Lack Germ Cells Research Articles

Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7Mb and 101.2Mb on rat chromosome 9. Nucleotide sequence analysis identified 2bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

The fate of germ cells in cryptorchid testis.

Cryptorchidism in males constitutes a notable risk factor for both infertility and testicular cancer. Infertility in adulthood is closely linked to the germ cell status in childhood. Furthermore, the significance of germ cell status is important as more than 95% of all reported testicular malignancies are germ cell tumors. The review aims to elucidate the pathogenesis of germ cells in cryptorchid testes concerning their association with infertility and testicular malignancies. Impaired germ cell numbers are evident in cryptorchid testes even during antenatal and neonatal stages. In cryptorchidism there is a rapid decline in germ cell number within the first year of life, partially attributed to physiologic gonocyte apoptosis. Additionally, germ cells fail to differentiate normally during mini-puberty leading to reduced germ cell proliferation and delayed clearance of gonocytes from the seminiferous epithelium. Absence of germ cells in testicular biopsies occurs already 10 months of age and germ cell deterioration progressively worsens with approximately 50% of persisting cryptorchid testes lacking germ cells during puberty. The deficient germ cell maturation and proliferation leads to later infertility. Elevated temperature in the cryptorchid testes and also hormonal deficiency contribute to this phenomenon. Germ cell neoplasia in situ (GCNIS) originating during fetal development may manifest in rare cases associated with disorders of sexual development, chromosomal abnormalities in boys, specific syndromes, and teratomas that include cryptorchidism. In adults, the presence of GCNIS predominantly represents a new histology pattern before invasive germ cell cancer is demonstrated and is neither congenital nor related to abnormal gonocyte transformation.

Induction of Reproductive Sterility in Coho Salmon (Oncorhynchus kisutch) by an Immersion-Based Gene Silencing Technology

Undesired maturation and reproduction are major challenges in fish aquaculture regarding genetic introgression, precocious maturation, and reproduction-related mortality, which can have profound ecological or economic impacts. Farming reproductively sterile fish can effectively mitigate these challenges. In this paper, we transferred and applied a novel immersion-based, non-transgenic gene silencing technology to sterilize coho salmon for the first time. Unfertilized eggs were bath immersion-treated with csdnd-MO-Vivo in different immersion media. Eyed rates of treated groups ranged from 0.9 to 63.5%. Sterile fish lacking germ cells, and those with arrested germ cells/atretic oocytes, were obtained at 14 and 20 months of age, albeit at a low percentage (2.3 to 10.0% based on females). Gonadal histology and vasa/nanos3 gene expression profile were provided for comparing fertile and sterile gonads, as well as retarded ovaries. Future directions and strategies for optimizing the technology and improving sterility induction were also proposed. The successful production of sterile coho salmon achieved in this study demonstrates the proof of principle for this new sterilization technology. As we continue to expand upon these findings and refine the technology, achieving coho salmon sterile population farming would facilitate the future transfer and application to other commercially important aquaculture fish.

Telomerase Activity in Somatic Tissues and Ovaries of Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Females.

Telomerase activity has been found in the somatic tissues of rainbow trout. The enzyme is essential for maintaining telomere length but also assures homeostasis of the fish organs, playing an important role during tissue regeneration. The unique morphological and physiological characteristics of triploid rainbow trout, when compared to diploid specimens, make them a promising model for studies concerning telomerase activity. Thus, in this study, we examined the expression of the Tert gene in various organs of subadult and adult diploid and triploid rainbow trout females. Upregulated Tert mRNA transcription was observed in all the examined somatic tissues sampled from the triploid fish when compared to diploid individuals. Contrastingly, Tert expression in the ovaries was significantly decreased in the triploid specimens. Within the diploids, the highest expression of Tert was observed in the liver and in the ovaries of the subadult individuals. In the triploids, Tert expression was increased in the somatic tissues, while the ovaries exhibited lower activity of telomerase compared to other organs and decreased compared to the ovaries in the diploids. The ovaries of triploid individuals were underdeveloped, consisting of only a few oocytes. The lack of germ cells, which are usually characterized by high Tert expression, might be responsible for the decrease in telomerase activity in the triploid ovaries. The increase in Tert expression in triploid somatic tissues suggests that they require higher telomerase activity to cope with environmental stress and maintain internal homeostasis.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

