Unveiling Molecular Effects of the Secondary Metabolite 2-Dodecanone in the Model Hymenopteran Nasonia vitripennis.

Over the past decade, multiple studies have suggested that the secondary metabolites produced by plants against herbivorous insects could be used as biopesticides. However, as the molecular mechanism of action of these compounds remains unknown, it is difficult to predict how they would affect non-target insects; thus, their innocuity needs to be clarified. Here, we investigate, from the molecular level to the organism, the responses of a useful parasitic insect Nasonia vitripennis (Walker, 1836) being exposed at the pupae stage for 48 h (up to 6 days) to sublethal doses (5 µg/L and 500 µg/L) of 2-Dodecanone. 2-Dodecanone altered the gene expression of genes related to ecdysone-related pathways, biotransformation, and cell homeostasis. A significant induction of ecdysone response-genes (EcR, usp, E78, Hr4, Hr38) was detected, despite no significant differences in ecdysteroid levels. Regarding the cell homeostasis processes, the gene l(2)efl was differentially altered in both experimental conditions, and a dose-dependent induction of hex81 was observed. 2-Dodecanone also triggered an induction of Cyp6aQ5 activity. Finally, 2-Dodecanone exposure had a significant effect on neither development time, energy reserves, nor egg-laying capacity; no potential genotoxicity was detected. For the first time, this study shows evidence that 2-Dodecanone can modulate gene expression and interfere with the ecdysone signalling pathway in N. vitripennis. This could lead to potential endocrine alterations and highlight the suitability of this organism to improve our general understanding of the molecular effects of plant defences in insects. Our findings provide new insights into the toxicity of 2-Dodecanone that could potentially be explored in other species and under field conditions for plant protection and pest management as a means to reduce reliance on synthetic pesticides.

Sublethal doses of pyriproxyfen stimulate reproduction and aggressive behavior in a non-target parasitoid wasp.

Insecticides are commonly used to control populations of pests and disease vectors. However, they can have multiple unintended effects on non-target species. Assessing their impacts on the physiology and behavior of beneficial insects, such as biological control agents, is thus necessary to gain insight into the diversity and nature of such side effects. Here, we investigated the effect of sublethal doses of the endocrine disrupting insecticide pyriproxyfen, a juvenile hormone agonist, on females of the solitary ectoparasitoid Eupelmus vuilleti (Hymenoptera: Eupelmidae). These parasitoid wasps can be used as biological control agents to control the cowpea weevil, Callosobruchus maculatus (Coleoptera: Bruchidae), that infests cowpea seeds, Vigna unguiculata (Fabacea). To do so, in addition to classical measures on female fecundity and survival, we focused on female behaviors that can have important consequences on female fitness and host exploitation. First, we showed that pyriproxyfen stimulated egg production without affecting female survival. Second, we observed that low doses of this insecticide had no effect on females' exploration and host discrimination ability but stimulated their aggressiveness when fighting for host access. Although a negative impact on other life-history traits cannot be ruled out at this point, these results showed low doses of pyriproxyfen can have unintended positive effects on ectoparasitoid E. vuilleti females, by enhancing at least temporarily their reproduction and host access in a situation of competition. Our work thus highlights the importance of studying the diversity of possible unintended sublethal effects of pesticides on beneficial insects.

Exposure to pyriproxyfen (juvenile hormone agonist) does not alter maternal care and reproduction in the European earwig.

