RNA-seq and cytological study of sex-specific meiosis response to Gsdf in medaka (Oryzias latipes)

doi:10.21203/rs.3.rs-1433778/v1

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RNA-seq and cytological study of sex-specific meiosis response to Gsdf in medaka (Oryzias latipes)

https://doi.org/10.21203/rs.3.rs-1433778/v1

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Sexually dimorphic meiosis is monitored by meiotic recombination to prevent the formation of abnormal gametes in males and females. However, the regulatory mechanism of sex-specific meiotic recombination is not completely understood. Here we report the role of gsdf, a unique BMP/TGF-b family member gene, in the regulation of meiotic recombination in medaka (Oryzias latipes) based on the RNA-seq and cytogenetic analysis of normal and gsdf deficient gametogenesis. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf deficient gonads. The escalation of alternative pre-mRNA isoforms of meiotic synaptonemal complex genes including sycp3 were visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. However, immunofluorescence analysis showed that Sycp3 protein products in gsdf deficient XY oocytes were significantly reduced. Transmission electron microscope observations showed that asynchronous cysts replaced normal synchronous cysts in gsdf-deficient testis. Breeding experiments revealed a skewed ratio of gsdf^−/− XY gamete fertility in a non-Mendelian manner resulting in XY chromosome non-disjunction. Taken together, our observations suggest that Gsdf signaling may directly or indirectly affect meiotic recombination in the process of sex-specific division of germline stem cells.

BMP/TGFβ: Bone Morphogenetic Protein/Transforming Growth Factor beta

gsdf: gonadal soma-derived factor

synaptonemal complex proteins Sycp1

Sycp3

AS: RNA alternative splicing

SAC: spindle assembly checkpoint

Male and female germ cells in vertebrates undergo different meiotic processes. Meiotic checkpoints strictly monitor these processes to prevent the formation of abnormal gametes (Hunt and Hassold, 2002). Germline stem cells (GSCs) are morphologically similar regardless of mitotic quiescence or activation after primordial germ cells (PGCs) reside in the genital ridge. Although the time courses of the sex-differentiated proliferation of GSCs vary among vertebrate species, male GSCs in testis usually arrest at the G0/G1 stage of the mitotic cell cycle, then undergo the progressive resumption of spermatogonia proliferation and meiotic differentiation to produce haploid spermatozoa. In contrast, most female GSCs enter meiosis and arrest at the end of the first meiotic prophase in the ovary during early oogenesis, forming a reserve of diplotene oocytes that are selectively recruited into follicular growth. Fully-grown oocytes resume the meiotic cell cycle and divide into haploid oocytes upon ovulation and fertilization after two cycles of polar body release (Gruhn et al., 2013). Cytological analysis has demonstrated the sex-specific differences in human meiotic recombination prior to the onset of double-strand breaks (DSBs) (Gruhn et al., 2013). Bone morphogenetic protein (BMP) signaling has been demonstrated to cooperate with retinoic acid (RA) to specify GSC female fate in mice (Miyauchi et al., 2017) and initiate meiosis through the upregulation of the female germline gene Stra8, while the male germline gene Nanos2 downregulates Stra8 (Sada et al., 2009). However, evidence of the normal meiosis of germ cells in mice that lack RA receptors suggests that RA may not be the pivotal factor (Vernet et al., 2008), but instead supports the feminizing effect of BMP alongside RA for germ cell sex specification (Miyauchi et al., 2017). In contrast to mammals, Nanos2 is not the sexual fate marker in medaka (Oryzias latipes) germ cells, whereas Stra8 is completely missing in medaka genomic databases (Nishimura and Tanaka, 2016). Although the location of PGCs relative to somatic structure has been pre-established, the post-transcriptional regulation of specific mRNA expression patterns can directly affect the fate of GSCs in the process of sexual differentiation (Herpin et al., 2007). In addition, overexpressing BMP2 or a dominant negative form of the BMP2/4 receptor in medaka caused no change in the number of PGCs (Herpin et al., 2007). These results suggest that BMP signaling may have an indirect role in the sex fate determination of GSCs, although the entire mechanism remains to be elucidated.

