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In Acanthocephala, the ovarian balls (floating ovaries) are distinctive structures found suspended in the fluid-filled metasoma of females and are responsible for egg production and maturation. Those structures have not been studied in Rhadinorhynchus niloticus. We aimed to investigate their ultra-structure by means of scanning and transmission electron microscopy. A total of 30 Lates niloticus fish individuals were collected by fishermen from the River Nile, Egypt, and the isolated adult female specimens were processed for electron microscopy studies. The ovarian balls are elongated and lobulated structures freely scattered in the metasoma. They exhibited three distinct primary structural zones, a central oogonial syncytium, a peripheral cellular zone and a surrounding somatic supporting syncytium. The oogonia, within the central syncytium, give rise to developing oocytes that transform into mature oocytes. The latter enclose some inclusions such as large yolk granules and smaller egg-shell granules. We also describe the process of fertilization within the ovarian ball. The structure of the ovarian ball and the steps of fertilization in R. niloticus are described, for the first time, in the present study.

We contest the recently reported findings that suggest the presence of female gametangia in the North American range of the invasive macroalga Nitellopsis obtusa (Harrow-Lyle, T.J., Ginn, B.K., Kirkwood, A.E., and Melles, S.J. 2023. First report of female gametangia in the invasive macroalga starry stonewort (Nitellopsis obtusa) in North America. Botany, 101: 61–64. doi:10.1139/cjb-2022-0096). The claims do not clearly show identifying morphology of N. obtusa female gametangia, are inconsistent with known characeaen developmental biology, and do not provide the evidence commensurate with such a claim. More convincing observational data are required. To date, only male thalli of N. obtusa have been confirmed either with herbarium vouchers or clear images. The appearance of female gametangia would have significant implications for the invasion biology of N. obtusa as well as approaches to managing its spread. [ABSTRACT FROM AUTHOR]

(1) Fshβ and Lhβ showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshβ and Lhβ at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E2 concentrations increased significantly at 174, 333, and 1435 dph, while T concentrations exhibited significant increases at 174 and 333 dph. These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis. [ABSTRACT FROM AUTHOR]

In some species, permanent curtailment of reproduction part‐way through the lifespan of adult females is a feature of their evolved life history. The existence of such a post‐reproductive life stage is apparently rare; reasonably robust evidence for this is confined to only six species (humans, Asian elephants and four whales). That it occurs at all appears to contradict our view of natural selection operating to maximize fitness and special circumstances must exist to explain its occurrence. We evaluate the main hypotheses posited to explain the evolution of this life stage, why it occurs in a restricted group of animals, and why only in females. We bring together literature from multiple biological disciplines and levels of enquiry, ranging through evolutionary ecology, developmental biology, physiology, neuroscience, molecular biology, and human medicine. We conclude that while time‐limited fertility is not in itself adaptive, the duration of subsequent survival is likely to be linked to inclusive fitness benefits. We present a new hypothesis which posits that the duration of female fertility in certain long‐lived, highly encephalised species, with no post‐natal oogenesis, is limited by the need for intense screening of oocyte mitochondria. This is required to support endothermy coupled with the very high energy requirement for the development and maintenance of the exceptionally large brain size required for complex social living. This limits the number and shelf‐life of oocytes, creating an antagonistically pleotropic effect that is beneficial to the production of high performing offspring but carries the later life cost of time‐limited female fertility. But the end of the fertile period is no time to die. Inclusive fitness benefits arising from protracted parental care of offspring, overlapping generations, and kin group structures means that continued survival of post‐reproductive females is favoured by selection. We suggest further lines of research to test these ideas. [ABSTRACT FROM AUTHOR]

