Single-cell transcriptome analysis of the germ cells and somatic cells during mitotic quiescence stage in goats.

The mitotic quiescence of prospermatogonia is the event known to occur during genesis of the male germline and is tied to the development of the spermatogenic lineage. The regulatory mechanisms and the functional importance of this process have been demonstrated in mice; however, regulation of this process in human and domestic animal is still largely unknown. In this study, we employed single-cell RNA sequencing to identify transcriptional signatures of prospermatogonia and major somatic cell types in testes of goats at E85, E105, and E125. We identified both common and specific Gene Ontology categories, transcription factor regulatory networks, and cell-cell interactions in cell types from goat testis. We also analyzed the transcriptional dynamic changes in prospermatogonia, Sertoli cells, Leydig cells, and interstitial cells. Our datasets provide a useful resource for the study of domestic animal germline development.

Somatic cell-derived BMPs induce premature meiosis in male germ cells during the embryonic stage by upregulating Dazl expression.

Although germ cell fate is believed to be determined by signaling factors from differentiated somatic cells, the molecular mechanism behind this process remains obscure. In this study, premature meiosis in male germ cells was observed during the embryonic stage by conditional activation of ß-catenin in Sertoli cells. Somatic and germ cell transcriptome results indicated that the BMP signaling pathway was enriched after ß-catenin activation. In addition, we observed a decreased DNA methylation within a reduction of DNMT3A in germ cells of ß-catenin activated testes and reversed increase after inhibiting BMP signaling pathway with LDN-193189. We also found that Dazl expression was increased in ß-catenin activated testes and decreased after LDN treatment. Taken together, this study demonstrates that male germ cells entered meiosis prematurely during the embryonic stage after ß-catenin activated in Sertoli cells. BMP signaling pathway involved in germ cell meiosis initiation by mediating DNA methylation to induce meiotic genes expression.

The function of Foxo1 in spermatogonia development is independent of PI3K/PTEN signaling.

Spermatogenesis is a highly coordinated process that initiates shortly after birth and continues throughout the lifespan of male animals. Foxo1 is a transcription factor and is involved in many biological processes. It has been reported that the inactivation of Foxo1 in gonocytes during the embryonic stage causes the defects of spermatogenesis. In the present study, we found that the inactivation of Foxo1 in spermatogonia after birth also caused germ cell loss and male infertility. We found that the initiation of meiosis was not affected; however, the germ cell development was arrested after meiosis and lack of mature spermatozoa in the cauda epididymis. We also found that the proliferation of Foxo1-deficient spermatogonia stem cells was significantly reduced under in vitro conditions. Further study revealed that inactivation of Pten in postnatal spermatogonia using Stra8-Cre did not affect germ cell development and the subcellular location of FOXO1 in Pten-deficient spermatogonia. This study demonstrated that Foxo1 was involved in the development of spermatogonia after birth and the function of Foxo1 was probably not regulated by PI3K/PTEN signaling.

Fancb deficiency causes premature ovarian insufficiency in mice†.

Fanconi anemia complementation group B (FANCB) protein is a major component of the Fanconi anemia (FA) core complex and plays an important role in hematopoiesis and germ cell development. Deletion of Fancb gene causes the defect of primordial germ cell (PGC) development and infertility in male mice. However, it remains unknown whether Fancb is required for female germ cell development. In this study, we found that the fertility of Fancb knockout male mice in C57/ICR mixed backgrounds was not affected. Female Fancb-/- mice were obtained by crossing Fancb+/- females with Fancb-/Y males. The number of PGCs was dramatically decreased in Fancb-/- females. Very few oocytes were observed after birth and the primordial follicle pool was completely depleted at 6 weeks of age in Fancb-/- females. However, the remained oocytes from Fancb-/- mice were normal in fertilization and embryonic development from 2-cell to the blastocyst stage. We also found that Fancb and Fancl double-knockout males were also fertile and the number of sperm in epididymis was not reduced as compared to that of Fancb-/- and Fancl-/- single-knockout mice. Taken together, these results showed that Fancb is also essential for female germ cell development. Inactivation of Fancb causes massive germ cell loss and infertility in adult females. We also found that Fancb and Fancl do not act synergistically in regulating germ cell development.

The functions of Wt1 in mouse gonad development and somatic cells differentiation†.

