Chronic treatment with an agonist of gonadotropin-releasing hormone enhances luteal function in cattle

Our hypothesis was that luteal function, as determined by plasma progesterone concentrations, and corpus luteum (CL) size is enhanced in cattle administered an agonist of GnRH when the CL is developing as compared with administration of an agonist when the CL is fully functional. Cattle were chronically administered a GnRH agonist, azagly-nafarelin, from Day 3 to Day 21 (D3) or Day 12 to Day 21 (D12) or served as untreated control females (Day 0 = behavioral estrus). Blood samples were serially collected on Days 7 and 14 to evaluate LH secretory patterns and twice daily to measure plasma progesterone. Ultrasonographic examinations were conducted daily to record the area of the CL. CL size and plasma progesterone concentrations were both enhanced in the D3 group as compared with the control group. Progesterone was increased in the D12 group on Days 16 and 17 as compared with the control females. Treatment with GnRH agonist increased basal and mean LH concentrations in both D3 and D12 groups as compared with the controls. We rejected our hypothesis because chronic administration of a GnRH agonist increased plasma progesterone when administered both when the CL was developing and when it was fully functional. The enhanced luteal function was likely due to increased basal LH.     

Expression and regulation of adrenodoxin and P450scc mRNA in rodent tissues

The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. This reaction occurs in steroidogenic tissue in the inner mitochondrial membrane, and is mediated by the cholesterol side-chain cleavage enzyme. This enzyme system transfers electrons from NADPH to cholesterol through its three protein components: adrenodoxin reductase, adrenodoxin, and the terminal oxidase, P450scc. We have previously shown that P450scc mRNA is regulated by tropic hormones and cAMP by a cycloheximide-independent mechanism in mouse Leydig tumor MA-10 cells. We now show that the mRNA for adrenodoxin, another component of the cholesterol side-chain cleavage enzyme system, is regulated by tropic hormones and cAMP in MA-10 cells. We cloned rat adrenodoxin cDNA to analyze adrenodoxin mRNA in various rat tissues and in MA-10 cells by RNase protection assays. Adrenodoxin mRNA is found in virtually all rat tissues examined, although it is most abundant in adrenals, ovaries, and testes. MA-10 cells synthesize two species of adrenodoxin mRNA, one of 1.2 kb and the other of 0.8 kb. Both of these adrenodoxin mRNAs are increased approximately six-fold by 1 mM 8-Br-cAMP, five-fold by 10 microM forskolin, and three-fold by both 25 ng/ml hCG and by 100 ng/ml LH. Maximal adrenodoxin mRNA accumulation occurs by 4 h of hormonal stimulation. The cAMP-mediated increase in adrenodoxin mRNA accumulation is independent of protein synthesis, since treatment with cycloheximide or puromycin in the absence or presence of cAMP does not inhibit, and even increases, adrenodoxin mRNA accumulation.     

Growth and pubertal development during and after treatment with a slow-release gonadotropin-releasing hormone agonist in central precocious puberty.

The auxological data of 25 patients (21 girls, 4 boys) with central precocious puberty (CPP), treated for 4 years with a slow-release gonadotropin-releasing hormone agonist [Decapeptyl-controlled release (D-CR) 3.75] every 4 weeks intramuscularly, and of 6 patients (3 girls, 3 boys), treated for 5 years, are presented. After 3 years of D-CR a stabilization of height velocity (HV) at about 4 cm/year was observed. Bone maturation (ratio of change in bone age to change in chronological age; delta BA/delta CA) slowed down to a mean delta BA/delta CA ratio of 0.5 +/- 0.2 (mean +/- SD) measured over 48 months. As a result, predicted adult height (PAH) improved from 156.3 +/- 7.4 to 162.2 +/- 6.8 cm in girls (p less than 0.001) and from 174.4 +/- 18.6 to 184.3 +/- 17.1 cm in boys after 4 years. In the 5th year an ongoing improvement of PAH was observed. 20 additional girls discontinued D-CR for at least 12 months after treatment with D-CR for 2 years or more. In 11 girls menses started after 10.6 +/- 3.1 months; 9 girls had no menarche after 12-16 months. HV increased in the first and second 6 months to a level of about 6.0 cm/year, decreased in the third 6 months after cessation to the level before discontinuing D-CR and decreased further afterwards. Bone maturation (delta BA/delta CA) increased progressively in the first 18 months after discontinuation, with a stabilization at about 1.3. PAH did not change in the first 12 months after discontinuation of D-CR, but showed a decrease afterwards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semi-quantitative RT-PCR analysis of environmental influence on P450scc and PNMT mRNA expression in rat adrenal glands.

