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A Microarray-Based Analysis of Gametogenesis in Two Portuguese Populations of the European Clam Ruditapes decussatus
research-article
Author(s):
Joana Teixeira de Sousa
1
,
2 ,
Massimo Milan
3 ,
Luca Bargelloni
3 ,
Marianna Pauletto
3 ,
Domitília Matias
2 ,
Sandra Joaquim
2 ,
Ana Margarete Matias
2 ,
Virgile Quillien
1 ,
Alexandra Leitão
2 ,
Arnaud Huvet
1
,
*
Publication date (Electronic):
18 March 2014
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.
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Most cited references29
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Hormad1 Mutation Disrupts Synaptonemal Complex Formation, Recombination, and Chromosome Segregation in Mammalian Meiosis
Yong-Hyun Shin, Youngsok Choi, Serpil Erdin … (2010)
Introduction Mammalian meiosis is unique to germ cells and a critical step in sexual reproduction. Meiosis reduces the chromosome complement to haploidy in preparation for fertilization. The first meiotic division is unique in pairing of homologous chromosomes, homologous recombination, and formation of chiasmata. The reduction in chromosome numbers happens when homologous chromosomes segregate to opposite poles during the first meiotic division. Proper disjunction (separation) requires crossovers (manifested cytologically as chiasmata). The sister chromatids organize along structures called axial elements (AEs) and transverse elements connect AEs to form the synaptonemal complex (SC) [1]. SC is a proteinaceous structure that connects paired homologous chromosomes during prophase I of meiosis, and SC is critical for wild-type levels of crossovers to occur during meiosis. AEs are critical part of the SCs and mutations in proteins that form AEs disrupt sister chromatid cohesion, recombination, and chromosome segregation [2]–[4]. Proteins with HORMA domain are critical components of the axial elements [5]. HORMA domain proteins are predicted to form globular structure that may sense specialized chromatin states, such as those associated with double strand breaks (DSBs) or other forms of DNA damage [5]. Several mammalian proteins that contain HORMA domain, such as mitotic arrest deficient protein 2, MAD2, are essential for mitosis [6]–[7]. Mice lacking MAD2 unsurprisingly die during early embryogenesis [7]. In lower organisms, several meiotic specific HORMA proteins are known and all are critical for meiosis. These HORMA proteins are: Hop1 [8] and Red1 [9] in yeast; Him-3 [10] in nematodes; and Asy1 [11] in plants. Him-3 localizes to the axial cores of both synapsed and unsynapsed chromosomes. C. elegans Him-3 mutants are deficient in chromosome pairing, synapsis, and the regulation of double strand break repair [10], [12]–[13]. Synapsis in both male and female Asy1 mutants is disrupted [14]–[15]. In yeast, plants, and nematodes, HORMA domain proteins are critical components of the synaptonemal complex and essential for meiosis I. Others and we identified a previously uncharacterized gene that we named Nohma, later re-named to Hormad1 [16]–[18]. Hormad1 encodes a protein that contains a HORMA domain, and unlike Mad2, Hormad1 expression is germ cell–specific [16]. Mouse and human HORMAD1 are highly conserved and share 77% amino acid identity overall, and share 89% amino acid identity in the HORMA domain. Moreover, mouse and human HORMAD1 HORMA domains share 28% amino acid identity with Hop1 HORMA domain. Hop1 in yeast appears to bind near or at the sites of DSB formation and may modulate the initial DSB cleavage [19]. Hop1 mutants in yeast have reduced number of DSBs [20], and Hop1 may participate in recruiting DMC1, RAD51 and other proteins that are required for DNA repair during meiotic synapsis and recombination [19]–[20]. Phosphorylation of Hop1 by Mec1/Tel1 yeast kinases is important for interhomologue recombination and prevents DMC1-independent repair of meiotic DSBs [21]. Here we report that HORMAD1 is likely the mammalian counterpart of Hop1, and that HORMAD1 deficiency disrupts mammalian synaptonemal complex formation, meiotic recombination, and chromosome segregation. Results HORMAD1 is essential for spermatogenesis We previously showed that Hormad1 RNA expression in testes began at postnatal day 10, with little expression detected at birth or postnatal day 5 [16]. Hormad1 RNA expression pattern coincided with the onset of meiosis, and appearance of primary spermatocytes in the developing testes. In situ hybridization with anti-sense Hormad1 riboprobe revealed that Hormad1 expression was confined to germ cells, and specifically spermatocytes, with no signal detected in spermatogonia or sertoli cells [16]. We generated antibodies against HORMAD1 and studied its protein localization pattern in testes. HORMAD1 localized exclusively in germ cells, specifically in zygotene, and early pachytene spermatocytes as previously described for the RNA expression [16]. Since HORMAD1 protein showed localization consistent with its potential role in meiosis I and contains the HORMA domain, we disrupted the Hormad1 gene to examine its requirement for germ cell development and meiosis in mouse. Hormad1 is located on chromosome 3 and composed of sixteen exons. We deleted