The advantages of the identified dmy/dmrt1bY sex determination gene (Volff and Schartl, 2002, Matsuda et al., 2002) and the well-known foxl3 (Forkhead box L3), the female germline sex determinator (Nishimura et al., 2015, Kikuchi et al., 2020), have made medaka fish a good experimental model for the study of germline proliferation and differentiation. Like most vertebrates, female germ cells in medaka enter oogenesis at hatching, while male germ cells continue in a state of mitotic arrest until they initiate spermatogenesis one month later in puberty medaka (Saito et al., 2007). XX germ cells are morphologically distinct from XY germ cells at 5 days post-hatching (dph) (Tanaka, 2019). The feminizing effect of germ cells has been demonstrated to be inherent and independent of somatic sex because the sex determination of germ cells occurs prior to the activation of the dmy master gene in somatic cells or the initiation of meiosis (Nishimura et al., 2018). The targeted disruption of gonadal soma-derived factor (gsdf), a unique BMP equivalent with a well-conserved transforming growth factor β (TGFβ) domain at its C-terminus, resulted in excessive germ cell proliferation and XY sexual reversal (Nishimura et al., 2018, Guan et al., 2017, Zhang et al., 2016, Imai et al., 2015). Nonetheless this XY feminization was suppressed in germ cell-deficient XY gsdf^−/− lines (Nishimura et al., 2018), which is consistent with the requirement of germ cells in male-to-female sex-reversal. Transgenic over-expression of gsdf produced XX males in medaka (Zhang et al., 2016, Myosho et al., 2012), while gsdf mutants developed hypertrophic XY testes in both HdrR (O. latipes) and HNI (Oryzias sakaizumii) medaka (Zhang et al., 2021). All of these findings indicate that gsdf affects the sex-specific proliferation of XX and XY germ cells rather than the initiation of spermatogenesis (Zhang et al., 2021). The Gsdf signal may affect the proliferation rate of XX and XY germ cells, or the rate of cyst formation and subsequent cyst rupture. However, the underlying mechanism remains to be determined.

Incorporation of the thymidine analog 5’-ethynyl-2’-deoxyuridine (EdU) into chromosomal DNA is a simple method used to identify the DNA synthesis and chromatin architecture at the S-phase during cell proliferation. EdU has been reported to visualize germline division progress in Caenorhabditis elegans (Kocsisova et al., 2018). To decipher the Gsdf regulatory mechanism of sexual cyst formation in vivo, molecular differences between normal gsdf-intact and abnormal gsdf-deficient gametogenesis were characterized by RNA-sequencing and cytogenetic analyses. EdU incorporation was used to identify the sex differences in proliferating germ cells in the S phase of pre-meiotic proliferation in gsdf-deficient gonads, and details were further elucidated by immunofluorescent and transmission electron microscope (TEM) observations. The consequent quality of gsdf-deficient gametes was evaluated by the fertilization and embryo development of the offspring of gsdf-deficient XY individuals mating with normal partners.

Fish and mating schemes

Normal and gsdf-knockout medaka fish were housed in re-circulating systems at 26°C–28°C with a light-dark cycle of 14 h daylight and 10 h darkness. All the experimental procedures used in this study were approved by the Committee for Laboratory Animal Research of Shanghai Ocean University. Phenotypic sex and genotypic sex (XX or XY) were assessed following the previously reported protocol (Matsuda et al., 2002). Genotyping of the gsdf allele was performed with primers as described previously (Zhang et al., 2016). The mating schemes were as follows: gsdf^−/− XY males (n = 3) paired with normal XX females (n = 3), gsdf^−/− XY females (n = 4) paired with gsdf-transgenic XX males (n = 6), and gsdf^−/− XX females (n = 3) paired with Sissy (a transgenic intersex line previously reported as Tg:cryG gsdf^−/− XY males, n = 3) (Zhang et al., 2021). The results of breeding experiments are summarized in Table 1. All offspring from 11 mating families were raised to 3-month-old adulthood for genetic sex and phenotypic sex assessment. In the progeny analysis of Family 7 (gsdf^−/− XY females paired with gsdf-transgenic XX males), dmy genotyping was carried out using embryos at stage 30.

Histological and ultrastructural observation

The protocol used for the histological section for hematoxylin and eosin (HE) staining was as previously described (Sun et al., 2017), and the results were viewed and photographed under the Nikon Ds-Ri2 (Nikon, Tokyo, Japan) or a confocal laser scanning microscope (DMi8 TCS SP8, Leica, Frankfurt, Germany). Ultrathin sections (70 nm thick) were prepared following a previously described method (Zhang et al., 2021) and examined with a FEI Tecnai G2 Spirit TEM (Thermo Fisher, Carlsbad, USA) at an acceleration voltage of 30 kV.