Environmental influence on brain function, particularly spatial learning and memory, has been extensively investigated, but little is known about the influence of environmental conditions on the functions of peripheral organs. In the present study, the effects of different housing conditions on the steady-state levels of mRNAs encoding cholesterol side-chain cleavage enzyme (cytochrome P450scc) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands was examined to investigate the environmental influence on both adrenocortical and adrenomedullary functions. Behavioral changes of the animals housed in different conditions were first examined to assess the relevance of environmental manipulation used. In consistent with previous findings, housing of the animals in enriched conditions resulted in the significant reduction of spontaneous motor activity (locomotor activity and rearing) in comparison with housing in isolated conditions, thus indicating the relevance of housing conditions used in this work for investigating the environmental influence on adrenal function. Then, the effects of these housing conditions on P450scc and PNMT mRNA levels in adrenal glands were examined using semi-quantitative RT-PCR method. In comparison with the isolated group, the enriched group showed significantly higher levels of P450scc mRNA. In contrast, PNMT mRNA levels in the enriched group were significantly lower than those in the isolated group. These results propose the possibility that the environmental conditions may cause differential alterations in adrenocortical and adrenomedullary functions, although their possible association with behavioral changes still remains to be elucidated.     

Genotype at the P450scc locus determines differences in the amount of P450scc protein and maximal testosterone production in mouse Leydig cells

A genetic difference in maximal testosterone production in Leydig cells relates to differences in the genotype at the P450scc locus. The genetic relationship between the P450scc gene, the amount of Leydig cell P450scc protein, and maximal testosterone production was determined in the F2 generation of mice derived from SWR/J mice (SWR), a high Leydig cell testosterone-producing strain, and from C3H/HeJ (C3H), a low Leydig cell testosterone-producing strain. A restriction fragment length polymorphism was identified in the P450scc gene between SWR and C3H mice. This restriction fragment length polymorphism was used to identify F2 mice homozygous for the SWR or the C3H alleles of the P450scc gene. The two types of homozygous mice were compared with regard to maximal testosterone production and the amounts of P450scc, P45017 alpha, and 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) proteins. Maximal testosterone production, amounts of P450scc and 3 beta HSD were significantly greater in the SWR than in the C3H progenitor mice. In the F2 mice, homozygous for either the SWR or the C3H allele of P450scc, the differences in maximal testosterone production and the amount of P450scc protein were comparable to the differences in the two progenitor strains. A significant correlation (r = 0.75; P less than 0.01) was found between the amount of P450scc protein and maximal testosterone production. No differences in the amounts of P45017 alpha or 3 beta HSD were observed in the F2 males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian suppression with gonadotropin-releasing hormone agonist reduces whole body protein turnover in women

The age-related decline in fat-free mass is accelerated in women after menopause. The role of ovarian hormone deficiency in the regulation of fat-free mass, however, has not been clearly defined. To address this question, we examined the effect of ovarian hormone suppression on whole body protein metabolism. Whole body protein breakdown, oxidation, and synthesis were measured using [(13)C]leucine in young, healthy women with regular menstrual patterns before and after 2 mo of treatment with gonadotropin-releasing hormone agonist (GnRHa; n = 6) or placebo (n = 7). Protein metabolism was measured under postabsorptive and euglycemic-hyperinsulinemic-hyperaminoacidemic conditions. Ovarian suppression did not alter whole body or regional fat-free mass or adiposity. In the postabsorptive state, GnRHa administration was associated with reductions in protein breakdown and synthesis (P < 0.05), whereas no change in protein oxidation was noted. Under euglycemic-hyperinsulinemic-hyperaminoacidemic conditions, a similar reduction (P < 0.05) in protein synthesis and breakdown was noted, whereas, protein oxidation increased (P < 0.05) in the placebo group. Testosterone, steroid hormone precursors, insulin-like growth factor I, and their respective binding proteins were not altered by GnRHa administration, and changes in these hormones over time were not associated with GnRHa-induced alterations in protein metabolism, suggesting that changes in protein turnover are not due to an effect of ovarian suppression on other endocrine systems. Our findings provide evidence that endogenous ovarian hormones participate in the regulation of protein turnover in women.     

Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat

We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 g/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.     

Suppression of luteal estradiol receptors and progesterone synthesis by a gonadotropin-releasing hormone agonist (WY-40972) during midgestation

Our recent studies demonstrated that the continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag: WY-40972) in early pregnancy or midpregnancy induces abortion in rats by suppressing the plasma levels of progesterone (P) within 24 h. This fall in P levels is not accompanied by a fall in ovarian vein plasma testosterone (T) or estradiol (E). To determine whether the suppression of P by GnRH-Ag at midpregnancy is due to decreased E present in the corpora lutea (CL) and/or a decrease in luteal receptors of E, rats were treated continuously on Days 11-14 of pregnancy with 5 micrograms/day of GnRH-Ag delivered by an osmotic minipump. Ovarian blood samples were obtained on Day 12; at autopsy, CL were harvested and incubated with Medium 199 for 4 h at 37 degrees C under an atmosphere of 95% O2:5% CO2. Additional rats were killed on Day 12 or 14; CL were isolated from the ovary and pooled within the group for measurement of nuclear and cytosolic E receptors. While the net synthesis of P by CL in the GnRH-Ag-treated rats decreased to 40 +/- 14 from 138 +/- 54 ng/CL in controls, T and E levels were not different from their respective controls. Steroid levels in the ovarian vein plasma reflected a similar response. Nuclear E receptors levels were 211 and 198 in controls and 62 and 61 fmoles/mg DNA in the treated group on Days 12 and 14, respectively. These results suggest that GnRH-Ag has no effect on the ability of the luteal synthesis of T and E and that the anti-pregnancy effect of GnRH-Ag may be at the level of the CL due to the direct inhibitory effect of GnRH-Ag on the luteal synthesis of P which, in turn, results in a fall in E receptors in the CL. Alternatively, GnRH-Ag treatment could suppress luteal receptors for rat placental lactogen that, in turn, lower luteal E receptors, leading to a fall in luteal synthesis and release of P.     

Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis

The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.     

Interferon-gamma inhibits steroidogenesis and accumulation of mRNA of the steroidogenic enzymes P450scc and P450c17 in cultured porcine Leydig cells

The inhibitory effects of recombinant porcine interferon-gamma (IFN gamma) on human CG (hCG)-stimulated testosterone production, and on mRNA concentrations of cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20lyase (P450c 17) were investigated using porcine primary Leydig cell culture as a model. After preincubation of Leydig cells for 24 h with 1000 pM IFN gamma, hCG-stimulated (10 ng/ml, 2 h) testosterone production was inhibited by 50%, whereas no significant changes were seen in hCG-stimulated cAMP production. Incubation with 10 microM 5-cholestene-3 beta,22(R)-diol or 10 microM 5-cholestene-3 beta,20 alpha-diol together with hCG (10 ng/ml, 2 h) reversed most of the inhibitory effect of IFN gamma, suggesting that IFN gamma inhibits P450scc activity, possibly by inhibiting the substrate (cholesterol) availability for P450scc. Incubation with IFN gamma also decreased basal concentrations of P450scc (45%) and P450c 17 (35%) mRNA, although these changes probably did not contribute to the decreased testosterone production. Long-term treatment with hCG (100 ng/ml, 24 h) increased P450scc mRNA (3- to 4-fold) and P450c 17 mRNA (4- to 5-fold) concentrations. Simultaneous treatment with IFN gamma attenuated these hCG-induced increases in P450scc mRNA (50%) and P450c 17 mRNA (40-100%) concentrations, as well as in testosterone production (77%). This inhibition of testosterone production could only be partly reversed by the hydroxylated cholesterol derivatives. This suggests that in addition to possible suppression of cholesterol availability, decreased P450scc and/or P450c 17 activities (through decreased mRNA concentrations) were also involved in the IFN gamma suppressed steroidogenic capacity of porcine Leydig cells during long-term hCG stimulation.     