exons 4 and 5 (Figure S1A), and this mutation is predicted to remove 33 amino acids from the highly conserved HORMA domain and to cause a frame shift mutation. Small amounts of truncated Hormad1 RNA transcripts were detectable on RT-PCR, and Western blots on testes extracts showed absence of HORMAD1 protein in knockout mice as expected (Figure S1B and S1C). Female and male heterozygote matings produced expected Mendelian ratios, averaged 8.1±2 pups per litter (n = 20 breeding pairs) over a 6-month period, and remained fertile for at least 9 months. The litter size was statistically not significantly different from the wild-type average (8.4±2 pups per litter). Male and female mice heterozygous for the mutation (Hormad1+/− ) were fertile with grossly normal male and female gonadal morphology and histology. However, both Hormad1−/− males and females were infertile with no pups produced over a period of 6 months from mating with wild-type female and male mice, respectively. While ovaries showed no gross morphologic differences between the knockout and wild-type mice, Hormad1 knockout adult testes were significantly smaller than the wild-type testes (Figure S2A). Testes in the 7-day-old Hormad1−/− mice were grossly normal and weighed 8.0±2.0 mg/pair and did not significantly differ from the wild-type, 9.0±3.0 mg/pair of testes. By 4 weeks of postnatal life, the knockout testes (47±6 mg/pair) were 50% of the wild-type weight (94±1.7 mg/pair), and by 8 weeks the knockout testes (60±7.7 mg/pair) were 27% of the wild-type weight (225±2.7 mg/pair) (Figure S2B). Histology at 6 weeks showed hypocellular seminiferous tubules with clumps of sertoli cells in the lumen. We observed spermatogonia and early spermatocytes, but no post-meiotic germ cells such as spermatids or spermatozoa (Figure 1A–1H). We therefore carefully examined spermatogenesis in Hormad1−/− mice. Spermatogenesis is a complex process that involves differentiating and proliferating self-renewing spermatogonia that differentiate into spermatozoa. Type A spermatogonia self-renew and can initiate differentiation into Type B spermatogonia which in turn differentiate into primary spermatocytes. Primary spermatocytes undergo meiosis I to form secondary spermatocytes. Secondary spermatocytes enter meiosis II and divide to produce haploid spermatids. We examined Hormad1 knockout testes histology during gonadal development to determine the stage at which spermatogenesis is disrupted. Identical testes weights at postnatal day 7, and similar histology between Hormad1−/− and wild-type testes argue that pre-spermatogonia in Hormad1−/− testes proliferate into Type A spermatogonia without major disruption. Immunohistochemistry with antibodies directed against PLZF and SOHLH1, markers that identify self-renewing (PLZF) and differentiating spermatogonia (SOHLH1), showed the presence of both proteins in the wild-type as well as the knockout animals, confirming that spermatogonia are unaffected (Figure 1J, 1K, 1N, 1O). At postnatal day 10, testes contain preleptotene/leptotene primary spermatocytes, and there was no gross difference between wild-type and Hormad1−/− testes. At 14 days, testes contain pachytene spermatocytes, and there was a significant difference between the wild-type and Hormad1−/− testes, with many apoptotic cells and few pachytene spermatocytes in Hormad1−/− testes (), and rising apoptotic index with age in the knockout as compared to the wild-type (Figure S3K). We counted leptotene, zygotene and pachytene spermatocytes in 6 week old wild-type and Hormad1−/− testes. Hormad1−/− testes showed declining number of spermatocytes beginning in stages II-III with 28±8 spermatocytes as compared to 52±12 in the wild-type (Figure 1A and 1E). No spermatocytes were noted in stages IV-IX in the Hormad1−/− testes (Figure 1F and 1G), and no significant difference was noted in the spermatocyte number in stages X-XII between the wild-type and knockout testes (Figure 1D and 1H). These results indicate that Hormad1 deficiency in the male gonad caused meiotic arrest at the pachytene stage. 10.1371/journal.pgen.1001190.g001 Figure 1 HORMAD1 is required for spermatogenesis. (A–H). Periodic acid/Schiff reagent (PAS) stained cross sections from 6 weeks old wild-type testes (A-D) and Hormad1 −/− (E-H) testes. Seminiferous tubule stages are shown above the panels. Lack of mature sperm and arrest in spermatogenesis is shown at different tubular stages. (I-P). Immunohistochemistry (IHC) of 6 weeks old wild type and Hormad1−/− testes with anti HORMAD1, SOHLH1, PLZF and DDX4 antibody. IHC with anti-HORMAD1 antibody shows HORMAD1 expression (brown) in wild-type stage IV pachytene, and stage XI zygotene spermatocytes (I), while no expression was observed in the knockout (M). SOHLH1 is expressed in differentiating spermatogonia and is present in wild-type and Hormad1−/− testes as shown by arrows (J and N). PLZF identifies differentiating and self-renewing spermatogonia and is expressed in both wild-type and Hormad1−/− testes as shown by arrows (K and O). DDX4 is an RNA binding protein specific for germ cells, and is under-expressed in Hormad1−/− spermatocytes (L and P). Arrows point to Spermatogonia (Sg) and Spermatocytes 1(Sc). Scale