Identification of proliferative germ cells by EdU incorporation

Normal and gsdf-deficient gonads were subjected to EdU (RIBOBio, Guangzhou, China) staining for the identification of proliferating germ cells. Briefly, gonads of 3-month-old mature adults were added to EdU solution (20 µg/ml), incubated for 4 h at 28°C, fixed with 4% paraformaldehyde (PFA), and then embedded in paraffin (Shenggong Co., Ltd., Shanghai, China) after being dehydrated in a graded ethanol series. Cross-sections were cut at 5 µm thickness, then rehydrated and labeled with microfluorescence for 30 min incubation with fresh EdU cocktail (1xApollo dyeing reaction solution) according to the manufacturer’s instructions (RIBOBio, Guangzhou, China), and then nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI). Apollo dye and DAPI fluorescent signals were photographed using a confocal laser scanning microscope (DMi8 TCS SP8, Leica, Germany).

Preparation of meiotic nuclear spreads and immunostaining

Germ cells were freshly collected by cutting adult gonads into small chunks. Cells were arrested by serum-free Dulbecco’s Modified Eagle Medium (Gibco DMEM, Sigma-Aldrich, USA) in addition to colchicines (10 ng/µm, Sigma-Aldrich, USA) prior to hypotonic 75 mM KCl treatment, and then fixed in Carnoy fix solution (3:1 methanol:acetic acid). Meiotic nuclear spreads were applied to clean slides and air dried. Slides were kept under − 20°C until use. Meiotic nuclear spreads were analyzed by Giemsa staining (Shenggong Co., Ltd., Shanghai, China), or incubated with primary antibodies and secondary antibodies (listed in Table S7) for immunofluorescence assay according to a previous report (Wu et al., 2019), and photographed with a Nikon Ds-Ri2 system (Nikon, Japan). All experiments were repeated at least three times with 3–5 individual fish.

Transcriptomic analysis

Total RNA was extracted from one-month-old puberty gonads of normal gsdf^+/+ testes (WTT) and ovaries (WTO), or gsdf^−/− testes (HmT) and ovaries (HmO). The gonads of 7–15 individuals from each group were collected and pooled to minimize the impact of individual differences according to our previous report (Zhang et al., 2021). RNAs were cDNA reverse sequenced as 2x100-bp paired-end reads on the HiSeq 2000 platform (Illumina) by Shenggong Biotech Co., Ltd. (Shanghai, China). Clean reads were mapped to the medaka genome (http://www.ensembl.org/index.html) using the Reads Mapper Tophat2 (version 2.0.13, http://ccb.jhu.edu/software/tophat/). Sequence alignment and assembly were conducted using Cufflinks (version 2.2.0, http://cole-trapnell-lab.github.io/cufflinks/cufflinks/) and Cuffmerge (http://cole-trapnell-lab.github.io/cufflinks/cuffmerge/) software, and assemblies were merged into a master transcriptome for the analysis of differentially expressed genes (DEGs). A Venn diagram was plotted against the reads of unigenes across samples. A cut-off of false discovery rate (FDR) < 0.1 (Benjamini-Hochberg method) was used for MA-plot comparisons. The Integrative Genomics Viewer (IGV) was used to view RNA-Seq read coverage across the entire Japanese medaka HdrR viral genome (Ensembl genome browser 104: Japanese medaka HdrR - Description). The reads in the IGV display an automatically generated coverage track that contains a histogram of read depth for each coordinate across the genome. RNA sequence data have been deposited in the National Center for Biotechnology Information (NCBI) with the accession number PRJNA759752.

Structural prediction of Sycp1 and Sycp3 variants

The protein structures of full-length and splicing variants of wild-type Sycp1 and Sycp3 were predicted by the Phyre2 web portal through assessment under the ZDOCK server and visualization with the PyMOL molecular graphic system (www.sbg.ic.ac.uk/phyre2/html).

Statistical analysis

Statistics were analyzed and graphs were generated using the IBM SPSS statistics software for Windows version 26.0 (IBM Corporation, New York, NY, USA). An ANOVA-test was used to determine significant differences between normal and null animal measurements. P-values less than 0.05 were considered significant. Error bars represent the standard error of mean (SEM), unless otherwise noted.

