Effects of cyp19a1a and cyp19a1b Knockout on Germ Cell Kinetics During Gonadal Sex Differentiation in Medaka.

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Gonadotropins play an important role in gametogenesis and reproduction in vertebrates. Their localization in the pituitary during gonadal sex differentiation has been studied mainly in southern (Oryzias latipes) strains of medaka fish, with that in northern medaka (O. sakaizumii) remaining poorly understood. Hence, in this study, we characterized gonadal differentiation and gonadotrophic cells during sex differentiation in two northern strains (HNI and Kaga) and two southern strains (Hd-rR and d-rR/Tokyo). All strains exhibited similar sex differentiation at hatching, such as (1) sex difference in germ cell number (XX > XY), and (2) the transition of XX germ cells into meiosis, and (3) presence of glycoprotein-α (Gpa)-positive cells. However, follicle-stimulating hormone-β (Fshb)-positive cells were first detected in the pituitary 1 day post-hatching in HNI. Exposure to high-temperature conditions and to cortisol in a dose dependent manner resulted in the localization of Fshb cells in the pituitary at hatching. This study demonstrates differences in gonadotropin subunit expression between northern and southern strains of medaka, and suggests that Fsh is not involved in early gonadal sex differentiation, such as the sex difference in germ cell number, and that high-temperature induce Fshb expression via cortisol production in medaka.

In this study, correlations between gonadal differentiation and gonadotrophic differentiation during early gonadal sex differentiation were examined in several strains from the Southern population of Japanese medaka, namely, Hd-rR, d-rR/Tokyo, d-rR/Shizuoka, and NIESR. Gonadal sex differentiation showed a similar pattern in all strains, that is, a sex-based difference in germ cell number was observed at hatching (XX > XY). In addition, a transition of XX germ cells into meiosis was observed at hatching in all strains. However, the ratio of meiotic transition of XX germ cells differed among strains, indicating that Hd-rR has fewer meiotic cells than other strains. Immunohistochemical analysis showed that glycoprotein α subunit (Gpa) and follicle-stimulating hormone β subunit (Fshb), but not luteinizing hormone β subunit (Lhb), localize to the pituitary at hatching in all strains examined. Although no sex differences were observed in the numbers of Gpa- and Fshb-positive cells in the pituitary at hatching in Hd-rR, d-rR/Tokyo, and NIESR strains, the d-rR/Shizuoka strain showed sex differences in the number of Fshb-positive cells at hatching (XX > XY); d-rR/Shizuoka is derived from d-rR/Tokyo. Even though d-rR/Shizuoka differs from its ancestral strain, d-rR/Tokyo, and Hd-rR in Fshb expression pattern, this suggests that d-rR/Shizuoka shows potential for use as a model to study the molecular regulatory mechanisms of fshb subunit gene expression and Fsh cell differentiation in the pituitary.

BackgroundEstrogen signaling is essential for ovarian differentiation in vertebrates, but its developmental onset and specific roles during early gonadogenesis remain unclear. In medaka (Oryzias latipes), the first morphological sex difference appears as a higher germ cell number in XX compared with XY embryos before hatching. Recently, we demonstrated that zygotically synthesized estrogen was dispensable for early germ cell sex difference but essential for subsequent oocyte meiotic progression and ovarian fate maintenance. Nevertheless, whether these phenomena depend on maternal estrogen or zygotic estrogen signaling mediated by nuclear estrogen receptors (nEsrs) is unknown.ObjectiveTo clarify the receptor-level requirement of estrogen signaling, we generated esr1/2a/2b triple knockout (ΔnEsrs) medaka using CRISPR/Cas9 genome editing. By comparing these receptor-deficient mutants with our previous ligand-deficient (Δcyp19a1a/1b double knockout) model, we aimed to determine whether estrogen signaling is involved in the establishment of the early germ cell number difference or acts later to control meiotic progression and ovarian maintenance.MethodsWe established ΔnEsrs medaka using CRISPR/Cas9 and analyzed gonadal histology, germ cell kinetics, and expression of steroidogenic enzyme genes, sex differentiation-related genes, and oocyte-specific expressed genes during early development.ResultsΔnEsrs mutants displayed normal early germ cell number and sex-specific differences at hatching (0 dph). At 10 dph, diplotene oocytes were markedly reduced, accompanied by significant downregulation of oocyte-specific genes, figa, 42sp50, as well as cyp19a1a and foxl2 mRNA. However, ΔnEsrs did not cause feedback regulation on other hypothalamus-pituitary-gonad (HPG) axis gene expression.ConclusionOur results demonstrate that estrogen signaling, both at the ligand and receptor levels, is dispensable for establishing the early germ cell sex difference but essential for subsequent oocyte meiotic progression and ovarian fate maintenance. This establishes a two-step estrogenic control model, redefining the developmental timing of estrogen action during the early phase of gonadal differentiation in vertebrate reproduction.

