Apelin (APLN) regulates progesterone secretion and oocyte maturation in bovine ovarian cells.

APLN and its G-protein coupled receptor APLNR are expressed in the bovine ovary. However their role in granulosa cells and oocytes is unknown. Here, we studied their expression in bovine ovarian cells and investigated their regulation in cultured luteinizing granulosa cells in response to IGF1 and FSH. We determined the effect and the molecular mechanism of APLN (isoforms 17 and 13) on bovine granulosa cell progesterone secretion and on oocyte maturation. By RT-qPCR and immunoblot, we showed that the expression of both APLN and APLNR in granulosa and oocytes significantly increased with ovarian follicles size whereas it was similar in theca interstitial cells. In vitro, in unstimulated luteinizing bovine granulosa cells and in response to IGF1 (10-8 M) but not to FSH (10-8 M), we observed that APLN (-17 and -13) (10-9 M) increased progesterone production; this was abolished in response to the APLNR antagonist ML221. These latter effects were dependent on the MAPK ERK1/2 kinase. Furthermore, we showed that APLN (-17 and -13) (10-9 M) increased cell proliferation through AKT signaling. Conversely, the addition of APLN-13 and APLN-17 to in vitro maturation medium containing IGF1 (10-8 M) but not FSH (10-8 M) arrested most oocytes at the germinal vesicle stage, which was associated with a decrease in progesterone secretion, an inhibition in MAPK ERK1/2 phosphorylation and an increase in PRKA phosphorylation in oocytes. Thus, APLN can increase progesterone secretion and cell proliferation in bovine luteinizing granulosa cells in vitro, while it blocks meiotic progression at the germinal vesicle stage during bovine oocyte in vitro maturation.

Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.

Timing associated with oviductal sperm storage and release after artificial insemination in domestic hens.

Female birds store sperm in sperm storage tubules (SSTs) in the uterovaginal junction of their reproductive tract for days or weeks (depending on species) before fertilization. Sperm are transported from the SSTs to the infundibulum where fertilization occurs immediately after ovulation of each ovum. The timing of sperm release from the SSTs relative to ovulation is unknown for any bird. Here, we show that, after artificial insemination of domestic fowl Gallus domesticus, sperm are not accepted into any region of the oviduct before sexual maturity. Once hens reach maturity, there is a temporal shift in the distribution of sperm throughout the oviduct. Sperm are first accepted into and accumulate in the SSTs 6 to 8 days before ovulation but are at this point significantly less numerous in the infundibulum. From 1 to 6 days before ovulation, approximately 10-fold more sperm (235 × 10(3) sperm) populate the infundibulum than at 6 to 8 days before ovulation (26 × 10(3) sperm; P < 0.001). Our results suggest that the mechanisms underlying sperm acceptance and release in the oviduct are under fine temporal control, most likely mediated by female hormones.

Automated versus manual sample inoculations in routine clinical microbiology: a performance evaluation of the fully automated InoqulA instrument.

The process of plate streaking has been automated to improve the culture readings, isolation quality, and workflow of microbiology laboratories. However, instruments have not been well evaluated under routine conditions. We aimed to evaluate the performance of the fully automated InoqulA instrument (BD Kiestra B.V., The Netherlands) in the automated seeding of liquid specimens and samples collected using swabs with transport medium. We compared manual and automated methods according to the (i) within-run reproducibility using Escherichia coli-calibrated suspensions, (ii) intersample contamination using a series of alternating sterile broths and broths with >10(5) CFU/ml of either E. coli or Proteus mirabilis, (iii) isolation quality with standardized mixed bacterial suspensions of diverse complexity and a 4-category standardized scale (very poor, poor, fair to good, or excellent), and (iv) agreement of the results obtained from 244 clinical specimens. By involving 15 technicians in the latter part of the comparative study, we estimated the variability in the culture quality at the level of the laboratory team. The instrument produced satisfactory reproducibility with no sample cross-contamination, and it performed better than the manual method, with more colony types recovered and isolated (up to 11% and 17%, respectively). Finally, we showed that the instrument did not shorten the seeding time over short periods of work compared to that for the manual method. Altogether, the instrument improved the quality and standardization of the isolation, thereby contributing to a better overall workflow, shortened the time to results, and provided more accurate results for polymicrobial specimens.

Metformin exposure affects human and mouse fetal testicular cells.