bars: 50 µm. HORMAD1 is critical for chromosome synapsis Previous studies on HORMA domain proteins indicate their specific involvement in cell division. MAD2 is a ubiquitously expressed mammalian HORMA domain protein involved in both meiosis and mitosis [7], while yeast HOP1, RED1, nematode HIM3 and plant ASY1 genes are specifically involved in meiotic segregation, synapsis and recombination [2], [10]–[11], [13], [15], [22]. No mammalian counterparts to Hop1, Red1, Him3 and Asy1 have been functionally evaluated up to date. We previously hypothesized that HORMAD1 is a functional counterpart to Hop1, Him3 and Asy1 [16]. Critical components of the synaptonemal complex include meiosis specific SYCP1, SYCP2 and SYCP3 proteins. SYCP1 is a major component of the transverse filaments, while both SYCP2 and SYCP3 are components of the axial lateral elements [23]–[26]. To determine HORMAD1 localization during meiosis, and whether HORMAD1 localizes to the axial elements, or transverse filaments, we used antibodies against SYCP1, SYCP2 and SYCP3 to study their respective co-localization with HORMAD1. HORMAD1 co-localized with SYCP3 and SYCP2 but did not co-localize with SYCP1, which indicates that HORMAD1 is located along the axial elements (Figure 2A, 2C, and 2E). Recent studies also show that HORMAD1 localizes to the axial elements [17]–[18]. We also studied whether absence of SYCP2 affected HORMAD1 localization along the chromosomes. HORMAD1 localization is independent of major germ cell–specific components of the axial elements of the synaptonemal complex because neither Sycp2 (Figure 3A–3F) nor Sycp3 mutation affected HORMAD1 localization to the axial elements [18]. 10.1371/journal.pgen.1001190.g002 Figure 2 HORMAD1 co-localizes with SYCP2 and SYCP3, but not with SYCP1. Chromosomal spread assay in wild-type (+/+) and Hormad1−/− (−/−) zygotene stage spermatocytes. (A–B) Immunofluorescence staining with anti-SYCP1 (Green) and anti-HORMAD1 (Red) antibody. HORMAD1 preferentially localizes to unsynapsed regions of chromosome axes (arrow head) and sex body (arrow) but does not co-localize with SYCP1 (A-A”). Hormad1 deficiency does not affect SYCP1 localization to the axes (B). HORMAD1 co-localizes with SYCP2 on the unsynapsed chromosome axes (C-C”), but HORMAD1 deficiency does not affect SYCP2 localization to the axes (D). SYCP3, another integral and critical component of the synaptonemal complex co-localizes with HORMAD1 on unsynapsed axes (E-E”). Similar to SYCP1 and SYCP2, SYCP3 localizes to the axes despite HORMAD1 deficiency (F). DNA was stained with DAPI. Scale bars: 10 µm. 10.1371/journal.pgen.1001190.g003 Figure 3 HORMAD1 localization in Sycp2 mutant spermatocytes. Chromosome spread assay was performed on the wild-type (+/+) (A–C) and Sycp2−/− (D–F) zygotene spermatocytes with anti-HORMAD1 (green) and anti-SYCP2 (red) antibodies. The truncated SYCP2 protein (SYCP2t) in Sycp2−/− mice is still made and localizes to axial chromosomal cores[48]. SYCP2t lacks the domain necessary to bind SYCP3. Scale bars: 10 µm. We examined localization of germ cell–specific synaptonemal complex proteins SYCP1, SYCP2 and SYCP3 in Hormad1−/− testes. SYCP1 is a major component of the transverse filaments and is known to form fibrillar structures in Sycp3 mutant spermatocytes [27]. However, the fibers are truncated, contain axial gaps, and do not associate with the centromeres of the meiotic chromosomes. We also observed truncated fibers with anti-SYCP1 antibodies in Hormad1−/− mice (Figure 2B). HORMAD1 is therefore not necessary for SYCP1 binding to the chromatin. We also examined localization of SYCP2 and SYCP3 proteins in Hormad1−/− mice. The localization of SYCP2 and SYCP3 to the chromatin was not significantly affected by the lack of HORMAD1 (Figure 2D and 2F). These results indicate that HORMAD1 is not necessary for SYCP2 and SYCP3 localization to the axial elements. The deficiency in synaptonemal complex proteins such as SYCP3 is known to affect chromosome synapsis [27]. In order to determine the effect of HORMAD1 deficiency on chromosome synapsis during meiosis I, we utilized CREST sera. CREST sera labels centromeres and allows the determination of the pairing status during meiosis [28]. In the wild type spermatocytes, prior to the synapsis, 40 centromeres are usually observed in the leptotene stage. The number of visible centromeres become reduced as the synapsis of homologues progresses. At the completion of the synapsis in pachytene, 20 centromeric foci are usually observed corresponding to 19 autosomal homologues and partially paired X-Y chromosomes. We examined CREST foci formation in Hormad1−/− spermatocytes. Examination of over 100 Hormad1−/− spermatocytes and oocytes in meiosis I, revealed greater than 20 centromeric foci in both male and female germ cells, most containing 40 CREST foci (Figure 4A, 4D, and data not shown). These results indicate that Hormad1 deficient germ cells cannot complete homologous chromosome pairing, and Hormad1 is therefore critical for chromosome synapsis during meiosis. 