The ovaries of foetal and neonatal rats (14·5 days post coitum to 2 days post partum ) have been studied in order to correlate morphological changes in oogonia and developing oocytes with fluctuations in their numbers. The chromosomal configurations of germ cells at different developmental stages were examined in squash preparations. A volumetric method was devised for the separate estimation of the populations of normal and degenerating germ cells. Counts were made of the numbers of mitoses in germinal and somatic cells up to 19·5 days p. c . Oogonia are mitotically active up to 17·5 days p. c . The onset of the leptotene phase of meiosis in the majority of oocytes (17·5 to 18·5 days p. c .) coincides with a sharp decline in the number of normal oogonial divisions. The duration of the zygotene phase appears to be considerably shorter (19·5 to 20·5 days p. c .) than that of pachytene (range 20·5 days p. c . to 2 days p. p .). Oocytes in the diplotene phase are typically seen at 2 days p. p . The number of germ cells attaining the dictyate or resting phase is small on the first day after birth, but increases on the second. Four different ‘waves’ of degeneration of germ cells were observed. The first, affecting a small number of oogonia, occurs before the onset of meiosis, and is characterized by pyknosis. The second is characterized by degeneration of dividing oogonia which probably sets in before or at mitotic prophase. Such ‘atretic divisions’ reach their peak at 18·5 days p. c ., when the frequency of normal mitotic divisions is relatively low. The third affects germ cells after they have entered meiotic prophase (particularly at the pachytene stage); the chromosomes condense and the cytoplasm becomes eosinophil (referred to as ‘ Z ’ cells). The fourth affects germ cells at the diplotene phase. Oogonial mitoses are associated with an increase in the population of germ cells from 12000 on day 14·5 to 71000 on day 17·5 p. c . The appearance of ‘atretic divisions’ is largely responsible for the reduction of about 3000 in the number of normal germ cells between days 17·5 and 18·5. Successful mitotic divisions, on the other hand, account for the increase of about 4000 in the total number of germ cells during the same interval. From day 18·5 onwards, both the total number and the number of normal germ cells decreases steadily, first due to the occurrence of ‘atretic divisions’ and later to degeneration of ' Z ' cells and atretic cells at diplotene. Thus by 2 days after birth, the peak number of normal germ cells (64000 at 17·5 days p. c .) has fallen to 19000, the total population (peak 75000 at 18·5 days p. c .) being reduced to 27000. The majority of germ cells appear to be eliminated from the ovary within 24 h of the onset of degeneration.

ABSTRACTPrimordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The development of the gonads has been studied in ammocoetes of the landlocked sea lamprey, Petromyzon marinus. Up to lengths of about 70 mm, the gonads remain undifferentiated: the germ cells proliferating to form first small groups and finally large cysts of germ cells. At lengths of 70 to 80 mm, over 70% of all gonads examined were of the cystic type. Measurements made on large numbers of animals at this stage indicate the presence of two distinct groups with smaller and larger gonads. These represent the future male and female gonads respectively. These size differences are thought to correspond to differences in germ cell numbers in the two sexes. In a few cases the sex of the gonads can be distinguished at lengths of 71 to 80 mm, but differentiation is not generally complete below lengths of 100 mm. Differentiation is preceded or accompanied in both sexes by the appearance of growing oocytes. The frequency distribution for oocyte counts in bimodal, the lower numbers representing the future male gonads. During auxocytosis and differentiation large numbers of germ cells undergo degeneration; the majority at the zygotene or pachytene stage. In definitive females, the surviving oocytes represent only 20 to 30% of the total number of germ cells in the cystic gonad. Oogenesis begins within the cysts which are broken up by the invasion of somatic elements. The presence of large cysts is therefore without relevance to the future sex of the gonad.The extensive regression of germ cells in the future male gonads results in an actual decrease in size during differentiation. The somatic characters that distinguish the earlier male gonads of Lampetra planeri are less developed in marinus. In size and histological structure these often resemble the undifferentiated gonads of earlier larval stages. The male germ cells are derived from residual undifferentiated elements which survive the extensive regression at the cystic stage. At first the male gonads contain only a few isolated germ cells which proliferate slowly throughout larval life. At metamorphosis there is a very marked acceleration in the division of the male germ cells. Male germ cells retain their undifferentiated character throughout the larval period. In regard to the mode,of sex differentiation marinus is more differentiated than planeri. This is supported by the existence of a sex dimorphism in gonad size (and probably germ cell numbers) preceding morphological and histological differentiation, and by the evidence of bimodality in oocyte numbers. The vastly reduced fecundity of the brook lamprey compared with that of the sea lamprey is associated with significant differences in the pattern and phasing of gonad development in the two species. In planeri, germ cell proliferation begins earlier than in marinus, but is shorter in duration, terminating in auxocytosis and differentiation. This results in an enormous reduction in the total numbers of germ cells. Expressed as a percentage of the total numbers, the proportion undergoing regression during sex differentiation is thought to be similar in both species. The difference in the number of germ cells which survive as oocytes in the female gonads of the two species is thus a direct consequence of precocious oocyte development and the earlier curtailment of the gonial divisions in the brook lamprey ammocoete.

BackgroundExposure to chemotherapy during childhood can impair future fertility. Studies using in vitro culture have shown exposure to platinum-based alkylating-like chemotherapy reduces the germ cell number in the human fetal testicular tissues. We aimed to determine whether effects of exposure to cisplatin on the germ cell sub-populations are dependent on the gestational age of the fetus and what impact this might have on the utility of using human fetal testis cultures to model chemotherapy exposure in childhood testis.MethodsWe utilised an in vitro culture system to culture pieces of human fetal testicular tissues (total n=23 fetuses) from three different gestational age groups (14-16 (early), 17-19 (mid) and 20-22 (late) gestational weeks; GW) of the second trimester. Tissues were exposed to cisplatin or vehicle control for 24 hours, analysing the tissues 72 and 240 hours post-exposure. Number of germ cells and their sub-populations, including gonocytes and (pre)spermatogonia, were quantified.ResultsTotal germ cell number and number of both germ cell sub-populations were unchanged at 72 hours post-exposure to cisplatin in the testicular tissues from fetuses of the early (14-16 GW) and late (20-22 GW) second trimester. In the testicular tissues from fetuses of mid (17-19 GW) second trimester, total germ cell and gonocyte number were significantly reduced, whilst (pre)spermatogonial number was unchanged. At 240 hours post-exposure, the total number of germ cells and that of both sub-populations was significantly reduced in the testicular tissues from fetuses of mid- and late-second trimester, whilst germ cells in early-second trimester tissues were unchanged at this time-point.ConclusionsIn vitro culture of human fetal testicular tissues can be a useful model system to investigate the effects of chemotherapy-exposure on germ cell sub-populations during pre-puberty. Interpretation of the results of such studies in terms of relevance to later (infant and pre-pubertal) developmental stages should take into account the changes in germ cell composition and periods of germ cell sensitivity in the human fetal testis.

