Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50 h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100 nM E2 (17β-oestradiol) or 100 nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2 pmol/cell) at similar rates during the 50 h. After 25 h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50 h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50 h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.
- energy metabolism
- Sertoli cell
Sertoli cells have been classified as the ‘nurse cells’ within the seminiferous epithelium and their main function is to provide the adequate environment for germ cells development [1–3]. Carbohydrate metabolism in Sertoli cells presents some unique characteristics , since the majority of glucose is converted into lactate and not oxidized via the citric acid cycle . On the other hand, germ cells are unable to use glucose and prefer lactate as an energy source . Mullaney et al.  showed that lactate production increases as the Sertoli cells differentiate during pubertal development, although the reasons why Sertoli cells preferentially export lactate for germ cells are not entirely understood . There is evidence that lactate has a crucial role in spermatogenesis , and also shows an anti-apoptotic effect on germ cells .
Lactate is produced, in Sertoli cells, from pyruvate following LDH (lactate dehydrogenase) catalysis and is transported across the plasma membrane to the germ cells by specific proton/MCTs (monocarboxylate transporters). The LDH enzyme family is responsible for the interconversion of pyruvate into lactate, with the concomitant oxidation/reduction of NADH to NAD+ . The pattern of LDH isoenzyme changes dramatically during testicular development. In immature testis, LDH A (LDH chain A) is highly expressed  and is responsible for the conversion of pyruvate into lactate .
Until recently, 14 members of the MCT family have been described in several cells and tissues . Galardo et al.  confirmed the presence of MCT1 and MCT4 in Sertoli cells. MCT1 has a higher affinity and a major role in lactate import from the extracellular milieu , while MCT4, which has a lower affinity for lactate, is primarily a lactate exporter [14,15], being mostly expressed in cells with high glycolytic capacity [13–15].
Glucose transport, the LDH isoenzyme system and lactate transport across the plasma membrane are key targets to be regulated for achieving an adequate lactate supply to germ cells. We aimed to examine the effect of E2 (17β-oestradiol) and 5α-DHT (dihydrotestosterone) on metabolite secretion or consumption in primary cultures of rat Sertoli cells, using 1H-NMR spectra analysis. Due to the importance of lactate to normal spermatogenesis, we also investigated the lactate metabolism and export in Sertoli cells and the possible role of hormonal treatment in LDH A and MCT4 mRNA expression.
MATERIALS AND METHODS
HBSS (Hanks balanced salt solution), DMEM:Ham's F12 (Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12), EDTA, soya-bean trypsin inhibitor, DNase, collagenase type I, E2, 5α-DHT, BSA, ExtrAvidin-Peroxidase Staining Kit, DAB (3,3′-diaminobenzidine) hydrochloride, trypsin-EDTA, ITS (insulin-transferrin-sodium selenite) supplement, TRI Reagent and other drugs were obtained from Sigma–Aldrich (St. Louis, MO, U.S.A.). FBS (fetal bovine serum) was obtained from Biochrom AG. Polyclonal antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). MMLV RT (Moloney-murine-leukaemia virus reverse transcriptase) and random hexamer primers were obtained from Invitrogen. dNTPs were obtained from GE Healthcare (Buckinghamshire, U.K.). 1×Buffer and Taq DNA Polymerase were obtained from Fermentas Life Sciences (Ontario, Canada).
Sertoli cell culture
Male Wistar rats (Charles River Laboratories, Barcelona, Spain) were housed in our accredited animal colony (Health Sciences Research Center, University of Beira Interior). Animals were maintained in specific environmental requirements with free access to standard rodent food and water. All the experiments complied with the ‘Guide for the Care and Use of Laboratory Animals’; published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and in accordance with the Institutional and National guidelines.
Ten male Wistar rats (20-day-old) were killed by cervical dislocation, the testis were immediately excised in aseptic conditions and washed twice in a 50 ml conical tube in 30 ml of ice-cold HBSS containing 10000 units/ml penicillin, 10 mg/ml streptomycin and 25 μg/ml amphotericin B (pH 7.4). Sertoli cells were isolated as previously described by Oliveira et al. [16,17].