Perspectives on past climate using lake sediments are critical for assessing modern and future climate change. These perspectives are especially important for water-stressed regions such as the western United States. One such region is northwestern California (CA), where Holocene-length hydroclimatic records are scarce. Here, we present a 9000-year, relative lake level record from Maddox Lake (CA) using a multi-indicator approach. The Early Holocene is characterized by variably low lake levels with a brief excursion to wetter climates/ relative highstand ca. 8.4-8.06 cal ka BP, possibly related to the 8.2 ka cold event and changing Atlantic Meridional Overturning Circulation (AMOC). From 5.2-0.55 cal ka BP, Maddox Lake experienced a long-term regression, tracking changes in summer-winter insolation, tropical and northeast Pacific SSTs, and the southward migration of the ITCZ. This gradual regression culminated in a pronounced relative lowstand during the Medieval Climatic Anomaly (MCA). A marked relative highstand followed the MCA, correlative to the Little Ice Age. The latter reflects a far-field response to North Atlantic volcanism, solar variability, and possibly changes in AMOC and Arctic sea ice extent. Our results further confirm the hydroclimatic sensitivity of northwest California to various forcings including those emanating from the North Atlantic. [ABSTRACT FROM AUTHOR]

Dexamethasone (DEX), which is a corticosteroid hormone (glucocorticoid), has been used to treat different conditions, such as immune system disorders, certain skin and eye disorders, as well as breathing problems. Cefotaxime sodium, also called Claforan, is synthesized from a naturally occurring material (semisynthetic). It is a broad-spectrum cephalosporin antibiotic that could be utilized for parenteral administration. The present study aimed to investigate histological changes occurring in the tissues and cells of the rats’ ovary (primordial, primary, secondary, antral, and mature follicle) treated with Cefotaxime sodium, as well as DEX, and evaluate the impacts of these medications on animals’ fertility. In total, 40 female adult Wistar rats were divided into four groups (n=10). The control group received 0.5 ml/kg of distilled water daily for five days as a placebo. The second group was injected with 0.5 mg/kg of DEX daily for five days. The same amount of Claforan (0.5 mg/kg) was injected into the third group daily for five days, and the fourth group received 0.5 mg/kg of both Claforan and DEX daily for five days. Afterward, the ovaries were prepared for histological examination. The ImageJ image analysis system was used to detect morphometric parameters and calculate the area of these organs. The findings of the present study showed that the DEX and Claforan brought changes to the ovarian area and the number of follicles. The ovarian area significantly increased (P

Nitellopsis obtusa (Desv.) J. Groves (starry stonewort) is a macroalga in the Characeae family first documented in North America c. 1974. Since initial introduction, N. obtusa clonal populations quickly established in inland lakes as early as 2005. Despite increased N. obtusa monitoring over the last decade, only sterile or male specimens were documented in North America; however, during routine monitoring in Lake Simcoe and Lake Scugog in 2022, we discovered the presence of female gametangia on N. obtusa. In addition, two other Characeae genera had prevalent antheridia and oogonia, co-occurring with oogonia-presenting N. obtusa, which had not been observed previously despite intensive monitoring since 2008. Further studies in North America are required to confirm the proportion of female populations present within invaded regions, as well as to identify plausible causes shifting gametangia development across non-native and native Characeae, especially within the context of climate change. The presence of oogonia on N. obtusa represents a major change to our understanding of this species and its reproductive ecology in North America. [ABSTRACT FROM AUTHOR]

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. • A vitrification procedure was optimized for sturgeon species for the first time. • Vitrification of sterlet and Russian sturgeon oogonia yielded ∼50% survival. • Vitrified/warmed oogonia incorporated into recipient gonads after transplantation. [ABSTRACT FROM AUTHOR]