Germ cell and Sertoli cell numbers were estimated in six normal adult monkeys (Macaca fascicularis) using a contemporary unbiased and efficient stereological method--the optical disector. The data was used to assess the efficiency of spermatogenesis from type B spermatogonia to elongated spermatids. Animals underwent orchidectomy, and the right testis (volume 17.5 +/- 1.7 cm3 [mean +/- SEM], range 13.2-25.1 cm3) was fixed in Bouin's fluid. Blocks were embedded in methacrylate resin and germ cells were counted in thick (25 microm) sections using the optical disector in conjunction with a systematic uniform random-sampling protocol. The total numbers of Sertoli cells and all germ cells per testis were 566 +/- 43 (419-683) million and 12.8 +/- 1.6 (9.0-20.2) billion, respectively. On average, one Sertoli cell supported 12.4 +/- 1.9 (range 8.2-18.4) step 1-12 spermatids, 3.1 +/- 0.4 (2.3-4.5) pachytene spermatocytes, and 23.7 +/- 4.1 (15.0-39.0) total germ cells. Sertoli cell number correlated poorly with both testicular size (correlation coefficient r = -0.12) and germ cell numbers (r = -0.35 with total germ cell number). However, testicular size had a consistent and significant correlation with germ cell numbers (r = 0.97 with total germ cell number). The conversion ratio of pachytene spermatocytes to step 1-12 spermatids was 3.94 +/- 0.19, which is close to the theoretical maximum of 4. Similarly, the conversion between other cell types was consistently close to the maximum theoretical value. We conclude that the efficiency of spermatogenesis in the adult monkey is high, with stepwise conversion being consistently close to the maximal values. The capacity of Sertoli cells to support a cohort of germ cells varies widely between monkeys. Although absolute number of cells per testis is always the preferred parameter, it cannot always be obtained in an experimental situation where cost and ethical constraints mean that biopsies, rather than whole testes, are collected. Thus, if absolute data on germ cell numbers are not available, experimental outcomes impacting on cells beyond preleptonene spermatocytes may be best expressed in terms of changes in germ cell conversion rather than the traditional germ cell: Sertoli cell ratio.

Effects of synthetic sex steroid hormone exposures on gonadal sex differentiation and dynamics of a male-related gene, Gonadal soma-derived factor (Gsdf) and an estrogen up-regulated gene, Choriogenine-H (ChgH) gene expression in the euryhaline Javafish medaka, Oryzias javanicus, based on genetic sexes

The developing gonads constitute a valuable model for studying developmental mechanisms because the testes and ovaries, while originating from the same primordia, undergo two different patterns of development. So far, gonadal development among birds has been described in detail in chickens, but literature on the earliest stages of gonadogenesis is scarce. This study presents changes in the structure of the gonads in three species of breeding birds (chicken, duck, and pigeon), starting from the first signs of gonadal ridge formation, that is, the thickenings of the coelomic epithelium. It appears that both gonads show asymmetry from the very beginning of gonadal ridge formation in both genetic sexes. The left gonadal ridge is thicker than the right one, and it is invaded by a higher number of primordial germ cells. Undifferentiated gonads, both left and right, consist of the primitive cortex and the medulla. The primitive cortex develops from the thickened coelomic epithelium, while the primitive medulla – by the aggregation of mesenchymal cells. This study also describes the process of sex differentiation of the testes and ovaries, which is initiated at the same embryonic stage in all three studied species. The first sign of gonadal sex differentiation is the decrease in the number of cortical germ cells and a reduction in cortical thickness in the differentiating testes. This is followed by an increase in the number of germ cells in the medulla. The cortical asymmetry and difference in size between the left and right testes diminishes during later development. However, the differentiating left ovary shows an increase in the number of cortical germ cells and cortical thickness. No regression is seen in the right ovary, although its development is slower. The right ovarian cortex undergoes testis-specific reduction, while the medulla undergoes ovary-specific development. The process of gonadogenesis is similar in the three studied species, with only slight differences in gonadal structure.

