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Surrogate production through germline stem cell (GSC) transplantation is becoming a very promising technology in species management and ex situ species conservation. In the present study, we have evaluated if the triploid rainbow trout is a suitable surrogate parent for the production of other salmonid gametes. In three independent trials conducted in two countries, we have transplanted brown trout and grayling GSCs into triploid rainbow trout larvae as recipients. Two months after transplantation, dissection of recipients displayed that both spermatogonial (SSCs) and oogonial (OSCs) stem cells of both species were able to colonize recipient gonads. After three years of rearing, neither male nor female recipients displayed signs of gametogenesis progression. Species-specific (for both donor and recipient species) amplification of mtDNA control region on the other hand displayed a presence of donor-derived germ cells within recipient gonads. This indicated that even though donor cells were able to colonize recipient gonads, they remained in a dormant or quasi-dormant state, and did not progress with gametogenesis. This study displays that the rainbow trout is not a suitable recipient for all salmonid species, and that careful selection of recipients is a crucial step in developing the surrogate production technology.

The objective of this study was to understand germ cell renewal and differentiation in the Yesso scallop Mizuhopecten yessoensis throughout its reproductive cycle. First, we identified a Yesso scallop vasa-like gene (my-vlg) as a useful marker for immature germ cells. Both in situ hybridization and immunohistochemistry confirmed the germ-cell-specific expression of my-vlg and protein, especially in spermatogonia, oogonia, and primary oocytes. An immunohistochemistry with anti my-Vasa antibody further revealed that immature germ cells (i.e., gonial cells) are located on the basal membrane of germinal acini and are present during all maturational stages in both the ovary and testis. Quantitative analysis revealed a shift in germ cell number and change in germ cell composition associated with the gonadal maturation process, indicating a persistence of my-Vasa-positive immature germ cells throughout the reproductive cycle. In the testis, a gradual increase in my-Vasa-positive immature germ cells was seen from November to March. In the ovary, an increase in my-Vasa-positive immature germ cells was observed from September until November. We also used gonad index (GI) to summarize and evaluate the maturational changes of scallops in the 2018 generation. This generation had a background of relatively high mortality and shell deformity in the previous year, and we noted a small irregular peak of GI in the summer (July–August). We confirmed that the germ cells proliferating in the summer were my-Vasa-positive, implying that germ cell developmental progression occurred in the 2018 generation during the non-reproductive period. In conclusion, this study establishes an immuno-identification method for my-Vasa-positive immature germ cells and reveals germ cell composition in a bivalve species throughout its reproductive cycle. These results will support monitoring techniques for gonad development and the reproductive status of brood stock in shellfish seed production hatcheries.

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me 2 SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me 2 SO in the ES, and 1.5 M MeOH and 5.5 M Me 2 SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me 2 SO in the ES, and 3 M PG and 3 M Me 2 SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me 2 SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN 2 at -80 °C displayed the highest OSC viability. Different cooling rates ranging from -1 to -40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me 2 SO) with VS3 (3 M PG and 3 M Me 2 SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species., (Copyright © 2020. Published by Elsevier Inc.)

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me 2 SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation., (Copyright © 2019. Published by Elsevier Inc.)

The conservation of endangered fish is an urgent issue. Although cryo-banking of fish gametes might ultimately help conserve endangered fish, cryopreservation of fish eggs or embryos is still not possible due to their large size and high yolk content. Therefore, as an alternative, we focused on undifferentiated germ cells, such as primordial germ cells, spermatogonia, and oogonia, as materials for cryopreservation. Transplantation of cryopreserved germ cells into the body cavity of allogeneic or xenogeneic recipients sterilized by triploidization or endogenous germ cell ablation resulted in migration of the transplanted germ cells toward the recipient genital ridges, where they were eventually incorporated. The donor-derived germ cells initiated either spermatogenesis or oogenesis in the recipient gonads, depending on the sex of the recipient. Furthermore, by mating the male and female recipients, viable offspring derived from the frozen germ cells were produced. Although this technology was established using salmonid fish, we found that it is applicable to a wide range of fish species. Thus, this method might represent a "silver bullet" for preserving the valuable genetic resources of endangered fish species., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me 2 SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me 2 SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Several sturgeon species are near extinction; therefore an efficient conservation strategy is required. Germ stem cells can be used for long-term storage and restoration of genetic information using surrogate reproduction. This study compared cryopreservation procedures of early stages of Siberian sturgeon Acipenser baerii testicular and ovarian cells. Whole gonad tissue or dissociated cells were frozen at a cooling rate of 1 °C/min in phosphate buffered saline with 0.5% bovine serum albumin, 50 mM glucose, and one of four different 1.5 M cryoprotectants: dimethyl sulfoxide, glycerol, ethylene glycol, or dimethyl sulfoxide with propanediol. The number of living cells obtained from 0.1 g of gonadal tissue after freeze/thaw of both whole tissue and dissociated cells was higher using ethylene glycol than with other cryoprotectants. Although there were no differences in the number of living cells in cryopreserved whole tissue vs. dissociated cells, the number of dead cells was lower with whole tissue cryopreservation, indicating that cells that died during freeze/thaw were digested during subsequent enzymatic dissociation. This resulted in more than 90% live cells after freeze/thaw and dissociation. The thawed tissue cryopreserved using ethylene glycol as protectant as well as fresh gonadal tissue were dissociated, and the cells were labelled by PKH26 and transplanted into larvae of sterlet Acipenser ruthenus. Ninety days post-transplant of both fresh and cryopreserved cells, introduced cells proliferated in more than half of the recipients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle., (Copyright © 2014. Published by Elsevier Inc.)