Amino acid transport system L activity in developing mouse ovarian follicles

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Amino acid transport system L activity in developing mouse ovarian follicles ARTICLE in HUMAN REPRODUCTION · SEPTEMBER 2011 Impact Factor: 4.57 · DOI: 10.1093/humrep/der298 · Source: PubMed

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2 AUTHORS: Ashwini Chand

Michael Legge

Olivia Newton-John Cancer Research Institute

University of Otago

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Human Reproduction, Vol.26, No.11 pp. 3102–3108, 2011 Advanced Access publication on September 13, 2011 doi:10.1093/humrep/der298

ORIGINAL ARTICLE Reproductive biology

Amino acid transport system L activity in developing mouse ovarian follicles Ashwini L. Chand1 and Michael Legge 2,* 1

Cancer Drug Discovery, Prince Henry’s Institute, Monash Medical Centre, Clayton, Melbourne 3168, Australia 2Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand *Correspondence address. Tel: +64-3-479-7845; Fax: +64-3-479-7866; E-mail: [email protected]

Submitted on April 14, 2011; resubmitted on July 26, 2011; accepted on August 11, 2011

methods: Mouse ovarian follicles were cultured in vitro for 5 days in increasing concentrations of IGF-1, and follicle diameter and atresia measured as endpoints for growth. Uptake of 3H-leucine was measured in follicles at different stages of development. In optimal IGF-1-mediated growth conditions, competitive inhibition of 3H-leucine uptake by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a non-metabolizable substrate analogue of L-leucine, was performed to demonstrate specificity of influx, via system L transporters. To test whether uptake rates were dependent on intracellular amino acid availability, follicles from in vitro cultures were pre-treated with L-phenylalanine prior to 3H-leucine uptake. results: Follicle development (P , 0.001) and survival (P , 0.001) increased with IGF-1 treatment. As pre-antral follicles progressed to late antral stage, we observed an increase in L-leucine uptake, which was reduced in pre-ovulatory follicles. BCH decreased L-leucine uptake rates in early antral (P , 0.05), antral (P , 0.001) and pre-ovulatory follicles (P , 0.01). L-leucine influx increased in follicles preloaded with phenylalanine (trans-stimulation). In follicles lacking free intracellular amino acids (zero-trans suppression), uptake rate was reduced (P , 0.05).

conclusions: These results demonstrate, for the first time, evidence of specific system L amino acid transport in intact, mouse ovarian follicles and profile L-leucine uptake during folliculogenesis. A better understanding of ovarian follicle metabolic pathways is necessary for improved in vitro maturation as well as determining the impact of altered metabolism on fertility. Key words: ovary / follicles / insulin-like growth factor 1 / amino acid transport / metabolism

Introduction Despite the detailed knowledge of carbohydrate metabolism in mouse ovarian follicles, oocytes and preimplantation embryos (Boland et al., 1994; Harris et al., 2007), there is little information on the uptake of amino acids by intact ovarian follicles. Most information regarding amino acid transport is related to mouse-ovulated oocytes (Haghighat et al., 1990) or preimplantation mouse embryos (Kaye et al., 1982; Hobbs et al., 1985; Van Winkle et al., 1990), where two amino acid transport systems have been described (Colonna and Mangia, 1983a;Colonna et al., 1983b, 1984): a sodium-independent L-transport system and a sodium-dependent A-transport system. Both transport systems operate in the ovulated oocyte, where the uptake and transfer of non-polar amino acids is mediated via granulosa cells and oocyte gap junctions by metabolic co-operativity (Colonna et al., 1984; Eppig et al., 2005). In contrast, metabolic co-operativity in ovulated oocytes only becomes apparent for amino acids such as