Study question Can the gas-liquid interface organ culture system that achieved in vitro spermatogenesis in mice also support in vitro spermatogenesis in human adult testis? Summary answer Although the progression of spermatogenesis was not observed, germ cells were maintained without the degeneration of the architecture in both fresh and cryopreserved testicular tissues. What is known already Although the research on in vitro spermatogenesis have been conducted for 100 years, only the organ culture system using gas-liquid interface method achieved in vitro spermatogenesis in mice. It has not been verified whether this culture system can be applied to other mammals including humans and induce spermatogenesis. Study design, size, duration Testicular tissue was obtained from the transgender patients receiving sex reassignment surgery. Testicular specimens were either immediately processed for cultivation or cryopreserved, using a vitrification freezing protocol. Organ culture of testicular fragments was performed in three different media for a maximum period of 3 weeks to evaluate the short-term changes in the cultured tissues (viability, proliferation and maintenance of germ and somatic cells). Participants/materials, setting, methods Fresh and cryopreserved-thawed testis fragments (1–2 mm3) were cultured using the organ culture system in alpha-MEM with knock-out serum replacement (K group), alpha-MEM with lipid-rich BSA (A group) and DMEM with FBS (D group). Luteinizing hormone, follicle stimulating hormone and testosterone were supplemented. The number of germ cells (using DDX4), proliferative activity of germ cells (using EdU assay) and intratubular cell apoptosis (by TdT-mediated dUTP Nick End Labeling) were evaluated by immunohistochemical staining weekly. Main results and the role of chance The architecture of the seminiferous tubules was maintained until the second week of culture in both the fresh and the cryopreserved culture group. The number of DDX4-positive germ cells per seminiferous tubule in groups D, K, and A was 49 ± 24, 55 ± 21, 50 ± 26 cells/tubule in 1 day, 32 ± 13, 42 ± 7, 36 ± 21 cells/tubule in 1week, respectively. The numbers gradually decreased to 26 ± 8, 24 ± 6 and 27 ± 18 cells/tubule, in 2 weeks, respectively, with no difference among the groups. The number of intratubular EdU-positive cells of groups D, K, and A was 0.2 ± 0.2, 2.8 ± 2.1, 1.1 ± 0.8 cells/tubule at 1 day, 0.1 ± 0.2, 0.5 ± 0.6, 0.3 ± 0.6 cells/tubule at 1 week, respectively. The values were 0.01, 0.05, and 0.03 at 2 weeks. Thus, EdU-positive cells drastically decreased from the first week of culture. The number of DDX4-positive germ cells and the intratubular EdU-positive cells in the cryopreserved culture group was not different from that in the fresh culture group. Limitations, reasons for caution Current organ culture systems are incomplete, being unable to induce human in vitro spermatogenesis. Further research is needed to improve culture condition with the aim of producing fertile sperm of infertile adult male patients. Wider implications of the findings: Our organ culture system could maintain testis structure and germ cells. By using the testis tissues of the transgender patients, which are available with their consent, we will promote the investigation of the culture condition necessary for germ cell proliferation and differentiation. Trial registration number Grant-in-Aid for Scientific Research on Innovative Areas 18H05546, Grant-in-Aid for Young Scientists (A) 17H05098 and Takeda Science Foundation

Medaka is a differentiated gonochoristic species with a male heterogametic sex determination. Here, we review recent studies on gonadal sex differentiation in medaka, as well as the experimental sex-reversal models available for this organism. The accumulated literature on teleost gonadal development facilitates comparative interspecies studies. Among these, comparison between medaka and zebrafish is of special interest, because zebrafish is an undifferentiated gonochorist that, nevertheless, shares many similarities with medaka and is also used as a small fish model for developmental biology. Accordingly, here we focus on the comparative aspects of gonadal development in medaka and zebrafish. In medaka, oogenesis begins in female gonads, whereas oogenesis is suppressed and germ cells remain in an undifferentiated state in male gonads. In zebrafish, oogenesis begins in all individuals, regardless of their future sex, while actual sex differentiation begins later in gonadal development, which means that degeneration of immature oocytes occurs in presumptive males, while oogenesis proceeds to completion in presumptive females. Despite these apparent differences between medaka and zebrafish, the process of gonadal development comprises similar stages: early oogenesis, early aromatase expression, later oocyte development or loss, sexually dimorphic expression of somatic genes, and spermatogenesis. We propose the concept of canalization as a key to gaining a comprehensive understanding of gonadal sex differentiation. In this respect, the possible role of the male-determining gene DMY/dmrt1bY is also discussed.