The differentially expressed genes regulated by Gsdf signaling

To identify the targeted genes responsible for Gsdf signaling before the initiation of spermatogenesis, RNA-seq was performed (transcriptome sequencing data were deposited in NCBI under accession number PRJNA759752).The differentially expressed genes (DEGs) were evaluated by comparing one-month-old gsdf^+/+ and gsdf^-/- gonads before the XY germ cells resume proliferation (Saito et al., 2007, Kanamori, 1985), with the threshold of a log₂ fold change (FC) ≥ 3 and a P-value of false discovery rate (FDR) < 0.05 as measures of statistical significance. FKB506-binding protein 7 (fkbp7), a molecular chaperone located in the endoplasmic reticulum (ER) as a suppressor of ATPase activity (Garrido et al., 2019), was remarkably upregulated (log₂ FC ≥ 3), and overlapped among four pair-wise comparisons of normal testes to normal ovaries (WTT vs WTO); gsdf-deficient ovaries to normal ovaries (HmO vs WTO); gsdf-deficient testes to normal testes (HmT vs WTT); and gsdf-deficient testes to gsdf-deficient ovaries (HmT vs HmO) as revealed by Venn diagram (Fig. 1a). The details are listed in Table S1. RNA transcripts of follicle-stimulating hormone receptor (fshr), microRNA 202 (miR202), a zinc-finger protein with a C-terminal domain of four cysteine and two histidine residues (zc4h2), and an immunoglobulin-like domain factor were significantly upregulated in a subset of three comparisons: WTT vs WTO, HmO vs WTO, and HmT vs HmO.

To assess the concordance of RNA-seq data on expression quantification, the relative transcripts per kilobase expression of exon model per million mapped reads (mean TPMs) and log₂MeanTPMs of sexually differentiated DEGs shown in Fig. 1b, were verified by real-time PCR quantification (qPCR) (Fig. 1c, details in Table S2 and Table S3). The sexually dimorphic expression of fshr, sycp1, sycp3, and miR202 was consistent with previous reports (Gay et al., 2018, Aoki et al., 2009, Herpin et al., 2007), and was higher in the testis than in the ovary as verified by qPCR (Fig. 1C, Table S3), with primers listed in Table S4. The log₂FC of sycp3 (HmO vs WTO comparison) was 2.07, whereas that of sycp1 (HmO vs WTO) was 4.3, indicating that the expression of sycp1 was more sensitive to the Gsdf signal (Fig. 1b, Table S1). The expression of sycp1 in the ovary was higher than that in the testis, resembling to the expression patterns of dynll2 and nanos3 (Fig. 1b). Nevertheless, the expression of sycp1 increased significantly in one-month-old gsdf-intact and gsdf-deficient testes (Fig. 1c), indicating the high proportion of sycp1-expressing germ cells in testes before the initiation of spermatogenesis. The expression of sycp3 was higher in normal or gsdf-deficient testes than in normal or gsdf deficient ovaries (Fig. 1b), resembling that of fkbp7 and dnaaf1 (dynein, axonemal, and assembly factor), and the structural maintenance of chromosome 1b (smc1b). nanos2 and nanos3 are related to stem-cell-like characteristics and are differentially expressed in medaka germ cells (Aoki et al., 2009). Notably, an increase of nanos3 was detected in both gsdf deficient ovaries and testes, compared to their normal counterpart gonads. In contrast to no difference between normal ovaries and gsdf deficient ovaries, nanos2 expression significantly increased in gsdf-deficient testes compared to normal testes (Fig. 1c). These results showed that the population of nanos2-expressing germ cells were sensitive to Gsdf and were mainly located in the testis, which was different from the population subsets of nanos3-expressing germ cells responsive to Gsdf in both the ovary and testis (Fig. 1c) (Zhang et al., 2021).

Meiotic recombination targeted by Gsdf signal

To assess the targeted transcriptomes and the related pathways responsive to Gsdf, Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis was performed to investigate common DEGs among three overlapped pair-wise comparisons (41 upregulated DEGs), or two overlapped pair-wise comparisons (44 downregulated DEGs), as revealed by Venn diagram (Fig. 1A). The KEGG-enriched pathways are displayed in Fig. 2A in a bubble map, while the full details are listed in Table S5. The peroxisome proliferator-activated receptor (PPAR) pathway is well characterized and activated by TGFβ signals (Pleniceanu et al., 2017). Glycerolipid metabolism, ECM-receptor interaction, cell adhesion molecules, and tight junction pathways were downregulated (Fig. 2A, right panel), which is consistent with the involvement of fatty acid synthesis in female-to-male sex reversal (Sakae et al., 2020). The pathway of DNA homologous recombination (HR) consisting of sycp3 (localized in chromosome18, Chr18) and sycp3 like (sycp3l) (localized in chromosome11, chr11) was significantly upregulated (Fig. 1a and Fig. 2A) (Pasquier et al., 2016). RNA alternative splicing (AS) variants of sycp3 lacking N-terminus transmembrane domains remarkably increased in gsdf-deficient gonads, which was confirmed by RT-PCR with primers flanking the region of sycp3 exon 2 and 5’ upstream adjacent phka1a (Fig. 2B, the right corner). The super-coil structure of normal Sycp3 was putatively replaced by the linear structure without N-terminus AS variants predicted by Phyre2 software (Fig. 2B, right). A high frequency of AS junction tracks was found in sycp3l and sycp1 region as shown by IGV Sashimi plot. This suggests that the active splicing machinery may promote the translation of defective Sycp1 and Sycp3l proteins, thus further interfering with DNA repair and meiotic HR events.