Medaka factor in the germline alpha (figα) is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor, figα, is one of the earliest marker genes of oocyte differentiation in vertebrates. In the present study, we made figα knockout medaka by CRISPR/Cas9, expecting aborted progress of oogenesis, to see if differentiation of somatic ovarian tissues is affected. Figα knockout male gonads differentiated normally into testes with functional sperm. The females, on the other hand, were sterile; there are oocytes only up to pachytene. No growing oocytes in diplotene were found. The phenotype was already apparent at 10 days after hatching, when diplotene oocytes start to develop in the control. Furthermore, several putative target genes of figα were not expressed in the mutant female gonads. Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred. Figα knockout female gonads differentiated morphologically into ovaries, suggesting that in medaka, figα knockout prevents oogenesis progress into diplotene stage, and ovarian differentiation do not need developed oocytes beyond pachytene stage. This ovarian phenotype reminded us of "pachytene checkpoint", which blocks meiotic progress into diplotene when chromosomal abnormalities are present. Therefore, we further analyzed phenotype of oocytes in the mutant ovaries. The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage. The number of apoptotic oocytes in the knockout ovaries was small. Finally, we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining. Taken together, figα knockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than "pachytene checkpoint" and arrested oocytes remained in the ovary. This is in contrast to figα knockout mice where all oocytes underwent apoptosis quickly after birth.

The Piwil1 N domain is required for germ cell survival in Atlantic salmon.

Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of piwil1 crispant males and females displayed a mosaic pattern of piwil1 mutations, exhibiting highly mutated alleles (53%–97%) in their fin gDNA samples. In general, piwil1 crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (−); functional in frame mutated alleles ( + ) and wt alleles (+). In F1, all piwil1 −/− fish lacked germ cells, while piwil1 +/+ siblings showed normal ovaries and testes. Yet, most juvenile F1 piwil1 +/−males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4–6 aa in this region Phe130-Ser136 may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of piwil1 leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.

Fertility potential in adult men treated for uncorrected bilateral cryptorchidism: A systematic literature review and analysis of case reports.

Reports of adult orchidopexy for bilateral undescended testicles (bUDT) are sparse, and fertility outcomes are not well established. Our aim was to determine prognosis for restoration of spermatogenesis among adult men (≥18 years) undergoing orchidopexy for bUDT. A systematic literature review, conforming to the PRISMA statement, was conducted using the PubMed/MEDLINE and EMBASE databases through March 2020 using search terms "adult" AND "bilateral orchidopexy" OR "bilateral cryptorchidism." Relevant referenced articles from non-indexed journal were identified by Google Scholar search and additionally included. Fifty-seven publications including adult men with uncorrected bilateral UDTs were identified. Baseline semen analysis was reported in 157 men, all of whom demonstrated azoospermia. Testosterone values were reported in 82 cases and were normal in 89%. Germ cells could not be identified in 72.6% of histologic specimens from 62 testicles. Abdominal testicles more frequently lacked germ cells (90%, p = 0.038) on univariate analysis. Eleven cases identified ejaculated spermatozoa following adult bilateral orchidopexy (8 publications). Sperm extraction (TESE) during orchidopexy or orchiectomy was reported in 13 men without success. Delayed TESE (median 10 months) was performed in 22 persistently azoospermic men with success in 10 (45.5%), none of whom had abdominal testicles prior to orchidopexy. Six men experienced successful paternity via natural conception (3) or assisted reproduction (3). Fertility is possible in adult men with inguinal bUDT following orchidopexy. Subsequent sperm retrieval may involve ejaculated specimens or delayed TESE. TESE performed prior to or at time of orchidopexy is unlikely to be of benefit.

Testes of DAZL null neonatal sheep lack prospermatogonia but maintain normal somatic cell morphology and marker expression.

Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem, we disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9-mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL null male neonatal sheep lacked germ cells on histological sections and showed greatly reduced germ cell markers. Somatic cells within their testes were morphologically intact and expressed normal levels of lineage-specific markers, suggesting that the germ cell niche remained intact. This extends the DAZL mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute germline transmitters in rapid livestock improvement.

Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon

Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.

Severe Degenerative Changes in Cryptorchid Testes in Japanese Black Cattle.

This is a histopathologic and endocrinologic study of 6 calves diagnosed with cryptorchidism. Cases 1-3 were diagnosed as resembling testicular regression syndrome. In cases 1 and 2, the extracted tissue was a small, firm, gray-white mass, and there was lack of obvious testicular tissue in case 3. Histopathologically, the excised tissue in cases 1-3 was a fibrotic testicular remnant with inflammation, mineralization, hemosiderin-laden macrophages or lipofuscin-laden macrophages, and lack of germ cells and interstitial endocrine cells. These findings were compared with cases 4-6, which were diagnosed as testicular hypoplasia due to cryptorchidism. These cases had small but otherwise grossly unremarkable intra-abdominal testicular tissue and histologically had a few germ cells and sustentacular cells with arrested spermatogenesis and an increase in interstitial endocrine cells. Cases 1-3 had more severe degenerative changes compared with cases 4-6. In case 2, the average diameter of the seminiferous tubules was much smaller than in cases 4-6, and there were few tubule cross sections. Anti-Müllerian hormone (214 pg/ml) was detected in the plasma of case 2. Based on the macroscopic and histopathologic findings as well as endocrinologic profiles, the testicular degeneration in cases 1-3 was considered similar to that of testicular regression syndrome. In this condition, it is thought that a normally developing intra-abdominal testis undergoes degeneration due to heat or a vascular disorder such as torsion.

Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation.

ABSTRACTSpermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5−/− mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5−/− mice. At 12 weeks of age, the tubules of Etv5−/− mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) Etv5−/− mice. Partially restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5−/− mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5−/− mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.

Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination.

The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte-transport along microtubules and cytoplasmic streaming-it is unclear how individual kinesin-1 activities contribute to the posterior determination. We examined Staufen, an RNA-binding protein that is colocalized with osk mRNA, as a proxy of posterior determination, and we used mutants that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. We demonstrated that late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. We propose a model whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V-based cortical retention cooperate in posterior determination.

Hybrid Sterility in Fish Caused by Mitotic Arrest of Primordial Germ Cells.

Sterility in hybrid animals is widely known to be due to a cytological mechanism of aberrant homologous chromosome pairing during meiosis in hybrid germ cells. In this study, the gametes of four marine fish species belonging to the Sciaenid family were artificially fertilized, and germ cell development was examined at the cellular and molecular levels. One of the intergeneric hybrids had gonads that were testis-like in structure, small in size, and lacked germ cells. Specification of primordial germ cells (PGCs) and their migration toward genital ridges occurred normally in hybrid embryos, but these PGCs did not proliferate in the hybrid gonads. By germ cell transplantation assay, we showed that the gonadal microenvironment in hybrid recipients produced functional donor-derived gametes, suggesting that the germ cell-less phenotype was caused by cell autonomous proliferative defects of hybrid PGCs. This is the first evidence of mitotic arrest of germ cells causing hybrid sterility in animals.

MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2018MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES Koji Shiraishi and Hideyasu Matsuyama Koji ShiraishiKoji Shiraishi More articles by this author and Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1907AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Non-obstructive azoospermia (NOA), particularly Sertoli cell-only (SCO), is the most severe form of male factor infertility and is classically thought to lack germ cells and spermatogenic stem cells (SSCs). However, evidence from microsurgical testicular sperm extraction (micro-TESE) on men with SCO has revealed the presence of focal areas of full spermatogenesis, suggesting the possible presence of SSCs in some patients with SCO lacking mature sperm. To determine whether gene products from SSCs could be detected in SCO, transcriptome analysis using next-generation sequencing (NGS) was performed using human biopsied testicular samples. METHODS Under IRB approval, testicular samples were obtained from 20 men (mean age: 35.7 years) with SCO in which no germ cells were found during laboratory examination and from five men with obstructive azoospermia (mean age: 38.5 years) with normal spermatogenesis. Men with sex chromosome or AZF abnormality were excluded. Transcriptomes on the Illumina platform were expressed as FPKM, and Cuffdiff analysis was performed to compare individual differences among testicular tissues from obstructive azoospermia (OA). Expression of mRNA and localization of targeted genes were confirmed by RT-PCR and immunohistochemical analyses, respectively. RESULTS From the 23,003 genes screened with using NGS with a significant signal after background correction and normalization, a total of 5,666 genes were analyzed. As representative markers of immature germ cells, UTF1, OCT4, DDX4, MMP1, GFRα, GPX1, SCP3, CD14, KLF9, GFRA1 and KIT mRNA were detected in SCO samples by transcriptome analysis. RT-PCR showed significant mRNA expression of UTF1, OCT4, DDX4, SCP3 and KIT from SCO samples. Immunohistochemistry showed the localization of DDX4 and/or KIT-positive cells in the seminiferous tubules and basement membrane in eight (40%) of the SCO samples. All the gene products evaluated in SCO samples were present in testes with obstructive azoospermia. There was no significant difference in the patients' background and hormonal data between the men with and without DDX4 and/or KIT-positive cells. CONCLUSIONS Germ cells are present in 40% of SCO samples as immature forms, namely SSCs. These observations help our understanding of the pathophysiology of spermatogenic failure where germ cell development stops during early stages of spermatogenesis in some men with SCO. The azoospermia seen in men with SCO appears to be, at least in part, secondary to a defect in proliferation and differentiation of SSCs. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e795 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Koji Shiraishi More articles by this author Hideyasu Matsuyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identification of a Critical Window for Ganciclovir-Induced Disruption of Testicular Development in Rats.

Ganciclovir (GCV) has been implicated in the development of testicular alterations. Exposure on gestational day (GD) 10 in rats induced permanent effects, including focal reduction or absence of germ cells (Sertoli cell-only tubules). Because the timing of exposure can be critical for testicular effects, we exposed rat dams to 300 mg/kg GCV (3 100 mg/kg subcutaneous injections) on GD10, 14 and 19, when germ cells have high rates of migration, proliferation and are mitotically quiescent, respectively. Males exposed to GCV in utero on GD10 and 14 were evaluated for androgenization markers, serum and fecal androgens, and testicular histomorphometry at adulthood. Double-labeling immunofluorescence for DAZL and Ki67 were used to assess gonocytes number and the proliferative activity of germ and somatic cells in fetal testes on GD15 and 20, ie, 24 h after GCV exposure. Adult rats exposed on GD14 showed delayed puberty onset, despite normal androgen levels. Also, there was a 50% reduction in testicular weight and about 30% of seminiferous tubules lacking germ cells. Effects on GD10 animals were less pronounced. In the fetal testis, the number of gonocytes was reduced by 50% in rats exposed on GD14, but normal in GD19 fetuses. GCV also reduced Sertoli cell proliferation immunolabeling in GD19 fetuses and Sertoli cell number in adults. In conclusion, GCV toxicity on germ cells seems to be linked to their proliferation rate and GD14 is a critical window in rats, when GCV exposure causes an acute massive loss of germ cells that persists until adulthood.

