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The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo-synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo-synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genome to early embryonic development. In this study, we analyzed different methodologies for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. The results showed that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although haploidy does not disturb early development up to around the 3rd mitotic division, androgenetic development is disturbed after the time of zygote genome activation those that reach the morula stage are less capable to become a blastocyst. Analysis of gene expression indicated abnormal levels of methyltransferase 3B and key long non-coding RNAs involved in X-chromosome inactivation and genomic imprinting of the KCNQ1 locus, which is associated to the methylation status of imprinted control regions of XIST and KCNQ1OT1. Thus, our results seem to exclude micromanipulation consequences and chromosomal abnormalities as major factors in developmental restriction, suggesting that their early developmental constraint is regulated at an epigenetic level.

The postacrosomal sheath (PAS) of the perinuclear theca (PT) is the first compartment of the sperm head to solubilize into the ooplasm upon sperm-oocyte fusion, implicating its constituents in zygotic development. This study investigates the role of one such constituent, glutathione-S-transferase omega 2 (GSTO2), an oxidative-reductive enzyme found in the PAS and perforatorial regions of the PT. GSTO2 uses the conjugation of reduced glutathione, an electron donor shown to be compulsory in sperm disassembly within the ooplasm. The proximity of GSTO2 to the condensed sperm nucleus led us to hypothesize that this enzyme may facilitate nuclear decondensation by reducing disulfide bonds before the recruitment of GSTO enzymes from within the ooplasm. To test this hypothesis, we utilized a cell permeable isozyme-specific inhibitor, which fluoresces when bound to the active site of GSTO2, to functionally inhibit spermatozoa before performing intracytoplasmic sperm injections (ICSI) in mice. The technique allowed for targeted inhibition of solely PT-residing GSTO2, as all that is required for complete zygotic development is the injection of the mouse spermatozoon head. ICSI showed that inhibition of PT-anchored GSTO2 caused a delay in sperm nuclear decondensation, and further resulted in untimely embryo cleavage, and an increase in fragmentation beginning at the morula stage. The confounding effects of these developmental delays ultimately resulted in decreased blastocyst formation. This study implicates PT-anchored GSTO2 as an important facilitator of nuclear decondensation and reinforces the notion that the PAS-PT is a critical sperm compartment harboring molecules that facilitate zygotic development.

The sperm-borne oocyte-activating factor (SOAF) resides in the sperm perinuclear theca (PT). A consensus has been reached that SOAF most likely resides in the postacrosomal sheath (PAS), which is the first region of the PT to solubilize upon sperm–oocyte fusion. There are two SOAF candidates under consideration: PLCZ1 and WBP2NL. A mouse gene germline ablation of the latter showed that mice remain fertile with no observable phenotype despite the fact that a competitive inhibitor of WBP2NL, derived from its PPXY motif, blocks oocyte activation when coinjected with WBP2NL or spermatozoa. This suggested that the ortholog of WBP2NL, WBP2, containing the same domain and motifs associated with WBP2NL function, might compensate for its deficiency in oocyte activation. Our objectives were to examine whether WBP2 meets the developmental criteria established for SOAF and whether it has oocyte-activating potential. Immunoblotting detected WBP2 in mice testis and sperm and immunofluorescence localized WBP2 to the PAS and perforatorium of the PT. Immunohistochemistry of the testes revealed that WBP2 reactivity was highest in round spermatids and immunofluorescence detected WBP2 in the cytoplasmic lobe of elongating spermatids and colocalized it with the microtubular manchette during PT assembly. Microinjection of the recombinant forms of WBP2 and WBP2NL into metaphase II mouse oocytes resulted in comparable rates of oocyte activation. This study shows that WBP2 shares a similar testicular developmental pattern and location with WBP2NL and a shared ability to activate the oocyte, supporting its consideration as a mouse SOAF component that can compensate for a WBP2NL., WBP2 and WBP2NL share sperm location, development origins, and oocyte-activating ability in mouse.

International audience; The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.

During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.

Although putative horse embryonic stem (ES)-like cell lines have been obtained recently fromin vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases,NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity andPOU5F1,SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may loseNANOG. Cystic embryoid body-like structures expressingPOU5F1,CDX2andEOMESwere produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressingPOU5F1and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.

The orphan nuclear receptor, liver receptor homolog-1 (aka Nuclear receptor subfamily 5, Group A, Member 2 (Nr5a2)), is widely expressed in mammalian tissues, and its ovarian expression is restricted to granulosa cells of activated follicles. We employed the floxed Nr5a2 (Nr5a2(f/f)) mutant mouse line and two granulosa-specific Cre lines, Anti-Mullerian hormone receptor- 2 (Amhr2Cre) and transgenic cytochrome P450 family 19 subfamily A polypeptide 1 (tgCyp19Cre), to develop two tissue- and time-specific Nr5a2 depletionmodels: Nr5a2(Amhr2-/-) and Nr5a2(Cyp19-/-). In the Nr5a2(Cyp19-/-) ovaries, Nr5a2 was depleted in mural granulosa, but not cumulus cells. We induced follicular development in mutant and wild-type (control, CON) mice with equine chorionic gonadotropin followed 44 h later treatment with human chorionic gonadotropin (hCG) to induce ovulation. Both Nr5a2(Amhr2-/-) and Nr5a2(Cyp19-/-) cumulus-oocyte complexes underwent a reduced degree of expansion in vitro relative to wild-type mice. We found downregulation of epiregulin (Ereg), amphiregulin (Areg), betacellulin (Btc) and tumor necrosis factor stimulated gene-6 (Tnfaip6) transcripts in Nr5a2(Amhr2-/-) and Nr5a2(Cyp19-/-) ovaries. Tnfaip6 protein abundance, by quantitative immunofluorescence, was likewise substantially reduced in the Nr5a2-depleted model. Transcript abundance for connexin 43 (Gja1) in granulosa cells was lower at 0 h and maximum at 8 h post-hCG in both Nr5a2(Amhr2-/-) and Nr5a2(Cyp19-/-) follicles, while Gja1 protein was not different prior to the ovulatory signal, but elevated at 8 h in Nr5a2(Amhr2-/-) and Nr5a2(Cyp19-/-) follicles. In both mutant genotypes, oocytes can mature in vivo and resulting embryos were capable of proceeding to blastocyst stagein vitro. We conclude that Nr5a2 is essential for cumulus expansion in granulosa cells throughout follicular development. The disruption of Nr5a2 in follicular somatic cells does not affect the capacity of the oocyte to be fertilized by intracytoplasmic sperm injection. Summary Sentence Liver receptor homolog-1 is necessary for cumulus expansion, but not fertilization of mouse oocytes.