Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.

DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.

C-Kit mutant mice have a selective loss of vagal intramuscular mechanoreceptors in the forestomach.

Intramuscular arrays are one of two major classes of vagal afferent mechanoreceptors that innervate the smooth muscle wall of the proximal gastrointestinal tract. They consist of rectilinear telodendria that distribute in the muscle sheets, parallel to the long axes of muscle fibers. Intramuscular arrays appear to make direct contact with the muscle fibers, but they also course on, and form appositions with, intramuscular interstitial cells of Cajal. These complexes formed by intramuscular arrays and intramuscular interstitial cells of Cajal suggest that intramuscular arrays might require either structural or trophic support of the interstitial cells of Cajal for normal differentiation and/or maintenance. To evaluate this hypothesis, we have examined the morphology and distribution of vagal afferent endings in the c-Kit mutant mouse that lacks intramuscular interstitial cells of Cajal. Vagal afferents were labeled by nodose ganglion injection of either wheat germ agglutinin-horseradish peroxidase conjugate or a tagged dextran, and the labeled afferent terminals in the stomach were mapped using a standardized quantitative sampling scheme. Intramuscular arrays were dramatically reduced (in circular muscle by 63%; in longitudinal muscle by 78%) in the c-Kit mutant mice relative to their wild-type littermates. Additionally, a substantial number of the surviving axons and terminals in the mutant stomachs were morphologically aberrant. Moreover, the loss of intramuscular arrays in mutants appeared to be selective: the structure, distribution and density of intraganglionic laminar endings, i.e., the other vagal mechanoreceptors in smooth muscle, were not significantly altered. Finally, the conspicuous decrease in intramuscular array density in mutants was associated with a non-significant trend toward loss of nodose ganglion neurons. Collectively these findings suggest that interstitial cells are required for the normal development or maintenance of vagal intramuscular arrays. Therefore, the c-Kit mutant mouse will be valuable for determining the role(s) of interstitial cells in intramuscular array development as well as for providing an animal model with the intramuscular array class of vagal afferents selectively ablated.

Gastrointestinal projection maps of the vagus nerve are specified permanently in the perinatal period.

The vagal innervation of the proximal gastrointestinal (GI) tract is lateralized. To determine whether this pattern is specified as early as the perinatal period, neonatal rat pups were given unilateral cervical vagotomies. Separate groups received (1) transections below the left nodose ganglion, (2) left cervical resections that included removal of the nodose ganglion, or (3) sham surgeries. At 4 months of age, each animal's vagal afferent projections from the unoperated side were mapped by injecting the nodose with WGA-HRP, preparing the stomach as wholemounts, and processing the tissue with tetramethyl benzidine. The two types of vagal afferent endings in GI smooth muscle, namely intraganglionic laminar endings and intramuscular arrays, were surveyed separately, and their regional distributions were mapped. Changes in the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMNX) were assessed with cell counts and area measurements. Neonatal loss of the vagus innervating one side of the GI tract, with or without ganglionectomy, did not cause the unoperated vagus to sprout to the denervated side. In addition, removal of the projections to the one side of the target organ did not produce a reorganization of the projection maps of the unoperated vagus within its normal or ipsilateral wall of the GI tract. Although the regional patterns of the unoperated ipsilateral vagus were not affected, the packing densities of both types of afferents supplied by this trunk were moderately reduced. The DMNX of the vagotomized side displayed extensive (approximately 83%) neuronal loss; the DMNX on the unoperated side as well as the NST on both sides exhibited limited (approximately 20--25%) losses. The lack of a peripheral projection field reorganization -- except for a moderate down-regulation -- after complete unilateral denervation suggests that both the laterality and the afferent terminal phenotypes (or target tissues) of the vagus in the proximal GI tract are specified by postnatal day one in the rat. The present results, taken together with other observations, also suggest that three different combinations of signals orchestrate the commitments of vagal afferents respectively to (1) the side of the organ, (2) the region within the organ wall, and (3) the accessory and innervated tissues that complex with the fully differentiated ending.

Sertoli cell-specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice.