valine, leucine and phenylalanine when the concentrations increase 5- to 10-fold above that in plasma. Colonna et al. (1983a,b, 1984) demonstrated that mouse granulosa cell-oocyte junctional channels transfer most amino acids across the plasma membrane with the same permeability factor (except phenylalanine and leucine), and are considered to be dependent on the granulosa cells-oocyte amino acid transport systems. Hillensyo¨ et al. (1992), using human granulosaluteal cells, identified insulin-like growth factor 1 (IGF-1)-stimulated uptake of the non-metabolizable a-aminoisobutyric acid and increased proliferation. In this study, we first investigated ovarian follicle growth response to IGF-1 using mouse ovarian follicle in vitro cultures. Then, using an optimal concentration of IGF-1, we investigated the uptake of the ketogenic amino acid leucine via the L-transport system in follicles. We provide evidence of specific system L transport activity that increases as pre-antral follicles mature into antral follicles; transport is reduced as the follicle becomes a pre-ovulatory follicle.

& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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background: Little is known about metabolic processes in the developing ovarian follicle. Using mouse ovarian follicles, we investigated uptake of L-leucine by follicles at varying stages of maturity in the presence of insulin-like growth factor (IGF)-1.

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As more work is undertaken with in vitro maturation (IVM) of ovarian follicles, a better understanding of the metabolic processes taking place in ovarian follicles is necessary both to enhance rates of IVM and to ensure normality (i.e. high quality) of the resulting mature follicles created in vitro. There is increasing evidence of obesity-related infertility and studies on metabolic processes in ovarian follicles may also provide important insights into underlying energy utilization pathways essential in folliculogenesis.

Materials and Methods Ovarian follicle collection and culture

Pre-antral follicles were cultured in media containing 0, 10, 50 and 100 ng ml21 IGF-1 (Sigma, Auckland, New Zealand), as described earlier and follicle diameter was measured at the same time each day for 5 days.

L-leucine uptake The ovarian follicle L-leucine uptake experiments were based on previously reported techniques for ovulated oocytes (Colonna and Mangia, 1983a;Colonna et al., 1983b, 1984 ) and for cultured smooth muscle cells (Low et al., 1994). The in vivo L-leucine uptake was measured immediately after dissection. For in vitro L-leucine uptake, follicles were isolated and maintained in culture as stated above. On the fifth day of culture, L-leucine uptake experiments were initiated. All L-leucine uptake experiments were performed in HEPES buffer (Low et al., 1994) containing 5.0 mM KCl, 0.9 mM CaCl2, 1.0 mM MgCl2, 5.6 mM D-glucose, 140 mM NaCl and 25 mM HEPES (pH 7.4). Groups of 10 follicles corresponding to each of the three follicle stages (early antral, antral and pre-ovulatory) were incubated in HEPES buffer containing 0.2 mM 3H-leucine (2 mCi/ml; Amersham, UK) under oil at 378C in a 5% carbon dioxide in air atmosphere. A time-course experiment was performed to establish ovarian follicle L-leucine uptake over time. Uptake experiments were terminated by a 10-fold dilution of the incubation mix with ice-cold HEPES buffer. The follicles were then rapidly washed twice with HEPES buffer to eliminate background binding of 3H-leucine. The labelled follicles were collected by centrifugation (3000g) for 1 min, lysed in 1.0 M sodium hydroxide for 4 h at 378C, or overnight at room temperature, before counting radioactivity using Opti Fluor-O scintillant in a scintillation counter.

Effect of substrate concentration on 3 H-leucine uptake rates Each of the three different follicle categories (7– 10 follicles/culture) were incubated in HEPES-buffered media (100 ml drops) under oil in the presence of increasing 3H-leucine concentrations for 1 h in Nunclon 4-well tissue culture plates at 378C in 5% carbon dioxide in air. The uptake system is the sum of a single saturable system (Vmax[S]/(Km + [S]) and a non-saturable, simple diffusion component V ¼ [Vmax[S])] ¼ Kd ¼ diffusion constant (fmol/min/ng protein), Km ¼ substrate affinity, Vmax ¼ maximal initial velocity (fmol/min/ng protein) and [S] ¼ substrate (3H-leucine) concentration. The kinetics of 3H-leucine uptake were determined using nonlinear regression analysis of Eadie-Hoftsee transformation. The specific rate of uptake was determined by subtracting the simple diffusion component (Kd[S]) from the rate of 3H-leucine uptake measured.