• Exogenous recombinant gonadotropins administered to meagre did not advance coordinated germ cell sexual differentiation. • Exogenous recombinant gonadotropins administered to 9-month-old meagre did not affect plasma estradiol levels. • Exogenous recombinant Fsh increased the proportion of meagre with isolated male germ cells. • Exogenous recombinant Fsh increased plasma 11-ketotestosterone in meagre treated for 6 weeks. Recombinant gonadotropins, follicle stimulating (rFsh) and luteinizing hormone (rLh), offer the potential to induce gametogenesis in prepubertal fish. This study aimed to determine the in vivo effect of the administration of Argyrosomus regius rFsh and rLh on the reproductive development of prepubertal meagre juveniles at the initial stages of sexual differentiation. Juvenile meagre, 9-months old with mean weight of 219 ± 3.9 g (mean ± SEM) were randomly distributed into nine groups (n = 8 per group). Experimental groups were treated weekly with an acute injection of either rFsh or rLh. Control groups were injected with saline solution. In a 3-week experiment, different groups were administered with different doses 6, 12 or 18 µg kg−1 of rFsh or rLh or saline solution. In a 6-week experiment a group was administered with 12 µg kg−1 of rFsh and a second group with saline solution. The fish were held in a single 10 m3 tank with natural photoperiod (Feb. – March) and temperature 16.1 ± 0.4 °C. At the start of the experiment (n = 8) and at the end of the 3-week experiment, fish were blood sampled and sacrificed. Blood was analysed for 17β-estradiol (E2) and 11-ketotestosterone (11-KT). Gonads and liver were dissected and weighed. Gonads were fixed in Bouińs solution and processed for histological analysis. Juvenile meagre at the start of the experiment were in the initial stages of sexual differentiation, indicated by the presence of the ovarian cavity or testes duct that was surrounded by undifferentiated embryonic germ stem cells and somatic cells. At the end of the 3-week experiment, there was no significant difference in gonadosomatic index (GSI) amongst control (initial and saline treated) and the experimental groups. After three weeks of application of rFsh, rLh or saline all fish presented a similar gonadal structure as at the start of the experiment. However, the incidence of sporadic developing germ cells (principally spermatogonia, spermatocytes, spermatids, but also perinucleolar stage oocytes) generally increased in rGth treated meagre. A mean of 44 % of meagre treated with rFsh or rLh presented sporadic isolated developing germ cells, mainly male cells. Plasma steroid levels of E2 decreased significantly from the start of the experiments to the end. At the end of the experiments there were no differences in plasma E2 amongst Control fish and rGth treated fish. Plasma 11-KT showed no change from the start of the experiment to week 3. However, a significant increase was observed in a proportion of the rFsh group after six weeks of treatment compared to the start of the experiment and the saline control group on week 6. The application of rFsh or rLh to meagre at the initial stages of sex differentiation did not stimulate steroid production until week six (11-KT) and had a limited, but evident effect on the development of sporadic isolated germ cells. However, we conclude that rGth, rFsh or rLh did not stimulate large developmental changes in sexually undifferentiated meagre gonads. [ABSTRACT FROM AUTHOR]

(1) Fshβ and Lhβ showed stronger signals and higher transcript levels from 590 to 1050 dph than at earlier stages, implying their active involvement during primary oocyte development. (2) Fshβ and Lhβ at lower levels were detected during the phases of ovarian differentiation and oogonial proliferation. (3) E2 concentrations increased significantly at 174, 333, and 1435 dph, while T concentrations exhibited significant increases at 174 and 333 dph. These findings suggest potential correlations between serum E2 concentrations and the phases of oogonial proliferation and pre-vitellogenesis.

Gametes in many species develop in cysts-clusters of germ cells formed by incomplete cytokinesis-that remain connected through intercellular bridges (ICBs). These connections enable sharing of cytoplasmic components between germ cells and, in the female germ line, enrich select cells in the cyst to become the oocyte(s). In mice, germline cysts of variable sizes are generated during embryonic development, thought to result from cyst fractures. Studies of fixed samples failed to capture fracture events, and thus, the mechanism remained elusive. Here, we use high-resolution live imaging of germ cells within their native tissue environment to visualize germline cyst dynamics. With this novel approach, we reveal a striking motile phenotype of gonad-resident germ cells and show that this randomly oriented cell-autonomous motile behavior during cyst formation underlies fracture events. Conversely, we show that stabilized ICBs help resist excessive fracturing. Additionally, we find that motility and thus fracture rates gradually decrease during development in a sex-dependent manner, completely ceasing by the end of cyst-forming divisions. These results lead to a model where the opposing activities of developmentally regulated cell motility and stable ICBs give rise to cysts of variable sizes. We corroborate these results by developing a model that uses experimentally measured fracture rates to simulate cyst formation and fracture and show that it can reproduce experimentally measured cyst sizes in both male and female. Understanding how variable cysts form will enable further studies of mammalian oocyte selection and establishment of the ovarian reserve., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes. • Development of a meiosis targeting culture model of the fetal ovary in sheep. • BPA reduced the expression of meiotic marker genes in exposed explants. • BPA altered expression of genes involved in cell cycle transition, meiosis, and spindle formation. • Long-term effects could result in a reduction of the ovarian reserve and an increased frequency of aneuploidy. [ABSTRACT FROM AUTHOR]

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.

