Serge Nef

Cryptorchidism in mice mutant for Insl3

Impaired testicular descent (cryptorchidism) is one of the most frequent congenital abnormalities in humans, involving 2% of male births. Cryptorchidism can result in infertility and increases risk for development of germ-cell tumours. Testicular descent from abdomen to scrotum occurs in two distinct phases: the trans-abdominal phase and the inguino-scrotal phase. Currently, little is known about the factors that regulate the trans-abdominal phase of testicular descent. Leydig insulin-like hormone (Insl3) is a member of the insulin hormone superfamily expressed in the developing testis. We show here that mice mutant for Insl3 are viable, but exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and infertility. Female homozygotes have impaired fertility associated with deregulation of the oestrus cycle. These findings reveal roles for Insl3 in the development of the urogenital tract and in female fertility. Insl3 may act as a hormone to regulate the growth and differentiation of the gubernaculum, thereby mediating intra-abdominal testicular descent.

Brain-Derived Neurotrophic Factor Conditional Knockouts Show Gender Differences in Depression-Related Behaviors

BACKGROUNDnIndirect evidence suggests that loss of brain-derived neurotrophic factor (BDNF) from forebrain regions contributes to an individuals vulnerability for depression, whereas upregulation of BDNF in these regions is suggested to mediate the therapeutic effect of antidepressants.nnnMETHODSnWe have tested this hypothesis by generating two independent lines of conditional BDNF knockout mice in which the BDNF gene is deleted selectively in forebrain.nnnRESULTSnWe show that male conditional knockouts exhibit hyperactivity but normal depression-related behaviors. In contrast, female conditional knockouts display normal locomotor activity but a striking increase in depression-like behavior. We also demonstrate that loss of BDNF in both male and female mice attenuates the actions of the antidepressant desipramine in the forced swim test.nnnCONCLUSIONSnThese gender differences in depression-related behaviors in BDNF conditional knockout mice provide direct evidence for a role of BDNF in depression. The results also support the view that forebrain BDNF may be essential in mediating antidepressant efficacy.

Conditional deletion of TrkB but not BDNF prevents epileptogenesis in the kindling model.

Epileptogenesis is the process whereby a normal brain becomes epileptic. We hypothesized that the neurotrophin brain-derived neurotrophic factor (BDNF) activates its receptor, TrkB, in the hippocampus during epileptogenesis and that BDNF-mediated activation of TrkB is required for epileptogenesis. We tested these hypotheses in Synapsin-Cre conditional BDNF(-/-) and TrkB(-/-) mice using the kindling model. Despite marked reductions of BDNF expression, only a modest impairment of epileptogenesis and increased hippocampal TrkB activation were detected in BDNF(-/-) mice. In contrast, reductions of electrophysiological measures and no behavioral evidence of epileptogenesis were detected in TrkB(-/-) mice. Importantly, TrkB(-/-) mice exhibited behavioral endpoints of epileptogenesis, tonic-clonic seizures. Whereas TrkB can be activated, and epileptogenesis develops in BDNF(-/-) mice, the plasticity of epileptogenesis is eliminated in TrkB(-/-) mice. Its requirement for epileptogenesis in kindling implicates TrkB and downstream signaling pathways as attractive molecular targets for drugs for preventing epilepsy.

TrkB Has a Cell-Autonomous Role in the Establishment of Hippocampal Schaffer Collateral Synapses

Neurotrophin signaling has been implicated in the processes of synapse formation and plasticity. To gain additional insight into the mechanism of BDNF and TrkB influence on synapse formation and synaptic plasticity, we generated a conditional knock-out for TrkB using the cre/loxp system. Using three different cre-expressing transgenic mice, three unique spatial and temporal configurations of TrkB deletion were obtained with regard to the hippocampal Schaffer collateral synapse. We compare synapse formation in mutants in which TrkB is ablated either in presynaptic or in both presynaptic and postsynaptic cells at early developmental or postdevelopmental time points. Our results indicate a requirement for TrkB at both the presynaptic and postsynaptic sites during development. In the absence of TrkB, synapse numbers were significantly reduced. In vivo ablation of TrkB after synapse formation did not affect synapse numbers. In primary hippocampal cultures, deletion of TrkB in only the postsynaptic cell, before synapse formation, also resulted in deficits of synapse formation. We conclude that TrkB signaling has a cell-autonomous role required for normal development of both presynaptic and postsynaptic components of the Schaffer collateral synapse.