Competitive inhibition of 3H-leucine uptake by 2-aminobicyclo-(2,2,1)-heptane-2carboxylic acid Competitive inhibition experiments were performed using the transport system L substrate inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which binds to the L-type amino acid transport system with high affinity (Shotwell et al., 1983). Follicles (n ¼ 5/incubation) from the three follicle groups (early antral, antral and pre-ovulatory stages) were incubated in 0.2 mM L-leucine together with 2 mM BCH for 3 h at 378C under oil. The labelled follicles were collected and processed as described above.

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All animal experiments were approved by the University of Otago, New Zealand, Animal Ethics Committee. The mice used in these experiments were 26 – 32 day old C57BL/6J × BALB/cJF1 hybrids, and were housed at constant temperature (228C), with controlled lighting (lights on 0700 h and off 1900 h) and at constant humidity. Food and water were provided ad libitum. Follicle isolation and culture were based on previously published procedures (Naydu and Osborn, 1992; Boland et al., 1993; Spears et al., 1994; Chand and Legge, 2011). Briefly, female mice were killed by cervical dislocation and ovaries aseptically removed in ice-cold Leibovitz-15 medium (L-15), supplemented with 3 mg ml21 bovine serum albumin (BSA) fraction V (Gibco-BRL, Auckland, New Zealand). The ovaries were then dissected free of fat and adherent tissues, and transferred to fresh L-15 medium. The follicles in each ovary were isolated using a pair of fine needles (0.45 × 12 mm) under an Olympus SZH141 dissecting microscope (Olympus, Auckland, New Zealand) and selected for culture using the following criteria; spherical shape, the presence of at least two pale translucent granulosa cell layers, an intact theca layer and a diameter of 200 – 220 mm (Hartshorne et al., 1994a,b). To minimize follicular damage, stromal tissues were left attached to the theca layer. Very small follicles were occasionally left attached but were deliberately damaged to prevent further growth. At collection, healthy ovarian follicles had pale, clear granulosa cells and a well-defined darker theca layer. The antral cavity could be identified as light patches and at pre-ovulatory stage a stigma structure was visible (Boland et al., 1994). Atretic follicles were identified as having a darkened appearance, the spherical shape was lost and the follicle had shrunk in size. These atretic follicles also were identified by the uptake of Trypan Blue solution (Invitrogen). Isolated follicles were removed from the dissection dish using a micropipette and washed in fresh, cold L-15 medium before being placed into culture media. The selected follicles were transferred, using glass micropipettes, into Nunclon 4-well tissue culture plates containing 20 ml droplets of a-minimum essential medium (Gibco-BRL). The follicles were cultured at 378C in an atmosphere of humidified air containing 5% carbon dioxide for all experiments unless otherwise stated. The culture media was prepared daily and follicles were transferred into fresh medium daily. For each experiment 30 follicles were isolated and cultured. Follicle diameters were measured at the start of each culture and at the same time daily thereafter using an eyepiece micrometer calibrated at ×64 magnification and an Olympus SZH141 dissecting microscope. The first day of culture was designated as Day 0. Follicle diameter was measured twice at right angles and the mean calculated. The theca layer was included in the measurement. If the follicle was not spherical then the longest visible diameter and one perpendicular to it was averaged (Hartshorne et al., 1994a,b; Chand and Legge, 2011). Follicular morphology was also noted and follicles classified as pre-antral (100– 125 mm diameter), early antral (156– 234 mm), antral (280– 313 mm), preovulatory (≥390 mm) or atretic. The experiments were concluded after 5 days.