DAX1 is an orphan member of the nuclear hormone receptor superfamily of transcription factors. Our recent characterization of Dax1 (Ahch)-deficient male mice revealed a primary testicular defect resulting in hypogonadism and sterility. The progressive degeneration of the germinal epithelium, independent of abnormal gonadotropin and testosterone production, suggested an intrinsic loss of Dax1 function in the Sertoli cells. To test this hypothesis, we assessed the effect of Sertoli cell-specific expression of a human DAX1 (AHC) transgene driven using the promoter of the Müllerian inhibiting substance (MIS) gene. The MIS-DAX1 transgene partially rescued the mutant phenotype of the Dax1-deficient male mice. Although testicular morphology remained abnormal, fertility was restored to levels matching that of wild-type littermates. Examination of several markers of sperm fertilizing capability revealed significant improvements in MIS-DAX1-rescued mice. Epididymal sperm count and sperm motility were greater in 12-week-old rescued mice than in age-matched Dax1-deficient mice. The ability of sperm to undergo an immediate acrosome reaction was impaired in Dax1-deficient animals, and sperm from Dax1-deficient mice fertilized only 8.2 +/- 6.8% of eggs in vitro, significantly less than rescue (67.8 +/- 19.1%) and wild-type (88.9 +/- 3.9%) sperm. These results indicate that Dax1 expression in Sertoli cells is adequate to overcome crucial thresholds related to sperm production and function. However, the failure to completely rescue the testicular pathology of Dax1-deficient mice suggests that Dax1 expression in other somatic cells is essential for normal testicular development.

Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography.

Intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs), the two putative mechanoreceptors that the vagus nerve supplies to the gastrointestinal smooth muscle, have been characterized almost exclusively in the rat. To provide normative inventories of these afferents for the mouse, the authors examined the endings in the stomach and small intestine of three strains used as backgrounds for gene manipulations (i.e., C57, 129/SvJ, and WBB6). Animals received nodose ganglion injections of wheat germ agglutinin-horseradish peroxidase or dextran-tetramethylrhodamine conjugated to biotin. The horseradish peroxidase tissue was processed with tetramethylbenzidine and was used to map the distributions and densities of the two endings; the dextran material was counterstained with c-Kit immunohistochemistry to assess interactions between intramuscular arrays and interstitial cells of Cajal. IGLEs and IMAs constituted the vagal innervation of mouse gastric and duodenal smooth muscle. IGLE morphology and distributions, with peak densities in the corpus-antrum, were similar in the three strains of mice and comparable to those observed in rats. IMAs varied in complexity from region to region but tended to be simpler (fewer telodendria) in mice than in rats. IMAs were most concentrated in the forestomach and sphincters in mice, as in rats, but the topographic distributions of the endings varied both between strains of mice (subtly) and between species (more dramatically). IMAs appeared to make appositions with both interstitial cells and smooth muscle fibers. This survey should make it practical to assay the effects of genetic (e.g., knockout) and experimental (e.g., regeneration) manipulations affecting visceral afferents and their target tissues.

Habituation of oral responding in adult rats.

The effects of repeated oral stimulation on ingestive responding were investigated in adult rats. A series of brief intraoral infusions of flavored diet was delivered to female rats once every minute through an oral cannula. When the flavor of the infused diet remained constant, significant decreases in mouthing behavior were observed by the end of testing, whereas switching the flavor of the diet during testing resulted in enhanced responding and infusions delivered through gastric cannulas produced minimal effects. Patterns of oral responding were also similar in food-restricted rats. These patterns of responding suggest that adult rats habituate to oral stimulation. Finally, oral habituation led to decreased ingestion, whereas gastric infusions had minimal effects. Thus, oral habituation may represent a mechanism influencing intake in rats at all ages.

MHC genotype controls the capacity of ligand density to switch T helper (Th)-1/Th-2 priming in vivo.

A quantitative mechanism for the differentiation of CD4 T cells into recognized subsets of Th1 and Th2 effectors is controversial. Here, we define the Ag dose more precisely to the density of a minimal immunogenic peptide presented on the surface of a specific APC type. Th1 and Th2 responder MHC genotypes differ by as much as an order of magnitude in the density of this peptide displayed on B7-2+ B cells. We asked whether such B cells presenting a low ligand density primed Th2 effectors in an MHC genotype with predisposed high-density presentation and Th1-type immunity, and whether high ligand density B cells primed Th1 effectors in an MHC genotype that normally presents a low density and the Th2 phenotype. While low ligand density had the capacity to switch phenotype in the Th1 responder, high-density presentation did not alter genetically determined Th2 responder status.