Sertoli cell culture purity was revealed by the immunoperoxidase technique. Briefly, cells were grown on six well culture plates, incubated overnight at 4°C with primary polyclonal antibody and processed by the labelled streptavidin–biotin method using an ExtrAvidin-Peroxidase Staining Kit (Sigma–Aldrich) giving a brown coloration to the Sertoli cells after reaction with DAB. The cell nuclei were then stained with haematoxylin. Negative-control incubations were performed by substituting the primary antibody by PBS.
Specific protein markers, anti-Mullerian hormone and vimentin were used to assess the purity of the rat Sertoli cells cultures . Cultures were examined by phase contrast microscopy and were selected if the cell contaminants were below 5% after 96 h.
Sertoli cells were allowed to grow until they reached 90–95% of confluence, and then washed thoroughly and the medium was replaced by serum-free medium (DMEM:Ham's F12, 1:1 with ITS supplement, pH 7.4). In order to evaluate the effects of sex hormones in metabolic secretion/production, and LDH A and MCT4 expression, Sertoli cells were treated with 100 nM E2 or 100 nM DHT. DHT was chosen as representative of the androgen family since it cannot be converted into oestrogens by the cells . The sex steroid hormone concentrations were chosen based on published data that reported that intratesticular interstitial fluid concentrations of those hormones are notably higher than those of circulating plasma, reaching values up to 200 nM [20–24]. Treatments were performed over 50 h, as in previous studies by our research group the presence of DHT and E2 in the culture medium was confirmed up to 72 h (results not shown). Control groups were treated with the same amount of solvent (ethanol) used in DHT and E2 groups (<0.025%, v/v). At the end of the 50 h treatment, the total number of cells per flask was determined with a Neubauer chamber and cells were collected for RNA extraction.
After the 50 h incubation period, the cells were detached with a trypsin-EDTA solution and a viability test was performed on the cells of the different experimental groups using the Trypan Blue Exclusion Test . Viability averaged 85–90%, always with values higher than 85%.
Prior to the stimulus, the cells were washed thoroughly with a HBSS and left for 30 min in DMEM:Ham's F12. The medium was then replaced with Control, E2- or DHT-containing medium. This was considered time zero of incubation. The cells were cultured in flasks containing 15 ml of medium. During the 50 h of incubation, 250 μl of medium was collected at the defined time intervals (5, 10, 25, 35 and 50 h) for 1H-NMR analysis.
To each sample 40 μl of a 10 mM sodium fumarate solution in 99.9% 2H2O was added. 1H-NMR spectra of the collected samples were acquired at 14.1 Tesla, 25°C, using a Varian 600 MHz spectrometer equipped with a 3 mm indirect detection probe with z-gradient (Varian, Palo Alto, CA, U.S.A.) by a method previously described by Alves et al. . Briefly, solvent-suppressed 1H-NMR spectra were acquired with a 6 kHz spectral width, using a 14 s delay for allowing total proton relaxation, 3 s of water pre-saturation, a 45° pulse angle, a 3.5 s acquisition time and at least 64 scans. The relative areas of 1H-NMR resonances were quantified using the curve-fitting routine supplied with the NUTSpro™ NMR spectral analysis program (Acorn, NMR Inc., Fremont, CA, U.S.A.).
1H-NMR spectroscopy was performed to determine lactate production, glucose consumption and variations in other substrates, such as pyruvate and alanine, during the 50 h of cell treatment. Sodium fumarate (final concentration of 2 mM) was used as an internal reference (6.50 ppm) to quantify metabolites in solution. The following metabolites were determined whenever present: lactate, doublet located at 1.33 ppm; alanine, doublet at 1.45 ppm; pyruvate, singlet at 2.36 ppm; and H1-α glucose, doublet at 5.22 ppm (Figure 1).
Metabolite consumption (or production) per cell was calculated by measuring the accumulated variation of the metabolite (versus time zero) at the defined time periods (5, 10, 25, 35 and 50 h) and dividing it by the total number of cells in each flask. The metabolite consumption (or production) rate was calculated for the various time intervals (0–5, 5–10, 10–25, 25–35 and 35–50 h) by dividing the metabolite variation in that time interval by the time and total number of cells in each flask. The concentrations obtained for the different metabolites were corrected for the removed volumes of media.