Wilms' tumor 1 (Wt1) encodes a zinc finger nuclear transcription factor which is mutated in 15-20% of Wilms' tumor, a pediatric kidney tumor. Wt1 has been found to be involved in the development of many organs. In gonads, Wt1 is expressed in genital ridge somatic cells before sex determination, and its expression is maintained in Sertoli cells and granulosa cells after sex determination. It has been demonstrated that Wt1 is required for the survival of the genital ridge cells. Homozygous mutation of Wt1 causes gonad agenesis. Recent studies find that Wt1 plays important roles in lineage specification and maintenance of gonad somatic cells. In this review, we will summarize the recent research works about Wt1 in gonadal somatic cell differentiation.

Safety of cholecystectomy in patients under antithrombotic Drugs: A systematic review and meta-analysis.

Objective: This review aimed to assess evidence on the safety of cholecystectomy in patients under antithrombotic therapy. Methods: PubMed, Embase, Science Direct, CENTRAL, and Google Scholar databases were searched from inception up to 20th January 2022 for studies comparing outcomes of patients undergoing cholecystectomy surgeries with or without concomitant antithrombotic therapy. Results: Nine studies were included. Meta-analysis revealed that the use of antithrombotic medications had no statistically significant effect on intra-operative blood loss in patients undergoing cholecystectomy (MD: 82.31 95% CI: -283.38, 448 I2=98% p=0.66). However, incidence of blood transfusion (OR: 5.65 95% CI: 1.10, 28.86 I2=83% p=0.04) and bleeding complications (OR: 8.02 95% CI: 1.71, 37.58 I2 = 71% p=0.008) were significantly increased in patients under antithrombotic therapy. Pooled analysis indicated that cholecystectomy patients under antithrombotic are at an increased risk of conversion to open surgery (OR: 2.02 95% CI: 1.21, 3.36 I2=0% p=0.007). Meta-analysis revealed significantly shorter LOS in patients under antithrombotic (MD: -5.01 95% CI: -8.29, -1.73 I2=97% p=0.03). Conclusion: Current evidence from a limited number of studies indicates that the use of antithrombotic may be associated with an increased risk of bleeding-related complications in patients undergoing cholecystectomies. Antithrombotic use may also increase the risk of conversion to open surgery in patients undergoing laparoscopic cholecystectomies.

Wt1 directs the lineage specification of sertoli and granulosa cells by repressing Sf1 expression.

Supporting cells (Sertoli and granulosa) and steroidogenic cells (Leydig and theca-interstitium) are two major somatic cell types in mammalian gonads, but the mechanisms that control their differentiation during gonad development remain elusive. In this study, we found that deletion of Wt1 in the ovary after sex determination caused ectopic development of steroidogenic cells at the embryonic stage. Furthermore, differentiation of both Sertoli and granulosa cells was blocked when Wt1 was deleted before sex determination and most genital ridge somatic cells differentiated into steroidogenic cells in both male and female gonads. Further studies revealed that WT1 repressed Sf1 expression by directly binding to the Sf1 promoter region, and the repressive function was completely abolished when WT1 binding sites were mutated. This study demonstrates that Wt1 is required for the lineage specification of both Sertoli and granulosa cells by repressing Sf1 expression. Without Wt1, the expression of Sf1 was upregulated and the somatic cells differentiated into steroidogenic cells instead of supporting cells. Our study uncovers a novel mechanism of somatic cell differentiation during gonad development.

Tracing the origin of the placental trophoblast cells in mouse embryo development†.

The placenta, which originates from the trophectoderm (TE), is the first organ to form during mammalian embryogenesis. Recent studies based on bioinformatics analysis have revealed that heterogeneous gene expression initiates cell-fate decisions and directs two distinct cell fates by modulating the balance of pluripotency and differentiation as early as the four-cell stage. However, direct developmental evidence to support this is still lacking. To address at which stage the cell fate of the TE and inner cell mass (ICM) is determined, in this study, we administered a microinjection of Cre mRNA into a single blastomere of the mTmG mouse at different cleavage stages before implantation to examine the distributions of the descendants of the single-labeled cell in the mouse fetus and the placenta at E12.5. We found that the descendants of the labeled cells at the two-cell stage contributed to both the placenta and the fetus. Notably, the derivatives of the labeled cells at the four-cell stage fell into three categories: (1) distributed in both embryonic and extraembryonic lineages, (2) distributed only in mouse placental trophoblast layers, or (3) distributed only in the lineage derived from the ICM. In addition, these results fell in line with single-cell studies focusing on gene expression patterns that characterize particular lineages within the blastocyst. In conclusion, this study shows that the four-cell blastomeres differ in their individual developmental properties insofar as they contribute to either or both the ICM and trophoblast fate.

ß-Catenin directs the transformation of testis Sertoli cells to ovarian granulosa-like cells by inducing Foxl2 expression.