Malformation of Balbiani body with abnormal expression of Sycp3-Piwi in gsdf deficient oocytes

Piwi, the major component of the meiotic nuage to silence the transposon activity during meiotic HR (Yashiro et al., 2018), was expressed predominantly in mitotic spermatogonia and oogonia as well as meiotic spermatocytes and oocytes, as shown by immunofluorescence analysis (Fig. 3). The anti-Sycp3 positive signals were detected in meiotic spermatocytes and oocytes (Fig. 3a–a’; Fig. 3b–b’). Strong anti-Sycp3 signals were detected in normal oocyte Balbiani bodis (Bbs), although they declined significantly in the small Bbs of gsdf-deficient XX and XY ovaries (Fig. 3c–c’; Fig. 3d–d’). The low level of Sycp3 protein may have been due to the blocked translation of defective sycp3 itself as a variant or the instability of protein products in gsdf deficient oocytes (Fig. 3c-3d). Compared with normal oocytes (stage III oocytes in Fig. 3b), gsdf deficient oocytes have fewer Piwi nuages in the cytoplasm (stage III oocytes in Fig. 3d), suggesting that the special transposons may become active without being inhibited, thereby promoting DSBs or HR events in gsdf^−/− oocytes.

Increase of HR errors in gsdf-deficient testes

Proliferative germ cells that undergo cyst-division are different between XY male and XX female (Saito et al., 2007), and are distinguishable by EdU (a thymidine analog) incorporation, while gonads without EdU treatment serve as a negative control (Figure S1). Although the spotted bars corresponding to chromosomes were obviously different between females (unevenly distributed in oogonia in Figure S2a₁–a₃) and males (evenly distributed in spermatogonia in Figure S2b₁–b₂), some spermatogonia were similar to oogonia in shape and pattern (SpgB_2a in Figure S2b₂–b₂’ vs Og in Figure S2a₃), but different from cystic spermatogonia (SpgB_2b in Figure S2b₁’). The identical shape may have been derived from the same cell cycle stage, similar DSBs, or HR events shared by these germ cells. Like humans (Capalbo et al., 2017, Gruhn et al., 2013), medaka germ cells in normal spermatogenesis undergo HR errors occasionally (e.g., non-exchange chromosomes or achiasmate chromosomes) to become oocyte-like (Ol) cells (Figure S2c). The evidence of a large number of Ol cells in Sissy gonads supports the hypothesis that impaired Gsdf signaling leads to high-frequency HR errors during meiosis (Zhang et al., 2021).

The effect of Gsdf on meiosis was further investigated by EdU incorporation. According to the ratio of SpgB₂ per total SpgB number for three testes, each with 14 count fields EdU-positive, SpgB_2a (ccOg alike) was double in gsdf-deficient testis (19.24%) compared to normal testis (10%) (Figure S3A–B). The counting results are summarized in Table S6. The separation of sister germ cells and cystic dividing cells are shown in Fig. 4.

Asynchronous cysts formation in gsdf-deficient testes

TEM observations showed that synchronous cysts containing cystic-proliferative spermatocytes (Spc) (Fig. 5a₁), spermatids (Spt) and sperm (Spm) (Fig. 5a₂), round and elongated spermatids (Fig. 5a₃₋ a₄), and densely packed sperm cells (Fig. 6a₅-6a₆) were present in the cross sections of normal testes (n = 3). However, many asynchronous cysts appeared in gsdf deficient XY testes, which showed that germ cells at different developmental stages were in the same cyst (Fig. 5b₁-b₄, n = 3) (Zhang et al., 2016). Spermatocytes (Spt), mitotic metaphase SpgB and sperm were present in a single cyst (Fig. 5b₁); dead Sertoli cells (*Sc) and ruptured cysts (Fig. 5b₂); round spermatids with loosened packaging of genomic DNA (Fig. 5b₃), and one cyst containing spermatozoa with irregular packaging of genomic DNA into tiny sperm heads (low magnification in Fig. 5b₄, and high magnification in Fig. 5b_4’-b_4”) were also found, supporting the theory that Piwi abnormality leads to the destruction of histone-protamine exchange during spermiogenesis (Gou et al., 2017).

