[Cell biology and physiology of male reproduction]. / Zellbiologie und Physiologie der männlichen Fortpflanzung.

BACKGROUND: An adequate and undisturbed generation of fertile sperm is a prerequisite for fatherhood. Therefore, spermatogenesis is of central importance for male fertility. The testes, however, not only hold the germinal epithelium as the sperm-generating organ but also acts as a gland releasing androgens to control male reproductive function. This dual testicular function provides options to couple and coordinate spermatogenesis and steroidogenesis. METHODS: The regulation of both processes via the hypothalamus-pituitary-gonadal axis is arranged via feedback loops, which are interconnected but also enable separate modulation of germ cell production and endocrine activity. Many parameters of gonadal function can be determined and provide information about physiological or pathological changes of testis function. OBJECTIVES: This article introduces the physiological basics of testis function and presents the repertoire of endpoints determined in clinical andrology to facilitate a deeper understanding for clinical diagnostics of male fertility.

Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta).

Kisspeptin-Kiss1R signalling in mammals has been implicated as an integral part of the reproductive cascade. Kisspeptinergic neurons upstream of GnRH neurons are involved in the activation of the hypothalamic GnRH pulse generator during pubertal onset. Thus, the major research focus has been on the central effects of kisspeptin. The demonstration of the presence of KissR expression in human testes suggests additional unknown actions of kisspeptin-KISS1R signalling at the distal component of the male reproductive axis. Here we explored the impact of kisspeptin at the testis in the adult male rhesus monkey. We employed the clamped monkey model to assess the intratesticular actions of kisspeptin. Plasma testosterone and LH levels were monitored in four adult male monkeys. The peripheral administration of human kisspeptin-10 (50 µg, iv bolus) caused a single LH pulse, which was followed by a robust increase in plasma testosterone levels sustained for at least 180 min. This response was abolished when kisspeptin was administered to GnRH receptor antagonist (acyline) pre-treated animals. However, kisspeptin administration significantly (P < 0.005) elevated hCG-stimulated testosterone levels in acyline pre-treated monkeys when compared with saline+ hCG treatment. These results revealed a novel peripheral facet of kisspeptin signalling.

A combined approach facilitates the reliable detection of human spermatogonia in vitro.

STUDY QUESTION: Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? SUMMARY ANSWER: Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. WHAT IS KNOWN ALREADY: The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. STUDY DESIGN, SIZE, DURATION: Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. MAIN RESULTS AND THE ROLE OF CHANCE: Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions. LIMITATIONS, REASONS FOR CAUTION: There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models. WIDER IMPLICATIONS OF THE FINDINGS: While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

Developmental expression of the pluripotency factor sal-like protein 4 in the monkey, human and mouse testis: restriction to premeiotic germ cells.

SALL4 (sal-like protein 4) is a pluripotency transcription factor, which is highly expressed in embryonic stem (ES) cells and which is essential for mouse preimplantation development. In adult mouse organs, Sall4 mRNA is highly expressed in the testis and ovary, while there is only little or no expression in other organs. There is also a high expression of SALL4 in human testicular germ cell tumors. However, there is as yet no detailed analysis of SALL4 expression during mammalian testicular development. We analyzed SALL4 expression in ES cells, preimplantation embryos, and the developing and adult testis of a nonhuman primate (NHP) species, the common marmoset monkey (Callithrix jacchus). Immunofluorescence revealed SALL4 in the nuclei of marmoset ES cells and preimplantation embryos. Marmoset SALL4 isoform analysis in ES cells and newborn and adult testis by RT- PCR and Western blotting showed two different isoforms, SALL4-A and SALL4-B. Immunohistochemistry localized this transcription factor to the nuclei of primordial germ cells and most gonocytes in the prenatal and early postnatal marmoset testis. In the pubertal and adult testis SALL4 was present in undifferentiated spermatogonia. In the developing and adult human and mouse testis SALL4 expression mimicked the pattern in the marmoset. Adult testes from additional NHP species, the treeshrew, the cat and the dog also exhibited SALL4 in undifferentiated spermatogonia, indicating a conserved expression in the mammalian testis. Taking into account the importance of SALL4 for mouse development, we conclude that SALL4 may play an important role during mammalian germ cell development and is involved in the regulation of spermatogonial proliferation in the adult testis.

