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1.
Genes Dev ; 35(9-10): 619-634, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33888561

RESUMO

Development of the ovary or testis is required to establish reproductive competence. Gonad development relies on key cell fate decisions that occur early in embryonic development and are actively maintained. During gonad development, both germ cells and somatic cells proliferate extensively, a process facilitated by cell cycle regulation. This review focuses on the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) in mouse gonad development. We particularly highlight recent single-cell RNA sequencing studies that show the heterogeneity of cyclin-dependent kinase inhibitors. This diversity highlights new roles for cell cycle inhibitors in controlling and maintaining female fertility.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Fertilidade/genética , Gônadas/crescimento & desenvolvimento , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Gônadas/metabolismo , Camundongos , Processos de Determinação Sexual/genética , Análise de Célula Única
2.
Biol Reprod ; 108(6): 854-865, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-36917225

RESUMO

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.


Assuntos
Organoides , Animais de Estimação , Humanos , Feminino , Animais , Gatos , Bovinos , Cavalos , Cães , Suínos , Fazendas , Tubas Uterinas , Diferenciação Celular
3.
Biol Reprod ; 106(4): 741-755, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35024820

RESUMO

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.


Assuntos
Caseínas , Infertilidade , Animais , Caseínas/metabolismo , Cromatografia Líquida , Feminino , Cavalos , Infertilidade/metabolismo , Masculino , Gravidez , Proteômica , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/metabolismo , Espectrometria de Massas em Tandem
4.
Mol Cell Proteomics ; 19(3): 444-455, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31848259

RESUMO

Male infertility is widespread and estimated to affect 1 in 20 men. Although in some cases the etiology of the condition is well understood, for at least 50% of men, the underlying cause is yet to be classified. Male infertility, or subfertility, is often diagnosed by looking at total sperm produced, motility of the cells and overall morphology. Although counting spermatozoa and their associated motility is routine, morphology assessment is highly subjective, mainly because of the procedure being based on microscopic examination. A failure to diagnose male-infertility or sub-fertility has led to a situation where assisted conception is often used unnecessarily. As such, biomarkers of male infertility are needed to help establish a more consistent diagnosis. In the present study, we compared nuclear extracts from both high- and low-quality spermatozoa by LC-MS/MS based proteomic analysis. Our data shows that nuclear retention of specific proteins is a common facet among low-quality sperm cells. We demonstrate that the presence of Topoisomerase 2A in the sperm head is highly correlated to poor head morphology. Topoisomerase 2A is therefore a potential new biomarker for confirming male infertility in clinical practice.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Infertilidade Masculina/metabolismo , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Adulto , Idoso , Biomarcadores/metabolismo , Cromatografia Líquida , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas em Tandem
5.
Mol Cell Proteomics ; 19(11): 1860-1875, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32839225

RESUMO

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC-MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.


Assuntos
Aconitato Hidratase/antagonistas & inibidores , Asparagina/metabolismo , Glicólise , Ácido N-Acetilneuramínico/metabolismo , Fosforilação Oxidativa , Capacitação Espermática , Espermatozoides/metabolismo , Aconitato Hidratase/química , Acrossomo/enzimologia , Acrossomo/metabolismo , Animais , Cromatografia Líquida , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Lipase/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Ácido N-Acetilneuramínico/química , Processamento de Proteína Pós-Traducional , Espermatozoides/enzimologia , Espectrometria de Massas em Tandem
6.
J Cell Sci ; 129(23): 4379-4387, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27802166

RESUMO

Ca2+-binding tyrosine-phosphorylation-regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause of and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR-Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. Cabyr-knockout male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr-knockout spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Fosfoproteínas/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epididimo/metabolismo , Epididimo/ultraestrutura , Feminino , Fertilidade , Deleção de Genes , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Fosforilação , Isoformas de Proteínas/metabolismo , Reprodutibilidade dos Testes , Espermatozoides/ultraestrutura , Especificidade por Substrato , Tirosina/metabolismo
7.
Biol Reprod ; 99(2): 395-408, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29228106