In vivo role of truncated trkb receptors during sensory ganglion neurogenesis

The mammalian trkB locus undergoes alternative splicing to produce two different types of brain-derived neurotrophic factor receptors. The first type is the full-length receptor tyrosine kinase (TrkB(Tk+); the second type is a truncated receptor lacking the intracellular tyrosine kinase domain (TrkB(Tk-)). To investigate the function of both types of TrkB receptor in vivo, we have generated knockout mice lacking all isoforms of the TrkB receptor (trkB-/-) and compared sensory neuron survival in these mice to that in the previously described TrkB kinase domain knockout mice (trkB(k)-/-). We observed that the presence of truncated TrkB receptors in trkB(k)-/- mice results in more severe sensory neuron losses. Increased neuron losses associated with the presence of truncated TrkB were most severe in regions where neuron survival is most dependent on brain-derived neurotrophic factor and neurotrophin-3. Our data suggest that truncated TrkB receptors negatively influence neuron survival by interfering with the function of catalytic TrkB receptors.

The Insulin-3 Gene: Lack of a Genetic Basis for Human Cryptorchidism

PURPOSEnThe etiology of cryptorchidism appears to be multifactorial and related to hormonal and mechanical factors. Recently, the insulin-3 gene (INSL3) was noted to have a role in mouse gubernacular development and testicular descent. Knockout male mice for the INSL3 gene show isolated bilateral cryptorchidism. This phenotype suggests that INSL3 may have a role in the development of human cryptorchidism. Using single strand conformational polymorphism analysis we detected mutations of the INSL3 gene in boys with cryptorchidism.nnnMATERIALS AND METHODSnGenomic DNA from 118 boys with cryptorchidism and 48 normal controls were obtained from 3 institutions. Using polymerase chain reaction with INSL3 sequence specific primers DNA fragments were analyzed using single strand conformational polymorphism reactions. Samples with band shifts were re-amplified and sequenced to detect mutations.nnnRESULTSnA single base substitution (G greater than A) causing an amino acid change (missense mutation) was identified in 27 of 118 cryptorchid (23%) samples and 12 of 48 normal (25%) samples. Two other base substitutions did not produce alterations in the amino acid sequence (silent mutations).nnnCONCLUSIONSnAlthough a common polymorphism was detected in the INSL3 gene, no specific mutations were detected in a large population of individuals with cryptorchidism. Therefore, mutations in the coding region of the INSL3 gene are not a common cause of human cryptorchidism.

LEYDIG INSULIN-LIKE HORMONE, GUBERNACULAR DEVELOPMENT AND TESTICULAR DESCENT

PURPOSEnTesticular descent is controlled by 2 morphological and hormonal steps. Transabdominal testicular descent is mediated by gubernacular swelling and regression of the cranial suspensory ligament. Müllerian inhibiting substance (MIS) has been proposed to stimulate the swelling but this remains controversial. Recently, a mouse mutant for Leydig insulin-like hormone (Insl3) was found to have undescended testis and deficient gubernaculum. We examine the testicular position of Insl3 mutant mice and the development of gubernacula.nnnMATERIALS AND METHODSnMice with Insl3 homozygotes (-/-), heterozygotes (+/-) and wild-types (+/+) were examined at embryonic day 16.5 and birth. Macroscopic dissections and measurements of the testicular position, as well as microscopic analysis (hematoxylin and eosin, and Massons trichrome) were performed.nnnRESULTSnOf the mice 11 Insl3 homozygote males had significantly impaired testicular descent at embryonic day 16.5 and birth (p <0.01), and the cord was thin and elongated, while 14 heterozygotes and 7 wild-types had normal testicular descent. Microscopically, the gubernaculum of Insl3 homozygotes was small with some muscle development but no central core of mesenchyme at embryonic day 16.5. On the other hand, heterozygotes and wild-types had normal gubernacular development with a swelling reaction.nnnCONCLUSIONSnInsl3 mutants show feminized gubernaculum with deficient mesenchymal core. Insl3 appears to have some role in the gubernacular swelling reaction in mice.

