– digoxigenin nick end-labelling (TUNEL) of sections of ovarian tissue showing atretic follicles in (a) wild-type (WT) and (b) low-density lipoprotein receptor-knockout (Ldlr–/–) mice. (c) Negative control for TUNEL staining (enzyme solution omitted). (d) Ratio of atretic to total follicles. The number of atretic and total follicles was determined in eight sections from four mice in each group. Data are the mean  s.e.m. **P , 0.01 compared with control (WT mice).

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(Jime´nez et al. 2010). When all three sources are compromised, the cholesterol esters stored in the CL could be used as a substrate for progesterone synthesis (Azhar et al. 1999; Jime´nez et al. 2010). Cholesterol is also used to synthesise androgen in theca cells and the androgen is then transferred to granulosa cells for oestrogen biosynthesis (McNatty et al. 1979; Azhar et al. 1999). Deletion of Ldlr in theca cells may lead to low oestrogen biosynthesis and secretion. Histochemical analysis of the ovaries demonstrated there a similar number of follicles on Day 4 in WT and Ldlr–/– mice; however, the total number of follicles in ovaries from Ldlr–/– mice was significantly lower than in WT mice on Days 18, 42 and 90. Specifically, Ldlr–/– mice had fewer preantral and antral follicles on Day 18, whereas on Days and 90 the number of primordial and antral follicles had decreased in Ldlr–/– compared with WT mice. These results indicate that the deletion of Ldlr has an obvious influence on ovarian follicle development. The fewer antral follicles in ovaries from Ldlr–/– mice may result in fewer CL. The decreased number of CL may also be one of the reasons why ovaries from Ldlr–/– mice have reduced levels TC and TG, as well as a lower total ovarian lipid content, than ovaries from WT mice, because the CL are a major site where lipids are found. Previous studies reported that oestrogen is not required for the initiation of follicle growth, primordial follicle differentiation and early activation, but is necessary for the later stages of follicular development (Britt et al. 2000, 2004a, 2004b). So, although there was no difference in total follicles on Day 4 between WT and Ldlr–/– mice, the low oestrogen level

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Fig. 7. Effects of low-density lipoprotein receptor (Ldlr) deletion on plasma hormone and reproductive physiology in wildtype (WT) and Ldlr–/– mice. Plasma (a) oestrogen and (b) progesterone concentrations were determined on Day 24 in WT and Ldlr–/– mice that had been stimulated with pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG; see text for details). Data are the mean  s.e.m. (n ¼ 10 in each group). (c) Time of vaginal opening in WT and Ldlr–/– mice. (d) The days in oestrus over a 21-day period in 3-month-old mice was determined by assessing vaginal smears. (e) The number of ova in mice. Mice were injected with 5 IU PMSG on the day indicated to stimulate follicle development, followed 48 h later by 5 IU hCG to induce ovulation. The morning after hCG administration, the oviducts were dissected, and the number of ova shed was determined. Data are the mean  s.e.m. *P , 0.05 compared with control.

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may have blocked subsequent follicular development. Primordial follicles are the stock from which all growing follicles and ovulatory ova are derived. In order to mature, follicles need to survive recruitment and selection; only 0.1% of primordial follicles that enter the growing phase will successfully pass all the selection processes and ovulate their oocytes, with the remainder undergoing atresia (Hsueh et al. 1994; McNatty et al. 2000). Within the ovary, Wilms tumour 1 (WT1) is expressed in granulosa cells of primary to secondary follicles, and then diminishes with further follicle development (Hsu et al. 1995; Chun et al. 1999). WT1 is a marker for immature follicles and it plays a role in suppressing the development of immature follicles by amplifying the actions of p53 in granulosa cells and by repressing the transcriptional activity of genes encoding several growth factors and their receptors (Hsu et al. 1995; Tilly et al. 1995; Chun et al. 1999; Kreidberg et al. 1999). A previous study reported increased WT1 levels in oestrogen-deficient mice (Britt et al. 2000). So, the low level oestrogen levels caused by abnormal cholesterol metabolism may limit the number of follicles entering the growing phase, resulting in more follicles undergoing atresia. This may explain why immature Ldlr–/– mice primed with a superovulatory regimen of PMSG and hCG ovulated fewer oocytes than control mice. Furthermore, the decreased ovulation potential appeared to result in fewer pups born to Ldlr–/– mice. The present study provides additional evidence for the role of Ldlr in ovarian function and further insights into its potential roles in the regulation of lipid metabolism, as well as hormone levels, ovarian follicular development, apoptosis and fertility during postnatal life in the mouse. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (NSFC; 31172040), Shandong Province Natural Science Foundation (ZR2011CM047) and Scientific Research Foundation for the Returned Overseas Chinese Scholars and State Education Ministry to CZ; NSFC (81370692) to WPL; and the Shanghai Commission of Science and Technology (12DZ2260600). The authors express their appreciation to Dr Zhilin Liu (Department of Molecular and Cellular Biology, Baylor College of Medicine) for critical reading of the manuscript and valuable advice.

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