10.1371/journal.pgen.1001190.g004 Figure 4 HORMAD1 is required for chromosome synapsis during male meiosis. (A and D) Immunofluorescence with CREST (red) and anti-SYCP3 (green) antibodies. Anti-CREST antibody recognizes chromosome centromeres. Synapsed wild-type zygotene spermatocytes contain about 20 CREST foci (n = 50) (A-A'). However, Hormad1−/− spermatocytes contain approximately 40 CREST foci (n = 50), an indication that synapsis is disrupted (D-D'). Arrow indicates chromosome synapses in the wild-type. Scale bars: 10 µm. (B–C, E–F) Electron microscopy analysis of synaptonemal complexes in 2 week old wild-type and Hormad1−/− spermatocyte at different magnifications. The typical, tripartite structure of the synaptonemal complex consists of one central element (CE) that connects with two axial elements (AE) (C). We did not identify normal tripartite synaptonemal complex structure in Hormad1−/− spermatocytes. Scale bars: 1 µm. Our experimental evidence strongly suggests that HORMAD1 localizes to the axial core and is yet another critical component of the synaptonemal complex. To determine the effect of Hormad1 deficiency on the structure of the synaptonemal complex, we visualized synaptonemal complexes during meiosis I in wild-type and Hormad1−/− spermatocytes using electron microscopy. In the wild-type, synaptonemal complexes were well visualized during the pachytene stage of meiosis (Figure 4B and 4C). Electron microscopy examination of one hundred and ten Hormad1−/− spermatocytes from three independent experiments revealed the lack of the typical tripartite synaptonemal complex structure (Figure 4E and 4F). Persistence of pre-synaptic number of centromeric foci (CREST staining) in Hormad1−/− spermatocytes, as well as non-visualization of the tripartite synaptonemal complex structure by electron microscopy, demonstrate that HORMAD1 is essential for chromosomal synapsis. Hormad1 deficiency disrupts localization of proteins important in early recombination Previous studies have indicated that Sycp3 deficiency has subtle effects on meiotic recombination [27]. Early recombination events do not seem to be disrupted in Sycp3, as similar number of DMC1 foci are present in Sycp3 mutant and wild-type meiosis [29]. DMC1 is a meiotic specific recombinase that together with ubiquitously expressed RAD51 catalyzes homologous pairing and DNA strand exchange [30]–[31]. These early steps in recombination are critical for establishing the physical connections between homologous chromosomes during meiosis. Hop1 has been implicated in modulating the formation and processing of double stranded breaks [19]. We examined formation of DMC1, RAD51, and RPA foci in zygotene stage Hormad1−/− spermatocytes. There is a dramatic decrease in the number of DMC1 foci as compared to the wild-type (Figure 5A and 5B). We counted a total of 98.9±28.2 DMC1 foci in the wild-type spermatocytes (n = 50) and 9.28±3.9 DMC1 foci in the Hormad1−/− spermatocytes (n = 45). The number of RAD51 foci was also decreased from 189.3±31.8 in the wild-type spermatocytes (n = 50), to 69.3±34.5 in the Hormad1−/− spermatocytes (n = 40) (Figure 5C and 5D). 10.1371/journal.pgen.1001190.g005 Figure 5 Hormad −/− spermatocytes are defective in early recombination. (A-B') Immunofluorescence assay with anti-SYCP2 (Green) and anti-DMC1 (Red) antibody in zygotene spermatocytes. (C-D') Immunofluorescence assay with anti-RAD51 (Red) and anti-SYCP2 (Green) antibody in zygotene spermatocytes. (E-F') Immunofluorescence assay with anti-RPA (Green) and anti-SYCP2 (Red) antibody in zygotene spermatocytes. DMC1 catalyzes DNA strand exchange during recombination, and DMC1, RAD51 and RPA foci mark early recombination events. DMC1, RAD51 and RPA foci are drastically decreased in Hormad1−/− spermatocytes. Scale bars: 10 µm. Following double-strand breaks formation by SPO11, RPA is recruited together with RAD51 to the single stranded DNA regions [32]. We counted a total of 194.5±64.5 RPA foci in the wild-type spermatocytes (n = 30) as compared to 70.1±38.5 foci in the Hormad1−/− spermatocytes (n = 20) (Figure 5E and 5F). These results indicate that early meiotic recombinational events were disrupted and not surprisingly, MLH1, a protein that forms foci in later stages of recombination and required for the formation of most of the crossovers (chiasmata) observed in mice [33], was dramatically reduced in Hormad1−/− spermatocytes (data not shown). We also examined DMC1, RAD51 and RPA foci formation in female meiocytes at embryonic day 15.5 (E15.5). Embryonic ovaries contain zygotene to early pachytene oocytes at E15.5 [34]. We counted a total of 208.7±117.1 DMC1 foci in the wild-type E15.5 oocytes (n = 50), and 79.1±81.5 foci in the Hormad1−/− oocytes (n = 30) (Figure 6A and 6B), a total of 197.9±46.0 RAD51 foci in the wild-type oocytes (n = 50) versus 85.1±37.6 foci in the Hormad1−/− oocytes (n = 40) (Figure 6C and 6D) and a total of 317.16±135.3 RPA in the wild-type oocytes (n = 50) and 51.7±48.8 in the Hormad1−/− oocytes (n = 50) (Figure 6E and 6F). DMC1, RAD51 and RPA foci are therefore, similar to our observations in spermatocytes, significantly decreased in Hormad1 deficient female meiocytes. 