RT–PCR (reverse transcription–PCR)
At the end of the 50 h treatment, RNAt (total RNA) was extracted from isolated Sertoli cells with TRI Reagent according to the manufacturer's instructions. RNA concentration and absorbance ratios (A260/A280) were spectrophotometry determined (Nanophotometer™, Implen, Germany). RNAt (1 μg) was reverse transcribed in a final volume of 20 μl with 200 units of MMLV RT according to the manufacturer's protocol, using 250 ng of random hexamer primers and 0.5 mM of each dNTP. The resulting cDNA was used with exon–exon spanning primer sets designed to amplify LDH A and MCT4 cDNA fragments. cDNA (1 μl) was amplified in a final volume of 25 μl, containing buffer (2 mM MgCl2 and 0.2 mM dNTP), 0.2 μM of each primer and 0.625 unit of Taq DNA polymerase. Both optimal annealing temperature and the number of cycles required for the amplification phase of fragments are shown in Table 1. PCRs were carried out in triplicate. The expression of LDH A and MCT4 mRNA was normalized with 18S gene expression. Densities from each band were obtained with BIO-PROFIL Bio-1D Software from Quantity One (Vilber Lourmat, Marne-la-Vallée, France) according to standard methods. The band density obtained was then divided by the respective 18S band density and expressed as the fold variation (induction/reduction) versus the control group. For instance, a given X-fold reduction for an mRNA expression indicates that the amount of the specific mRNA, in that situation, is X times smaller than that of the control situation.
The statistical significance of differences in glucose and pyruvate consumption, lactate and alanine production, and in LDH A and MCT4 expression among the experimental groups was assessed by two-way ANOVA, followed by Bonferroni post-test (GraphPad Software). All experimental data are shown as means±S.E.M (n=5 for each condition). P<0.05 was considered statistically significant.
DHT treatment increases glucose consumption
The glucose consumption in the first hours is lower in DHT-treated Sertoli cells; however, after 50 h, the Sertoli cells cultured in control conditions presented a glucose consumption of 28±2 pmol/cell, while E2-treated and DHT-treated cells consumed 29±2 and 36±2 pmol/cell of glucose respectively. The treatment of cultured Sertoli cells with DHT resulted in significantly higher glucose consumption after 50 h, although glucose consumption increased after only 25 h of treatment (Figure 2A).
The glucose consumption rate is smaller in the first 5 h in DHT-treated Sertoli cells (0.20±0.04 pmol·h−1·cell−1) when compared with E2-treated (1.09±0.21 pmol·h−1·cell−1) and cells in the control condition (1.65±0.23 pmol·h−1·cell−1). Between 25 and 35 h of incubation, the glucose consumption rate highly increased in DHT-treated cells that presented a rate of 1.89±0.22 pmol·h−1·cell−1, while cells in the control condition and E2-treated cells consumed glucose at a rate of 1.03±0.22 and 0.44±0.30 pmol·h−1·cell−1 respectively (Figure 2B).
There were no hormonal-related effects in pyruvate consumption
The pyruvate consumption was not dependent on hormonal treatment. After 50 h, the pyruvate consumption was 1.58±0.08, 1.46±0.14 and 1.82±0.05 pmol/cell in control, E2-treated and DHT-treated cells respectively (Figure 2C).
There were also no differences in the pyruvate consumption rate between control, E2-treated and DHT-treated Sertoli cells over the 50 h (Figure 2D).
DHT treatment decreases lactate production
The amount of lactate produced after the 50 h of treatment was similar for control cells and E2-treated cells (18±0.5 and 17±0.5 pmol/cell respectively). During the first 15 h, the lactate production was similar in all conditions; however, after the 50 h treatment with DHT, the cells secreted significantly lower amounts of lactate (15±0.9 pmol/cell; Figure 3A).
