AZFa Y gene, DDX3Y, evolved novel testis transcript variants in primates with proximal 3´UTR polyadenylation for germ cell specific translation.

Translational control is a major level of gene expression regulation in the male germ line. DDX3Y located in the AZFa region of the human Y chromosome encodes a conserved RNA helicase important for translational control at the G1-S phase of the cell cycle. In human, DDX3Y protein is expressed only in premeiotic male germ cells. In primates, DDX3Y evolved a second promoter producing novel testis-specific transcripts. Here, we show primate species-specific use of alternative polyadenylation (APA) sites for these testis-specific DDX3Y transcript variants. They have evolved subsequently in the 3´UTRs of the primates´ DDX3Y transcripts. Whereas a distal APA site (PAS4) is still used for polyadenylation of most DDX3Y testis transcripts in Callithrix jacchus; two proximal APAs (PAS1; PAS2) are used predominantly in Macaca mulatta, in Pan trogloydates and in human. This shift corresponds with a significant increase of DDX3Y protein expression in the macaque testis tissue. In chimpanzee and human, shift to predominant use of the most proximal APA site (PAS1) is associated with translation of these DDX3Y transcripts in only premeiotic male germ cells. We therefore assume evolution of a positive selection process for functional DDX3Y testis transcripts in these primates which increase their stability and translation efficiency to promote its cell cycle balancing function in the human male germ line.

Deep brain stimulation as a possible treatment of hyperthermia in patients with serotonin syndrome.

Maintaining a body temperature within a narrow range is vital for the survival of all mammals, including humans. With the help of optogenetics, a better understanding of the thermoregulatory organs and pathways is achieved. Optogenetic activation of the GABAergic neurons in the ventral part of the lateral preoptic nucleus (VLPO) leads to decrease in the body temperature. On the other hand, number of drugs could alter the thermoregulatory balance, leading to a hyperthermic state, such as serotonin syndrome (SS). SS is a potentially life-threatening clinical condition that occurs as a result of a drug-induced increase in the intrasynaptic serotonin (5-hydroxytryptamine, 5-HT) levels due to overdose of a single drug or due to interaction between two or more drugs with serotonergic mechanism of action. In this hypothesis, we propose a novel method for the treatment of hyperthermia, a core clinical sign of serotonin syndrome, through deep brain stimulation (DBS). An electrode is stereotactically placed in the VLPO, which may lead to reduction of the core body temperature. If proven effective, this technique should be left as a salvage method for reduction of hyperthermia, where the drug treatment is insufficient or ineffective. This technique could be used for the treatment of other syndromes, where hyperthermia takes a central place, including malignant hyperthermia, neuroleptic malignant syndrome, etc. DBS, on the other hand, could be used alone to induce hyperthermia in patients with malignant diseases. Hyperthermia improves the immune response, improves the drug penetration and stop the repair of already damaged tumor cells after chemotherapy or radiotherapy.

A possible novel technique for intraoperative imaging of the vertebral artery during arthrodesis of the upper cervical spine.

In elderly, the fractures in C1-C2 are a common entity. Poor bone quality and wide range of motion hamper the natural bone fusion, thus making surgery often the only possible way to deal with the underlying pathology. The proximity to important neurovascular structures represents the stabilization in this segment a challenge to the surgical team. There are two major techniques, which are used to achieve a dorsal fusion in the C1-C2 Segment: Goel/Harms and Magerl techniques. The reported risk for damaging the vertebral artery in both techniques lies between 8% and 9,5% using a C-Arm. In Goel/Harms technique lateral mass screws in C1 and pedicle screws in C2 are placed. A transarticular screw is placed on both sides C1-C2 in Magerl technique in order to achieve stabilization of the C1-C2 Segment. By using the new navigational methods for a better imaging of the bony structures (O-Arm), this risk could be reduced further down. The risk for injury of the vertebral artery using the O-Arm navigation depends on the pathology, which is operated, ranging from 0, 3% to 2%. A further problem represents the anatomical variations of the vertebral artery, of which the high-riding vertebral artery being the most important one, reported between 10 and 14,5% of the cases according to the literature review. The novel technique for intraoperative imaging of the vertebral artery represents a fusion between an intraoperative O-Arm and intraoperative application of contrast, thus intraoperatively seeing the exact way of the vertebral artery. Also, after the insertion of the screws, a second CT scan with the O-Arm could be performed, yet again with contrast, to see whether the perfusion of both vertebral arteries is preserved. The significance of this method could bring the injuries of the vertebral artery to 0% independently on the technique, which has been used. This method could be used not only for craniocervical stabilization but also for removal of complex tumors in craniocervical junctions, whereas the vertebral artery is encompassed.

Primordial germ cells do not migrate along nerve fibres in marmoset monkey and mouse embryos.