10.1371/journal.pgen.1001190.g006 Figure 6 Hormad −/− fetal oocytes are defective in early recombination. Chromosome spread assay in the wild-type and Hormad1−/− E15.5 fetal ovary. A-E represent zygotene stage, G and H represent leptotene stage. (A-B') Immunofluorescence assay with anti-SYCP2 (Red) and anti-DMC1 (Green) antibody. (C-D') Immunofluorescence assay with anti-SYCP2 (Red) and anti-RAD51 (Green) antibody. (E-F') Immunofluorescence assay with anti-SYCP2 (Red) and anti-RPA (Green) antibody. (G-H') Immunofluorescence assay with anti-SYCP2 (Green) and anti-γH2AX (Red) antibody. DMC1, RAD51, RPA and γH2AX signals were significantly decreased in Hormad1−/− fetal oocyte. DNA was stained with DAPI (Blue). Scale bars: 10 µm. These results indicate that homologous recombination is significantly affected in Hormad1−/− mammalian germ cells, as previously reported for HOP1 [35]. We also observed effects of Hormad1 deficiency on γH2AX staining (a phosphorylated form of histone H2AX), a well known surrogate marker for DSB formation [36]. In the leptotene stage, phosphorylation of H2AX is induced by SPO11 catalyzed DSBs in meiotic DNA, and γH2AX appears as large, cloud-like patterns thatdisappearat the pachytene stage [37]. At the leptotene stage, γH2AX staining in Hormad1−/− spermatocytes was significantly decreased (76% decrease in signal intensity) as compared to the wild-type (Figure 7A–7D). γH2AX staining was also significantly decreased in Hormad1− /− fetal oocytes (71% decrease in signal intensity) (Figure 6G and 6H). These results suggest that similar to Hop1 mutants, DSBs do not efficiently form in Hormad1 mutants. 10.1371/journal.pgen.1001190.g007 Figure 7 HORMAD1 disrupts γH2AX, BRCA1, and ATR localization to the XY chromosomes. Chromosome spread assay was performed on wild-type and Hormad1−/− leptotene (A-D), zygotene (E-H) and pachytene (I-L) spermatocytes to determine effects of HORMAD1 deficiency on γH2AX, BRCA1 and ATR localization to the sex chromosomes. (A-D) Immunofluorescence assay with anti-SYCP2 (Green) or anti-HORMAD1 (Green) and anti-γH2AX (Red). (E-H) Immunofluorescence assay with anti-SYCP1 (Green) or anti-HORMAD1 (Green) and anti-γH2AX (Red) antibody. (C and D) At the leptotene stage, γH2AX and SYCP2 localization to chromatin can be detected in Hormad1−/− spermatocytes, however, γH2AX is substantially reduced. (F and H) γH2AX staining localizes preferentially to the sex chromosome in the wild-type pachytene stage spermatocytes but no preferential localization to the sex chromosomes was observed in the Hormad1−/− spermatocytes, which is not surprising since sex body does not form in Hormad1 mutants. (G) Arrow head indicates truncated axial fiber. (I, M-M”) BRCA1 and HORMAD1 co-localize to the sex chromosomes, but Hormad1 deficiency disrupts BRCA1 localization (J). Similarly, ATR co-localizes with HORMAD1 to the XY chromosomes (K, N-N”), and Hormad1 deficiency disrupts ATR localization (L). These results indicate that HORMAD1 is upstream of the currently known critical components of the meiotic sex chromosome inactivation complex, γH2AX, BRCA1 and ATR. DNA was stained with DAPI (Blue), and arrows indicate sex chromosomes. Scale bars: 10 µm. HORMAD1 is a germ cell–specific component of the meiotic sex chromosome inactivation complex HORMAD1 protein localization along autosomes was previously shown to be transient [17] (Figure 7B and 7F). HORMAD1 staining was highest along unsynapsed chromosome axis in the zygotene to pachytene stage and diminished significantly along autosomes in the pachytene [17]–[18] (Figure 7F and 7I). Interestingly, HORMAD1 localized strongly along desynapsed autosomes in diplotene meiocytes [17]. During the pachytene stage, HORMAD1 is faintly visible along the autosomes, but co-localizes strongly with γH2AX on the XY chromosomes, and specifically along the axial elements [17]–[18]. γH2AX is a phosphorylated form of histone H2AX, and a marker for DSBs [36], [38]. H2AX is phosphorylated throughout the chromatin in leptotene spermatocytes and by the pachytene stage [17]–[18], γH2AX staining is undetectable on autosomes and restricted to the sex body (Figure 7E and 7F) [39]. H2ax knockout shows the essential role for H2AX in sex body formation and meiotic sex chromosome inactivation [39]. Meiotic sex chromosome inactivation also involves ATR and BRCA1 dependent phosphorylation of H2AX [18], [40]. Interestingly, Hormad1 deficiency, similar to H2ax deficiency, abolishes the formation of the sex body (Figure 7G and 7H, and data not shown). The lack of sex body formation is most likely due to the disruption of Hormad1−/− spermatocytes prior to the pachytene. We examined the γH2AX localization in the wild-type and Hormad1−/− spermatocytes. Chromatin in Hormad1−/− spermatocytes stained with anti-γH2AXantibodies, but no preferential localization to the sex chromosomes was observed (Figure 7G and 7H). BRCA1 and HORMAD1 have recently been shown to co-localize in the sex body [18]. We determined whether BRCA1 localization is dependent on HORMAD1. BRCA1 protein could not be localized in Hormad1−/− spermatocytes (Figure 7I–7J, 7M). This finding is unlike H2ax knockout, where BRCA1 is still detected on the sex chromosomes despite the lack of the sex body [40]. We also examined ATR localization in wild-type and Hormad1−/− testes. In wild-type testes, HORMAD1 and ATR co-localized in the sex body, but we could not detect ATR in Hormad1−/− spermatocytes (Figure 7K–7L, 7N). Above data suggest that HORMAD1 may be involved in the recruitment of BRCA1, ATR and γH2AX to the sex chromosome. Since ATR, BRCA1 and γH2AX are involved in the transcriptional silencing of sex chromosomes, we examined whether Hormad1 deficiency affects transcriptional repression. Previous studies have shown that H2ax and Brca1 deficiencies individually, lead to the over-expression of genes exclusively expressed from the X or Y chromosome [40]. We examined whether X-linked germ cell–specific genes were over-expressed in Hormad1−/− testes compared to wild-type testes. We performed quantitative real-time PCR on select autosomal genes (Hormad2, Rnh2 and Mov10l1) as well as X-chromosome derived genes (Usp26, Fthl17, Pramel3, Tex11, and Tex13) on wild-type and Hormad1−/− testes (Figure 8A–8H). Rnh2 and Mov10l1 are germ cell–specific transcripts, derived from autosomes, and were not differentially expressed between wild-type and Hormad1−/− testes (Figure 8B and 8C). In contrast, all of the germ cell–specific transcripts transcribed from the X chromosome were significantly elevated in Hormad1−/− testes over the wild-type. These include Usp26 (4.5 fold increase), Fthl17 (6.5 fold increase), Pramel3 (3.5 fold increase), Tex11 (2.2 fold increase), and Tex13 (2.8 fold increase) (Figure 8D–8H). Moreover, RNA expression microarray analyses comparing two week old Hormad1 deficient testes with corresponding wild-type, indicate that almost 20% of the up-regulated genes derive from the X chromosome (Figure 8I). Our results are remarkably similar to transcriptional de-repression observed in H2ax and Brca1 mutants [39]–[40], and indicate that HORMAD1 is a germ cell and meiosis specific factor critical in meiotic sex chromosome inactivation and transcriptional silencing. 10.1371/journal.pgen.1001190.g008 Figure 8 Sex chromosome expressed transcripts escape meiotic sex chromosome inactivation in Hormad −/− spermatocytes. (A–H) Quantitative real time PCR analyses show that expression of testes specific genes derived from the autosome, Hormad2, Rnh2, and Mov10l1, did not significantly differ between the wild-type and the Hormad1−/− testes (A-C). However, X chromosome linked, testis specific genes, Usp26, Fthl17, Pramel3, Tex11 and Tex13 transcript were 2–6 fold increased (D-H). (I) Microarray analysis shows preponderance of X and Y derived transcripts among the top 38 up-regulated genes in Hormad −/− testes. Data are normalized to β-actin expression and presented as the mean relative quantity (compared to wild-type) with error bars representing the standard error of mean. Student's t test was used to calculate the P values. Hormad1 deficiency abrogates ATM autophosphorylation HORMAD1 is a likely mammalian counterpart to the yeast HORMA domain meiotic protein, Hop1. Hop1 is phosphorylated by Mec1/Tel1, the budding yeast homologue to the mammalian ATR and ATM kinases, and this phosphorylation is thought to play an important role in inter-homologous recombination [21]. We therefore examined HORMAD1 expression in Atm deficient mice as well as ATM expression in Hormad1−/− animals. ATM is a serine/threonine-specific protein kinase that has been associated with cell cycle regulation, apoptosis, and response to DNA damage repair. ATM kinase activation is associated with increased auto-phosphorylation of ATM at multiple sites including serine 1981 [41]. We examined HORMAD1 protein expression in testes between postnatal days 5–21 (Figure 9A). Eight to ten day old testes contain spermatogonial cells as well as preleptotene/leptotene spermatocytes [34]. At postnatal day 14–18, testes contain pachytene spermatocytes and visible sex bodies [34]. HORMAD1 is known to be phosphorylated [17] (Figure 9B). Western blot analysis on testes extracts detected phospho-HORMAD1 beginning at postnatal day 14 (Figure 9A). Phospho-HORMAD1 protein was decreased in postnatal day 18 and 21 wild-type testes (Figure 9A). Phospho-HORMAD1 appearance correlates temporally with sex body formation. HORMAD1 phosphorylation was not affected by Atm deficiency (Figure 9A). These results indicate that ATM is not responsible for HORMAD1 phosphorylation. We also examined HORMAD1 localization in Atm−/− spermatocytes. We observed HORMAD1 localization to chromosomal axes in Atm deficient spermatocytes, as previously described by others [17] (Figure 9C and 9D). Since synaptonemal complexes do not form in Atm mutants, HORMAD1 association with unsynapsed chromosomes does not require ATM. The anti-ATM phospho-S1981 antibody did not detect phosphorylated ATM in Hormad1−/− testes (Figure 9E and 9F). These results suggest that HORMAD1 is upstream of ATM auto-phosphorylation, and therefore likely upstream of ATM kinase activation. 