Male Somatic Cells Research Articles

Assessing testicular morphofunctionality under Roundup WG® herbicide exposure in zebrafish.

Glyphosate-based herbicides, like Roundup WG® (RWG) used for a range of crops, such as corn, soybean, coffee, sugarcane, rice, apple, and citrus, can reach aquatic ecosystems and impact non-target organisms like fish. Thus, the fish were exposed to three RWG concentrations plus one negative control, which represents the concentration allowed for inland Brazilian waters and concentrations found in surface water worldwide (0.0, 0.065, 0.65, and 6.5mg a.i./L) for 7 and 15days. Morphological analysis revealed significant alterations in the testicular structure, particularly in Sertoli cell extensions and cytoplasmic bridges between germ cells. Subcellular compartments also displayed alterations, including dilated mitochondria and the loss of electron density and autophagic vesicles. Gene transcript levels related to autophagy and steroidogenic regulation were upregulated in exposed fish. Germ cell quality was also affected, increasing ROS (reactive oxygen species) production and DNA fragmentation. The study highlighted the RWG reproductive toxicity, providing valuable insights into understanding the morphofunctional alterations in somatic and germ cells of Danio rerio. In conclusion, the environmental relevant concentrations used in this study were toxic to male somatic and germ cells, which raises a concern about the concentrations considered safe for human and animal use.

Dosage Compensation in Drosophila: Its Canonical and Non-Canonical Mechanisms.

Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila.

Single-cell RNA-sequencing reveals the dynamic process and novel markers in porcine spermatogenesis

BackgroundSpermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported.ResultsTo characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively.ConclusionsThe present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.

Transcriptomic analysis of feminizing somatic stem cells in the Drosophila testis reveals putative downstream effectors of the transcription factor Chinmo.

One of the best examples of sexual dimorphism is the development and function of the gonads, ovaries and testes, which produce sex-specific gametes, oocytes, and spermatids, respectively. The development of these specialized germ cells requires sex-matched somatic support cells. The sexual identity of somatic gonadal cells is specified during development and must be actively maintained during adulthood. We previously showed that the transcription factor Chinmo is required to ensure the male sexual identity of somatic support cells in the Drosophila melanogaster testis. Loss of chinmo from male somatic gonadal cells results in feminization: they transform from squamous to epithelial-like cells that resemble somatic cells in the female gonad but fail to properly ensheath the male germline, causing infertility. To identify potential target genes of Chinmo, we purified somatic cells deficient for chinmo from the adult Drosophila testis and performed next-generation sequencing to compare their transcriptome to that of control somatic cells. Bioinformatics revealed 304 and 1549 differentially upregulated and downregulated genes, respectively, upon loss of chinmo in early somatic cells. Using a combination of methods, we validated several differentially expressed genes. These data sets will be useful resources to the community.

The histone demethylase KDM2B regulates human primordial germ cell-like cells specification

Germline specification is a fundamental step for human reproduction and this biological phenomenon possesses technical challenges to study in vivo as it occurs immediately after blastocyst implantation. The establishment of in vitro human primordial germ cell-like cells (hPGCLCs) induction system allows sophisticated characterization of human primordial germ cells (hPGCs) development. However, the underlying molecular mechanisms of hPGCLC specification are not fully elucidated. Here, we observed particularly high expression of the histone demethylase KDM2B in male fetal germ cells (FGCs) but not in male somatic cells. Besides, KDM2B shared similar expression pattern with hPGC marker genes in hPGCLCs, suggesting an important role of KDM2B in germ cell development. Although deletion of KDM2B had no significant effects on human embryonic stem cell (hESC)'s pluripotency, loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect, indicating this defect was due to KDM2B's loss in hPGCLC specification. Mechanistically, as revealed by the transcriptional profiling, KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification. These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification, shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation.

Gonad development examination of high-temperature–treated genetically female Nile tilapia