Regulatory role of cytochrome P450scc and pregnenolone in myelination by rat Schwann cells

To investigate the production of steroid hormones by Schwann cells and to examine the regulation of steroid hormone production during myelination, cultures of rat Schwann cells were differentiated into their myelinating phenotype in the absence of neurons with dibutyryl cAMP (db-cAMP). During this process, the expression of P450scc (involved in steroid biosynthesis) was elevated at both the mRNA and protein levels as evident in RT-PCR, Western blots, and immunostaining. Labeling of the cells with [14C] acetate revealed enhanced production of pregnenolone during differentiation into the myelinating phenotype. Disruption of P450scc's activity with an inhibitor diminished the extent of differentiation into the myelinating phenotype as levels of mRNA and protein expression of myelin protein zero (P0) declined. However, the effect was reversed with the addition of pregnenolone. Furthermore, when the differentiating cultures were treated with pregnenolone, mRNA expression of P0 was upregulated, suggesting the stimulation of the differentiation process. Together, these results provide evidence for Schwann cells as a major producer of steroid hormones and pregnenolone production by P450scc as an important regulatory step during myelination.     

Suppression in the expression of a male-specific cytochrome P450, P450-male: difference in the effect of chemical inducers on P450-male mRNA and protein in rat livers

Hypophysectomy of male adult rats caused a 70% decrease in the hepatic level of mRNA hybridized to two specific oligonucleotide probes for the sequence of coding and 3'-noncoding regions of P450(M-1) (H. Yoshioka et al., (1987) J. Biol. Chem. 262, 1706-1711), which corresponds to P450-male. Treatment of hypophysectomized male and female rats with subcutaneous injection of human growth hormone twice a day for 7 days increased the mRNA to a level similar to that of normal male rats. In contrast, the mRNA was decreased by treatment with continuous infusion. These results correlated well with those on the amounts of P450-male protein, indicating that growth hormone regulates the hepatic level of P450-male protein mainly by acting at the pretranslational step. Treatment of adult male rats with phenobarbital (PB), dexamethasone (Dex), or 3-methylcholanthrene (MC) decreased the content of P450-male protein by 68, 36, and 46%, respectively. The content of P450-male protein was also decreased to 65% in Dex-treated hypophysectomized male rats, but was not changed by treatment of hypophysectomized male rats with PB or MC, suggesting that PB and MC decrease P450-male protein through a pituitary growth hormone-mediated process. However, the level of mRNA hybridizable to the P450-male oligonucleotide probe was not decreased, but rather it increased in PB- or Dex-treated hypophysectomized male rats. A similar inconsistent change in protein and mRNA was also observed in PB-treated normal rats. These results indicate that PB and Dex have an additional effect of increasing the hepatic level of the specific mRNA of P450-male/(M-1) or a closely related form. Noncoordinate changes in the level of P450-male protein and mRNA also suggest that the hepatic level of P450-male protein is regulated by plural mechanisms: pretranslational and translational regulation in which pituitary growth hormone and/or other endocrine factors are involved.     

The effects of orchidectomy and replacement therapy on the ultrastructure and gonadotropin-releasing hormone content of the median eminence of the rat

The effects of 2 weeks of orchidectomy and replacement therapy with testosterone upon the content and distribution of gonadotropin-releasing hormone (GnRH) in the median eminence were determined by means of radioimmunoassay and electron microscopy. Photographic montages were prepared from electron micrographs of the lateral median eminence at the point of deepest invagination of the tuberoinfundibular sulcus. Morphometric analysis of photographs of tissues immunohistochemically stained for GnRH was performed to determine changes in the volume density of GnRH-containing axon profiles following the experimental treatments. A decrease in GnRH content after orchidectomy was observed both by morphometric analysis of axon volume density and radioimmunoassay of total GnRH content. Testosterone treatment of orchidectomized animals prevented the postorchidectomy loss of GnRH. Morphometric analysis of conventional electron micrographs revealed an increase in the number of axons containing no dense-core vesicles following orchidectomy, but no decrease in volume density of the neuropil. The results indicate that the change in volume density of immunostained axons was related to the loss of immunostainable dense-core vesicles and not to a change in the size or number of axons. The area corresponding to the location of the highest concentration of GnRH-containing axons was observed to be largely avascular and separated from the vessels of the tuberoinfundibular sulcus by a "border zone" composed of glial foot processes. The unique morphology of the GnRH area has suggested the name "compact zone" to distinguish it from the palisade zone with which it is continuous medially. GnRH axons in this region are probably part of a tract extending farther caudally rather than a terminal field.     