IGF-1 dose response

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Effect of trans-stimulation in cultured ovarian follicles Following 5 days of culture, the follicles were washed in HEPES buffer and then incubated in amino acid free HEPES buffer for 3 h at 378C to deplete free intracellular amino acids (Christensen, 1989). The follicles were then incubated for 1-h in HEPES buffer either in the presence of 1.0 mM phenylalanine or in the absence of amino acids, in 140 mM Li+ and 10 mM sucrose. Incubation in 0.2 mM 3H-leucine was carried out for 1, 2 and 3-h periods prior to uptake rates being measured as described above.

Protein assays

Statistical analysis Data are presented as mean + SEM of three separate experiments. Statistical comparisons were made using Student’s paired t-test or analysis of variance and correlation statistics and best fit plots were also analysed. All statistical analysis used GraphPad Prism software. A P-value of , 0.05 was considered significant in data comparisons.

Results Effect of IGF-1 on follicle development in vitro In the presence of IGF-1 (10 –100 ng ml21), follicles exhibited lower rates of atresia compared with follicles cultured without IGF-1 (Fig. 1A). This was also reflected in an increase in follicular maturation rates with 50 and 100 ng ml21 IGF treatment (P , 0.001) (Fig. 1B). An IGF-1 concentration of 100 ng ml21 yielded the highest proportion (64%) of follicles growing to pre-ovulatory stage, or releasing oocytes. In comparison 58, 23 and 17% reached pre-ovulatory follicle stage with 50, 10 and 0 ng ml21, respectively. Furthermore, measurement of follicular diameter demonstrated a greater increase with IGF-1 treatment (Fig. 1C). Follicles grown in 50 ng ml21 were larger on Day 3 and 4 of culture when compared with controls (P , 0.001). In the absence of IGF-1, after the second day in culture follicles demonstrated no increase in follicular diameter, while steady increments in diameter were observed at all time points with IGF-1 treatments (Fig. 1C).

Amino acid concentration on L-leucine uptake rates 3

H-leucine uptake rates (i.e. immediately after dissection) at 0.01 mM leucine was 12.77 + 2.45 fmol/min/ng protein in freshly isolated large antral follicles. The uptake rate increased to 31.68 + 6.44 fmol/

Figure 1 Effect of IGF-1 on mouse follicle maturation in vitro. Immature follicles (160– 190 mm in diameter) were cultured in the absence or presence of increasing concentrations of IGF-1 (0– 100 ng/ml). (A) Follicule atresia rates (B) Percentage of follicles that reached maturation in culture after 4 days. (C) Follicle diameter when cultured in the absence or presence of 50 ng/ml IGF-1. Data are mean + SEM of triplicate experiments, n ¼ 10 – 15 follicles per treatment group, *P , 0.05, **P , 0.01, ***P , 0.001, versus no added IGF (paired Student’s t-test).

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Amino acid uptake data were normalized to protein content. Follicles were pooled in groups of 10 corresponding to the three different stages and lysed by repeated freezing and thawing. The tissue was then homogenized and suspended in 100 ml HEPES buffer. Aliquots of 20 ml were used to determine total protein using the bicinchoninic acid assay (Smith et al., 1985). The reaction was carried out in 1 ml volumes at 378C for 30 min and the absorbance measured at 562 nm using an LKB Ultraspec II spectrophotometer. BSA was used as the protein standard.

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min/ng protein when leucine concentration was increased 10-fold to 0.1 mM (Fig. 2A). A further 10-fold increase in leucine concentration resulted in an uptake rate of 76.76 + 7.76 fmol/min/ng protein. Equilibrium was reached at 3 mM of L-leucine with an uptake of 170.13 + 2.35 fmol/min/ng protein. Maximal initial velocity was 31.47 fmol/ min/ng protein and Km was calculated at 0.018 mM.

