Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes.

STUDY QUESTION: Could whole-exome sequencing (WES) be useful in clinical practice for men with maturation arrest (MA) after a first testicular sperm extraction (TESE)? SUMMARY ANSWER: WES in combination with TESE yields substantial additional information and may potentially be added as a test to predict a negative outcome of a recurrent TESE in patients with MA. WHAT IS KNOWN ALREADY: At present, the only definitive contraindications for TESE in men with non-obstructive azoospermia (NOA) are a 46,XX karyotype and microdeletions in the azoospermia factor a (AZFa) and/or AZFb regions. After a first negative TESE with MA, no test currently exists to predict a negative outcome of a recurrent TESE. STUDY DESIGN, SIZE, DURATION: In a cohort study, we retrospectively included 26 patients with idiopathic NOA caused by complete MA diagnosed after a first TESE. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-six men with MA at the spermatocyte stage in all seminiferous tubules, according to a histopathological analysis performed independently by two expert histologists, and a normal karyotype (i.e. no AZF gene microdeletions on the Y chromosome) were included. Single-nucleotide polymorphism comparative genomic hybridization array and WES were carried out. The results were validated with Sanger sequencing. For all the variants thought to influence spermatogenesis, we used immunohistochemical techniques to analyse the level of the altered protein. MAIN RESULTS AND THE ROLE OF CHANCE: Deleterious homozygous variants were identified in all seven consanguineous patients and in three of the 19 non-consanguineous patients. Compound heterozygous variants were identified in another 5 of the 19 non-consanguineous patients. No recurrent variants were identified. We found new variants in genes known to be involved in azoospermia or MA [including testis expressed 11 (TEX11), meiotic double-stranded break formation protein 1 (MEI1), proteasome 26s subunit, ATPase 3 interacting protein (PSMC3IP), synaptonemal complex central element protein 1 (SYCE1) and Fanconi anaemia complementation group M (FANCM) and variants in genes not previously linked to human MA (including CCCTC-binding factor like (CTCFL), Mov10 like RISC complex RNA helicase 1 (MOV10L1), chromosome 11 open reading frame 80 (C11ORF80) and exonuclease 1 (EXO1)]. LARGE SCALE DATA: Data available on request. LIMITATIONS, REASONS FOR CAUTION: More data are required before WES screening can be used to avoid recurrent TESE, although screening should be recommended for men with a consanguineous family background. WES is still a complex technology and can generate incidental findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirmed the genetic aetiology of MA in most patients: the proportion of individuals with at least one pathologic variant was 50% in the overall study population and 100% in the consanguineous patients. With the exception of MEI1 (compound heterozygous variants of which were identified in two cases), each variant corresponded to a specific gene-confirming the high degree of genetic heterogeneity in men with MA. Our results suggest that WES screening could help to avoid recurrent, futile TESE in men with MA in general and in consanguineous individuals in particular, but these results need to be confirmed in future studies before clinical implementation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Fondation Maladies Rares (Paris, France), Merck (Kenilworth, NJ, USA), IRSF (Montigny le Bretonneux, France) and Agence de la Biomédecine (Saint Denis, France). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

NLRP7 is increased in human idiopathic fetal growth restriction and plays a critical role in trophoblast differentiation.