Sertoli and granulosa cells are two major types of somatic cells in male and female gonads, respectively. Previous studies have shown that Sertoli and granulosa cells are derived from common progenitor cells and that differentiation of these two cell types is regulated by sex differentiation genes. The signaling pathway including the adhesion and transcription factor Ctnnb1 (cadherin-associated protein, ß1, also known as ß-catenin) regulates differentiation of granulosa cells in the absence of the transcription factor Sry, and overactivation of ß-catenin in the presence of Sry leads to granulosa prior to sex determination. Surprisingly, our previous study found that ß-catenin overactivation in Sertoli cells after sex determination can also cause disruption of the testicular cord and aberrant testis development. However, the underlying molecular mechanism was unclear. In this study, we found that constitutive activation of Ctnnb1 in Sertoli cells led to ectopic expression of the granulosa cell-specific marker FOXL2 in testes. Co-staining experiments revealed that FOXL2-positive cells were derived from Sertoli cells, and Sertoli cells were transformed into granulosa-like cells after Ctnnb1 overactivation. Further studies demonstrated that CTNNB1 induced Foxl2 expression by directly binding to transcription factor Tcf/Lef-binding sites in the FOXL2 promoter region. We also found that direct overexpression of Foxl2 decreased the expression of Sertoli cell-specific genes in primary Sertoli cells. Taken together, these results demonstrate that repression of ß-catenin (CTNNB1) signaling is required for lineage maintenance of Sertoli cells. Our study provides a new mechanism for Sertoli cell lineage maintenance during gonad development.

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation.

Meiotic recombination is essential for fertility in most sexually reproducing species, but the molecular mechanisms underlying this process remain poorly understood in mammals. Here, we show that RNF20-mediated H2B ubiquitination is required for meiotic recombination. A germ cell-specific knockout of the H2B ubiquitination E3 ligase RNF20 results in complete male infertility. The Stra8-Rnf20-/- spermatocytes arrest at the pachytene stage because of impaired programmed double-strand break (DSB) repair. Further investigations reveal that the depletion of RNF20 in the germ cells affects chromatin relaxation, thus preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. The gametogenetic defects of the H2B ubiquitination deficient cells could be partially rescued by forced chromatin relaxation. Taken together, our results demonstrate that RNF20/Bre1p-mediated H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation, and suggest an old drug may provide a new way to treat some oligo- or azoospermia patients with chromatin relaxation disorders.

Reprogramming of Sertoli cells to fetal-like Leydig cells by Wt1 ablation.

Sertoli and Leydig cells, the two major somatic cell types in the testis, have different morphologies and functions. Both are essential for gonad development and spermatogenesis. However, whether these cells are derived from the same progenitor cells and the mechanism regulating the differentiation between these two cell types during gonad development remains unclear. A previous study showed that overactivation of Ctnnb1 (cadherin-associated protein, beta 1) in Sertoli cells resulted in Sertoli cell tumors. Surprisingly, in the present study, we found that simultaneous deletion of Wilms' Tumor Gene 1 (Wt1) and overactivation of Ctnnb1 in Sertoli cells led to Leydig cell-like tumor development. Lineage tracing experiments revealed that the Leydig-like tumor cells were derived from Sertoli cells. Further studies confirmed that Wt1 is required for the maintenance of the Sertoli cell lineage and that deletion of Wt1 resulted in the reprogramming of Sertoli cells to Leydig cells. Consistent with this interpretation, overexpression of Wt1 in Leydig cells led to the up-regulation of Sertoli cell-specific gene expression and the down-regulation of steroidogenic gene expression. These results demonstrate that the distinction between Sertoli cells and Leydig cells is regulated by Wt1, implying that these two cell types most likely originate from the same progenitor cells. This study thus provides a novel concept for somatic cell fate determination in testis development that may also represent an etiology of male infertility in human patients.

Protein arginine methyltransferase 5 (Prmt5) is required for germ cell survival during mouse embryonic development.

In mammals, germ cells undergo massive epigenetic remodeling during fetal development. However, the physiological functions of epigenetic modification in germ cell development remain unclear. In this study, we found that protein arginine methyltransferase 5 (Prmt5) was abundantly expressed in the germ cells of both male and female gonads. Deletion of Prmt5 by crossing with Tnap-Cre mice resulted in germ cell depletion in adult mice. Germ cell loss was first observed between Embryonic Days 12.5 and 13.5 (E12.5 and E13.5), and very few of these cells remained at birth. Oct4, Sox2, and Nanog were abundantly expressed in Prmt5-deficient germ cells at E13.5 and E15.5, whereas the expression of these genes was dramatically decreased in control germ cells. Interestingly, the expression of meiosis-associated genes was virtually absent in Prmt5-deficient female germ cells at E13.5, whereas the expression of other germ cell-specific genes was not changed. Further study revealed that H4R3me2s was completely absent after Prmt5 inactivation, whereas the level of H3R2me2s was not changed in Prmt5-deficient germ cells. Collectively, this study demonstrated that Prmt5 plays critical roles in germ cell development that are required for germ cell survival during embryonic stages.

Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction.

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn(-/-) males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn(-/-) males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn(-/-) mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.

Wt1 is involved in leydig cell steroid hormone biosynthesis by regulating paracrine factor expression in mice.

Wilms tumor gene, Wt1, is abundantly expressed in testis Sertoli cells. Our recent study demonstrated that Wt1 is involved in spermatogenesis by regulating Sertoli cell polarity. In the present study, we found that Wt1 is also required for steroidogenesis in Leydig cells and that deletion of the Wt1 gene resulted in defects in testosterone biosynthesis and downregulation of steroidogenic gene expression, including cytochrome P450 side-chain cleavage (P450scc), steroidogenic acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase I (3beta-HSD), and cytochrome P450 17A1 (Cyp17a1). The expression of LHR was significantly decreased in Wt1(-/flox); Cre-ER(TM) testes after tamoxifen induction, whereas the luteinizing hormone level in serum was unchanged. Further studies revealed that desert hedgehog (Dhh) expression was regulated by Wt1 in Sertoli cells and that its expression was significantly reduced in Wt1-deficient testes. In vitro study demonstrated that the defect in testosterone production and decreased expression of several steroidogenic genes in Wt1-deficient testis explants was partially rescued by smoothened agonist (SAG), a hedgehog pathway agonist. These results indicate that Wt1 is most likely involved in Leydig cell steroidogenesis by regulating the expression of paracrine factors in seminiferous tubules. Dhh probably had important roles in this process, but we could not exclude the possibility that other factors were also required for Leydig cell steroidogenesis. Loss of Wt1 leads to downregulation of paracrine factors, which in turn causes a decrease in steroidogenic enzyme expression and reduces testosterone production in Leydig cells. The results of this study further confirm that the cross talk between Sertoli cells and Leydig cells has important roles in Leydig cell steroidogenesis.

CCDC189 affects sperm flagellum formation by interacting with CABCOCO1.

Multiple morphological abnormalities of the sperm flagella (MMAF) are one of the major causes of male infertility and are characterized by multiple defects. In this study, we found that the coiled-coil domain-containing 189 (Ccdc189) gene was predominantly expressed in mouse testes and that inactivation of the Ccdc189 gene caused male infertility. Histological studies revealed that most sperm from Ccdc189-deficient mice carried coiled, curved or short flagella, which are typical MMAF phenotypes. Immunoelectron microscopy showed that the CCDC189 protein was located at the radial spoke of the first peripheral microtubule doublet in the sperm axoneme. A CCDC189-interacting protein, CABCOCO1 (ciliary-associated calcium-binding coiled-coil protein 1), was discovered via co-immunoprecipitation and mass spectrometry, and inactivation of Cabcoco1 caused malformation of sperm flagella, which was consistent with findings obtained with Ccdc189-deficient mice. Further studies revealed that inactivation of CCDC189 caused downregulation of CABCOCO1 protein expression and that both CCDC189 and CABCOCO1 interacted with the radial-spoke-specific protein RSPH1 and intraflagellar transport proteins. This study demonstrated that Ccdc189 is a radial-spoke-associated protein and is involved in sperm flagellum formation through its interactions with CABCOCO1 and intraflagellar transport proteins.

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.

Integration of single-cell transcriptome and chromatin accessibility of early gonads development among goats, pigs, macaques, and humans.

The early gonads of mammals contain primordial germ cells (PGCs) and somatic cell precursors that are essential for sex determination and gametogenesis. Although it is extensively documented in mice, the development of early gonads in non-rodents remains to be delineated. Because molecular differences between mouse and human gonadal cells have been reported, it warrants the study of the key markers and regulatory features that are conserved or divergent between non-rodent species and human. Here, we integrate single-cell transcriptome and chromatin accessibility analysis to identify regulatory signatures of PGCs and somatic cells in the early gonads of goats, pigs, macaques, and humans. We identify the evolutionarily conserved and species-specific events, including genes expression, signaling pathways, and cell-cell interactions. We also uncover potential cis-regulatory elements and key transcription factors in PGCs and somatic cells. Our datasets provide important resources for better understanding the evolutionary programs of PGCs and gonadal somatic cell development in mammals.