Follicle size is dependent on L-leucine uptake rates

To assess if the metabolic activity of follicles differed in vitro and in vivo, uptake studies were carried out with follicles of similar sizes either freshly isolated from ovaries or on the fourth day of culture. Results demonstrate an in vitro uptake rate of 29.63 + 2.02 fmol/min/ng protein. Follicles at the same stage of development (late antral) in vivo demonstrated a similar mean L-leucine uptake of 25.83 + 1.09 fmol/min/ng protein which was not significantly different (P . 0.05).

Competitive inhibition of L-leucine uptake by BCH in vivo In early antral follicles, the presence of BCH decreased the L-leucine uptake rate from 22.54 + 1.05 to 10.65 + 1.04 fmol/min/ng protein, representing an inhibition of 53% of the total rate (Fig. 2C). In follicles at a later stage of antral development (280–313 mm), the uptake rate decreased from 43.19 + 2.66 to 7.41 + 1.57 fmol/min/ng protein with BCH, an inhibition of 83% of total uptake (Fig. 2C). Pre-ovulatory follicles exhibited a L-leucine uptake rate 31.26 + 2.96 fmol/min/ng protein, which decreased to 10.57 + 1.69 fmol/min/ng protein with BCH, an inhibition of 65% (Fig. 2C). The inhibitory effect of BCH

Figure 2 System L amino acid transport rates in developing mouse follicles. (A) Freshly isolated, large antral follicles were incubated in HEPES buffer with increasing concentrations of 3H-leucine for 2 h to determine uptake rates. (B) Comparative rates of 3H-leucine (0.2 mM) uptake in follicles (freshly isolated) at different stages of development. (C) Competitive inhibition of 3H-leucine uptake by (BCH) in follicles. (D) Follicles maintained in culture for 4 days were incubated in HEPES buffer containing 140 mM Li+ and sucrose (zero-trans suppression) or 10 mM phenylalanine (transstimulation) for varying time periods (x-axis). Data are mean + SEM of triplicate experiments, n ¼ 5 – 10 follicles per treatment group, *P , 0.05, **P , 0.01, ***P , 0.001, versus pre-antral 3H-leucine uptake rate (B) or 3H-leucine in non-BCH-treated controls (C).

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We hypothesized that a variation would be observed in the amino acid transport rate of follicles at different stages of development. Freshly isolated follicles with diameters of 100 –125; 156– 234; 280– 313 mm and those larger than 330 mm were classified as pre-antral, early antral, late antral and pre-ovulatory, respectively (Naydu and Osborn, 1992; Oktem and Urman, 2010). Pre-antral follicles displayed the lowest leucine uptake rate of 4.67 + 1.0 fmol/min/ng protein (Fig. 2B). Early antral follicles had an uptake rate of 17.95 + 2.53 fmol/min/ng protein, while follicles at the later stages of antrum formation had an uptake rate of 36.33 + 2.89 fmol/min/ng protein. Pre-ovulatory follicles displayed reduced mean uptake rate of 29.58 + 3.53 fmol/min/ng protein compared with rates of uptake in antral follicles; however, mean rate of uptake was significantly greater than pre-antral uptake rates (P , 0.001, Fig. 2B).

Comparison of uptake rates in vivo and in vitro

3106 on leucine uptake, versus control, was significant at each stage of follicle development.