Dexamethasone (DEX), which is a corticosteroid hormone (glucocorticoid), has been used to treat different conditions, such as immune system disorders, certain skin and eye disorders, as well as breathing problems. Cefotaxime sodium, also called Claforan, is synthesized from a naturally occurring material (semisynthetic). It is a broad-spectrum cephalosporin antibiotic that could be utilized for parenteral administration. The present study aimed to investigate histological changes occurring in the tissues and cells of the rats' ovary (primordial, primary, secondary, antral, and mature follicle) treated with Cefotaxime sodium, as well as DEX, and evaluate the impacts of these medications on animals' fertility. In total, 40 female adult Wistar rats were divided into four groups (n=10). The control group received 0.5 ml/kg of distilled water daily for five days as a placebo. The second group was injected with 0.5 mg/kg of DEX daily for five days. The same amount of Claforan (0.5 mg/kg) was injected into the third group daily for five days, and the fourth group received 0.5 mg/kg of both Claforan and DEX daily for five days. Afterward, the ovaries were prepared for histological examination. The ImageJ image analysis system was used to detect morphometric parameters and calculate the area of these organs. The findings of the present study showed that the DEX and Claforan brought changes to the ovarian area and the number of follicles. The ovarian area significantly increased (P<0.007) in the DEX-treated group (mean±SEM=7.3±0.5 mm2), compared to the control group (mean±SEM=4.6±0.20 mm2). However, DEX was found to decrease body weight. Furthermore, the ovarian area significantly increased in the Claforan-treated group (mean±SEM=8.6±0.6 mm2); however, their body weight significantly decreased (P<0.008), in comparison with the control and DEX-treated groups. The combination treatment (i.e., DEX + Cefotaxime sodium) significantly increased (P<0.009) the area of ovaries even more, compared to single treatments (mean±SEM=9.6±0.4 mm2). Overall, both DEX and Claforan brought histological changes to ovaries. However, the effect of DEX on ovaries was less than that of Claforan. The concurrent administration of both medications was found to have more significant effects on rats' ovaries. [ABSTRACT FROM AUTHOR]

Echinoderms produce functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes remains a mystery. Here, we present the first single cell RNA sequencing data sets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus [Sp] and Lytechinus variegatus [Lv]), and one sea star species (Patiria miniata [Pm]). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum., (© 2023 Wiley Periodicals LLC.)

