Targeted disruption of the Insl3 gene causes bilateral cryptorchidism.

The sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (originally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.

Steroidogenic capacity of isolated adult mouse Leydig cells does not decrease with age.

Leydig cells were isolated from the testes of adult young (5--7 months), old (21 months), and senile (27 months) mice using a highly preservative Percoll procedure. The yield in Leydig cells per testis from young animals was slightly lower than from the old age groups. hCG-induced testosterone synthesis proceeded in a linear fashion for at least 5 h in all three groups. The maximal steroidogenic capacity of cells from old animals was identical to that from young adults. There was no significant difference between Leydig cells from young and old mice with respect to hCG-induced cAMP accumulation and protein kinase activation. Determination of hCG concentrations required for half-maximal stimulation of testosterone synthesis and cAMP accumulation showed identical, or even lower, values in the old age groups. The phenomenon may be connected with the significant augmentation with age of the DNA content per cell (polyploidization), possibly acting as a compensatory mechanism of age-induced deficiencies. Detailed kinetic studies of cAMP accumulation, protein kinase activation, protein kinase activation, and steroidogenesis as well as ultrastructural analyses support the findings of unimpaired or increased capacities of the testosterone-forming cells in old animals. Thus, the aging of Leydig cells appears to differ from that of other tissues of the mouse (e.g. skeletal muscle), which exhibit decreasing abilities to respond to stimuli.

Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels of the human testis.

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor L-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.

Endothelin and endothelin receptors A and B in the human testis.

Human testicular capillaries interconnect Leydig cells and seminiferous tubules. Microcirculation and blood flow are therefore essential for the maintenance of spermatogenesis. The expression and the localisation of ET (endothelin) and its receptors in testicular tissue, in seminiferous tubules and in human testicular capillaries were studied. ET-1 mRNA was detected in whole testicular tissue and in seminiferous tubules whereas isolated testicular capillaries were negative. Big ET-1 (Big endothelin 1) and ET peptides were localised in Leydig and Sertoli cells whereas interstitial and intramural capillaries (within the lamina propria) remained unstained. ET was also found in mature spermatids. ET-A (endothelin receptor A) mRNA was detected in seminiferous tubules and whole testicular tissue whereas testicular blood vessels were negative. ET-A immunostaining was displayed in Leydig and Sertoli cells and in spermatids. ET-B (endothelin receptor B) mRNA was detected in whole testicular tissue, seminiferous tubules and in testicular capillaries. ET-B peptide was prominent in Leydig cells, peritubular cells, endothelial cells and pericytes of interstitial and intramural capillaries as well as in vascular endothelial and smooth muscle cells. From these results we conclude that ET produced in Leydig and Sertoli cells can act in a paracrine manner via ET-B on the human testicular microvasculature and the peritubular cells. The presence of both ET-A and ET-B in Leydig cells and of ET-A in Sertoli cells leads to the assumption that ET could influence these cells as an autocrine factor.

New aspects of Leydig cell function.

Previous studies indicated that the Leydig cells of the human testes show similarities to neuroendocrine cells. In this context, the local synthesis of two neuroactive signaling molecules, namely nitric oxide (NO) and C-type natriuretic peptide (CNP), both acting via the second messenger, cyclic guanosine monophosphate (cGMP), might be of physiological relevance. By immunoblotting, immunohistochemical analyses and affinity crosslinking experiments, respectively, the presence of soluble guanylate cyclase (sGC), the NO receptor, and of guanylate cyclase B (GC-B), representing the CNP receptor, was demonstrated in Leydig cells, seminiferous tubules and blood vessels of the human testis. Moreover, cGMP and its binding protein cGMP-dependent protein kinase type I (GK I) were found in these structures. The functional activity of the two receptors was proved by generation of cGMP in response to treatments with the NO donor, sodium nitroprusside (SNP), and with CNP, respectively. As indicated by immunohistochemical analyses and by treatments of cells with either SNP or CNP, human Leydig tumour cells and MA10 cells, representing a mouse Leydig tumour cell line, were found to be distinguished by a reduced expression of the receptors for NO and CNP. Furthermore, expression levels of the components of the two cGMP-generating systems were found to be widely unchanged in Leydig cells during different ontogenetic stages. Though cGMP has been shown to influence testosterone release, the constant developmental expression patterns of NO and CNP apparently independent of differences in androgen production, the down-regulation of their receptors in tumorous cells, and the presence of GK I, may point to additional autocrine functions of these factors and of cGMP in Leydig cells. Moreover, possible paracrine actions of NO and CNP may include relaxation of seminiferous tubules and blood vessels in order to modulate sperm transport and testicular blood flow, respectively. These findings suggest that Leydig cell-derived factors may exert activities different from or in addition to those involved in the regulation of testosterone production.

[Human spermatogenesis: basic research and clinical issues]. / Spermatogenese beim Menschen: Grundlagenforschung und Klinik.

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.

Reinvestigation of the transitional epithelium (urothelium) of the human ureter.