Immature rhesus monkey (Macaca mulatta) testis xenografts show increased growth, but not enhanced seminiferous differentiation, under human chorionic gonadotropin treatment of nude mouse recipients.

Prepubertal male cancer patients facing gonadotoxic therapy cannot be offered a procedure to create a fertility reserve, in contrast to the options available for men. Sperm production by testis xenografting has been proposed for boys, but as the efficacy of sperm production in animal trials is low, hormonal stimulation of recipients carrying xenografts has been proposed to enhance graft development. We confirm that spermatogonia are the only germ cells present in immature rhesus testis. We xenografted immature tissues into nude mice and treated them with human chorionic gonadotropin (hCG) at a low (1IU) and high (10IU) dose twice weekly for 3months. We observe significantly larger grafts in treated recipients, and significantly larger recipient body weight and seminal vesicle weight in the high dose group. However, histological analysis demonstrates that no significant increase in seminiferous maturation is induced by hCG treatment. Moreover, grafts in control recipients develop spermatozoa within 5months. Thus, although hCG treatment of hosts enhances the growth of xenografted prepubertal primate testis tissue and stimulates androgen production in the grafts, the treatment does not enhance the differentiation of the seminiferous epithelium.

Reduced expression of DNMT3B in the germ cells of patients with bilateral spermatogenic arrest does not lead to changes in the global methylation status.

DNA methylation events during spermatogenesis have important implications for gamete integrity and transmission of epigenetic information to the next generation. However, the role of DNA methyltransferases in the disorders of human spermatogenesis has not been elucidated. The aim of the present study was to evaluate the expression of DNMT3B, crucial for full germ cell methylation, in testicular germ cells of patients with spermatogenic arrest and to determine whether or not there is an association with the global methylation status. In order to determine the DNMTs expression status at various stages of spermatogenesis, immunohistochemical localization was performed on 16 fertile controls having normal spermatogenesis and 11 patients with bilateral spermatogenic arrest. DNMT3B was expressed in most of the germ cell types in both controls and patients with bilateral spermatogenic arrest. The number of DNMT3B positive preleptotene/zygotene cells and pachytene spermatocytes was significantly lower in patients with bilateral arrest. However, evaluation of 5-methylcytosine, a global methylation marker, in the few matured germ cells of these patients did not reveal altered methylation. In conclusion, the global methylation status of germ cells is not affected by spermatogenic defects in spite of aberrant DNMT3B expression indicating the necessity of proper methylation for full spermatogenesis.

Germ cell dynamics in the testis of the postnatal common marmoset monkey (Callithrix jacchus).

The seminiferous epithelium in the nonhuman primate Callithrix jacchus is similarly organized to man. This monkey has therefore been used as a preclinical model for spermatogenesis and testicular stem cell physiology. However, little is known about the developmental dynamics of germ cells in the postnatal primate testis. In this study, we analyzed testes of newborn, 8-week-old, and adult marmosets employing immunohistochemistry using pluripotent stem cell and germ cell markers DDX4 (VASA), POU5F1 (OCT3/4), and TFAP2C (AP-2γ). Stereological and morphometric techniques were applied for quantitative analysis of germ cell populations and testicular histological changes. Quantitative RT-PCR (qRT-PCR) of testicular mRNA was applied using 16 marker genes establishing the corresponding profiles during postnatal testicular development. Testis size increased during the first 8 weeks of life with the main driver being longitudinal outgrowth of seminiferous cords. The number of DDX4-positive cells per testis doubled between birth and 8 weeks of age whereas TFAP2C- and POU5F1-positive cells remained unchanged. This increase in DDX4-expressing cells indicates dynamic growth of the differentiated A-spermatogonial population. The presence of cells expressing POU5F1 and TFAP2C after 8 weeks reveals the persistence of less differentiated germ cells. The mRNA and protein profiles determined by qRT-PCR and western blot in newborn, 8-week-old, and adult marmosets corroborated the immunohistochemical findings. In conclusion, we demonstrated the presence of distinct spermatogonial subpopulations in the primate testis exhibiting different dynamics during early testicular development. Our study demonstrates the suitability of the marmoset testis as a model for human testicular development.