Aneuploidy and low fertility of gsdf-deficient gametes

Meiosis in normal and gsdf deficient gametes was investigated using a cytological approach (Giemsa staining, Fig. 6). A total of 1312 prophase cells from normal and gsdf^−/− testicular or ovarian biopsies were examined. The details are listed in Table S8. The defects were obvious as early as during the transition between leptotene/zygotene stages; instead of a bouquet configuration (Fig. 6a_1 − 2), loosened chromosome loops were observed in gsdf-deficient testes (arrows in Fig. 6a₃, enlarged in 6a₃’) and ovaries (arrows in Fig. 6a₇–a_7’). Aneuploid chromosomes (n = 25) were found in gsdf-deficient spermatocytes (Fig. 6a₄, a₅), in contrast to normal chromosomes in gsdf-intact XY spermatocytes (n = 24 shown in Fig. 6a₂). Incomplete synapsis formation may ultimately lead to pachytene prolongation and the failure of pachytene completion. The lack of Gsdf destroys the proportion of normal prophase stages through the early leptotene/zygotene transition, and prolongs the pachytene of gsdf^−/− testis and ovary, suggesting that Gsdf may affect the initiation of meiosis and the formation of SC. The proportional distribution of prophase stages shown in Fig. 6A₈.

The qualities of gsdf^−/− gametes were evaluated by breeding experiments. The fertilization rate of normal XY or transgenic gsdf XX sperm and normal XX oocytes ranged from 94.4–98.9% (Table 1, Family 9–12), whereas the fertilization rates of gsdf deficient sperm (Table 1, Family 1–3) and eggs (Table 1, Family 4–7) ranged from 56.6–71.9% and 41.7–76.4%, respectively. These results indicated that aneuploid chromosomes in gsdf-deficient gamete led to a serious decline in the fertility of oocyte and sperm. The fertilization rate of gsdf-deficient XX oocytes and Sissy gsdf^−/− sperm were the lowest at 39.3% and 37.5%, respectively, as shown in Table 1 (Family 8). The embryogenesis was severely damaged and delayed from one-cell fertilization throughout the entirety of embryogenesis. Two-cell division was delayed and asymmetric, and embryo development was severely hindered (Fig. 6b–d). In contrast to normal embryos, which reached stage 18 (St18 late neurula stage, 26 h after fertilization) according to the embryogenesis stages (Iwamatsu, 2004), the offspring embryos of gsdf-deficient males and normal females were delayed in St13 (early and middle gastrula) (Fig. 6d). This result indicated that the embryogenesis was damaged at the beginning of development, which may affect the development of multiple tissues. To our surprise, progeny produced from mating families between gsdf-deficient XY males (n = 3) and normal XX females developed as all XY males, while the offspring of gsdf^−/− XY females (n = 4) mated with gsdf transgenic XX males were XX, with seven males and 49 females derived from three mating families. Fry fish of Family 7 (gsdf-deficient XY female bred with Tg XX male) were dissected and dmy-genotyped at hatching and showed the ratio of four XY males to eight XX females (Table 1). These results suggest that XX and XY germ cells respond differently to Gsdf signaling. gsdf-deficient X-bearing oocytes and Y-bearing sperm have more survival advantages during the processes of oogenesis and spermatogenesis, respectively.

In summary, we describe the sex chromosomes susceptible to HR errors in gsdf^−/− gametogenesis from the aspects of X-Y non-disjunction and aneuploidy abnormalities. The loss of gsdf leads to defective gametogenesis, stimulates FKBP7-mTOR activity and promotes female cytoskeleton assembly through the downstream robust mRNA transcripts and splicing variants. Nevertheless, gsdf deficiency does not block all male cascades. Aberrant expression of sycp1 and sycp3 eventually leads to mis-segregation between homologous chromosomes (Fig. 6a₃–a_3’), which is likely to result in the occurrence of aneuploidy gametes caused by the non-disjunction of X-Y chromosomes, This can also explain the low fertility of gsdf^−/− gametes and the sex bias of gsdf deletion offspring.