Sublethal exposure to pesticides can alter the survival and reproduction of a wide range of non-target organisms. However, it remains unclear whether this exposure can alter behaviours that are often essential for long-term population dynamics and maintenance, such as parental care. In this study, we tested the effect of pyriproxyfen exposure (an insect growth regulator) on maternal care in the European earwig, an insect that is both used in pest control in pip-fruit orchards and considered a pest in stone fruit orchards. We exposed 424 females at doses either 10 times lower, equivalent or 10 times higher than normal application rates in French orchards. As maternal care can change over the weeks of family life, we exposed the earwig mothers at five different days before and after egg hatching. We then measured the expression of ten forms of maternal care towards eggs and juveniles, six non-caring behaviours, eggs and juvenile development, metabolic reserves in mothers at egg hatching and females' production of a terminal clutch. First, our results revealed that the three tested doses of pyriproxyfen were non-lethal and confirmed that maternal care decreased throughout both pre- and post-hatching family life. However, we did not detect any effect of pyriproxyfen on maternal care and non-care behaviours, eggs and juvenile development, quantities of lipids, proteins and glycogen in mothers at egg hatching, and on the production of a future clutch. Overall, these findings suggest that the maximal doses of pyriproxyfen authorized in French orchards is likely to have limited effects on the short- and long-term maintenance of populations of the European earwig and raises fundamental questions about the nature of the link between juvenile hormone and parental care in insects.

A comparative genomic approach using mouse and fruit fly data to discover genes involved in testis function in hymenopterans with a focus on Nasonia vitripennis.

BACKGROUND: Spermatogenesis appears to be a relatively well-conserved process even among distantly related animal taxa such as invertebrates and vertebrates. Although Hymenopterans share many characteristics with other organisms, their complex haplodiploid reproduction system is still relatively unknown. However, they serve as a complementary insect model to Drosophila for studying functional male fertility. In this study, we used a comparative method combining taxonomic, phenotypic data and gene expression to identify candidate genes that could play a significant role in spermatogenesis in hymenopterans. RESULTS: Of the 546 mouse genes predominantly or exclusively expressed in the mouse testes, 36% had at least one ortholog in the fruit fly. Of these genes, 68% had at least one ortholog in one of the six hymenopteran species we examined. Based on their gene expression profiles in fruit fly testes, 71 of these genes were hypothesized to play a marked role in testis function. Forty-three of these 71 genes had an ortholog in at least one of the six hymenopteran species examined, and their enriched GO terms were related to the G2/M transition or to cilium organization, assembly, or movement. Second, of the 379 genes putatively involved in male fertility in Drosophila, 224 had at least one ortholog in each of the six Hymenoptera species. Finally, we showed that 199 of these genes were expressed in early pupal testis in Nasonia vitripennis; 86 exhibited a high level of expression, and 54 displayed modulated expression during meiosis. CONCLUSIONS: In this study combining phylogenetic and experimental approaches, we highlighted genes that may have a major role in gametogenesis in hymenopterans; an essential prerequisite for further research on functional importance of these genes.

Sublethal exposure to deltamethrin stimulates reproduction and has limited effects on post-hatching maternal care in the European earwig.

Although pesticides are typically used to limit pest population, the diversity and nature of their unintentional effects on non-target organisms remain unclear. Better understanding these effects requires to carry out risk assessments on key physiological and behavioral processes specific to beneficial insects. In this study, we addressed this question by exposing mothers of the European earwig (a beneficial insect) to two sublethal doses of deltamethrin (a common pesticide in agriculture) during family life and measured the short- and long-term effects on a series of behavioral, physiological, and reproductive traits. Somewhat surprisingly, our results first revealed that high and low doses of deltamethrin enhanced mothers' future reproduction by augmenting their likelihood to produce a second clutch, shortening the number of days until its production, and increasing the resulting number of eggs and their hatching rate. Conversely, the high dose of deltamethrin was detrimental, as it limited maternal brood defence, and reduced food consumption and expression of self-grooming. Finally, other traits were independent of deltamethrin exposure, such as three proxies of family interactions (i.e., distance to the brood, occurrence, and duration of mother-offspring contacts), mothers' walking distance, and mother weight gain during family life. Our study overall demonstrates that sublethal exposure to a pesticide such as deltamethrin can have both positive and negative effects on non-target beneficial insects. It thus emphasizes that focusing on narrow parameters can lead to misleading conclusions about the unintended impacts of pesticides in treated agro-ecosystems and call for better considering this parameters diversity in integrated pest management programs.

Ecdysteroids affect female reproductive status and outcome of contest over hosts in the parasitoid wasp Eupelmus vuilleti.