Trans stimulatory effect on L-leucine transport system

Discussion We have demonstrated a significant effect of IGF-1 in promoting follicle survival and growth in vitro. Follicle morphology, such as increase in diameter, the presence of an antral cavity and visual changes commonly associated with atresia were used as endpoints in monitoring follicle development in vitro. Previous studies have demonstrated a positive relationship between follicle diameter and follicular processes, such as estradiol production and glucose uptake (Boland et al., 1994; Vitt et al., 1998). We have recently shown significant correlations between follicular diameter, antral volume and granulosa cell number by stereological methods of individual follicles that were cultured in vitro (Chand and Legge, 2011). Furthermore, atretic features, such as a darkened appearance, loss of spherical shape and reduced follicle size, were correlated with the presence of pyknotic nuclei within granulosa cells (Chand and Legge, 2011). IGF-1 has a synergistic interaction with FSH on mouse ovarian follicle development (Adashi et al., 1997; Louhio et al., 2000) and has been proposed as a survival factor for early stage ovarian follicles (Louhio et al., 2000). This has been clearly demonstrated with the data presented in the current study, where increasing concentrations of IGF-1 demonstrated a corresponding increase in both follicle growth and survival rate. IGF-1 is also a potent inhibitor of apoptosis and is important in cellular growth promotion, especially via the AKT signalling pathway, stimulating transport and metabolism of glucose, signalling the conversion of cells from oxidative phosphorylation to aerobic glycolysis and the transport of amino acids, which in turn support the mammalian target of rapamycin (mTOR) signalling system (Richards et al., 2002; Plas and Thompson, 2005; Kimball and Jefferson, 2006). In addition, an IGF-1/AKT/mTOR signalling system has been identified which specifically targets L-leucine as a nutrient regulator (Richards et al., 2002). These reports are consistent with our findings that IGF-1 is growth promoting in follicle cultures, and that the uptake of L-leucine is associated with growth. Throughout oogenesis, the L-transport system is predominant (Kaye et al., 1982; Van Winkle et al., 1990). There is a change in the nature of amino acid uptake in the embryo once it reaches the compacted morula stage, with emergence of a Na+-dependent

A-system of transport (Van Winkle et al., 1990). L-leucine is an essential amino acid and its deprivation is sufficient enough on its own to cause enhancement to transport system L (adaptive regulation). L-leucine is also a ketogenic amino acid that is degraded to acetyl Coenzyme A (CoA) or acetoacyl CoA, giving rise to ketone bodies. The current study demonstrated the existence of an L-transport system in the mouse ovarian follicle. Kinetic studies showed the system L-transporters had a Km value of 0.018 mM. This is a low value, which reflects a high affinity to leucine. The maximal initial velocity (Vmax) of a value of 3.03 pmol/h/oocyte has been reported for L-leucine transport in mature, unfertilized mouse oocytes (Colonna et al., 1984). In the current study, Vmax was calculated as 20.76 pmol/h/follicle. In contrast to transport in oocytes, a higher value for follicular Vmax observed reflects higher numbers of L-system transporters and a higher transport activity in the follicle when compared with L-leucine transport in denuded oocytes. We considered that a variation would be observed in the transport rates for leucine uptake at different stages of follicle development. Experimental data proved this to be correct. Pre-antral follicles displayed the lowest uptake rate and when a follicle was committed to growth (early antral) the rate of L-leucine uptake increased markedly, reaching a maximum rate as the follicle progressed to the later antral phase. However, this rate decreased in pre-ovulatory follicles. This uptake pattern corresponds to other cellular uptake systems within the follicle, asserting that once a follicle is committed to growth, there is an increase in both proliferative and metabolic activity: this is supported by the initial increase in L-leucine uptake rates in antral follicles. Increases in granulosa cell mass increase the efficiency of transport owing to the presence of additional plasma membrane transporters and gap junctions. Follicles at pre-ovulatory stages display a lower mean uptake of L-leucine compared with follicles at the mid-antral phase. Bra¨nnstro¨m and Norman (1993) have shown that the induction of interleukin (IL)-b, IL-8, tumour necrosis factor-a and various cytokines stimulated by the LH signal causes an increase in follicular collagenases and gelatinases, enzymes essential for the breakdown of extracellular matrix. A decrease in L-leucine uptake rates may reflect a decrease in granulosa- and oocyte-associated amino acid transporters as the pre-ovulatory follicle prepares for ovulation. To further determine whether L-leucine was being transported by a specific transporter and not by simple diffusion, cis-inhibition experiments with BCH, a non-metabolizable substrate analogue of L-leucine, were performed. The binding of BCH to the transport protein competitively inhibits the transport route of L-leucine and other L-system-specific amino acids (McGiven, 1996). Co-incubation of BCH at a 10-fold excess with L-leucine showed an inhibition of the uptake system, as expected. Results demonstrate that BCH inhibited the uptake of L-leucine by up to 80% depending on the stage of follicle development, suggesting that the transporter specific to the L-system is present in the follicle and is actively involved in the transport of L-leucine. Cis-inhibition was observed in follicles at early antral through to pre-ovulatory stages, suggesting that the L-system transporters are present throughout the developmental process. A comparison of L-leucine uptake rates in vivo and in vitro showed no significant difference between follicles with comparable development, indicating the success of follicle culture at modelling in vivo growth. The model proposed for trans-stimulation suggests that the