Nile tilapia (Oreochromis niloticus) is a kind of GSD + TE (genotypic sex determination with temperature effects) fish. It is known that in some reptiles with temperature dependent sex determination (TSD), the gonads are morphologically undifferentiated and bipotential at the start of both Male- and Female-producing temperature (MPT and FPT) cultivation and develop into ovaries and testes while incubating at FPT and MPT, respectively. However, we did not know whether the gonads of high-temperature–treated GSD + TE fish developed in the same way as those of some reptiles with TSD. Here, the 9 dpf (days post fertilization) Nile tilapia fry was cultured at 36 °C for 12 days for the induction of XX female sex reversal, and the gonads from 21, 39 and 99 dpf high-temperature–treated Nile tilapia were used for immunohistochemical analysis. VASA immunohistochemistry indicated that there were oogonia in 21 dpf XX and XX + HT (XX with high-temperature treatments) gonads and gonocytes in 21 dpf XY gonads. CYP19A1A was expressed in 21 dpf XX and XX + HT gonads but was not expressed in 21 dpf XY gonads. These results indicate that the gonads of XX + HT fish are ovary-like at 21 dpf. The immunohistochemistry results for VASA, CYP19A1A and DMRT1 showed that 39 dpf XX + HT gonads contained male germ cells and pre-Sertoli cells but did not contain female somatic cells. Therefore, we concluded that XX + HT gonads transformed from ovary-like to testis-like during 21–39 dpf. In 99 dpf XX + HT gonad, germ cells, such as spermatogonia, spermatocytes and spermatids, and male somatic cells, such as Sertoli cells surrounding the spermatogonia, could be observed. The gonads of high-temperature–treated Nile tilapia became true testes at 99 dpf. This is the first report regarding the gonad cell transformation process during high-temperature–induced sex reversal in fish species. The present study may provide an important foundation for understanding the mechanisms of high-temperature–induced Nile tilapia female sex reversal.

Differential Cytotoxicity of Different Sizes of Graphene Oxide Nanoparticles in Leydig (TM3) and Sertoli (TM4) Cells.

Graphene oxide (GO) is an common nanomaterial and has attracted unlimited interest in academia and industry due to its physical, chemical, and biological properties, as well as for its tremendous potential in applications in various fields, including nanomedicine. Whereas studies have evaluated the size-dependent cytotoxicity of GO in cancer cells, there have been no studies on the biological behavior of ultra-small graphene nanosheets in germ cells. To investigate, for the first time, the cyto- and geno- toxic effects of different sizes of GO in two different cell types, Leydig (TM3) and Sertoli (TM4) cells, we synthesized different sized GO nanosheets with an average size of 100 and 20 nm by a modification of Hummers’ method, and characterized them by various analytical techniques. Cell viability and proliferation assays showed significant size- and dose-dependent toxicity with GO-20 and GO-100. Interestingly, GO-20 induced significant loss of cell viability and cell proliferation, higher levels of leakage of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) generation compared to GO-100. Both GO-100 and GO-20 induced significant loss of mitochondrial membrane potential (MMP) in TM3 and TM4 cells, which is a critical factor for ROS generation. Furthermore, GO-100 and GO-20 caused oxidative damage to DNA by increasing the levels of 8-oxo-dG, which is formed by direct attack of ROS on DNA; GO-100 and GO-20 upregulate various genes responsible for DNA damage and apoptosis. We found that phosphorylation levels of EGFR/AKT signaling molecules, which are related to cell survival and apoptosis, were significantly altered after GO-100 and GO-20 exposure. Our results showed that GO-20 has more potent toxic effects than GO-100, and that the loss of MMP and apoptosis are the main toxicity responses to GO-100 and GO-20 treatments, which likely occur due to EGFR/AKT pathway regulation. Collectively, our results suggest that both GO-100 and GO-20 exhibit size-dependent germ cell toxicity in male somatic cells, particularly TM3 cells, which seem to be more sensitive compared to TM4, which strongly suggests that applications of GO in commercial products must be carefully evaluated.

Chinmo prevents transformer alternative splicing to maintain male sex identity.

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to “feminize”, resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution.

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014-1044, 2016. © 2016 Wiley Periodicals, Inc.

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features.

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans.

Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion).

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP-expressing cells were fused with immature oocytes, matured invitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after invitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two-cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP-expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose "prematuration somatic cell fusion" as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.

Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance.

Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs.

Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells.

BackgroundSilver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs).MethodsHere, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins.ResultsAgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs.ConclusionOur results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required.

Reactivation of the inactive X chromosome and post-transcriptional reprogramming of Xist in iPSCs

Direct reprogramming of somatic cells to pluripotent stem cells entails the obliteration of somatic cell memory and the reestablishment of epigenetic events. Induced pluripotent stem cells (iPSCs) have been created by reprogramming somatic cells through the transduction of reprogramming factors. During cell reprogramming, female somatic cells must overcome at least one more barrier than male somatic cells in order to enter a pluripotent state, as they must reactivate an inactive X chromosome (Xi). In this study, we investigated whether the sex of somatic cells affects reprogramming efficiency, differentiation potential and the post-transcriptional processing of Xist RNA after reprogramming. There were no differences between male and female iPSCs with respect to reprogramming efficiency or their differentiation potential in vivo. However, reactivating Xi took longer than reactivating pluripotency-related genes. We also found that direct reprogramming leads to gender-appropriate post-transcriptional reprogramming - like male embryonic stem cells (ESCs), male iPSCs expressed only the long Xist isoform, whereas female iPSCs, like female ESCs, expressed both the long and short isoforms.