Primordial germ cells (PGCs) are the embryonic precursors of spermatozoa and eggs. In mammals, PGCs arise early in embryonic development and migrate from their tissue of specification over a significant distance to reach their destinations, the genital ridges. However, the exact mechanism of translocation is still debated. A study on human embryos demonstrated a very close spatial association between migrating PGCs and developing peripheral nerves. Thus, it was proposed that peripheral nerves act as guiding structures for migrating PGCs. The goal of the present study is to test whether the association between nerves and PGCs may be a human-specific finding or whether this represents a general strategy to guide PGCs in mammals. Therefore, we investigated embryos of different developmental stages from the mouse and a non-human primate, the marmoset monkey (Callithrix jacchus), covering the phase from PGC emergence to their arrival in the gonadal ridge. Embryo sections were immunohistochemically co-stained for tubulin beta-3 chain (TUBB3) to visualise neurons and Octamer-binding protein 4 (OCT4 (POU5F1)) as marker for PGCs. The distance between PGCs and the nearest detectable neuron was measured. We discovered that in all embryos analysed of both species, the majority of PGCs (>94%) was found at a minimum distance of 50 µm to the closest neuron and, more importantly, that the PGCs had reached the gonads before any TUBB3 signal could be detected in the vicinity of the gonads. In conclusion, our data indicate that PGC migration along peripheral nerves is not a general mechanism in mammals.

Characterization of a non-human primate model for the study of testicular peritubular cells-comparison with human testicular peritubular cells.

STUDY QUESTION: Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? SUMMARY ANSWER: MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. WHAT IS KNOWN ALREADY: Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. STUDY DESIGN, SIZE, DURATION: We established a culture method for MKTPCs and characterized these cells from six young adult animals (2-3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. MAIN RESULTS AND THE ROLE OF CHANCE: We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. LARGE SCALE DATA: List of identified proteins in MKTPCs by liquid chromatography-tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). LIMITATIONS, REASON FOR CAUTION: This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. WIDER IMPLICATIONS OF THE FINDINGS: Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. STUDY FUNDING AND COMPETION OF INTEREST(S): This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests.

The involvement of gonadotropin inhibitory hormone and kisspeptin in the metabolic regulation of reproduction.

Recently, kisspeptin (KP) and gonadotropin inhibitory hormone (GnIH), two counteracting neuropeptides, have been acknowledged as significant regulators of reproductive function. KP stimulates reproduction while GnIH inhibits it. These two neuropeptides seem to be pivotal for the modulation of reproductive activity in response to internal and external cues. It is well-documented that the current metabolic status of the body is closely linked to its reproductive output. However, how reproductive function is regulated by the body's energy status is less clear. Recent studies have suggested an active participation of hypothalamic KP and GnIH in the modulation of reproductive function according to available metabolic cues. Expression of KISS1, the KP encoding gene, is decreased while expression of RFRP (NPVF), the gene encoding GnIH, is increased in metabolic deficiency conditions. The lower levels of KP, as suggested by a decrease in KISS1 gene mRNA expression, during metabolic deficiency can be corrected by administration of exogenous KP, which leads to an increase in reproductive hormone levels. Likewise, administration of RF9, a GnIH receptor antagonist, can reverse the inhibitory effect of fasting on testosterone in monkeys. Together, it is likely that the integrated function of both these hypothalamic neuropeptides works as a reproductive output regulator in response to a change in metabolic status. In this review, we have summarized literature from nonprimate and primate studies that demonstrate the involvement of KP and GnIH in the metabolic regulation of reproduction.

Primordial germ cell development in the marmoset monkey as revealed by pluripotency factor expression: suggestion of a novel model of embryonic germ cell translocation.

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the 'dogma' of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

The neonatal marmoset monkey ovary is very primitive exhibiting many oogonia.

Oogonia are characterized by diploidy and mitotic proliferation. Human and mouse oogonia express several factors such as OCT4, which are characteristic of pluripotent cells. In human, almost all oogonia enter meiosis between weeks 9 and 22 of prenatal development or undergo mitotic arrest and subsequent elimination from the ovary. As a consequence, neonatal human ovaries generally lack oogonia. The same was found in neonatal ovaries of the rhesus monkey, a representative of the old world monkeys (Catarrhini). By contrast, proliferating oogonia were found in adult prosimians (now called Strepsirrhini), which is a group of 'lower' primates. The common marmoset monkey (Callithrix jacchus) belongs to the new world monkeys (Platyrrhini) and is increasingly used in reproductive biology and stem cell research. However, ovarian development in the marmoset monkey has not been widely investigated. Herein, we show that the neonatal marmoset ovary has an extremely immature histological appearance compared with the human ovary. It contains numerous oogonia expressing the pluripotency factors OCT4A, SALL4, and LIN28A (LIN28). The pluripotency factor-positive germ cells also express the proliferation marker MKI67 (Ki-67), which has previously been shown in the human ovary to be restricted to premeiotic germ cells. Together, the data demonstrate the primitiveness of the neonatal marmoset ovary compared with human. This study may introduce the marmoset monkey as a non-human primate model to experimentally study the aspects of primate primitive gonad development, follicle assembly, and germ cell biology in vivo.