Immature rat seminiferous tubules reconstructed in vitro express markers of Sertoli cell maturation after xenografting into nude mouse hosts.

Sertoli cells undergo a maturation process during post-natal testicular development that leads to the adult-type Sertoli cell, which is required for spermatogenesis. Understanding Sertoli cell maturation is therefore necessary to gain insight into the underlying causes of impaired spermatogenesis and male infertility. The present study characterized the cellular and molecular differentiation of Sertoli cells in a xenograft model of mammalian testicular development. Immature rat Sertoli cells were cultured in a three-dimensional culture system to allow the formation of cord-like structures. The in vitro Sertoli cell cultures were then grafted into nude mice. Sertoli cell proliferation, morphological differentiation and mRNA expression of Sertoli cell maturation markers were evaluated in xenografts. Sertoli cell proliferation significantly decreased between 1 and 4 weeks (6.7 +/- 0.9 versus 1.2+/- 0.1%, P < 0.001), and was maintained at low levels thereafter. Sertoli cell cord-like structures significantly decreased between 1 and 4 weeks (59.6 versus 21%, P < 0.05), whereas Sertoli cell tubules were more frequently observed after 4 weeks (13.3 versus 73.1%, P < 0.05). Furthermore, expression of androgen binding protein, transferrin and follicle stimulating hormone receptor, markers for mature Sertoli cells, was detected after 1 week of grafting and increased significantly thereafter. We conclude from these results that rat Sertoli cells continue maturation after xenografting to the physiological environment of a host. This model of in vitro tubule formation will be helpful in future investigations addressing testicular maturation in the mammalian testis.

Limited survival of adult human testicular tissue as ectopic xenograft.

BACKGROUND: Grafting of testicular tissue into immunodeficient mice has become an interesting and promising scientific tool for the generation of gametes and the study of testicular function. This technique might potentially be used to generate sperm from patients whose testes need to be removed or are destroyed due to therapeutic intervention or as a consequence of disease. Here we explore whether adult human testicular tissue from patients with different testicular pathologies survives as xenograft. METHODS AND RESULTS: Testis tissue from adult patients with varying degrees of spermatogenesis was grafted into two strains of immunodeficient mice (severe combined immunodeficiency, Nu/Nu). Tissue with active spermatogenesis prior to grafting largely regressed. However, testicular tissue survival was better in cases where spermatogenesis was suppressed prior to grafting and occasionally spermatogonial stem cells survived. Cases with spermatogenic disruption were not corrected by the xenografting. CONCLUSION: Superior survival of the germinal epithelium and spermatogonia when spermatogenesis was suppressed prior to grafting could provide a novel strategy for germline preservation in pre-pubertal cancer patients. This approach could also be valuable to study the early stages of human spermatogenesis.

Complex secretory products in the oviduct of the plethodontid salamander Bolitoglossa dofleini (Amphibia, Urodela).

The ultrastructure of the secretory granules in the cells of the subdivisions of the oviduct in the neotropical plethodontid salamander Bolitoglossa dofleini was studied by transmission electron microscopy. In addition, we applied the cationic dye Cuprolinic Blue (CB) at different electrolyte concentrations to demonstrate proteoglycans, and the pyrogallol red-copper (PR-C) method to stain proteins at the ultrastructural level. The entire oviduct is lined by a simple epithelium that contains ciliated and microvillous cells in the first subdivision, the aglandular pars recta; microvillous cells show a moderate secretory activity. The following pars convoluta is differentiated into five glandular subdivisions and the aglandular "uterine portion". Especially in the glandular parts, the epithelium is arranged in longitudinal folds. At their crests ciliated and microvillous cells similar to those in the pars recta occur. Gland cells are crowded with secretory granules that differ in their structural complexity (with and without electron-dense spheres or masses; elaborated, homogeneous or granular matrix; spherical; distorted) along the various subdivisions. Further, as suggested by the CB-technique, the cranial subdivisions contain large amounts of sulphated proteoglycans that decrease in the caudal direction. Carboxylated proteglycans appear to be present in all subdivisions examined. Electron-dense spheres of secretory granules are largely free of CB-precipitates, but stain more or less intensely with PR-C. The ultrastructure of the pars recta, and especially the "uterine portion" indicates transporting capability. The epithelial cells of the "uterus" have coated pits and a considerable amount of lysosome-like bodies.

