Adolf F. Holstein

Progenitor cells of the testosterone-producing Leydig cells revealed

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.

The neuroendocrine leydig cells and their stem cell progenitors, the pericytes

The Leydig cells of the testis represent the main source of androgens. The idea of Leydig cells as endocrine cells has been the leading characteristic of this interesting cell population till now. Our studies of the last 2 decades allowed us to reveal a new important feature of Leydig cells that is their obvious similarity with structures of the central and peripheral nervous system. This includes the expression of neurohormones, neurotransmitters, neuropeptides and glial cell antigens. In this way, it became evident that in addition to the well established control by steroids and systemic hormones, important local auto- and paracrine control me chanisms of testicular functions exist. Thus, the Leydig cells represent a specialized cell population with both endocrine and neuroendocrine properties. The discovery of the neuroendocrine features of Leydig cells gave rise to the hypothesis of a potential neuroectodermal and/or neural crest origin of testicular Leydig cells. In an experimental animal model we revealed that adult Leydig cells originate by transdifferentiation from stem/progenitor cells (pericytes and smooth muscle cells), underlying the close relationship of Leydig cells with testis microvasculature. This and the supporting data from the literature provided the basis for revealing the pericytes as a common adult stem cell type of mammalian species. Distributed by the microvasculature through the entire body, the pericyte, acting as a resting early pluripotent adult stem cell, provides an ingenious system to assure the maintenance, physiological repair and regeneration of organs, each under the influence of specific local environmental factors.

Generation of Cyclic Guanosine Monophosphate by Heme Oxygenases in the Human Testis—A Regulatory Role for Carbon Monoxide in Sertoli Cells?

Abstract Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.

Catecholamine-synthesizing enzymes in the adult and prenatal human testis

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.

Muskulatur und Motilität des Nebenhodens beim Kaninchen

SummaryIn rabbits the arrangement of smooth muscle cells and the motility of the epididymis was observed in vivo and investigated by means of light microscopy. 1.The tubules of the epididymis are surrounded by thin layers of smooth muscle cells. In the ductuli efferentes they are arranged circularly; in the other segments there is also a layer of circular muscle cells and, in addition, longitudinal muscle cells are lying on the outside.2.Fine groups of smooth muscle cells are found in the connective tissue capsule of the epididymis and the testis. These cells are continous with the circular layers of smooth muscle surrounding the testis.3.In vivo there are peristaltic and peristolic movements of the tubular system of the epididymis. In the initial segment of the head of the epididymis the tubules contract with a frequency of 12/min. In the other segments of the epididymis the waves of contractions are less frequent.4.The muscular tissue in the capsule of the epididymis and testis shows rhythmic contractions, which may be regular or irregular.ZusammenfassungDie Anordnung der glatten Muskulatur und die Bewegungen im Nebenhoden des Kaninchens wurden lichtmikroskopisch und in vivo untersucht. 1.Dünne Zellagen glatter Muskulatur umgeben die Nebenhodenkanälchen. In den Ductuli efferentes sind die Muskelzellen zirkulär angeordnet, in den anderen Nebenhodenabschnitten liegen außen auf den zirkulären zusätzlich längsverlaufende Zellzüge.2.Zarte längsverlaufende Zellstränge glatter Muskulatur liegen in der Kapsel von Nebenhoden und Hoden. Sie gehen im Bereich des Hodens in zirkuläre Muskelbündel über.3.Peristaltische und peristolische Bewegungen lassen sich in vivo an den Kanälchen beobachten. Im Eingangsabschnitt des Nebenhodenkopfes haben die Kanälchen 12 Kontraktionswellen pro Minute. In den übrigen Nebenhodenabschnitten sind die Kontraktionswellen seltener.4.Die glatte Muskulatur in der bindegewebigen Kapsel von Nebenhoden und Hoden zeigt rhythmische Kontraktionen, die regelmäßig oder unregelmäßig sein können.