Fetal growth restriction (FGR) the leading cause of perinatal mortality and morbidity is highly related to abnormal placental development, and placentas from FGR pregnancies are often characterized by increased inflammation. However, the mechanisms of FGR-associated inflammation are far from being understood. NLRP7, a member of a family of receptors involved in the innate immune responses, has been shown to be associated with gestational trophoblastic diseases. Here, we characterized the expression and the functional role of NLRP7 in the placenta and investigated its involvement in the pathogenesis of FGR. We used primary trophoblasts and placental explants that were collected during early pregnancy, and established trophoblast-derived cell lines, human placental villi, and serum samples from early pregnancy (n = 38) and from FGR (n = 40) and age-matched controls (n = 32). Our results show that NLRP7 (i) is predominantly expressed in the trophoblasts during the hypoxic period of placental development and its expression is upregulated by hypoxia and (ii) increases trophoblast proliferation ([3H]-thymidine) and controls the precocious differentiation of trophoblasts towards syncytium (syncytin 1 and 2 and ß-hCG production and xCELLigence analysis) and towards invasive extravillous trophoblast (2D and 3D cultures). We have also demonstrated that NLRP7 inflammasome activation in trophoblast cells increases IL-1ß, but not IL-18 secretion. In relation to the FGR, we demonstrated that major components of NLRP7 inflammasome machinery are increased and that IL-1ß but not IL-18 circulating levels are increased in FGR. Altogether, our results identified NLRP7 as a critical placental factor and provided evidence for its deregulation in FGR. NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies. KEY MESSAGES: NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies.

PreImplantation Factor (PIF*) endogenously prevents preeclampsia: Promotes trophoblast invasion and reduces oxidative stress.

Preeclampsia is a unique pregnancy disorder whose patho-physiology is initiated early in gestation, while clinical manifestations typically occur in mid-to-late pregnancy. Thus, prevention should optimally be initiated in early gestation. The intimate interaction between PIF, secreted early by viable embryos, and its host-mother provides insight into putative mechanisms of preeclampsia prevention. PIF is instrumental at the two critical events underlying preeclampsia. At first, shallow implantation leads to impaired placentation, oxidative stress, protein misfolding, and endothelial dysfunction. Later in gestation, hyper-oxygenation due to overflow of maternally derived oxygenated blood compromises the placenta. The first is likely involved in early preeclampsia occurrence due to reduced effectiveness of trophoblast/uterus interaction. The latter is observed with later-onset preeclampsia, caused by a breakdown in placental blood flow regulation. We reported that 1. PIF promotes implantation, endometrium receptivity, trophoblast invasion and increases pro-tolerance trophoblastic HLA-G expression and, 2. PIF protects against oxidative stress and protein misfolding, interacting with specific targets in embryo, 3. PIF regulates systemic immunity to reduce oxidative stress. Using PIF as an early preventative preeclampsia intervention could ameliorate or even prevent the disease, whose current main solution is early delivery.

EG-VEGF controls placental growth and survival in normal and pathological pregnancies: case of fetal growth restriction (FGR).

Identifiable causes of fetal growth restriction (FGR) account for 30 % of cases, but the remainders are idiopathic and are frequently associated with placental dysfunction. We have shown that the angiogenic factor endocrine gland-derived VEGF (EG-VEGF) and its receptors, prokineticin receptor 1 (PROKR1) and 2, (1) are abundantly expressed in human placenta, (2) are up-regulated by hypoxia, (3) control trophoblast invasion, and that EG-VEGF circulating levels are the highest during the first trimester of pregnancy, the period of important placental growth. These findings suggest that EG-VEGF/PROKR1 and 2 might be involved in normal and FGR placental development. To test this hypothesis, we used placental explants, primary trophoblast cultures, and placental and serum samples collected from FGR and age-matched control women. Our results show that (1) EG-VEGF increases trophoblast proliferation ([(3)H]-thymidine incorporation and Ki67-staining) via the homeobox-gene, HLX (2) the proliferative effect involves PROKR1 but not PROKR2, (3) EG-VEGF does not affect syncytium formation (measurement of syncytin 1 and 2 and ß hCG production) (4) EG-VEGF increases the vascularization of the placental villi and insures their survival, (5) EG-VEGF, PROKR1, and PROKR2 mRNA and protein levels are significantly elevated in FGR placentas, and (6) EG-VEGF circulating levels are significantly higher in FGR patients. Altogether, our results identify EG-VEGF as a new placental growth factor acting during the first trimester of pregnancy, established its mechanism of action, and provide evidence for its deregulation in FGR. We propose that EG-VEGF/PROKR1 and 2 increases occur in FGR as a compensatory mechanism to insure proper pregnancy progress.