The lactate production rate is similar in all conditions during the first 15 h, but it highly increased, between 15 and 25 h, in control cells (0.72±0.11 pmol·h−1·cell−1) when compared with E2-treated (0.44±0.09 pmol·h−1·cell−1) and DHT-treated cells (0.25±0.07 pmol·h−1·cell−1) (Figure 3B). As for glucose consumption, in DHT-treated cells, the lactate production rate peaked between 25 and 35 h of incubation, but after the 50 h treatment they secreted significantly lower amounts of lactate.
E2 treatment highly increases alanine production
The alanine production was very similar in all conditions during the first 25 h of treatment. Throughout that period the control cells, E2-treated and DHT-treated cells secreted 1.05±0.08, 1.22±0.08 and 1.00±0.10 pmol/cell alanine respectively. After the first 25 h, the production of alanine significantly increased in E2-treated cells (1.93±0.08 pmol/cell) when compared with control conditions and DHT-treated cells that produced similar amounts of alanine (1.32±0.9 and 1.16±0.05 pmol/cell respectively). At the end of the 50 h, E2-treated cells secreted significantly higher amounts of alanine than the cells in the other conditions (Figure 3C).
Alanine production rate was very similar in the first 5 h in E2-treated cells (0.39±0.06 pmol·h−1·cell−1), DHT-treated cells (0.41±0.06 pmol·h−1·cell−1) and in cells in the control condition (0.35±0.03 pmol·h−1·cell−1). Between 25 and 35 h, the alanine production rate highly increased in E2-treated cells, which presented a rate of 0.07±0.01 pmol·h−1·cell−1, while control and DHT-treated cells consumed produced alanine at a rate of 0.03±0.002 and 0.02±0.01 pmol·h−1·cell−1 respectively (Figure 3D).
DHT treatment strongly decreases LDH A and MCT4 mRNA levels
After observing a decrease in the lactate levels produced by the Sertoli cells of hormonally treated groups, we investigated the possibility of an effect of DHT and E2 on the mRNA expression levels of LDH A and MCT4. RNAt was extracted from Sertoli cells after 50 h of hormonal treatment and a semi-quantitative RT–PCR was performed to quantify the mRNA levels of LDH A and MCT4.
In DHT-treated cells, a significant decrease in LDH A mRNA levels was observed after 50 h (Figure 4). The levels of LDH A in DHT-treated cells were 0.62-fold reduced when compared with control conditions. With regards to E2-treated cells, a significant decrease of LDH A mRNA levels was also observed after 50 h of treatment (0.81-fold reduction), when compared with the control group (Figure 4). In addition, the results obtained for LDH A expression also show a significantly difference between DHT- and E2-treated cells.
DHT- and E2-treated cells also presented a decrease in MCT4 mRNA levels being 0.83-fold reduced in E2-treated cells and 0.71-fold reduced in DHT-treated cells when compared with the control group after 50 h treatment (Figure 5).
Glucose is the most widely used substrate for ATP production in cell cultures either by oxidative phosphorylation or by glycolysis. It has been described that testes show an unusual dependence on glucose as a source of energy [5,26,27]. However, when subjected to glucose deprivation, Sertoli cells adapt their metabolism to ensure an adequate lactate concentration in the microenvironment where germ cells develop  as germ cells are unable to use glucose as a source of energy. Instead, lactate is pivotal in germ cells fate since it is used as a substrate for ATP production  and exerts an anti-apoptotic effect .
In our experimental conditions, glucose consumption was significantly higher in DHT-treated Sertoli cells than in control or E2-treated cells after 50 h. However, this increase in glucose consumption was not followed by an increase in lactate production, as would be expected. Surprisingly, DHT-treated cells produced less lactate than E2-treated or control cells. This may be explained by a reduced transport of lactate to the extracellular medium, via MCTs, or a decrease in the conversion of pyruvate into lactate catalysed by LDH A.