BACKGROUND: Metformin is a drug used in the treatment of diabetes and of some disorders related to insulin resistance, such as polycystic ovary syndrome. Gestational diabetes can cause complications for both mother and child, and some studies have shown a beneficial effect of metformin during pregnancy without an increase in perinatal complications. However, the effects on the gonads have not been properly studied. Here we investigated the effect of metformin administered during pregnancy on the development and function of the fetal testis. METHODS: A dual approach in vitro and in vivo using human and mouse models was chosen. Cultures of human and murine organotypic testes were made and in vivo embryonic testes were analysed after oral administration of metformin to pregnant mice. RESULTS: In human and mouse organotypic cultures in vitro, metformin decreased testosterone secretion and mRNA expression of the main factors involved in steroid production. In vitro, the lowest observed effect concentration (LOEC) on testosterone secretion was 50 µM in human, whereas it was 500 µM in mouse testis. Lactate secretion was increased in both human and mouse organotypic cultures with the same LOEC at 500 µM as observed in other cell culture models after metformin stimulation. In vivo administration of metformin to pregnant mice reduced the testicular size of the fetal and neonatal testes exposed to metformin during intrauterine life. Although the number of germ cells was not affected by the metformin treatment, the number of Sertoli cells, the nurse cells of germ cells, was slightly yet significantly reduced in both periods (fetal period: P = 0.007; neonatal period: P = 0.03). The Leydig cell population, which produces androgens, and the testosterone content were diminished only in the fetal period at 16 days post-coitum. CONCLUSIONS: This study showed a potentially harmful effect of metformin treatment on the development of the fetal testis and should encourage future human epidemiological studies.

Characterization of chicken Sertoli cells in vitro.

In the testis, Sertoli cells play a key physiological role in that they support, nourish, and protect germ cells. Because of the importance of Sertoli cells, several laboratories have established a culture system of Sertoli cells. These cultures have been well developed in mammalian species, but to our knowledge no purified avian Sertoli cells culture has been described. The aim of this study was to isolate avian Sertoli cells and to investigate their function using a chicken model in an in vitro test system. Immature chicken Sertoli cells in culture present morphology similar to that of mammalian cells and conserve expression of the specific Sertoli marker, anti-Müllerian hormone. Furthermore, in contrast to mammals, they express the 3ß-hydroxysteroid dehydrogenase enzyme. Stimulation of Sertoli cells with ovine follicle-stimulating hormone rapidly activates the 3 main downstream signaling pathways of the follicle-stimulating hormone receptor: cyclic( )adenosine monophosphate/protein kinase A, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase pathways. In vitro, Sertoli cells are able to secrete lactate and inhibin and have conserved the phagocytosis property. Finally, avian Sertoli cells present 3 interesting characteristics: they actively proliferate in vitro, can be passaged several times, and are suitable for freezing in nitrogen. A direct consequence of these properties is to use this cell culture test system as an alternative method to bird reprotoxicity studies.

An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential.

We have developed an in vitro model that replicates the composition, organization, and barrier and spermatogenesis functions of the in vivo rat blood-testis barrier. This engineered blood-testis barrier (eBTB) is based on a three-dimensional (3-D) culture in a bicameral chamber of testicular cells isolated from 18-day-old rats. Peritubular cells were cultured on the bottom of the insert. On the top of the insert, a mixture of Sertoli and germ cells were coated within an artificial extracellular matrix, thereby mimicking the basement membrane. The matrix composition was defined to obtain a cord-like organization. This structure was revealed depending on morphogenetic gradients, and was made of polarized Sertoli cells and germ cells in the center of the structure. The in vivo functionality of the BTB was characterized by tight junctions between Sertoli cells. Claudin-11 protein immunodetection suggests that these junctions were also implicated in vitro in the cord-like structure, suggesting the presence of a physical compartment with apical and basal spaces. Measurement of the trans-epithelial electrical resistance characterized the relationship between the Sertoli cells, peritubular cells, and matrix/cells that influenced the tightness of their junctions during the course of the culture. In vitro germ cell differentiation was confirmed with the detection of haploid cells. The development of the eBTB under optimum conditions addresses the involvement of new models, testing the barrier and spermatogenesis functions that are sensitive to chemical compounds from the environment. In this way, the eBTB could be used as an alternative method to animal reprotoxicity studies, and would be of high interest in the scope of regulatory requests for chemical risk assessment.

Role of adiponectin receptors, AdipoR1 and AdipoR2, in the steroidogenesis of the human granulosa tumor cell line, KGN.