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum.

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.

The structure and development of the skull of Costa Rican plethodontid salamanders (Amphibia: Urodela).

We describe the structure of the skulls of the Costa Rican plethodontid salamanders Bolitoglossa subpalmata, Oedipina uniformis and Nototriton abscondens, and the characteristic sequences of development and ossification of the bony elements resulting from direct development using mainly cleared and stained specimens. Significant differences between the species studied are observed. N. abscondens possesses the broadest premaxillary pars dentalis and O. uniformis the narrowest one. The premaxillary dorsal processes are fused over their rostral third only in B. subpalmata; over half their extention in N. abscondens and almost completely in O. uniformis. A prefrontal is always present in N. abscondens; it is hidden underneath the nasal or missing in B. subpalmata, and it is always absent in O. uniformis. The skull bones, with the exception of the orbitosphenoid, develop and ossify sequentially from caudal to rostral in these directly developing species. A more massive pars dentalis of a generally narrower premaxillary are found as typical characters in males.

Teeth and their sex-dependent dimorphic shape in three species of Costa Rican plethodontid salamanders (Amphibia: Urodela).

The shape of the teeth and their sex-dependent dimorphic expression in three species of Costa Rican plethodontids (Bolitoglossa subpalmata, Oedipina uniformis and Nototriton abscondens) were studied using light microscopy and scanning electron microscopy. The teeth of the vomerine tooth patches are about one third larger than the teeth of the jaws in B. subpalmata and O. uniformis, whereas all teeth of N. abscondens are of about uniform size. The occurrence of bicuspid tooth germs in the fetus proves that primary teeth are bicuspid in these directly developing plethodontids. Females possess only bicuspid teeth consisting of a pedicel and a crown, as is considered characteristic for urodeles after metamorphosis. Adult males possess conical monocuspid teeth on the premaxillary. These teeth--which are similar to the typical late larval tooth of salamanders presenting a larval stage--are about twice as big as the neighbouring bicuspid maxillary teeth. N. abscondens males possess some monocuspid teeth and teeth of aberrant shapes on the premaxillary and the maxillaries. A tendency to build more monocuspid teeth in the premaxillary region than in the maxillary region can be observed in this species. We suppose that different degrees of sensitivity to androgens in each section of the dental lamina of the upper jaw cause the secondary occurrence of conical monocuspid teeth predominantly on the premaxillary section.

Development of the pattern of dentition and dental laminae of Costa Rican plethodontid salamanders (Amphibia: Urodela).

The pattern of development of teeth and dental laminae of three Costa Rican plethodontids (Amphibia, Urodela, Plethodontidae) was investigated using transparent preparations, light microscopy and scanning electron microscopy. The teeth of the jaws are monostichously positioned, those of the posterior vomeral parts are polystichously arranged. The anterior vomeral parts carry monostichously positioned teeth at the caudal margin; yet, the adult Bolitoglossa subpalmata possesses two lines. As a sex dimorphism adult males display long monocuspid premaxillary teeth which protrude to the outside of the mouth cavity. All species studied possess paired dental laminae in the lower jaw. Nototriton abscondens possesses an unpaired dental lamina in the upper jaw, which is constricted between the unpaired premaxillary and the maxillaries. In contrast, the dental laminae in the upper jaw of B. subpalmata and Oedipina uniformis are segmented into a premaxillary and two maxillary laminae. All species possess a pair of anterior vomeral and a pair of posterior vomeral dental laminae in the adults, whereas the vomeral dental laminae of the subadults are unsegmented. The pattern of dentition is compared with that of Gyrinophilus and Eurycea.