Interspecific hybridization of sturgeon species affects differently their gonadal development

Gonad development in fish is generally assumed to be negatively influenced by interspecific hybridization, resulting in sterility or sub-sterility. However, this is not the case in sturgeons (Acipenseridae), in which fertile hybrids are common. In the present study, we investigated gonad development in several sturgeon interspecific hybrids and purebred species. Six interspecific hybrid groups and three purebred groups were analyzed including 20 hybrid specimens with even ploidy, 40 specimens having odd ploidy levels, and 30 purebred specimens. Hybrids of species with the same ploidy (even ploidy – 2n, 4n) exhibited normally developed gonads similar to those seen in purebred specimens. In contrast, hybrids of species differing in ploidy (odd ploidy – 3n) did not display fully developed gonads. Ovaries were composed of oocytes or nests of differentiating oocytes that ceased development in early stages of meiosis (pachytene to zygotene) with a higher content of adipose and apoptotic tissue. Testes contained single spermatogonia along with Sertoli cells and spaces lacking germ cells. The obtained results showed that gonad development was influenced by genetic origin and ploidy of the sturgeon hybrids and were consistent with full fertility of hybrids with even ploidy. Sterility of females, but possibly limited fertility of males, is suggested for hybrids with odd ploidy.

Application of dead end-knockout zebrafish as recipients of germ cell transplantation.

Germ cell transplantation is a promising technology for the propagation of endangered or valuable fishes. In this technique, sterile male and female recipient fish are injected with donor germ cells so they can produce viable gametes derived from the donor cells. The dead end (dnd) gene is involved in the migration of primordial germ cells; therefore, dnd-knockout zebrafish are expected to be germ-cell-free, making them suitable recipients for germ cell transplantation. dnd mutants were produced by microinjecting 2 nl of 10 ng/μl cRNAs encoding zinc finger nucleases against dnd into the blastodisc of zebrafish embryos before the cell- cleavage stage. One of the resulting founder males was mated with a wild-type female, and produced heterozygous mutants in the F1 generation. Mating of these F1 mutants produced an F2 generation with approximately 25% of the clutch being homozygous mutant (dnd-knockout) male, and lacking germ cells (as confirmed by expression analyses of vasa). The resulting dnd-knockout zebrafish males were tested for suitability as germ cell transplantation recipients by intraperitoneal transplantation of testicular cells prepared from vasa-gfp zebrafish. GFP-positive germ cells incorporated into the germ-cell-free gonads of the dnd-knockout recipients matured into functional sperm. Progeny tests revealed that the sperm from these dnd-knockout recipients were derived entirely from donor cells. Thus, we demonstrated that homozygous dnd mutants became germ-cell-free males that are able to nurse donor-derived germ cells.

Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.

Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signaling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-associated phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny production and somatic aging and identify the loss of lysosomal acidity-a critical regulator of the proteolytic output of these organelles-as a novel biomarker of aging in C. elegans The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reproduction, and correlates with the reduction in proteostasis in early adult life. Our results further implicate the steroid signaling pathway and DAF-16 in dynamically regulating lysosomal pH in the intestine of wild-type worms in response to the reproductive cycle. In the intestine of reproducing worms, DAF-16 promotes acidic lysosomes by upregulating the expression of v-ATPase genes. These findings support a model in which protein clearance in the soma is linked to reproduction in the gonad via the active regulation of lysosomal acidification.

Bmp15 Is an Oocyte-Produced Signal Required for Maintenance of the Adult Female Sexual Phenotype in Zebrafish.

Although the zebrafish is a major model organism, how they determine sex is not well understood. In domesticated zebrafish, sex determination appears to be polygenic, being influenced by multiple genetic factors that may vary from strain to strain, and additionally can be influenced by environmental factors. However, the requirement of germ cells for female sex determination is well documented: animals that lack germ cells, or oocytes in particular, develop exclusively as males. Recently, it has been determined that oocytes are also required throughout the adult life of the animal to maintain the differentiated female state. How oocytes control sex differentiation and maintenance of the sexual phenotype is unknown. We therefore generated targeted mutations in genes for two oocyte produced signaling molecules, Bmp15 and Gdf9 and here report a novel role for Bmp15 in maintaining adult female sex differentiation in zebrafish. Females deficient in Bmp15 begin development normally but switch sex during the mid- to late- juvenile stage, and become fertile males. Additionally, by generating mutations in the aromatase cyp19a1a, we show that estrogen production is necessary for female development and that the function of Bmp15 in female sex maintenance is likely linked to the regulation of estrogen biosynthesis via promoting the development of estrogen-producing granulosa cells in the oocyte follicle.