Morphology of the Leydig Cells

Several updated reviews on the Leydig cell structure (Russell 1996; Pudney 1996) and structural aspects related to Leydig cell development and functional activities have already been published (Chemes 1996; Pelliniemi et al. 1996; Ge et al. 1996; Haider 2004; Haider et al. 2007; Prince 2007). Here we will address only certain structural features that are characteristic for human and rodent Leydig cells. The Leydig cells represent a heterogeneous cell population. There are significant differences in the organization, number, shape and other cellular properties of the Leydig cells between species (Pudney 1996; Russel 1996; Prince 2007). In addition to the species-related differences, peculiarities based on developmental, metabolic, functional and seasonal influences have to be considered (Callard 1996; Haider et al. 2007).

Fetal and Adult Leydig Cells Are of Common Orig

In the developing testis, fetal Leydig cells appear in mice at embryonic day 12.5, in rats at embryonic day 14.5, and in human at weeks 7–8 of pregnancy (Huhtaniemi and Pelliniemi 1992; Pelliniemi et al. 1996; Majdic et al. 1998; Yao and Barsoum 2007). The fetal Leydig cells form clusters that are surrounded by a basal lamina that becomes discontinuous after birth (Kuopio and Pelliniemi 1989; Kuopio et al. 1989a). According to O’Shaugnessy et al. (2006) and Yao and Barsoum (2007), the first visible Leydig cells could be recognized by staining for 3β-hydroxysteroid dehydrogenase or cytochrome P450 side chain cleavage enzyme (CytP450scc) at 12.5 days post coitus (dpc). This occurs after the formation of the testicular cords and the coelomic vessel. At this time, it is not clear whether the first Leydig cells transdifferentiate directly from their stem/progenitors within the gonadal mesenchyme or via stem/progenitor Leydig cells (pericytes) in the testicular vasculature. After initial differentiation and proliferation of fetal Leydig cells around day 12 of gestation (Gondos 1980), the fetal Leydig cell population remains virtually unchanged in mice, and, for example, the number of Leydig cells is stable between day 16 of gestation and day 5 after birth (Baker and O’Shaughnessy 2001a). This suggests that the fetal Leydig cells are terminally differentiated and do not divide (Zhang et al. 2008). However, there are also reports indicating that the number of fetal Leydig cells increases between embryonic days 17 and 21 or immediately before birth (Kerr et al. 1988). The latter would not be compatible with the idea that fetal Leydig cells arise by transdifferentiation from stem/progenitor Leydig cells in analogy with the adult Leydig cell population. After birth, the number of fetal Leydig cells significantly decreases. In contrast, Kerr and Knell (1988) proposed that 50–75% of the original “fetal-type” Leydig cell population present at birth persists in the adult testis. In this context, observations (Davidoff et al. 2004) that “fetal-type” Leydig cells are generated during postnatal development are significant, since these cells could be hardly distinguished from the prenatal fetal Leydig cells.

Development of the Testis

The mammalian gonad develops within the urogenital ridge as a thickening along the ventromedial cranial area of the mesonephros. In mice, this occurs at day 10.0–10.5 post coitus (dpc) (Byskov 1986; Brennan and Capel 2004; Kim and Capel 2006; Wilhelm et al. 2007; Tang et al. 2008). This thickening results from both the proliferation of the coelomic epithelium and the allocation of cells from the mesonephros (Yao and Capel 2002; Ross and Capel 2005; Cool et al. 2008). At the beginning, the structure of this gonad anlage is identical in XX and XY mice embryos, and either ovary or testis can develop from this bipotential primordium. For testis development, a member of the Sox (Sry-related high-mobility group box) family of transcription factors, SRY (sex-determining region of the Y chromosome; Sry in mice) is expressed as a primary trigger by the supporting cell lineage, the precursor cells to the Sertoli cell lineage (DiNapoli and Capel 2008). Between 11.5 and 12.5 dpc in mice, the following events were established in the XY gonad: increased proliferation of coelomic epithelial cells, migration of cells from the mesonephros, structural organization of the testis cords, appearance of a male-specific coelomic vessel and differentiation of the steroidogenic Leydig cells (Brennan and Capel 2004).