Estrogen-related receptor gamma modulates energy metabolism target genes in human trophoblast.

Placenta growth and functions depend on correct trophoblast migration, proliferation, and differentiation. The placenta has a critical role in gas and nutrient transport. To accomplish these numerous functions, the placenta depends on a highly efficient energy metabolism control. Recent studies showed that the orphan nuclear receptor Estrogen-Related Receptor gamma (ERRγ) is highly expressed in human placentas. As ERRγ has been described as a major energy metabolism regulator, we investigated ERRγ expression and putative roles on energy homeostasis in human trophoblast from first trimester placentas. First, we showed that ERRγ expression level increased during pregnancy and that ERRγ was more abundant in villous than in extravillous trophoblasts. We also observed that ERRγ expression increased during trophoblast differentiation. Second, we demonstrated that mitochondrial biogenesis and expression of some energy metabolism target genes decreased when ERRγ expression was impaired. Altogether, these results suggest that ERRγ could be implicated in the energy metabolism regulation of human trophoblasts.

Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

A sequence variation in the promoter of the placental alkaline phosphatase gene (ALPP) is associated with allele-specific expression in human term placenta.

Placental alkaline phosphatase (PLAP), encoded by the ALPP gene, is produced by the fetal side of the placenta. This enzyme displays strong genetic variability. Some of the variants were reported to be associated with pathology of pregnancy. We show here that the two most common ALPP allelic variants, Pl(1) and Pl(2), differ in mRNA expression level. This differential expression was independent of the parental origin and probably results from linkage disequilibrium with the sequence variation rs2014683G>A in the ALPP gene promoter that was shown to have allele-specific binding patterns to placental nuclear proteins. The possible role of this allelic-specific expression in placenta-related pathology is discussed.

Effects of 17beta-estradiol on preadipocyte proliferation in human adipose tissue: Involvement of IGF1-R signaling.

Estrogens are known to stimulate the proliferation of human preadipocytes. However, the molecular mechanisms underlying the increased cell growth by these steroids are poorly understood. In the present study, we have demonstrated that the proliferative effect of 17beta-estradiol involves the induction of both cell cycle gene expressions, c-myc and cyclin D1. Moreover, the mitogenic effects of 17beta-estradiol are suppressed by the pure antagonist ICI 182780 suggesting that estradiol action is mediated by estrogen receptor (ER). We have also shown that 17beta-estradiol is able to inhibit human preadipocyte apoptosis capacity as reflected by DNA fragmentation experiments and the mRNA expression of the pro- and antiapoptotic genes. Finally, 17beta-estradiol significantly induces both mRNA and protein expression of RIGF1 in human preadipose cells via ER and thus reinforces the signaling pathway of the proliferative factor, IGF1. Taken together, these data reinforce the concept of cross-talk between IGF1- and ER-signaling pathways in preadipocytes and indicate that IGFI may be a critical regulator of estrogen-mediated preadipose growth.

Evidence for functional estrogen receptors alpha and beta in human adipose cells: regional specificities and regulation by estrogens.

Adipocytes are estrogen-responsive cells, but the quantitative expression and transcriptional regulation of the estrogen receptors (ER-alpha and ER-beta) in human adipocytes and their precursor cells are unclear. Using real-time quantitative PCR, we have demonstrated that both ER-alpha and ER-beta mRNA are expressed in human mature adipocytes with a large predominance of ER-alpha mRNA. Moreover, ER-alpha mRNA is identically expressed whatever the anatomic origin (intraabdominal and subcutaneous) of the adipocytes and the gender. ER-beta mRNA levels are higher in women compared with men, without regional differences. 17beta-Estradiol in vitro upregulates expression of both ER-alpha and ER-beta mRNA in subcutaneous adipocytes from women but only the ER-alpha mRNA in subcutaneous and intra-abdominal adipocytes from men. In preadipocytes, only the ER-alpha subtype was present. In the latter cells, estrogens in vitro had no influence on ER-alpha expression (mRNA and protein). The present study also shows that estrogens in vitro increase the AP-1, SP-1, and estrogen response element DNA binding activities in differentiated but not in confluent preadipocytes, suggesting that ER become functional during the course of adipogenesis. On the whole, these data are consistent with a predominant role of the ER-alpha subtype in mediating the effects of estrogens on human adipose tissue development and metabolism.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways.