Control conditions induced the cells to consume more glucose in the first 5 h of incubation, but afterwards this consumption was significantly decreased. Sertoli cells in this basal condition are not subjected to a variety of stimuli, namely from androgens and oestrogens, which are known to have a major role in its physiological functioning and male fertility [29,30]. The lack of these stimuli (when compared with E2- or DHT-treated cells) could be the reason for this behaviour. Androgens and oestrogens interact with their respective receptors [ARs (androgen receptors) and ERs (oestrogen receptors)], which are known to be present in Sertoli cells [29,31], and the primary mechanism of action for these receptors is the direct up- or down-regulation of specific gene transcription, which results in changes of specific protein levels. More recently, it has been shown that the binding to cytoplasmic receptors can cause rapid changes in cell functioning, such as changes in membrane transport rates  that are independent of gene transcription . Hence, Sertoli cells in the absence of these stimuli will probably have their metabolic functioning altered and consume or produce dissimilar amounts of the various metabolites.
Although a change in mRNA level is not a direct measure of the variation on the protein quantity or functioning, it can be a clear indication of the effectiveness of the regulation exerted on the studied protein. Hence, to identify alterations in the mRNA expression of MCT4 and LDH A associated with the presence of the sex steroids, we quantified the mRNAs levels of these enzymes under the different experimental conditions.
The mRNA levels of MCT4, a membrane transporter mainly involved in the export of lactate to the extracellular medium in cells with high glycolytic capacity [13,14], were also significantly decreased after 50 h in DHT- and E2-treated cells, which certainly contribute to the decrease in lactate accumulation in the extracellular medium. The significant decrease in lactate production of DHT-treated cells may also be a consequence of a lower cellular conversion of pyruvate into lactate catalysed by LDH A. In fact, the mRNA levels of LDH A were also significantly decreased in both, E2 and DHT-treated cells, although the decrease was more pronounced after DHT treatment.
These results lead us to conclude that DHT and E2 are key modulators of lactate production/export in Sertoli cells and DHT is more capable of inhibiting the lactate production/export under our experimental conditions. A few reports have studied the hypothesis that sex steroids might regulate LDH A and MCT4 expression in Sertoli cells . Nevertheless, it has been shown that in mice testes, specifically in differentiated germ cells, the expression of MCT2 is under hormonal (follicle-stimulating hormone and testosterone) control , and testosterone reduced MCT2 mRNA levels in a dose-dependent manner in those cells. Also, there is a report in the lizard Hemidactylus flaviviridis, in which it has been described that DHT inhibited the lactate production by Sertoli cells in a dose- and time-dependent manner and E2 markedly suppressed the lactate production also in a dose-dependent manner . In primary cultures of rat Sertoli cells, testosterone has been described as a modulator of fatty acid biosynthesis [3,37]. Conversely, it has been described that lactate production is decreased in Sertoli cells obtained from rats exposed to flutamide, an antagonist of the AR . Although the results described by these authors  seem to be in clear contrast with our results, in which DHT reduces lactate production, one must be conscious that the experimental model used in that report is quite distinct from ours. They subjected the animals to in utero exposure to flutamide, prior to the establishment of primary Sertoli cell cultures. The prolonged and generalized pre-natal exposure to the antagonist used by Goddard et al.  certainly caused a variety of dysfunctions in the organism that must have directly and indirectly influenced testis and Sertoli cells development/functioning. For that reason, their results cannot be directly correlated with the data in the present study, which were obtained using an in vitro approach and in which Sertoli cells were subjected separately and directly to the various experimental conditions. Fix et al.  also presented some divergent results from ours. They used the non-hydrolysable AR agonist R1881 to stimulate Sertoli cells isolated from 15-day-old Sprague–Dawley rats and verified LDH A gene expression was induced within 6 h of stimulation . However, R1881 does not exactly duplicate the effects of testosterone or DHT  and it may exhibit some features that can be called as ‘side effects’ , which could be at least partly responsible for these divergent results.
Nevertheless, all these studies suggest that sex hormones are key factors in Sertoli cells energy metabolism, however, none accurately measured the metabolite production/secretion by Sertoli cells under hormonal treatment.
Grootegoed et al.  described that cultured Sertoli cells oxidized exogenous pyruvate in the presence of glucose. In our experimental conditions, pyruvate consumption was increased not only after treatment with DHT and E2 but also in control conditions that confirm that cultured Sertoli cells highly consume pyruvate when this metabolite is available. According to our results, the cells of the different experimental groups consumed almost all the available pyruvate in the first 5 h of incubation. It seems to be a preferential substrate in our experimental conditions.