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Follicles that been maintained in culture for 4 days were preloaded with L-phenylalanine, another L-system amino acid (trans-stimulation), or were incubated in an amino acid free media (zero-trans suppression) to clear the intracellular amino acid pool. Follicles, preloaded with phenylalanine, when incubated in 3H-leucine, demonstrated a higher uptake rate of 59.67 + 1.02 fmol/min/ng protein within the first 30 min of incubation, compared with follicles in amino acid deficient media, which had an uptake rate of 32.75 + 3.19 mol/ min/ng protein (P , 0.05) (Fig. 2D). Uptake rates decreased rapidly over the 2 h incubation; however, uptake rates for trans-stimulation were higher when compared with the zero-trans suppression experimental groups (P , 0.05, Fig. 2D).

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Authors’ roles M.L. and A.C. jointly designed the study, A.C. performed the data analysis. M.L. wrote the manuscript with contribution from A.C.

Funding This work was supported in part by funding from the University of Otago Bequest funds, Lottery Health. A.C. was the recipient of a New Zealand Vice-Chancellors Committee award.

References Adashi EY, Resnick CE, Payne DW, Rosenfeld RG, Matsumoto T, Hunter MK, Gargosky SE, Zhou J, Bondy CN. The mouse intraovarian insulin-like growth factor 1 system: departures from the rat paradigm. Endocrinology 1997;138:3881 – 3890. Boland NI, Humperson PG, Leese HJ, Gosden RG. Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in-vitro. Biol Reprod 1993;48:798 – 806. Boland NI, Humperson PG, Leese HJ, Gosden RG. Characterization of follicular energy metabolism. Hum Reprod 1994;9:604 – 609. Bra¨nnstro¨m M, Norman RJ. Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function. Hum Reprod 1993; 8:1762 – 1775. Chand AL, Legge M. Stereological assessment of developing mouse ovarian follicles in an in-vitro culture system. Anat Rec 2011; 294:379– 383. Christensen HN. Distinguishing amino acid transport systems of a given cell or tissue. Meth Enzymol 1989;173:576 – 616. Colonna R, Mangia F. Mechanisms of amino acid uptake in cumulus-enclosed mouse oocytes. Biol Reprod 1983a;28:797 – 803. Colonna R, Cecconi S, Buccione R, Mangia F. Amino acid transport in growing mouse oocytes. Cell Biol Int Rep 1983b;7:1007 – 1015. Colonna R, Cecconi S, Buccione R, Mangia F. Stage-dependent modifications of amino acid uptake by antral and metaphase II mouse oocytes. Cell Biol Int Rep 1984;8:3 – 9. Eppig JJ, Pendola FL, Wigglesworth K, Pendola JK. Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport. Biol Reprod 2005;73:351 – 357. Gannon MC, Nuttal FQ, Saeed A, Jordan K, Hoover H. An increase in dietary protein improves blood glucose in persons with type 2 diabetes. Am J Clin Nutr 2003;78:734– 741. Haghighat N, Van Winkle LJ. Developmental change in follicular cell-enhanced amino acid uptake into mouse oocytes that depends on intact gap junctions and transport system Gly1. J Exp Zool 1990; 253:71– 82. Harris SE, Adriaens I, Leese HJ, Gosden RG, Picton HM. Carbohydrate metabolism by murine ovarian follicles and oocytes in vitro. Reproduction 2007;134:415 – 424. Hartshorne GM, Sargent IL, Barlow DH. Meiotic progression of mouse oocytes throughout follicle growth and ovulation in vitro. Hum Reprod 1994a;9:352– 359. Hartshorne GM, Sargent IL, Barlow DH. Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine. Hum Reprod 1994b;9:1003 – 1012. Hillensyo¨ T, Bergh C, Selleskog U. Insulin-like growth factor-1 stimulates amino acid accumulation in cultured granulosa cells. Hum Reprod 1992;7:1094 – 1097. Hinault C, Mothe-Satney I, Gautier N, Lawerence JC, Van Obberghen E. Amino acids and leucine allow insulin activation of the PKB/mTOR pathway in normal adipocytes treated with wortmannin and in adipocytes from db/db mice. FASEB J 2004;18:1894 –1896. Hobbs JG, Kaye PL. Glycine transport in mouse eggs and preimplantation embryos. J Reprod Fert 1985;74:77 –86.