Study Question: How are germ cell numbers and initiation of folliculogenesis affected in fetal Turner syndrome (TS) ovaries?Summary Answer: Germ cell development was severely affected already in early second trimester pregnancies, including accelerated oogonia loss and impaired initiation of primordial follicle formation in TS ovaries, while the phenotype in TS mosaic ovaries was less severe.What Is Known Already: Females with TS are characterized by premature ovarian insufficiency (POI). This phenotype is proposed to be a consequence of germ cell loss during development, but the timing and mechanisms behind this are not characterized in detail. Only few studies have evaluated germ cell development in fetal TS and TS mosaic ovaries, and with a sparse number of specimens included per study.Study Design, Size, Duration: This study included a total of 102 formalin-fixed and paraffin-embedded fetal ovarian tissue specimens. Specimens included were from fetuses with 45,X (N = 42 aged gestational week (GW) 12-20, except one GW 40 sample), 45,X/46,XX (N = 7, aged GW 12-20), and from controls (N = 53, aged GW 12-42) from a biobank (ethics approval # H-2-2014-103).Participants/materials, Setting, Methods: The number of OCT4 positive germ cells/mm2, follicles (primordial and primary)/mm2 and cPARP positive cells/mm2 were quantified in fetal ovarian tissue from TS, TS mosaic and controls following morphological and immunohistochemical analysis.Main Results and the Role Of Chance: After adjusting for gestational age, the number of OCT4+ oogonia was significantly higher in control ovaries (N = 53) versus 45,X ovaries (N = 40, P < 0.001), as well as in control ovaries versus 45,X/46,XX mosaic ovaries (N = 7, P < 0.043). Accordingly, the numbers of follicles were significantly higher in control ovaries versus 45,X and 45,X/46,XX ovaries from GW 16-20 with a median range of 154 (N = 11) versus 0 (N = 24) versus 3 (N = 5) (P < 0.001 and P < 0.015, respectively). The number of follicles was also significantly higher in 45,X/46,XX mosaic ovaries from GW 16-20 compared with 45,X ovaries (P < 0.005). Additionally, the numbers of apoptotic cells determined as cPARP+ cells/mm2 were significantly higher in ovaries 45,X (n = 39) versus controls (n = 15, P = 0.001) from GW 12-20 after adjusting for GW.Limitations, Reasons For Caution: The analysis of OCT4+ cells/mm2, cPARP+ cells/mm2 and follicles (primordial and primary)/mm2 should be considered semi-quantitative as it was not possible to use quantification by stereology. The heterogeneous distribution of follicles in the ovarian cortex warrants a cautious interpretation of the exact quantitative numbers reported. Moreover, only one 45,X specimen and no 45,X/46,XX specimens aged above GW 20 were available for this study, which unfortunately made it impossible to assess whether the ovarian folliculogenesis was delayed or absent in the TS and TS mosaic specimens.Wider Implications Of the Findings: This human study provides insights about the timing of accelerated fetal germ cell loss in TS. Knowledge about the biological mechanism of POI in girls with TS is clinically useful when counseling patients about expected ovarian function and fertility preservation strategies.Study Funding/competing Interest(s): This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC).Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Compact blankets of free-floating plants generate stressful aquatic environments. The response of submerged macrophytes remains largely elusive. Will they rush toward the light or rather speed up reproductive efforts and escape using propagules—the macrophyte equivalent of lifeboats? We studied the effects of complete duckweed (Lemna minor) cover on growth and reproductive fitness of macroalga Chara globularis in a pond mesocosm experiment. C. globularis growing in Lemna-covered plots lost biomass and developed longer internodes, indicating an elongative reflex to escape stress. Densities of reproductive organs per biomass unit evolved positively in open plots and negatively in covered plots, suggesting a trade-off between reproductive effort and vegetative elongation. Reproductive potential correlated significantly with incident radiation. Lemna cover, however, did not affect oospore rain—at least within the limited time span of propagule trapping. C. globularis thus displayed an ability to modify phenology in response to floating plant stress, allocating resources to internodes instead of gametangia. Nevertheless, duckweed dominance clearly suppressed the overall reproductive performance of C. globularis. The regenerative capacity of many submerged macrophytes will likely suffer from increased floating plant dominance due to global warming—unless efforts are made to reduce nutrient levels in vulnerable waterbodies. [ABSTRACT FROM AUTHOR]

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.

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. • No effective cryopreservation technique exists for fish eggs or embryos. • Transplantation of cryopreserved immature germ cells can generate eggs and sperm. • However, the resultant offsprings generated were unnatural sex-biased fish. • Masu salmon produced by spermatogonia-derived sperm and oogonia-derived eggs show a normal sex ratio. • This approach could be practically applied in conservation programs. [ABSTRACT FROM AUTHOR]