Germ cell proliferation is an energy-consuming and sexually dimorphic process. The discovery of the strong response of fkbp7 expression to Gsdf signaling in this study, in addition to our previous report on the physical interaction between Gsdf and eukaryotic elongation factor 1α (eEF1α) (Zhang et al., 2021), suggests that Gsdf may directly or indirectly inhibit the fkbp7-mediated eEF1α−elF4F eukaryotic mRNA translation machinery (Firczuk et al., 2013). Members of the FKBP family form rapamycin induced ternary complexes that inhibit mTOR-kinase activity, and affect microtubule stability via associated tau protein (Cioffi et al., 2011). They are also involved in protein folding, apoptosis, and transcription/translation balance. Co-localization of Fkbp6 with Sycp1 and Sycp3 is required for the formation of the synaptonemal complex during meiotic prophase to allow the paired homologous chromosomes to cross over (Noguchi et al., 2008). Impairment of the above molecules will lead to abnormal chromosome pairing, which will further arrest these meiotic germ cells in the pachytene stage and remove them through apoptosis (Noguchi et al., 2008). As a meiotic monitor of the fidelity of homologous chromosome pairing in spermatogenesis (Crackower et al., 2003). Fkbp6^−/− and Sycp3^−/− mice displayed sex-specific differences in male and female fertility with male-specific infertility (Crackower et al., 2003, Ayarza et al., 2016). Although the physical binding or co-localization of medaka Fkbp7 and Sycp1/Sycp3 remains to be elucidated, the asynchronous cysts in gsdf-deficient testes, probably resulted from the incomplete meiosis of germ cells and progressive somatic cell apoptosis (Fig. 5). The activated splicing regulators, particularly small nuclear ribonucleoproteins (snRNPs) in gsdf deficiency (Zhang et al., 2021) may specifically escalate the pre-mRNA AS variants of sycp3, sycp3l, and sycp1 (Fig. 2). However, the reduction of Sycp3 protein products resulted in the failure of the normal assembly framework of rigid SC (Fig. 2B), thereby resulting in synaptic instability and non-crossover DSBs, or the chromosomal mis-segregation found by cytological observations (Fig. 6a3-3’). This could further weaken the mechanical force to promote the mitosis-meiosis transition (Elkouby, 2017). The impairment of chromosome assembly and recombination by abnormal sycp3 and sycp1 in mice meiotic cells produced aneuploid gametes, whereas heterogenous germ cells were blocked at different stages, such as mitotic cessation and chromosomal desynapsis to the incomplete diplotene/embryonic stage (Ayarza et al., 2016). This occurred in gsdf deficient gametogenesis, as the cysts composed of differentiated germ cells were fused abnormally as the result of germ and somatic cell apoptosis in gsdf-deficient spermatogenesis (Fig. 7). In addition, heterogenous germ cells with positive or negative expression of Igf2bp3 and H3K27me3 coexisted in a single cyst of the gsdf^−/− XY ovary (Wu et al., 2019).

The developmental arrest of primary oocytes in gsdf-deficient XY ovaries partially overlapped the phenotype of fshr and miR202 disruption in medaka (Gay et al., 2018, Murozumi et al., 2014). However, the expression levels of fshr and miR202 were significantly upregulated in gsdf deficient germ cells (Fig. 1). Human FSHR variants lacking exon 2 and/or exon 3 are unable to stimulate the FSH signaling cascade and are associated with subfertility in women (Karakaya et al., 2014). Alternative spliced variants of fshr may interfere with the development of gsdf-deficient oocytes, in addition to the canonical Smad3 dependent activation of Fshr promoter to increase the cell division rate of rat granulosa cells (Gong and McGee, 2009). In fact, the irreversible synergistic effect of TGFβ and FSH in the expression of Fshr mRNA, as demonstrated in cultured rat granulosa cells (Inoue et al., 2003), may explain the prevalence of 100% ovotestis development in Sissy individuals (the line that restores the exogenous gsdf expression in endogenous gsdf-targeted disruption) (Zhang et al., 2021) by 10% compared to that of hotei (amhr2 mutant) (Morinaga et al., 2007).

Sycp1 has been demonstrated to be necessary for the complete pairing of homologs in meiosis of zebrafish meiosis (Imai et al., 2021). The large-scale replacement of the normal SCs by defective SCs may have been detected by meiotic checkpoints, thereby triggering apoptosis, as demonstrated in Sycp3 and Sycp1 knockout mice (Ayarza et al., 2016). Males form fewer DSBs than females, and sex-specific differences are noted in HR during human meiosis prior to the establishment of DSBs (Gruhn et al., 2013). Sex chromosomes are particularly susceptible to paternal meiotic errors (Hall et al., 2006), and the deduced aneuploid cells will be eliminated by checkpoint mechanisms acting either at pachytene, to eliminate cells with synaptic errors or at metaphase I to eliminate cells with univalent chromosomes (Hall et al., 2006). X and Y spermatozoa are produced at a 50:50 ratio during normal spermatogenesis according to Mendelian segregation, but this ratio is skewed by a set of differentiated expression proteins and genes, which has been reviewed (Rahman and Pang, 2019). It was noteworthy that gsdf-deficient XY germ cells produced a seriously skewed sex ratio in the offspring of three gsdf-deficient XY males mated with normal XX females, as well as gsdf-deficient XY females mated with gsdf-transgenic XX males, according to our previous report (Zhang et al., 2016). This finding suggests that gsdf is involved in monitoring the progression and maintaining the fidelity of homologous chromosome pairing in meiosis through the regulation of sycp1 and sycp3 transcription and splicing. However, oocytes adopt a synaptic configuration in which the single X chromosome ‘self pairs’ and thereby avoids detection. The effect of gsdf on the preferential survival of X-chromosomal oocytes and Y-chromosome sperm may provide some clues to explain why men with more brothers are more prone to have sons, while men with more sisters have a higher probability of having daughters (Taketo, 2015); this phenomenon may be due to ligand-receptor interaction and responsiveness, assuming that a common role is conserved between mammalian BMP/TGFβ function and medaka Gsdf.