Reactivation of the inactive X chromosome and post-transcriptional reprogramming of Xist in iPSCs.

Direct reprogramming of somatic cells to pluripotent stem cells entails the obliteration of somatic cell memory and the reestablishment of epigenetic events. Induced pluripotent stem cells (iPSCs) have been created by reprogramming somatic cells through the transduction of reprogramming factors. During cell reprogramming, female somatic cells must overcome at least one more barrier than male somatic cells in order to enter a pluripotent state, as they must reactivate an inactive X chromosome (Xi). In this study, we investigated whether the sex of somatic cells affects reprogramming efficiency, differentiation potential and the post-transcriptional processing of Xist RNA after reprogramming. There were no differences between male and female iPSCs with respect to reprogramming efficiency or their differentiation potential in vivo. However, reactivating Xi took longer than reactivating pluripotency-related genes. We also found that direct reprogramming leads to gender-appropriate post-transcriptional reprogramming - like male embryonic stem cells (ESCs), male iPSCs expressed only the long Xist isoform, whereas female iPSCs, like female ESCs, expressed both the long and short isoforms.

Direct reprogramming of human somatic cells to meiotic germ cells

To achieve direct conversion of human male somatic cells to meiotic germ cells by in vitro reprogramming with key genes implicated in germ cell development. Lentiviral transfection of human fibroblasts with 12 germ cell related genes and genetic, epigenetic and meiosis assessment including ploidy of resulting cells. Lentiviral transduction to overexpress germ cell related genes (i12F pool) was performed in human primary fibroblasts (46, XY). After transfection,cells were cultured in DMEM/F12 with leukemia inhibitory factor (LIF), glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), stem cell factor (SCF) and retinoic acid (RA). Characterization of reprogrammed cells included gene expression analysis by qRT-PCR, immunostainning and epigenetic analysis by bisulfite sequencing. Meiotic spreads were analyzed with anti-synaptonemal complex protein 3 (SCP3) primary antibody. Ploidy analysis was performed by DNA-content FACS by propidium iodide (PI) intensity followed by FISH testing chromosomes 18, X, Y. Finally, a comparison among the different combinations of genes was performed. After lentiviral transduction of fibroblast with the i12F pool, a cell morphology change was obvious, showing different early, meiotic and post-meiotic markers expression. Meiotic progression was observed by the correct assembly of the synaptonemal complex as well as haploid cell progression and germ cell-like epigenetic status acquisition. Finally, the screening among the i12F showed that only 6 factors were necessary to obtain a similar phenotype. Our results suggest that it is possible to achieve an in vitro direct conversion of human somatic cells into germ cell-like cells using this reprograming strategy. These results represent a future promise for the generation of artificial gametes.

Testis development requires the repression of Wnt4 by Fgf signaling

The bipotential gonad expresses genes associated with both the male and female pathways. Adoption of the male testicular fate is associated with the repression of many female genes including Wnt4. However, the importance of repression of Wnt4 to the establishment of male development was not previously determined. Deletion of either Fgf9 or Fgfr2 in an XY gonad resulted in up-regulation of Wnt4 and male-to-female sex reversal. We investigated whether the deletion if Wnt4 could rescue sex reversal in Fgf9 and Fgfr2 mutants. XY Fgf9/Wnt4 and Fgfr2/Wnt4 double mutants developed testes with male somatic and germ cells present, suggesting that the primary role of Fgf signaling is the repression of female-promoting genes. Thus, the decision to adopt the male fate is based not only on whether male genes, such as Sox9, are expressed, but also on the active repression of female genes, such as Wnt4. Because loss of Wnt4 results in the up-regulation of Fgf9, we also tested the possibility that derepression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we found that the relationship between these two signaling factors is not symmetric: loss of Fgf9 in XX Wnt4−/− gonads does not rescue their partial female-to-male sex-reversal.

Generation of Viable Male and Female Mice from Two Fathers1

In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These male-derived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.

Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal

BackgroundHormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development.ResultsWe examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad.ConclusionsWe have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein.See commentary: http://www.biomedcentral.com/1741-7007/8/110

73 IMPROVEMENT OF CANINE CLONING EFFICIENCY BY OPTIMIZED DONOR CELL PREPARATION

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al. (2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1 for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author.