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.

Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2.

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17beta-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (x2) of 17beta-estradiol (0.01 microM) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 microM) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17beta-estradiol (0.01 microM), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17beta-estradiol significantly increased (x1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor gamma2 was up-regulated by 17beta-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 beta-estradiol (0.01 microM) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 microM) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor gamma2 expression (estrogens).

In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones.

As a sexual dimorphism appears in plasma leptin levels, the aim of the present study was to investigate, in vivo and in vitro, the influence of sex steroid hormones on ob messenger RNA (mRNA) and leptin expressions in rat fat cells from various anatomical localizations. In male rats, castration resulted in a modulation of ob gene mRNA expression which was increased by 2-fold in perirenal and half-reduced in sc adipocytes. Moreover, in isolated fat cells from both perirenal and s.c. fat depots, ob gene mRNA expression was reduced by 20% after a 24-h in vitro exposure to dihydrotestosterone (10(-8) M). This effect of dihydrotestosterone on ob mRNA was prevented by exposure to the antiandrogen cyproterone acetate and also by actinomycin D. In contrast, leptin secretion from both perirenal and sc adipocytes was unchanged after 24 h exposure to dihydrotestosterone. In female rats, ovariectomy induced a 25% decrease in ob gene mRNA expression in perirenal fat cells. In vitro studies revealed that a 24-h exposure to 17-beta estradiol (10(-8) M) induced a 1.4-, 1.2-, and 1.75-fold increase in ob mRNA expression and a 3.8-, 1.65- and 2-fold increase in leptin secretion in sc, perirenal and parametrial adipocytes, respectively. Moreover, these effects were prevented by the antiestrogen ICI182780 and also by actinomycin D. Altogether, these results demonstrate that in rat adipocytes, estrogens, and androgens modulate ob gene expression at the mRNA level through sex steroid receptor-dependent transcriptional mechanisms.

Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids.

Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.

Estrogen binding sites in hamster white adipose tissue: sex- and site-related variations; modulation by testosterone.

Estrogen binding sites (ER) were studied, using 17 beta-[3H]estradiol as the ligand, in epididymal or parametrial and subcutaneous adipose tissues of male and female hamsters. Compared with other mammalian fat deposits, intact male and female hamsters possess abundant estrogen binding sites with moderate affinity for estradiol and which occur as a single class of receptor in males but as two populations in females. The levels of estrogen receptors depend on both sex and tissue localization. In males, receptor densities are higher in both localizations when compared to those of females and ER are more abundant in superficial adipose deposits than in the deep fat tissue. In females, there are two estrogen binding populations; the one with the highest affinity is similar to the classical estrogen receptor and both populations are more abundant in deep fat than in subcutaneous deposits, in contrast to male hamsters. These characteristics depend on androgen status: in male adipose tissues, testosterone (TP) up-regulates the ER levels. Conversely, in female fat deposits, TP down-regulates the highest affinity estrogen receptors and the lowest affinity population disappears. Binding affinities are never affected by testosterone. These results suggest that, in hamster adipose tissue, estrogen receptors exhibit site- and sex-related differences, as previously described for androgen receptors. Furthermore, estrogen receptor expression is modulated by the androgen status, depending on gender, which could be related to some physiological situations observed in the hamster.

Enhancement of the expression of the alpha 2-adrenoreceptor protein and mRNA by a direct effect of androgens in white adipocytes.