In summary, our results suggest that Gsdf signaling is required for precise homologous chromosome pairing, recombination, and synapsis formation. through Sycp1–Sycp3 splicing variants in vertebrate pre-meiotic and meiotic differentiation. These mechanisms in gsdf deletion gametogenesis will help to elucidate the meiotic dysfunctions associated with infertility.

Supplementary data

This is linked to the online version of the paper at XXX.

Declaration of competing interests

All authors declare that they have no conflicts of interest regarding the contents of this article.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

GuanG was the primary corresponding author and Liangbiao Chen was the secondary corresponding author, both designed the overall studies; GuanG, LiX, LiXW, LiW, GuoH, WangG, HuR, LiY, WangS, WuX and WangT performed methodological investigations; GuanG, LiX, LiXW, and LiW undertook writing and review; and GuanG, ChenL and ZhaoX performed funding acquisition and manuscript editing. All authors have read and approved the final manuscript.

Acknowledgments

This work was supported by the First Class Discipline Program for Fisheries from the Shanghai municipal government, a grant from the National Natural Science Foundation of China (81771545), the Guangdong Basic and Applied Basic Research Foundation (2020B1515020001), and a grant from the National Key Research and Development Program of China (2018YFD0900601). We also thank LetPub LLC (www.letpub.com) for language-editing services and NBRP medaka for providing the HdrR and HNI strains.

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Table 1 Genotypic and phenotypic analysis of progenies from mutant and normal lines
Types	Family	Fertilized eggs/total eggs	Hatching	3-month-old adults
		(Fertilization rate)	(Survival rate)	♂:♀
gsdf^-/-XY♂ x gsdf^+/+XX♀	1	82/145 (56.6%)	47/82 (57.3%)	15:0 (all XY)
	2	91/148 (61.5%)	35/91 (38.5%)	22: 0 (all XY)
	3	87/121 (71.9%)	32/87 (36.8%)	24: 0 (all XY)
gsdf^-/-XY♀ x Tg: gsdf^+/+XX♂	4	73/170 (42.9%)	38/73 (52.1%)	2:16 (all XX)
	5	87/127 (68.5%)	42/87 (48.3%)	4:22 (all XX)
	6	55/72 (76.4%)	28/55 (50.9%)	1:11 (all XX)
	7	30/72 (41.7%)	12/30 (40%)	(embryos St30 4XY:8XX)
gsdf^-/-XX♀ x Sissy gsdf^-/-XY⚥	8	48/122 (39.3%)	18/48 (37.5%)	4XY:12 XX
				(SissyXY⚥:XX♀)

gsdf^+/+XY♂ x gsdf^+/⁺XX♀	9	198/209 (94.7%)	179/198 (90.4%)	62:75
				(XY♂:XX♀)

	10	205/212(96.7%)	186/205 (90.7%)	80:95
Tg: gsdf^+/+XX♂				(XY♂:XX♀)
x
gsdf^+/+XX♀	11	170/180 (94.4%)	154/170 (90.6%)	28:114
				(XX♂:XX♀)
	12	138/143 (98.9%)	122/138 (88.4%)	21:87
				(XX♂:XX♀)

No competing interests reported.

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RNA-seq and cytological study of sex-specific meiosis response to Gsdf in medaka (Oryzias latipes)

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Abstract

Figures

Introduction

Materials And Methods

Fish and mating schemes

Histological and ultrastructural observation

Identification of proliferative germ cells by EdU incorporation

Preparation of meiotic nuclear spreads and immunostaining

Transcriptomic analysis

Structural prediction of Sycp1 and Sycp3 variants

Statistical analysis

Results

The differentially expressed genes regulated by Gsdf signaling

Meiotic recombination targeted by Gsdf signal

Malformation of Balbiani body with abnormal expression of Sycp3-Piwi in gsdf deficient oocytes

Increase of HR errors in gsdf-deficient testes

Asynchronous cysts formation in gsdf-deficient testes

Aneuploidy and low fertility of gsdf-deficient gametes

Discussion

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References

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