Ecdysteroids are a family of insect hormones that may play a role in modulating aggressive behavior in reproductive contexts. In Hymenoptera, the few studies investigating the link between ecdysteroid titers, reproduction and aggressiveness during contests concern solely eusocial species. Here, we explored whether ecdysteroid titers influenced female reproductive status as well as aggressiveness and resolution of conflict in a solitary ectoparasitoid, Eupelmus vuilleti (Hymenoptera: Eupelmidae). Eupelmus vuilleti females parasitize and feed upon juvenile stages of Callosobruchus maculatus (Coleoptera: Bruchidae). When two E. vuilleti females are simultaneously present on a patch, they tend to protect the host they exploit by displaying aggressive behaviors towards conspecific competitors. To our knowledge, nothing is known about the association between ecdysteroids and aggressiveness or the outcome of contests for host access in solitary insects. First, we quantified ecdysteroid titers that naturally circulate in females without fighting experience and after a contest over host access. Ecdysteroid titers measured after the contest did not correlate with female aggressiveness during the contest, but winner wasps had higher titers than both losers and females that did not fight. Then, we manipulated hormone titers via injection: ecdysone favored egg maturation (i.e., gonadotropic effect) within 24 h and increased almost immediately the females' probability of winning host access without affecting their aggressiveness. Our results represent an important step in understanding how hormones, such as ecdysteroids, mediate insect behavior during intraspecific competition.

Sublethal exposure to deltamethrin impairs maternal egg care in the European earwig Forficula auricularia.

The application of pesticides typically leads to lethal and sublethal exposure of non-target insects. Whereas our current understanding of these sublethal effects typically focuses on reproductive and physiological parameters, recent works emphasize that sublethal effects on behaviors such as maternal care could be of major importance in non-target species. However, it remained unknown whether these sublethal effects occur in insects. Here, we tested if exposure to sublethal doses of deltamethrin - a pyrethroid insecticide commonly used in crops - alters the expression of maternal egg care in females of the European earwig Forficula auricularia, a predator insect and pest control. Our results first reveal that deltamethrin exposure impaired the expression of three forms of maternal egg care: It decreased the likelihood of mothers to gather their otherwise scattered clutch of eggs, increased the time during which the female abandoned the clutch after a predator attack and reduced egg grooming duration. These sublethal effects did not reflect a lower activity of deltamethrin-exposed females, as these females increased their expression of self-grooming, and deltamethrin exposure did not affect females' exploration and mobility. Finally, we found that the negative effects of deltamethrin on egg care did not modify egg development, hatching rate and juvenile weight, possibly due to the transient effects of deltamethrin on maternal behaviors. Overall, our results reveal that sublethal exposure to a pesticide may diminish maternal egg care in a natural pest control and call for the integration of this measurement in assays on pesticides application.

An evolutionary approach to recover genes predominantly expressed in the testes of the zebrafish, chicken and mouse.

BACKGROUND: Previously, we have demonstrated that genes involved in ovarian function are highly conserved throughout evolution. In this study, we aimed to document the conservation of genes involved in spermatogenesis from flies to vertebrates and their expression profiles in vertebrates. RESULTS: We retrieved 379 Drosophila melanogaster genes that are functionally involved in male reproduction according to their mutant phenotypes and listed their vertebrate orthologs. 83% of the fly genes have at least one vertebrate ortholog for a total of 625 mouse orthologs. This conservation percentage is almost twice as high as the 42% rate for the whole fly genome and is similar to that previously found for genes preferentially expressed in ovaries. Of the 625 mouse orthologs, we selected 68 mouse genes of interest, 42 of which exhibited a predominant relative expression in testes and 26 were their paralogs. These 68 mouse genes exhibited 144 and 60 orthologs in chicken and zebrafish, respectively, gathered in 28 groups of paralogs. Almost two thirds of the chicken orthologs and half of the zebrafish orthologs exhibited a relative expression ≥50% in testis. Finally, our focus on functional in silico data demonstrated that most of these genes were involved in the germ cell process, primarily in structure elaboration/maintenance and in acid nucleic metabolism. CONCLUSION: Our work confirms that the genes involved in germ cell development are highly conserved across evolution in vertebrates and invertebrates and display a high rate of conservation of preferential testicular expression among vertebrates. Among the genes highlighted in this study, three mouse genes (Lrrc46, Pabpc6 and Pkd2l1) have not previously been described in the testes, neither their zebrafish nor chicken orthologs. The phylogenetic approach developed in this study finally allows considering new testicular genes for further fundamental studies in vertebrates, including model species (mouse and zebrafish).