In vivo, testosterone-treatment of female hamsters for 4 days promotes a doubling of alpha 2-adrenoreceptor protein in parametrial adipocytes, with a concomitant accumulation of the alpha 2A-adrenoreceptor subtype mRNA. During in vitro incubation of minced parametrial fat pads for 6 to 48h with testosterone or dihydrotestosterone (100 nM), alpha 2A-adrenoreceptor protein and mRNA levels were also increased and remained to control levels when an antiandrogen or actinomycin D were added in the medium. It is concluded that in hamster adipocytes, androgens upregulate alpha 2A-adrenoreceptor subtype expression at the mRNA level by an androgen receptor-dependent transcriptional activation.

Androgen receptors in cultured rat adipose precursor cells during proliferation and differentiation: regional specificities and regulation by testosterone.

Different studies suggest that sex hormones affect adipose tissue metabolism and deposition. To investigate the possibility that androgens may play a role in adipose tissue development, we have studied androgen receptors (AR) in rat adipose precursor cells from two different anatomical fat deposits, one deep intraabdominal (epididymal) and one subcutaneous (inguinal) during the proliferation and differentiation processes. AR were quantified by [(3)H]R1881 specific binding in whole cells and the nuclear fraction and were localized by immunocytofluorimetry in both the cytosol and the nucleus. During the proliferative phase, total AR level decreased from D3 to D6. At confluence (D5), AR were higher in epididymal (64±4 fmol/mg protein) than in subcutaneous (33±3 fmoles/mg proteins) preadipocytes and were up-regulated by testosterone but not by 5α-dihydrotestosterone or by 17ß-estradiol. At differentiation (D10-11), nuclear AR decreased by 50% in both precursor fat cell populations when compared to the confluent state (D5) and AR were no more up-regulated but rather down-regulated by testosterone. Because AR are present in preadipocytes and are differently regulated by testosterone depending on the stage of proliferation and differentiation, this study suggests that testosterone may play a role in the control of the adipogenic process.

Protein kinase in rat adipocytes: influence of androgenic status and regional fat distribution.

The effects of castration and testosterone treatment on insulin- and phorbol ester (TPA)-stimulated lipogenic responses, phorbol dibutyrate-specific binding to protein kinase C (PKC) in the cytosol and the beta-PKC isoform level quantified by immunoblotting were compared in rat fat cells from femoral subcutaneous (SC) and deep intra-abdominal (epididymal) fat deposits. In control rats, the PKC content was lower in SC than in epididymal fat cells. After castration, the difference in PKC content between SC and epididymal fat cells was reduced and restored by testosterone treatment. However, androgenic status failed to modify the PKC content in SC fat cells. The lipogenic response to insulin was also differently regulated by the androgenic status in the two fat deposits. After castration, the response was increased in SC fat cells, while it was blunted in epididymal fat cells. These effects were corrected by testosterone administration. These results demonstrate that, in white adipocytes, PKC is an additional biological parameter which varies according to the anatomical origin of the fat cells. They also provide evidence that PKC is controlled by androgens in vivo and emphasize the regional site specificity of such a control.

Regulation of white adipocyte guanine nucleotide binding proteins Gs alpha and Gi alpha 1-2 by testosterone in vivo: influence of regional fat distribution.

The influence of the androgenic status on the steady-state amounts of Gi alpha 1-2 and Gs alpha subunits was compared in hamster fat cell membranes from the femoral subcutaneous (FSC) and epididymal (EP) adipose tissues, using immunoblotting experiments. In sham-operated hamsters, Gi alpha 1-2 and Gs alpha steady-state amounts found in FSC fat cells were 38% and 40% reduced, respectively, as compared to EP adipocytes. In EP fat cells, castration induced a down-regulation of both Gi alpha 1-2 (-39%) and Gs alpha (-33%), whereas testosterone replacement restored Gs alpha, but not Gi alpha 1-2 levels, to control values. In contrast, these G protein alpha-subunits were insensitive to the androgenic status in FSC fat cells. These data provide the first evidence that the androgenic status can modulate the expression of both the Gi alpha 1-2 and Gs alpha subunits of the fat cell adenylate cyclase regulatory Gi and Gs proteins and that this modulation depends on the anatomical origin of these cells.