PRMT5 regulates ovarian follicle development by facilitating Wt1 translation.

Protein arginine methyltransferase 5 (Prmt5) is the major type II enzyme responsible for symmetric dimethylation of arginine. Here, we found that PRMT5 was expressed at high level in ovarian granulosa cells of growing follicles. Inactivation of Prmt5 in granulosa cells resulted in aberrant follicle development and female infertility. In Prmt5-knockout mice, follicle development was arrested with disorganized granulosa cells in which WT1 expression was dramatically reduced and the expression of steroidogenesis-related genes was significantly increased. The premature differentiated granulosa cells were detached from oocytes and follicle structure was disrupted. Mechanism studies revealed that Wt1 expression was regulated by PRMT5 at the protein level. PRMT5 facilitated IRES-dependent translation of Wt1 mRNA by methylating HnRNPA1. Moreover, the upregulation of steroidogenic genes in Prmt5-deficient granulosa cells was repressed by Wt1 overexpression. These results demonstrate that PRMT5 participates in granulosa cell lineage maintenance by inducing Wt1 expression. Our study uncovers a new role of post-translational arginine methylation in granulosa cell differentiation and follicle development.

Infertility in women can be caused by many factors, such as defects in the ovaries. An important part of the ovaries for fertility are internal structures called follicles, which house early forms of egg cells. A follicle grows and develops until the egg is finally released from the ovary into the fallopian tube, where the egg can then be fertilised. In the follicle, an egg is surrounded by other types of cells, such as granulosa cells. The egg and neighbouring cells must maintain healthy contacts with each other, otherwise the follicle can stop growing and developing, potentially causing infertility. The development of a follicle depends on an array of proteins. For example, the transcription factor WT1 controls protein levels by activating other genes and their proteins and is produced in high numbers by granulosa cells at the beginning of follicle development. Although WT1 levels dip towards the later stages of follicle development, insufficient levels can lead to defects. So far, it has been unclear how levels of WT1in granulose cells are regulated. Chen, Dong et al. studied mouse follicles to reveal more about the role of WT1 in follicle development. The researchers measured protein levels in mouse granulosa cells as the follicles developed, and discovered elevated levels of PRMT5, a protein needed for egg cells to form and survive in the follicles. Blocking granulosa cells from producing PRMT5 led to abnormal follicles and infertility in mice. Moreover, mice that had been engineered to lack PRMT5 developed abnormal follicles, where the egg and surrounding granulosa cells were not attached to each other, and the granulosa cells had low levels of WT1. Further experiments revealed that PRMT5 controlled WT1 levels by adding small molecules called methyl groups to another regulatory protein called HnRNPA1. The addition of methyl groups to genes or their proteins is an important modification that takes place in many processes within a cell. Chen, Dong et al. reveal that this activity also plays a key role in maintaining healthy follicle development in mice, and that PRMT5 is necessary for controlling WT1. Identifying all of the intricate mechanism involved in regulating follicle development is important for finding ways to combat infertility.

Abnormal Meiosis Initiation in Germ Cell Caused by Aberrant Differentiation of Gonad Somatic Cell.

The interaction between germ cell and somatic cell plays important roles in germ cell development. However, the exact function of gonad somatic cell in germ cell differentiation is unclear. In the present study, the function of gonad somatic cell in germ cell meiosis was examined by using mouse models with aberrant somatic cell differentiation. In Wt1R394W/R394W mice, the genital ridge is absent due to the apoptosis of coelomic epithelial cells. Interestingly, in both male and female Wt1R394W/R394W germ cells, STRA8 was detected at E12.5 and the scattered SYCP3 foci were observed at E13.5 which was consistent with control females. In Wt1-/flox; Cre-ERTM mice, Wt1 was inactivated by the injection of tamoxifen at E9.5 and the differentiation of Sertoli and granulosa cells was completely blocked. We found that most germ cells were located outside of genital ridge after Wt1 inactivation. STRA8, SYCP3, and γH2AX proteins were detected in germ cells of both male and female Wt1-/flox; Cre-ERTM gonads, whereas no thread-like SYCP3 signal was observed. Our study demonstrates that aberrant development of gonad somatic cells leads to ectopic expression of meiosis-associated genes in germ cells, but meiosis was arrested before prophase I. These results suggest that the proper differentiation of gonad somatic cells is essential for germ cell meiosis.

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

BACKGROUND: Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation. RESULTS: By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels. CONCLUSION: Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.