Effects of Orchiopexy on Congenitally Cryptorchid Insulin-3 Knockout Mice

PURPOSEnInsulin-3 (Insl3) knockout mice exhibit isolated, bilateral, high intra-abdominal cryptorchidism. If left in this position until adulthood, these testes will deteriorate to the Sertoli-cell-only state, leading to infertility in 100%. We examined the effect of orchiopexy in this genetically engineered animal model.nnnMATERIALS AND METHODSnA total of 160 testes from 80 male offspring of Insl3 F crosses underwent either no orchiopexy (29 testes), sham surgery (25 testes), 1-stage Fowler-Stephens scrotal orchiopexy (57 testes) or primary orchiopexy into a low abdominal, subcutaneous pouch (49 testes). A group of postoperative mice underwent fertility testing 3 months postoperatively. At 6 to 9 months postoperatively the testes were harvested and histologically analyzed.nnnRESULTSnTestes were atrophic in 100% (25 of 25) of the sham group, 91% (52 of 57) of the Fowler-Stephens orchiopexy group and 33% (16 of 49) of the subcutaneous pouch group (testis weight less than 50 mg.). Fertility testing was done in 30 mice (sham 5, Fowler-Stephens orchiopexy 17 and subcutaneous pouch 8). Infertility was secondary to bilateral testicular atrophy (sham 5 of 5, Fowler-Stephens orchiopexy 13 of 17), spermatogenic maturation arrest (subcutaneous pouch 8 of 8) and ductal obstruction [normal spermatogenesis with epididymis devoid of sperm] (Fowler-Stephens orchiopexy 2 of 17). Fertility with normal spermatogenesis was observed in 2 of 17 Fowler-Stephens orchiopexy mice, both of which were Insl3 knockout mice.nnnCONCLUSIONSnIntrascrotal orchiopexy can rescue these congenitally cryptorchid Insl3 knockout testes from their intra-abdominal fate of Sertoli-cell-only and lead to fertility. The results suggest that orchiopexy has a crucial and central role in preservation of spermatogenesis.

Altered regulation of brain-derived neurotrophic factor protein in hippocampus following slice preparation

Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially punctate pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.

Loss of Insl3: A Potential Predisposing Factor for Testicular Torsion

PURPOSEnThe testicular hormone Insl3 is critical for mouse gubernacular development. Knockout mice exhibit bilateral intra-abdominal cryptorchidism with absent gubernaculum. Prior studies described torsion of the vas deferens in Insl3 mutant mice. We performed a detailed anatomical analysis of the vas deferens and testis in Insl3 mutant mice to characterize associated anomalies further.nnnMATERIALS AND METHODSnInsl3 wild-type (Insl3(+/+)), heterozygous (Insl3(+/-)) and knockout (Insl3(-/-)) male mice were examined either prepubertally (postnatal day 23) or in adulthood (postnatal day 90 or later). The macroscopic appearance, characteristics, and mobility of the testes and spermatic cord were recorded.nnnRESULTSnWe examined 56 prepubertal and 33 adult mice (175 testes, 28 [20:8] Insl3(+/+), 97 [60:37] Insl3(+/-), 50 [32:18] Insl3(-/-)). Unlike normal Insl3(+/+) testes, 94% of Insl3(-/-) testes were located intra-abdominally at all ages. Delayed descent occurred in Insl3((+/-)) testes, since 37% of postnatal day 23 and 8% of P90 or later testes were intra-abdominal. Vas elongation/convolution and spermatic cord twisting were noted in 65% of Insl3(-/-), 27% of Insl3((+/-)) and 0% of Insl3(+/+) testes. While all Insl3(+/+) testes were normal, 5% of Insl3((+/-)) and 32% of Insl3(-/-) testes showed significant testicular pathology, including torsion, atrophy and vanished testis, which statistically increased with age.nnnCONCLUSIONSnPoorly formed gubernacula and increased testicular mobility in Insl3 mutant mice result in spermatic cord anomalies, delayed/absent testicular descent and subsequent testicular torsion in a gene dose dependent manner. Prepubertal testicular torsion in the mutant mice predisposes to testicular atrophy and vanishing testes in adulthood. Thus, Insl3 is a candidate signaling molecule in human delayed testicular descent and torsion.