BACKGROUND: Adiponectin is involved in the regulation of energy homeostasis and more recently in the reproductive functions. We have previously shown that adiponectin receptors (AdipoR1 and AdipoR2) are expressed in human granulosa cells. However, it remains to be investigated whether both AdipoR1 and AdipoR2 or only one of these receptors serve as the major receptor(s) for adiponectin in human granulosa cells. METHODS: The RNA interference (RNAi) technology was used to specifically knockdown the expression of either AdipoR1 or AdipoR2. Progesterone and estradiol levels in the conditioned media were measured by radioimmunoassay, and determination of cell proliferation by tritiated thymidine incorporation. The levels of adiponectin receptors and proteins involved in the steroidogenesis and in the signalling pathways were examined by western blot. RESULTS: We generated AdipoR1 (R1) and AdipoR2 (R2) knockdown KGN cell lines. R1 cells were apoptotic and had increased expression levels of cleaved caspase 3 and decreased levels of BAD phosphorylation and PCNA as compared with control or parental KGN cells. R2 cells had similar morphology to control or KGN cells. However, they produced less progesterone and estradiol and expressed lower levels of StAR protein in response to FSH or IGF-1 stimulation compared with control cells. Furthermore, the increase of MAPK ERK1/2 phosphorylation in response to human recombinant adiponectin and FSH was lower in R2 than control cells. CONCLUSIONS: In the human granulosa KGN cell-line, AdipoR1 seems to be involved in the cell survival whereas AdipoR2, through MAPK ERK1/2 activation, may be implicated in the regulation of steroid production.

[Role of the fatty acids in ovarian functions: involvement of peroxisome proliferator-activated receptors (PPAR) and adipokines]. / Rôle des acides gras sur les fonctions ovariennes : implications des Peroxisome Proliferator-Activated Receptors (PPAR) et des adipocytokines.

The impact of nutrition and energy reserves on the reproductive functions is known for a very long time. However, the metabolic factors involved in the interactions between nutrition and reproduction are still poorly understood. These factors may be hormones or nutrients (glucose, protein and fatty acids). However, it remains to determine whether these factors act directly or indirectly on the reproductive tissues. In this issue, we briefly summarize the impact of fatty acids on the development of ovarian follicles, oocyte and embryo. We then discuss the current hypotheses about the mechanisms of action of these fatty acids on the ovarian functions. We describe more particularly the role of some receptors of fatty acids, Peroxisome Proliferator-Activated Receptors (PPAR) and Liver X Receptors (LXR) and two adipokines, leptin and adiponectin on ovarian cells.

Inactivation of the IGF-I receptor gene in primary Sertoli cells highlights the autocrine effects of IGF-I.

IGF-I regulates pituitary and gonadal functions, and is pivotal for sexual development and fertility in mammalian species. To better understand the function of autocrine IGF-I in Sertoli cell physiology, we established a system for Cre-mediated conditional inactivation of the IGF-I receptor (IGF-IR) in cultured Sertoli cells. We show here that loss of IGF-IR decreased the number of viable Sertoli cells as a consequence of diminished Sertoli cell proliferation and increased Sertoli cell death. Furthermore, the lack of IGF-IR altered the morphology of cultured Sertoli cells and decreased lactate and transferrin secretions. Collectively, our data indicate that autocrine IGF-I contributes significantly to Sertoli cell homeostasis. The described in vitro system for loss-of-function analysis of the IGF-IR can be readily transposed to study the role of other intratesticular growth factors involved in spermatogenesis.

Middle mesenteric artery: contraindication to endovascular repair of an abdominal aortic aneurysm?

Middle mesenteric artery has been described in 1923. We report the observation of a patient with an abdominal aortic aneurysm who had this rare artery arising from the anterior wall of the aneurysmal sac. His inferior mesenteric artery was occluded at its origin from the aorta and the middle and the distal colon was vascularized only by the middle mesenteric artery. Occlusion of this artery would have been necessary before endovascular repair of the aneurysm. We were concerned about the risk of colic ischemia after the occlusion of the middle mesenteric artery, so we abandoned this approach and operated on the patient via a laparotomy. Based on a case report, we here report a literature overview on the repair of abdominal aortic aneurysm in the presence of a middle mesenteric artery.

Peroxisome proliferator-activated receptors in reproductive tissues: from gametogenesis to parturition.