[Cerebral venous thrombosis and subdural haematoma: complications of spontaneous intracranial hypotension]. / Sinus-/Hirnvenenthrombose und Subduralhämatome als Komplikationen eines spontanen Liquorunterdrucksyndroms.

We report on the case of a spontaneous intracranial hypotension with subdural hygroma, as well as cerebral venous thrombosis (CVT), both known complications of intracranial hypotension. The 45-year-old patient was subsequently treated - according to current guidelines for CVT - with anticoagulation, but developed subdural haematoma (SDH), which required neurosurgical treatment. Our case highlights the complex pathophysiological sequelae of intracranial hypotension, as well as the occasionally difficult treatment decisions. Subdural hygroma probably predisposes patients to SDH during anticoagulation. Thus, the potential benefit of anticoagulation needs to be weighed against the risk of SDH on an individual basis.

False-positive antibody signals for the pluripotency factor OCT4A (POU5F1) in testis-derived cells may lead to erroneous data and misinterpretations.

Octamer-binding protein 4 (OCT4) is a key player in pluripotent embryonic stem (ES) cells and is essential for the generation of induced pluripotent stem cells. Recently, several reports indicated the spontaneous recovery of pluripotency in cultured adult human testis-derived cells. This was evidenced also by the detection of OCT4 using antibodies. However, the soundness of some data was recently put into question. During our attempts to derive pluripotent cells from the common marmoset monkey (Callithrix jacchus) testis, we obtained inconsistent data which prompted us to analyze deeper the characteristics of three independent OCT4 antibodies that were used in numerous published studies that received greatest attention. All antibodies detected OCT4 by immunofluorescence (IF) in a marmoset monkey ES cell line. Two of the three OCT4 antibodies also gave robust nuclear signals in testis-derived cells. However, the latter cells expressed no OCT4 mRNA as revealed by quantitative RT-PCR and turned out to be mesenchymal cells. When tested in western blot analyses, all antibodies detected heterologously expressed marmoset monkey OCT4 protein. But, importantly, those antibodies that resulted in non-specific signals in IF also showed additional non-specific bands in western blots. In summary, some commercially available OCT4 antibodies result in false-positive signals which may provoke erroneous conclusions when used in studies aiming at the generation of pluripotent cells in vitro. We conclude that (i) antibodies must be carefully characterized before use to prevent misleading observations and (ii) OCT4 expression must be monitored by a second antibody-independent method.

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.

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus).

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.

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.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture.

BACKGROUND: Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS: We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS: Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.

Protamine-1 represents a sperm specific gene transcript: a study in Callithrix jacchus and Bos taurus.

Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone.

BACKGROUND: Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. METHODS: To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. RESULTS: Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED(50) values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. CONCLUSION: High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin.

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.

Krüppel-like factor 4 is widely expressed in the mouse male and female reproductive tract and responds as an immediate early gene to activation of the protein kinase A in TM4 Sertoli cells.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critically involved in cell proliferation, differentiation, and carcinogenesis. Recently, KLF4 has also been used for the generation of induced pluripotent stem cells. In this study, we analyzed Klf4 expression in different mouse tissues using northern blot analysis and immunohistochemistry. Focusing on the male and female reproductive tract, we showed for the first time that KLF4 is expressed in the epithelia of the murine uterus and the vagina. In the male reproductive tract, we detected KLF4 in the epithelia of the epididymis, ductus deferens, coagulating gland, and the penis. As KLF4 is strongly inducible by FSH signaling in Sertoli cells and as this transcription factor is also involved in Sertoli cell development, we employed the mouse Sertoli cell line TM4 as a model system to investigate i) the induction kinetics of Klf4 upon activation of the cAMP/protein kinase A pathway by forskolin and ii) the effects of Klf4 induction on TM4 cell cycle progression. Interestingly, Klf4 mRNA and protein were rapidly but transiently induced, reaching peak levels after 90-120 min and declining to basal levels within 4 h. Compared with the inducible cAMP early repressor, an immediate early response gene, the induction kinetics of Klf4 is much faster. In conclusion, Klf4 is an immediate early gene in TM4 cells and its expression in several epithelia of the male and female reproductive tract suggests an important role of Klf4 in mouse reproductive functions.

The pluripotency transcription factor Krüppel-like factor 4 is strongly expressed in intratubular germ cell neoplasia unclassified and seminoma.

Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises like the embryonal carcinoma from an intratubular germ cell neoplasia unclassified (IGCNU), i.e. fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma (or embryonal carcinoma). The molecular causes for the development of an IGCNU are still unknown. However, IGCNU cells share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). This study examined KLF4 expression data from 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information. Immunohistochemistry was used to detect KLF4 protein expression in IGCNU (n = 6), seminoma (n = 14) and fetal human testes (n = 14). Microarray data from three independent sources suggest higher mRNA expression in seminoma than in normal testis. Normal spermatogonia, which are the stem cells of spermatogenesis, controlled by their stem cell niche, do not express KLF4. In contrast, IGCNU and seminoma cells strongly express KLF4. In conclusion, this finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of IGCNU as well as for the malignancy of seminoma.