Effect of gonadotropin-releasing hormone agonist treatment in boys with central precocious puberty: final height results

OBJECTIVE: The small number of boys present in most studies on final height (FH) after gonadotropin-releasing hormone agonist (GnRHa) treatment for central precocious puberty (CPP) offers difficulties in the evaluation of the effects of treatment on FH in males. METHOD: We therefore combined FH data from The Netherlands, Italy and France to study the effect of GnRHa treatment in a large group of 26 boys with CPP. RESULTS: The mean chronological age at the start of treatment was 7.6 +/- 2.0 (SD) years, bone age (BA) was 11.0 +/- 2.1 years. All boys were treated with depot formulations of the GnRHa triptorelin with established gonadal suppression for a mean treatment period of 4.7 +/- 2.1 years. FH was 172.9 +/- 6.6 cm. FH standard deviation score (SDS) was -0.66 +/- 1.22, not significantly different from the target height SDS of -0.23 +/- 0.75. FH-SDS was significantly lower in the subgroup of 12 patients with organic CPP compared to patients with idiopathic CPP (-1.34 +/- 1.06 vs. -0.08 +/- 1.06, respectively; p = 0.01), but no difference in height gain was observed. The mean estimated height gain, defined as the difference between predicted and actual adult height was 6.2 +/- 8.7 cm using the average tables of Bayley and Pinneau, and 0.3 +/- 8.6 cm using the BA advance adjusted tables. Regional differences in height gain were observed between the different countries, reflecting different local practices. CONCLUSION: We conclude that GnRHa treatment in boys results in a FH close to target height.     

Reversible reproductive control in harbour seals (Phoca vitulina) with a gonadotropin-releasing hormone agonist

Reproductive control in captive pinnipeds is an important management subject for many facilities. To date reproductive control in harbour seals (Phoca vitulina) has been achieved using anti-androgens, progestagen preparations, castration, and physical separation of the sexes. The harbour seal group at the seal station in Friedrichskoog, Germany consists of three mature females (all >10 years), one older mature male (13 years of age in 2000) and one male who reached maturity during the study (3 years of age in 2000). In 2000 the older mature male received for the first time a 3-month depot injection of a gonadotropin-releasing hormone agonist (buserelin acetetate, 9.9 mg) by subcutaneous injection. This male was subsequently given the gonadotropin-releasing hormone agonist in 2001, 2004 and 2005. The younger male reached maturity during the investigation and received burserelin for the first time in 2004 and again in 2005. No pups were born in 2001, 2002, 2005 or 2006. No reproductive control was performed in 2002 and 2003, resulting in three newborns in 2003 and 2004. Serum levels of testosterone were measured by a routine liquid chromatography coupled with mass spectometry. Pre-burserelin testosterone levels varied between 0.02 and 2.18 ng/ml. Post-burserelin levels were under the detection limit except for the first year of the investigation. No behavioural changes such as changes in social ranking and no clinical side effects were observed. This study shows that the gonadotropin-releasing hormone agonist, burserelin acetate, can be used for reversible reproductive control in harbour seals without observed side effects or detrimental behavioural changes.     

Increased content of mRNA for a precursor of S-adenosylmethionine decarboxylase in rat prostate after treatment with 2-difluoromethylornithine

Total poly(A)-containing mRNA was isolated from rat ventral prostate and translated in a reticulocyte lysate system. The proteins corresponding to S-adenosylmethionine decarboxylase were precipitated with a specific antiserum to this protein. Two proteins were found; one having an Mr of 32,000, which corresponded to the subunit of this enzyme, and a larger protein of Mr 37,000. Immunopurification of polysomes with the antiserum to S-adenosylmethionine decarboxylase followed by mRNA extraction yielded an mRNA preparation which was 10-30% pure mRNA for S-adenosylmethionine decarboxylase. The translation of this mRNA showed clearly that the protein of Mr 37,000 was a precursor of the Mr 32,000 S-adenosylmethionine decarboxylase subunit. Treatment with 2-difluoromethylornithine, which depletes cellular spermidine and is known to increase the content of S-adenosylmethionine decarboxylase protein, led to a 9-fold increase in the content of its mRNA, but treatment with methylglyoxal bis(guanylhydrazone) did not change the mRNA content.