Physiological and biochemical responses to thermal stress vary among genotypes in the parasitic wasp Nasonia vitripennis.

In ectotherm species such as insects, thermal fluctuations represent a major environmental factor driving development, survival and reproduction of individuals. Reproductive traits are particularly sensitive to heat stress that can induce a permanent sterility, or at least hypofertility, of adult males. This study aims to compare physiological and biochemical responses associated to male performances to an exposure of 24 h to moderately high temperature (36 °C) among three inbred lines of N. vitripennis (AsymC, Cor, Oul). Cor males showed very specific metabolic adjustments compared to the two other lines. By contrast, Oul males showed stronger phenotypic adjustment of its life cycle, and produced metabolic water to compensate water loss by heat stress. Finally, AsymC males had probably more difficulties to acclimate at 36 °C, even for a short period, as their adult longevity was significantly reduced. Thus, the ability of developmental plasticity in N. vitripennis males exposed to heat stress appears to be dependent of their genotypes.

Sperm limitation affects sex allocation in a parasitoid wasp Nasonia vitripennis.

Insect reproduction is influenced by various external factors including temperature, a well-studied constraint. We investigated to what extent different levels of sperm limitation of males exposed to different heat stresses (34 and 36 °C) affect females' offspring production and sex allocation in Nasonia vitripennis. In this haplodiploid parasitoid wasp attacking different species of pest flies, we investigated the effect of the quantity of sperm females received and stored in their spermatheca on their sperm use decisions, hence sex allocation, over successive ovipositions. In particular, we compared the sex allocation of females presenting three levels of sperm limitation (i.e., mated with control, 34 °C heat-stressed or 36 °C heat-stressed males) on each host they parasitized. To disentangle the potential reduction of sperm quality after a heat stress exposure from that of sperm quantity, we also explored the clutch size and sex ratio produced by females that were partially sperm limited after copulating with multiply mated males. Independently of their sperm numbers, all types of females produced a similar total number of offspring, but the more limited ones had fewer daughters. Sperm limitation further affected the distribution of daughters' production across time. In addition to constraints acting on female physiology, male fertility should therefore be considered in studies measuring reproductive outputs of insects submitted to heat stresses.

The spatio-temporal partitioning of sperm by males of the prospermatogenic parasitoid Nasonia vitripennis is in line with its gregarious lifestyle.

Male fitness depends on the number of lifetime progeny of their mates and could be constrained by the chance of finding a mate, lifespan and temporal patterns of sperm production and allocation. Here, we used the parasitic wasp Nasonia vitripennis with a two-week lifespan and a gregarious lifestyle, to analyze how the reproductive system is organized to allocate spermatozoa over consecutive matings. Results show that spermatogenesis is synchronized and completed one day before emergence so that males emerge with a full sperm complement. We also found a regulation of spermatozoa transfer between testis and seminal vesicles that allows males to partition small ejaculates over multiple matings. Overall, this study shows that for N. vitripennis, male fertilization potential is determined (1) at the pupal stage, when spermatogenesis takes place to generate a complete life-long stock, (2) on emergence, when transport of spermatozoa from testes to seminal vesicles is initiated and (3) in adulthood, during which spermatozoa are partitioned over successive copulations. Such life history-traits are consistent with the gregarious lifestyle of N. vitripennis.

A combined approach to heat stress effect on male fertility in Nasonia vitripennis: from the physiological consequences on spermatogenesis to the reproductive adjustment of females mated with stressed males.