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L- and y+-system transporters act as exchangers (Low and Grigor, 1995). The rate-limiting step in this cycling process is the return of the transporter to the outer side of the cell membrane for binding to new substrate. A high concentration of substrate thus increases the rate of the binding, causing an increase in transport exchange rates. The transport system of L-leucine in the mouse follicle displayed trans-stimulation properties similar to those documented in other cell types, such as for vascular smooth muscle cells (Low and Grigor, 1995) and chinese hamster ovary cell lines (Shotwell et al., 1982). When follicles were preloaded with phenylalanine, an L-system substrate, uptake rates increased owing to increased cycling of the transporter between inner and outer cell membranes. When follicular cells were lacking free intracellular amino acids (zero-trans suppression), the uptake rate was comparatively lower. This suggests that in altered metabolic states, such as insulin insensitivity, changes in amino acid transport could arise owing to trans-stimulation of L- and y+-system transporters, which could potentially impact folliculogenesis and fertility. Many hormones and growth factors have a stimulatory effect on the transport activity of system L (reviewed by Shotwell et al., 1982). Future investigations on the effect of hormones and growth factors on system L transport would be another step towards understanding amino acid transport and utilization in ovarian follicles. To better understand follicle metabolism, the fate of L-leucine after uptake has to be determined. For example, the possibility of the role of de novo protein synthesis of transport carriers could be explored with the use of protein synthesis inhibitors, such as cycloheximide, or blockers of RNA synthesis, such as actinomycin. In summary, the current study provides evidence of a functional system L amino acid transport system in the mouse ovarian follicle. We demonstrate increased follicular influx of L-leucine, within increased follicular maturation, in IGF-1 stimulated follicle cultures. Leucine activates cellular mTOR signalling (Kimball et al., 1999; Lynch et al., 2000) impacting several metabolic pathways. The current rise in infertility caused by metabolic syndrome, encompassing obesity and type 2 diabetes, calls for the study of follicular metabolism and its consequences on follicle development and maturation. Recent investigations demonstrate a role for leucine in altering cellular metabolism. Leucine has been shown to rescue insulin signalling via activation of the mTOR pathway in insulin-resistant db/db mice (Hinault et al., 2004) and increasing dietary leucine intake can restore many metabolic abnormalities in mice fed on a high-fat diet (Macotela et al., 2011). Furthermore human studies demonstrate that a high-protein diet, which increases leucine levels, can improve insulin sensitivity (Gannon et al., 2003; Kalogeropoulou et al., 2008). In the context of human reproduction, this study demonstrates the presence of an influx and efflux mechanism of transport of amino acid substrates across the follicle barriers. Our data provide a profile of the uptake of an essential amino acid during folliculogenesis and suggest that the study of amino acid-dependent regulation of granulosa cell metabolism is vital for understanding the impact of environmental substrates, such as dietary protein intake in the regulation of human fertility.

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