10.1371/journal.pgen.1001190.g009 Figure 9 HORMAD1 phosphorylation is unaffected by Atm deficiency while Hormad1 deficiency disrupts ATM autophosphorylation. (A) Western blots analyses with anti-HORMAD1 specific antibodies show that HORMAD1 protein and its phosphorylated form (*) appear circa post-natal day 14. Atm1 deficient testes (Atm− /− ) express both forms of HORMAD1 and HORMAD1 is therefore unlikely to be ATM1 substrate. (B) The presumed phosphorylated form of HORMAD1 (higher molecular weight band indicated by asterisk) decreases in intensity after protein phosphatase I (PPI) treatment. (C and D) Chromosome spread assay in wild-type (Atm+/+ ), and Atm deficient spermatocytes (Atm−/− ). HORMAD1 preferentially localizes to unsynapsed regions of chromosome axes (C), and HORMAD1 antibody stains unsynapsed chromosome axes in Atm−/− spermatocytes (D) more intensely than in the wild-type spermatocytes. Scale bars: 10 µm. (E and F) Immunohistochemistry analysis in 6 week old wild-type and Hormad1−/− testes with anti-phospho ATM-S1981 antibody. Phospho-ATM S1981 was detected in wild-type zygotene, but not detected in Hormad1−/− zygotene spermatocytes. Scale bars: 50 µm. Hormad1 deficiency does not affect gross ovarian development We have previously shown that Hormad1 RNA expression in the ovary was confined to the germ cell [16]. Meiosis I in the female gonad commences circa E13.5 and most oocytes arrest at the dictyate stage by the time of birth. Antibodies against HORMAD1 recognized HORMAD1 protein at E14.5 (leptotene) and E18.5 (arrest in diplotene) oocytes (Figure 10A and 10B), but little HORMAD1 protein was detected in the newborn ovary oocytes (Figure 10C), at the time when oocytes are arrested in diplotene. Deficiency in genes critical in meiosis can disrupt early ovarian development, as is the case for Dmc1, Msh5, Spo11 and Atm [42]–[44]. 10.1371/journal.pgen.1001190.g010 Figure 10 HORMAD1 is expressed in embryonic but not post-natal oocytes. (A–C) HORMAD1 protein was detected by immunofluorescence in the embryonic ovary at E14.5 (A), E18.5 (B), but not in the newborn ovary (C). (D–F) Oocytes in Hormad1−/− ovary (−/−) were stained with antibodies against the germ cell–specific transcriptional regulator SOHLH1 at E14.5 (D), E18.5 (E), and newborn ovary (F). DNA was stained with DAPI (Blue). Scale bars: 20 µm. We therefore examined ovarian development in Hormad1−/− females. Antibodies against germ cell–specific transcriptional regulator SOHLH1 [45] stained wild-type and knockout oocytes throughout embryonic gonadal development with no significant differences noted (Figure 10A–10F). Moreover, the numbers of primordial, primary and secondary follicle counts did not significantly differ between the mutant and wild-type ovaries at post-natal day 8 (Figure S4). These results indicate that Hormad1 deficiency does not affect embryonic ovarian development, germ cell cyst breakdown, and primordial follicle formation. We also examined the histology of wild-type and Hormad1−/− ovaries between 2 and 30 weeks of life. Mice reach sexual maturity around 6 weeks of life, and mouse ovaries at this time consist of all of the follicular types including corpora lutea, an indication that the ovaries are ovulating. Postnatal Hormad1−/− ovaries were grossly indistinguishable from wild-type mice between 2 and 30 weeks of life, with abundant corpora lutea in the Hormad1−/− ovaries indicating that the normal process of oocyte maturation was not disrupted, and ovulation has occurred (Figure 11A–11H). We induced superovulation in knockout and wild-type mice with exogenous gonadotropins to determine whether subtler ovarian defects contributed to infertility in Hormad1−/− mice. Hormad1−/− females super-ovulated 28±11 eggs (n = 22), while wild-type animals superovulated 29±14 eggs (n = 13). We therefore did not observe significant difference between the number of eggs superovulated from wild-type versus knockout mice. These results indicate that ovarian development is grossly normal in Hormad1−/− mice, and that ovarian defects are unlikely to account for observed infertility. 10.1371/journal.pgen.1001190.g011 Figure 11 Histological analysis of wild-type and Hormad1 –/– ovaries. (A–H) PAS staining of wild-type (+/+) and Hormad1−/− (−/−) ovaries does not show gross histological differences. Wild-type and Hormad1−/− ovaries are shown at different post-natal ages that show the full range of ovarian follicles: primordial follicles (PF), primary follicles (PrF), secondary follicles (SF), antral follicles (AF) and corpus luteum (CL). Scale bars: 50 µm. Hormad1 deficient eggs fertilize and embryonic development arrests at blastocyst stage due to aneuploidy We studied early embryonic development of fertilized Hormad1−/− oocytes because ovarian defects were unlikely to explain observed infertility. We recovered embryos from wild-type male matings with Hormad1−/− females at E0.5, E1.5, E2.5, and E3.5. Comparable numbers of morphologically indistinct 1-cell zygotes were recovered from oviducts of control and mutant female mice at E0.5 (Figure 12A–12C), and little difference was noted at the 2-cell stage, except for an increased number of 1-cell embryos in the knockouts, indicating a lag in the progression from the 1-cell to 2- cell stage (Figure 12D–12F). By E2.5, the number of normal appearing 8-cell stage embryos was significantly less in Hormad1−/− fertilized eggs as opposed to the wild-type (Figure 12G–12I), and no morphologically normal blastocysts were observed in the Hormad1−/− fertilized eggs at E3.5. It is interesting to note that at E3.5, a significant number of 4 and 8 cell stage embryos were observed in the knockout while only blastocysts were observed in the wild-type E3.5 embryos (Figure 12J–12L). We also tested, using Chicago Sky Blue 6B dye (Sigma, MO, USA) injection into the tail vein [46], whether blastocysts derived from Hormad1−/− fertilized eggs could implant. We did not detect implantation of Hormad1−/− fertilized eggs (Figure 12M). These results indicate that a defect in early embryogenesis led to premature loss of embryos. 