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell‐like cells (hPGCLCs) derived from human‐induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106‐fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC‐like transcriptome and preserve their genome‐wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50‐fold) and persist only around 1 week, yet undergo cell‐autonomous genome‐wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic‐reconstituted ovaries, expanded hPGCLCs initiate genome‐wide DNA demethylation and differentiate into oogonia/gonocyte‐like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage. Synopsis: In vitro reconstitution of germ‐cell development is a key for analyzing and enhancing reproductive biology. This resource reports a culture method for robust propagation of human primordial germ cell‐like cells (hPGCLCs) under defined conditions, establishing a tractable paradigm for human germ‐cell expansion. Induced pluripotent stem cell‐derived hPGCLCs expand ~106‐fold under forskolin/SCF/bFGF in vitro over a period of four months.During expansion, hPGCLCs maintain an early hPGC(LC) transcriptome and DNA methylome.hPGC(LC) specification and epigenetic reprogramming are genetically dissociable.Expanded hPGCLCs differentiate into oogonia with epigenetic reprogramming in xenogeneic reconstituted ovaries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Primordial follicles have been formed before birth in mammals, and the reserve in follicle pool no longer increase after birth. With the activation of the primordial follicles, the follicles which were originally dormant gradually enter the growth stage, and gradually evolve from primary follicles and secondary follicles to antral follicles. Ovulation occurs in the dominant follicles with the stimulation of gonadotropins. Oocyte-derived and exogenous factors which regulate the maturation of oocytes, the proliferation and differentiation of the cells and granulosa cells through paracrine, autocrine, and endocrine ways. Besides, the communication between oocytes and granulosa cells through gaps junction allows synchronous development of all compartments of the follicle to maintain the follicle integrity. The thorough research on the mechanism of follicle activation and folliculogenesis will be beneficial to the solution of oocyte-related infertility in the field of reproductive medicine [ABSTRACT FROM AUTHOR]

We analysed the ovarian dynamics of the anadromous semelparous allis shad Alosa alosa for which our working hypothesis was that mature pre‐spawning females would have very low or even exhausted primary growth (PG) oocyte reserves; semelparity has been linked with the depletion of the pool of PG oocytes. To test this hypothesis, the PG oocytes were enumerated, their recruitment pattern to the secondary growth (SG) phase was analysed and their potential replenishment from the pool of oogonia was examined in females caught very close to the Mondego River mouth, in central Portugal and along the river. The development of the SG oocytes was also analysed, the fecundity (batch, total and annual) values were estimated and the intensity of atresia was quantified. Ovarian samples and histological sections were investigated in parallel. A dynamic recruitment pattern of PG oocytes to the SG phase was revealed, where all PG oocytes were recruited and were not replenished by oogonia. Annual fecundity was subject to down‐regulation due to atresia prior to spawning and its size was multiple times higher than the size of batch fecundity. Lack of population synchronicity in ovarian development and spawning migration was also observed. This multifaceted analysis of the ovarian dynamics of this species will contribute to management efforts for this critically endangered and economically important fish throughout its geographical distribution. The results reported in this study will also assist in unravelling the complexity of the early processes of oogenesis in fish. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Slow-rate freezing of common carp OSCs yielded ∼65% post-thaw survival. • Hypothermic storage presented a valuable alternative to cryopreservation. • Cryopreserved and fresh OSCs were recovered in surrogate goldfish recipients. • Identity of donor-derived cells was confirmed by fluorescent labelling and RT-PCR. [ABSTRACT FROM AUTHOR]

Teleost sex differentiation largely depends on the number of undifferentiated germ cells. Here, we describe the generation and characterization of a novel transgenic zebrafish line, Tg(piwil1:egfp-UTRnanos3)ihb327Tg, which specifically labels the whole lifetime of germ cells, i.e., from primordial germ cells (PGCs) at shield stage to the oogonia and early stage of oocytes in the ovary and to the early stage of spermatogonia, spermatocyte, and spermatid in the testis. By using this transgenic line, we carefully observed the numbers of PGCs from early embryonic stage to juvenile stage and the differentiation process of ovary and testis. The numbers of PGCs became variable at as early as 1 day post-fertilization (dpf). Interestingly, the embryos with a high amount of PGCs mainly developed into females and the ones with a low amount of PGCs mainly developed into males. By using transient overexpression and transgenic induction of PGC-specific bucky ball (buc), we further proved that induction of abundant PGCs at embryonic stage promoted later ovary differentiation and female development. Taken together, we generate an ideal transgenic line Tg(piwil1:egfp-UTRnanos3)ihb327Tg which can visualize zebrafish germline for a lifetime, and we have utilized this line to study germ cell development and gonad differentiation of teleost and to demonstrate that the increase of PGC number at embryonic stage promotes female differentiation. [ABSTRACT FROM AUTHOR]

Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells. [ABSTRACT FROM AUTHOR]

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia—27%; grayling spermatogonia—28%; grayling oogonia—23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species. [ABSTRACT FROM AUTHOR]

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation., (© 2023. Springer Nature Limited.)