In recent years, several studies have shown a decline in reproductive success in males in both humans and wildlife. Research on male fertility has largely focused on vertebrates, although invertebrates constitute the vast majority of terrestrial biodiversity. The reduction of their reproductive capacities due to environmental stresses can have strong negative ecological impacts, and also dramatic consequences on world food production if it affects the reproductive success of biological control agents, such as parasitic wasps used to control crop pests. Here Nasonia vitripennis, a parasitic wasp of various fly species, was studied to test the effects of 24h-heat stress applied during the first pupal stage on male fertility. Results showed that only primary spermatocytes were present at the first pupal stage in all cysts of the testes. Heat stress caused a delay in spermatogenesis during development and a significant decrease in sperm stock at emergence. Females mated with these heat-stressed males showed a reduce sperm count stored in their spermatheca. Females did not appear to distinguish heat-stressed from control males and did not remate more frequently to compensate for the lack of sperm transferred. As a result, females mated with heat-stressed males produced a suboptimal lifetime offspring sex ratio compared to those mated with control males. This could further impact the population dynamics of this species. N. vitripennis appears to be an interesting biological model to study the mechanisms of subfertility and its consequence on female reproductive strategies and provides new research perspectives in both invertebrates and vertebrates.

Signaling via class IA Phosphoinositide 3-kinases (PI3K) in human, breast-derived cell lines.

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, ß, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110ß>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not ß- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not ß- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110ß, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110ß function in the absence of PTEN.

Cellular and molecular effect of MEHP Involving LXRα in human fetal testis and ovary.

BACKGROUND: Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10(-4)M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. CONCLUSIONS/SIGNIFICANCE: We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.

GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

[Parasitic wasps, a model for studying male subfertility]. / Les hyménoptères parasitoïdes: des modèles pour l'étude de l'hypofertilité mâle.

Many studies have reported an increase in male reproductive disorders due to the environment. Despite their ecological importance invertebrates have been poorly studied; they are yet affected in the same way as vertebrates by changes in the environment. Different stresses made on parasitic wasps are able to induce a male subfertility. These insects are easy to rear in the laboratory, used to control insect pests, and have other benefits presented in this article. Parasitic wasps are good models for understanding the mechanisms inducing subfertility and may provide new areas for research in vertebrates.

Differential effects of bisphenol A and diethylstilbestrol on human, rat and mouse fetal leydig cell function.

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10(-12) to 10(-5) M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5-10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1-3 days. BPA concentrations as low as 10(-8) M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10(-5) M BPA were required. Similarly, 10(-8) M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10(-5) and 10(-6) M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10(-8) M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.

FSH-stimulated PTEN activity accounts for the lack of FSH mitogenic effect in prepubertal rat Sertoli cells.

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells.

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.

Testis cord differentiation after the sex determination stage is independent of Sox9 but fails in the combined absence of Sox9 and Sox8.

Sox9 and Sox8 are transcription factors expressed in embryonic and postnatal Sertoli cells of the mouse testis. Sox9 inactivation prior to the sex determination stage leads to complete XY sex reversal. In contrast, there is normal embryonic testis development in Sox8 mutants which are initially fertile, but later develop progressive seminiferous tubule failure and infertility. To determine whether Sox9 is required for testis development after the initial steps of sex determination, we crossed Sox9(flox) mice with an AMH-Cre transgenic line thereby completely deleting Sox9 in Sertoli cells by E14.0. Conditional Sox9 null mutants show normal embryonic testis development and are initially fertile, but, like Sox8(-/-) mutants, become sterile from dysfunctional spermatogenesis at about 5 months. To see whether Sox8 may compensate for the absence of Sox9 during embryonic testis differentiation, we generated a Sox9 conditional knockout on a Sox8 mutant background. In the double mutants, differentiation of testis cords into seminiferous testis tubules ceases after P6 in the absence of one Sox8 allele, and after P0 in the absence of both Sox8 alleles, leading to complete primary infertility. Sox9,Sox8 double nullizygous testes show upregulation of early ovary-specific markers and downregulation of Sertoli intercellular junctions at E15.5. Their very low Amh levels still cause complete regression of the Müllerian duct but with reduced penetrance. This study shows that testis cord differentiation is independent of Sox9, and that concerted Sox9 and Sox8 function in post E14.0 Sertoli cells is essential for the maintenance of testicular function.