10.1371/journal.pgen.1001190.g012 Figure 12 Hormad1–/– oocyte derived embryos arrest at blastocyst stage. Embryos derived from wild-type matings (M+/+-F+/+) and matings involving Hormad1−/− oocytes fertilized by wild-type males (M+/+-F − /− ), were collected at E0.5, E1.5, E2.5 and E3.5 for the analysis of the early embryonic development. (A-C) No significant differences were noted between wild-type and Hormad1−/− at E 0.5, with normal appearing one cell embryos and polar bodies visible in both. (D-F) At E1.5, there is an excess of 1 cell embryos in Hormad1−/− fertilized oocytes, indicating a lag in embryo development at this stage. (G-I) At E2.5 there is a significantly less number of eight cell embryos formed in Hormad1−/− fertilized oocytes as opposed to the wild-type. A large proportion of 8-cell embryos appear disorganized and apoptotic. (J-L) Blastocysts form at E3.5 in wild-type but very few normal blastocysts are visible in Hormad1−/− embryos. The count only includes morphologically normal blastocysts (L). Error bars represent standard error of the mean. Student's t test was used to calculate P values (*:P 0.5, and therefore not statistically significant. Bars, 50 µm. (1.85 MB TIF) Click here for additional data file. Table S1 BAC clones used in FISH experiments. (0.03 MB DOC) Click here for additional data file.
- Record: found
- Abstract: found
- Article: not found
A profile of fertilization in mammals.
Eveline Litscher, L Jovine, Paul M Wassarman (2001)
Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.
- Record: found
- Abstract: found
- Article: not found
Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo developmental competence.
Jennifer Watson (2007)
Efforts have intensified to successfully mature and inseminate oocytes in vitro and then culture ensuing embryos to transferable stages from a large number of mammalian species. Success varies, but generally even for the most successful species it is only possible to obtain a maximum of a 40 to 50% development of zygotes to the blastocyst stage. Reduced oocyte developmental competence is suggested as a primary reason for the reduced potential of in vitro-produced embryos. The vast majority of in vitro-matured oocytes are meiotically competent; however, many do not attain an optimal oocyte diameter before insemination. Variations in oocyte in vitro maturation media can influence embryo development, blastocyst cell number, and apoptosis. In addition, studies have indicated that cytoplasmic donation from so-called competent to incompetent oocytes can improve developmental outcomes. Oocyte cytoplasmic maturation includes those events that instill upon the oocyte a capacity to complete nuclear maturation, insemination, early embryogenesis and thus provide a foundation for implantation, initiation of pregnancy, and normal fetal development. Although we can define oocyte cytoplasmic maturation, we are only now beginning to understand the molecular steps that underlie this process. In general terms, oocyte cytoplasmic maturation involves the accumulation of mRNA, proteins, substrates, and nutrients that are required to achieve the oocyte developmental competence that fosters embryonic developmental competence. Collectively we are beginning to specify oocyte cytoplasmic maturation, and eventually a coherent understanding of this critical event in oocyte biology will emerge.
Author and article information
Contributors
Michael Schubert: Role: Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
(San Francisco, USA
)
ISSN
(Electronic):
1932-6203
Publication date Collection: 2014
Publication date
(Electronic):
18
March
2014
Volume: 9
Issue: 3
Electronic Location Identifier: e92202
Affiliations
Author notes
* E-mail:
ahuvet@
123456ifremer.fr.
Article
Publisher ID: PONE-D-13-46612DOI: 10.1371/journal.pone.0092202
PMC ID: 3958495
PubMed ID: 24643002
SO-VID: e43252f0-91c6-4bbe-b1de-d0da643ad2b0
Copyright statement:
Copyright @
2014
License:
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 8
November
2013
Date
accepted
: 19
February
2014
Page count
Pages: 12
Funding
This study was supported by the European program REPROSEED EU Grant No. 245119 (
http://www.reproseed.eu/). The first author is a Ph.D. student financially supported by the same program.
The authors are grateful to the REPROSEED partners JL Nicolas, R Robert and P Boudry
for their support during the course as well as P Sourdaine, P Favrel and C Lelong
for helpful discussion. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
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Animal Types
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Research and Analysis Methods
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Bioassays and Physiological Analysis
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Microarrays
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