Macrobrachium amazonicum is a species of economic interest with a wide distribution in the Americas and high morphological and reproductive variability. Three phenotypes can be observed in this species: i) large-size amphidromous, ii) large-size and iii) small-size hololimnetic prawns. In the present work, the morphological, histochemical and ultrastructural aspects of ovarian development in the three phenotypes were comparatively analyzed. In addition, the interaction between the ovary and the hepatopancreas was investigated in these phenotypes through the use of gonadosomatic (GSI) and hepatosomatic (HSI) indices. Despite the morphological differences and different reproductive strategies adopted by the females, the macroscopic, histochemical and ultrastructural patterns of ovarian development showed no differences between the phenotypes. The ovaries were macroscopically classified into five stages of development (I to V). In early stages (I and II), the ovaries are full of oogonia, previtellogenic oocytes and oocytes in primary or endogenous vitellogenesis. At these stages, the rough endoplasmic reticulum (RER) produces a granular electron-dense material and sends it to the Golgi apparatus, where it will be modified, compacted and transformed into immature yolk granules. From stage III, secondary or exogenous vitellogenesis begins (with no interruption of endogenous vitellogenesis), where follicular cells nourish the oocytes and extracellular material is absorbed by endocytic vesicles, which fuse with immature yolk granules (forming mature granules) or with existing mature yolk granules. In stages IV and V, secondary vitellogenesis continues and mature yolk granules progressively occupy the cytoplasm. In M. amazonicum, the patterns of increase in oocyte diameter are quite similar between phenotypes, being greater in the small-size phenotype. This is related to the formation of larger oocytes/eggs and the production of large lipid reserves for their larvae. Changes in GSI and HSI during ovarian development show strong similarity between phenotypes, supporting the results obtained by histology and ultrastructure. Females in stages III and IV mobilize hepatopancreas reserves for ovarian maturation, which justifies the higher HSI values recorded in these stages. On the other hand, females in stage V show higher GSI and lower HSI values, indicating a mobilization of resources for the end of ovarian development as the females are ready to spawn., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Abstract: The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L‐15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The 2‐week trial demonstrated that storage media have a profound influence, while regular medium exchange does not have a positive effect on cell viability. Viability of SSCs and OSCs after two weeks was approximately 40% and 25%, respectively; however, survival of ~70% was obtained after 10 days of storage for SSCs and 7 days for OSCs. Hypothermic storage developed in this study has many practical applications during the development of surrogate broodstock technologies for common carp, but also in carp hatcheries and for the conservation of genetic resources of closely related cyprinid species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively. [ABSTRACT FROM AUTHOR]