Peroxisome proliferator-activated receptors (PPARalpha, PPARbeta/delta and PPARgamma) are a family of nuclear receptors that are activated by binding of natural ligands, such as polyunsaturated fatty acids or by synthetic ligands. Synthetic molecules of the glitazone family, which bind to PPARgamma, are currently used to treat type II diabetes and also to attenuate the secondary clinical symptoms frequently associated with insulin resistance, including polycystic ovary syndrome (PCOS). PPARs are expressed in different compartments of the reproductive system (hypothalamus, pituitary, ovary, uterus and testis). Conservative functions of PPARs in mammalian species could be suggested through several in vivo and in vitro studies, especially in the ovary and during placental development. Several groups have described a strong expression of PPARgamma in ovarian granulosa cells, and glitazones modulate granulosa cell proliferation and steroidogenesis in vitro. All these recent data raise new questions about the biologic actions of PPARs in reproduction and their use in therapeutic treatments of fertility troubles such as PCOS or endometriosis. In this review, we first describe the roles of PPARs in different compartments of the reproductive axis (from male and female gametogenesis to parturition), with a focus on PPARgamma. Secondly, we discuss the possible molecular mechanisms underlying the effect of glitazones on PCOS. Like other 'insulin sensitizer' molecules, such as metformin, glitazones may in fact act directly on ovarian cells. Finally, we discuss the eventual actions of PPARs as mediators of environmental toxic substances for reproductive function.

Stenting of a spontaneous dissection of the superior mesenteric artery: a new therapeutic approach?

Spontaneous dissection of the superior mesenteric artery (SMA) is rare and has been reported only sporadically. Therapeutic options are either a surgical approach, which is the more frequently adopted, or a simple observation. We report a case of spontaneous dissection of the SMA with a review of the literature and present a new therapeutic approach.

The insulin-like growth factor axis in cell cycle progression.

Emerging evidence suggests that members of the Insulin-like Growth Factors (IGFs) family, including IGF-I, IGF-II, the IGF-I receptor (IGF-IR), and the IGF-binding proteins (IGFBPs) play a central role in the development and progression of cancer. Cancer cells exhibit an increased and deregulated proliferative activity. Abnormalities in many positive and negative modulators of the cell cycle are also frequent in many cancer types. Recent advances in the understanding of cell-cycle control mechanisms have been applied to outline the molecular mechanism through which IGFs regulate cell cycle progression. In this review, we will provide a brief overview of the role of the IGF system as a regulator of some components of the cell cycle.

[Hepatic lesions after effective external cardiac massage--an iatrogenic complication not to be overlooked]. / Lésions hépatiques après massage cardiaque externe efficace--une complication iatrogène à ne pas négliger.

After external cardiac massage, 3-8% of patients present potentially dangerous liver lacerations. Here we discuss the case of a patient who presented a major haemodynamic instability following external cardiac massage and thrombolytic therapy. Falsely attributed to a cardiac complication, this was actually consecutive to a hepatic haemorrhage originating from liver lacerations caused by the cardiac massage. Haemodynamic stability was obtained by reestablishing blood volume and coagulation factors. Following this the patient evolved favorably. We discuss the importance of evoking the possible existence of abdominal lacerations after external cardiac massage and the therapeutic attitude which must be adopted towards these, particularly in the light of thrombolytic therapy used in interventional cardiology and its effect on these complications.

[Closed injuries of the intrahepatic bile ducts--clinical presentation, diagnosis and treatment]. / Traumatismes fermés des voies biliaires intrahépatiques--présentation clinique, diagnostic et traitement.

Two cases of blunt trauma of the intrahepatic bile ducts are described: one illustrates the difficulty to establish a diagnosis that is often delayed, the other emphasizes the usefulness of the peroperative cholangiography to precise the diagnosis and define the treatment. In the two cases a simple drainage either perihepatic or of the common bile duct resulted in complete healing of the biliary wound. When liver rupture and bleeding complicates a biliary injury, efficient packing allows to reoperate the patient under better conditions the next day. According to the extent and location of the biliary injury, simple drainage, direct suture, bilio-digestive anastomosis and even hepatectomy in rare cases will be indicated.

[Isolated splenic trauma in adults: value of Resciniti's CT scoring system to define the indications for surgery]. / Traumatismes isolés de la rate chez l'adulte. Intérêt de la classification scanographique de Resciniti dans l'indication opératoire.

The data of 62 adult patients with isolated blunt splenic trauma were retrospectively analysed to determine the value of a CT score-system in the choice of treatment: nonoperative treatment versus surgical management. 22 patients (35%) without hemodynamic instability presenting with pain localized in the left flank were primarily managed conservatively. 3 of them subsequently required splenectomy. 40 patients (65%) were operated immediately, 32 on the basis of clinical criteria and 8 on the basis of laboratory criteria. 45 patients with no initial haemodynamic disorders were investigated by abdominal CT-scan. Splenic injuries were retrospectively classified according to Resciniti's CT-scoring system. 13 patients had a splenic injury score > or = 5.5. All of them were operated, 11 early and 2 after failure of conservative management. According to our study this score > or = 5.5, which concerns 21% of our patients, can be considered to be an indication for surgery; in this case, a conservative approach should not be at tempted, even in the absence of immediate clinical and laboratory operative criteria.