The exogenous pyruvate is a substrate in intermediary metabolism that can be converted into lactate by a NADH-dependent reduction. Pyruvate may also be converted into alanine via a transaminase reaction . The ratio of lactate to alanine is an index of the redox state of the cell  as the reduction of pyruvate into lactate or its conversion into alanine is related to the re-oxidation of cytosolic NADH into NAD+, and the lactate/alanine ratio reflects the NADH/NAD+ ratio . The appearance of high alanine can be associated with a reduced redox cytosolic state since the conversion of pyruvate into lactate by LDH is less extensive when lower levels of NADH are present. In our experimental conditions, alanine production was significantly decreased in DHT-treated cells after 50 h when compared with E2-treated cells. The lactate production was also decreased after DHT treatment. This may suggest that DHT is redirecting glucose metabolism to the Krebs cycle and not to lactate or alanine production. The cells under this condition become metabolically more efficient. Results from Gupta et al.  obtained in the epididymis are in accordance with this hypothesis. They investigated the role of sex steroids in the regulation of energy metabolism of epididymis of Rhesus monkey and measured the activity of succinate dehydrogenase and malate dehydrogenase in castrated oestrogen- and DHT-treated animals . Their results indicated that DHT alone stimulated the activities of those enzymes, whereas E2 failed to stimulate any of the enzymes in castrated animals . Thus, at least in the epididymis of monkey testis, sex steroids have been proven to stimulate the activity and expression of enzymes involved in the Krebs cycle and in intermediate pathways coupled with the Krebs cycle. Further works will be needed to accurately measure the influence of sex hormones in the aerobic respiration of Sertoli cells. Nevertheless the results of the present study are a step further in understanding the cellular and biochemical mechanisms associated with dysfunctions at the testicular levels of sex steroid hormones. Some pathological conditions such as Klinefelter's syndrome have been associated with altered androgen and oestrogen levels , a striking feature of the pathology that certainly has a preponderant role in the spermatogenesis dysfunction observed.
In conclusion, glucose metabolism was regulated by hormonal treatment since both DHT and E2 were able to modulate cultured Sertoli cell metabolism and DHT proved to have a greater contribution. Nevertheless, we cannot exclude the utilization of ketone bodies and fatty acids by Sertoli cells, since they are also described as a substrate for cultured rat Sertoli cells . The glycogenolysis pathway may also be hormonally regulated, for it has been described in the presence of glycogen and of glycogen phosphorylase activity in Sertoli cells, indicating the possibility that glycogen hydrolysis fuels the glycolytic pathway in these cells [47,48]. Although observations in Sertoli cell cultures may not represent an in vivo situation exactly, the results of the present study are a step further to identify key mechanisms by which hormones can regulate Sertoli cell metabolism and ultimately spermatogenesis thus the hormonal control of Sertoli cell metabolism has an indirect influence in the reproductive capacity of individuals.
Luís Rato and Marco Alves participated in the experimental design, execution and analysis. Rui Carvalho participated in the 1H-NMR experimental analysis and discussion. Silvia Socorro and José Cavaco participated in the critical discussion of the paper. Pedro Oliveira participated in the overall study design, execution, analysis, paper drafting and discussion.
This work was partially supported by the Pluriannual Programme of the Portuguese Foundation for Science and Technology (FCT). P.F.O. was financed by the FCT (Programme Ciência 2008).
Abbreviations: AR, androgen receptor; DAB, 3,3′-diaminobenzidine; DMEM:Ham's F12, Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12; DHT, dihydrotestosterone; E2, 17β-oestradiol; HBSS, Hanks balanced salt solution; ITS, insulin-transferrin-sodium selenite; LDH, lactate dehydrogenase; LDH A, LDH chain A; MCT, monocarboxylate transporter; MMLV RT, Moloney-murine-leukaemia virus reverse transcriptase; RNAt, total RNA; RT–PCR, reverse transcription–PCR
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