Study Question: Does ibuprofen use during the first trimester of pregnancy interfere with the development of the human fetal ovary?Summary Answer: In human fetuses, ibuprofen exposure is deleterious for ovarian germ cells.What Is Known Already: In utero stages of ovarian development define the future reproductive capacity of a woman. In rodents, analgesics can impair the development of the fetal ovary leading to early onset of fertility failure. Ibuprofen, which is available over-the-counter, has been reported as a frequently consumed medication during pregnancy, especially during the first trimester when the ovarian germ cells undergo crucial steps of proliferation and differentiation.Study Design, Size, Duration: Organotypic cultures of human ovaries obtained from 7 to 12 developmental week (DW) fetuses were exposed to ibuprofen at 1-100 μM for 2, 4 or 7 days. For each individual, a control culture (vehicle) was included and compared to its treated counterpart. A total of 185 individual samples were included.Participants/materials, Setting, Methods: Ovarian explants were analyzed by flow cytometry, immunohistochemistry and quantitative PCR. Endpoints focused on ovarian cell number, cell death, proliferation and germ cell complement. To analyze the possible range of exposure, ibuprofen was measured in the umbilical cord blood from the women exposed or not to ibuprofen prior to termination of pregnancy.Main Results and the Role Of Chance: Human ovarian explants exposed to 10 and 100 μM ibuprofen showed reduced cell number, less proliferating cells, increased apoptosis and a dramatic loss of germ cell number, regardless of the gestational age of the fetus. Significant effects were observed after 7 days of exposure to 10 μM ibuprofen. At this concentration, apoptosis was observed as early as 2 days of treatment, along with a decrease in M2A-positive germ cell number. These deleterious effects of ibuprofen were not fully rescued after 5 days of drug withdrawal.Large Scale Data: N/A.Limitations, Reasons For Caution: This study was performed in an experimental setting of human ovaries explants exposed to the drug in culture, which may not fully recapitulate the complexity of in vivo exposure and organ development. Inter-individual variability is also to be taken into account.Wider Implications Of the Findings: Whereas ibuprofen is currently only contra-indicated after 24 weeks of pregnancy, our results points to a deleterious effect of this drug on first trimester fetal ovaries ex vivo. These findings deserve to be considered in light of the present recommendations about ibuprofen consumption pregnancy, and reveal the urgent need for further investigations on the cellular and molecular mechanisms that underlie the effect of ibuprofen on fetal ovary development. [ABSTRACT FROM AUTHOR]

Thysanoessa macrura (Sars, 1883) is a small, albeit highly abundant, Southern Ocean euphausiid. It has been reported that T. macrura reproduces during late winter, with the reproductive cycle decoupled from periods of high primary productivity in the highly seasonal Southern Ocean. Despite this uncommon reproductive strategy, there has been little work to describe this cycle and quantify the reproductive potential of this species. Thysanoessa macrura collected from Antarctic waters during early September to early October 2007 were used to establish sexual developmental stages, for females and males, to provide a standard means for assessing the reproductive maturity of this species. Seven sexual developmental stages were established for females based upon the organisation of ovaries and their constituent oogonia and oocytes. Four sexual developmental stages were identified in males using secondary sexual characters. A large lipid deposit was identified in the carapace of both sexes, reducing in size with progressive gonadal development, likely fuelling egg and sperm production. Egg-batch size was determined by direct ovarian counts and scaled allometrically with total wet weight. Predicted egg-batch size ranged from 34 to 746 eggs batch-1 for the range of individual sizes studied (30.5-165.6 mg). Using the ratios of the different oocytes remaining after the first-spawning event of females, it was predicted that up to three egg batches can be produced by T. macrura in a single reproductive season. Consequently, the fecundity of T. macrura is estimated at 60 to > 2200 eggs female-1 year-1. A model of T. macrura reproductive cycle confirms a winter reproductive period and further highlights the reproductive potential of this small-size Southern Ocean euphausiid, providing the first estimates of fecundity reported for this species. [ABSTRACT FROM AUTHOR]

The reproductive strategy of diatoms includes asexual and sexual phases, but in many species, including the model centric diatom Thalassiosira pseudonana, sexual reproduction has never been observed. Furthermore, the environmental factors that trigger sexual reproduction in diatoms are not understood. Although genome sequences of a few diatoms are available, little is known about the molecular basis for sexual reproduction. Here we show that ammonium reliably induces the key sexual morphologies, including oogonia, auxospores, and spermatogonia, in two strains of T. pseudonana, T. weissflogii, and Cyclotella cryptica. RNA sequencing revealed 1,274 genes whose expression patterns changed when T. pseudonana was induced into sexual reproduction by ammonium. Some of the induced genes are linked to meiosis or encode flagellar structures of heterokont and cryptophyte algae. The identification of ammonium as an environmental trigger suggests an unexpected link between diatom bloom dynamics and strategies for enhancing population genetic diversity. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Study Question: How can pre-meiotic germ cells persist in the human foetal ovary?Summary Answer: Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis.What Is Known Already: The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive.Study Design Size, Duration: In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization.Participants/materials Setting, Methods: Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures.Main Results and the Role Of Chance: Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells.Large Scale Data: N/A.Limitations Reason For Caution: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis.Wider Implications Of the Findings: We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex.Study Funding/competing Interest(s): This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]