The transitional epithelia (urothelia) of the ureters of 30 patients of different ages were studied by means of light and electron microscopical, histochemical and immunocytochemical methods. A great variability of the normal structural appearance of the urothelium was established. Structural features and the uptake of exogenous peroxidase by the surface epithelial cells provided high endocytotic activity. Urothelial cells take up many low and high molecular weight substances from the urine and further metabolize and transport these toward the subepithelial connective tissue. Lymphocytes, macrophages, monocytes, plasma cells and rarely polymorphonuclear leukocytes are distributed intra- and extraepithelially and are involved in the immunological response to agents which enter the intercellular spaces of the epithelium. These cells are also responsible for the elimination of aged and degenerating superficial squamous cells. The present investigation establishes the existence of immunological defence mechanisms in the adult human urothelium. The results obtained suggest that the human ureter contains three functional barriers directed against aggressive components of the urine: the first represented by structures of the superficial squamous cells, the second by the upper cells of the ureteric intermediate layer and the third comprising epithelial and immunological cells involved in immune defence mechanisms.

[Spermatogenesis in the aged--a borderland between normal and pathologic anatomy]. / Spermatogenese im Alter--ein Grenzgebiet zwischen normaler und pathologischer Anatomie.

Aging of the testis is a normal physiological process, which gradually proceeds with increasing age. Under normal conditions, an abrupt breaking off the spermatogenetic and hormonal functions of the testis does not occur. Already in the testis of every adult fertile man single degenerating germ cells are recognizable, the occurrence of which is understood as physiological germ cell loss. In men, older than 65 years, further degenerative changes of the germ cells and of the germinal epithelium regularly can be found, which have negative effects on the spermatogenetic activity of the germinal epithelium. These changes concern disturbances of the kinetics of spermatogenesis, disturbances of spermatogoniogensis and of meiosis, malformations of the spermatids, the release of immature germ cells from the germinal epithelium, the concentration of lipids within the Sertoli cells, the appearance of diverticles of the seminiferous tubules and possibly also atrophy of seminiferous tubules. In the course of regular testicular aging these degeneration phenomena can be observed to a relatively small extent diffusely distributed in the testis and exhibiting individual differences. A phatological alteration of the testicular tissue is under consideration as soon as a definitive pattern of damage predominates in the whole organ or when a focal degeneration of testicular tissue has appeared. The borderland between normal and pathological anatomy of testis tissue during aging is characterized.

[Morphology of syncytiotrophoblast cells in testicular tumors]. / Zur Morphologie von Syncytiotrophoblast-Zellen in Hodentumoren.

Syncytiotrophoblastic cells were identified in a teratocarcinoma of the testis by light microscopy using the semi-thin sectioning technique. An electron microscope study confirmed them to be syncytial. Many irregularly shaped nuclei lying in a honeycombed cytoplasm were observed, while the free margins contained microvilli. These cells are morphologically similar to syncytiotrophoblasts of the normal placenta. The presence of syncytiotrophoblastic cells in testicular tumours means a worse prognosis than would otherwise be concluded on the basis of the particular tumour concerned.

[Spermatogenesis in patients with traumatic transverse paralysis]. / Spermatogenese bei Patienten mit traumatischer Querschnittlähmung.

Testicular biopsies from 22 paraplegic men were studied by light and electron microscopy. The investigations of semithin sections by a special standardized method revealed that normal spermatogenesis occurred in only one patient, whereas in 21 patients severe disturbance of spermatogenesis was found. The germinal epithelium displayed a decrease of spermatogenic cells, an increase of degenerating cells, especially of spermatids, and a release of large groups of premature germ cells. Cross sectioned tubules often showed no patent lumina. As revealed by electron microscopical investigations the spermatids show malformations in most cases of the head and acrosome, rarely of the tail. Decreased spermatogenic activity occurred in various degrees in each of the 21 men. There is no evidence that any specific form of spermatogenic disturbance exists typical of spinal cord injury. No obvious relationship was found between the degree of spermatogenic disturbance and duration or level of the spinal cord lesion or incidence of urinary tract infection.

[Early detection of a contralateral second carcinoma in patients with testicular tumors caused by testicular carcinoma in situ]. / Früherkennung des kontralateralen Zweitkarzinoms bei Hodentumorpatienten durch das Carcinoma in situ testis.

Carcinoma in situ (CIS) of the testis is considered to be a precursor of germ cell cancer. Diagnosis is made by the conventional biopsy technique. Only for patients at risk is a screening biopsy justifiable. This group includes patients with testicular cancer in whom the incidence of contralateral second germ cell tumors is increased. In a prospective study we found three cases of testicular CIS in biopsies from the contralateral testes of 61 such patients. All cases with a diagnosis of CIS presented with testicular atrophy (volume less than 12 ml), associated with necrozoospermia in one patient and with azoospermia in two patients. Treatment consisted in local irradiation (20 Gy) of the remaining testis to preserve Leydig cell function. In control biopsies no evidence of CIS or germ cells was found. More than 3 months after therapy, plasma testosterone levels were normal and LH and FSH levels were increased. None of the patients with negative biopsy (n = 49) who were followed up was found to have a second cancer of the contralateral testis. The average observation time so far is 17.2 months.