RESUMO

Male infertility is a complex condition, and for the most part, all men produce defective spermatozoa, but infertile men have a tendency to produce more. Despite attempts to classify infertility, there is no definitive test. One approach would be to use protein biomarkers; however as yet, we still do not understand proteins that are differentially expressed within defective spermatozoa. As such, we took nine men (fertility status unknown) and used Percoll density gradients to isolate a population of good- and poor-quality sperm. For four of these men, we also obtained multiple ejaculations. The most noticeable differences between the Percoll-isolated fractions were motility and CMA3 staining. While the good sperm fraction produced cells with at least 80% forward progressive motility and low levels of CMA3 staining, the poor-quality sperm demonstrated less than 10% forward progressive motility and higher levels CMA3 staining. Using the technique of sequential window activation of all theoretical mass spectra, we quantified 2774 proteins and found 171 proteins to be significantly more abundant in the good sperm fraction, while 104 proteins were significantly more abundant in the lower sperm fraction (adjusted Benjamini-Hochberg significance of P < 0.018, minimum 2-fold difference).


Assuntos
Infertilidade Masculina/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Adulto , Idoso , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Análise do Sêmen , Contagem de Espermatozoides , Adulto Jovem
8.
Biol Reprod ; 99(4): 789-797, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29733335

RESUMO

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.


Assuntos
Proteínas de Ligação a Calmodulina/fisiologia , Proteínas de Plasma Seminal/fisiologia , Motilidade dos Espermatozoides/fisiologia , Animais , Cálcio/fisiologia , Proteínas de Ligação a Calmodulina/genética , Feminino , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese , Oviductos/citologia , Oviductos/fisiologia , Gravidez , Proteínas de Plasma Seminal/genética , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/fisiologia , Transporte Espermático/genética , Transporte Espermático/fisiologia
9.
Mol Cell Proteomics ; 15(12): 3685-3693, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27770032

RESUMO

Globally, ∼1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.


Assuntos
Proteínas de Choque Térmico/metabolismo , Infertilidade Masculina/metabolismo , Fosfoproteínas/análise , Proteômica/métodos , Sêmen/fisiologia , Adulto , Cromatografia Líquida , Regulação para Baixo , Humanos , Masculino , Análise do Sêmen , Cabeça do Espermatozoide/metabolismo , Motilidade dos Espermatozoides , Espectrometria de Massas em Tandem
10.
Proteomics ; 17(17-18)2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28782881

RESUMO

Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blastocoel fluid and embryo capsule. Analyses revealed 72 (24 h) and 97 (48 h) unique protein IDs in the embryo secretome, 732 protein IDs in blastocoel fluid, and 11 proteins IDs in the embryo capsule. Novel findings of interest include secretion of a pregnancy specific proteinase (PAG) by the equine embryo at day 10, along with detection of a prostaglandin receptor inhibiting protein (PTGFRN) and a progesterone potentiating factor (FKBP4) in blastocoel fluid. This is the first comprehensive proteomic analysis of the equine embryo secretome, and provides new insights into the unique physiology of early pregnancy in this species.


Assuntos
Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Cavalos/embriologia , Cavalos/metabolismo , Fragmentos de Peptídeos/metabolismo , Manutenção da Gravidez , Animais , Embrião de Mamíferos/citologia , Feminino , Gravidez , Proteômica/métodos
11.
Biol Reprod ; 94(4): 91, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26962120

RESUMO

Mammalian spermatozoa acquire fertilizing potential as they undergo a series of changes during epididymal transit. One major facet of such is the alterations in the sperm glycome. Modifications of the sialic acid content within glycan moieties are known to regulate epitope presentation and cellular adhesion and signaling, all of which may be critical for sperm to successfully reach and fertilize the egg. To date, there is paucity of information regarding the sialic acid changes that occur on spermatozoa during epididymal transit. Therefore, the aim of this study was to identify N-linked sialylated glycoproteins in rat epididymal sperm and investigate whether they are regulated during epididymal transit. Sialylated glycopeptides from caput, corpus, and cauda spermatozoa were enriched using titanium dioxide beads. Bound N-linked glycopeptides were released by enzymatic deglycosylation using PNGase F and then analyzed by liquid chromatography tandem-mass spectrometry. A total of 92 unique N-linked sialylated glycopeptides were identified from 65 different proteins. These included members of the disintegrin and metalloproteinase domain-containing protein family (ADAM), Basigin, and Testis-expressed protein 101 (TEX101). Remarkably, label-free quantification showed that more than half of these peptides (48/92) were regulated during epididymal transit. Of interest, the protein TEX101 exhibited PNGase F-resistant deglycosylation under the conditions used in this study. The results from this study showed that changes in the N-linked sialoglycoprotein profile is a major hallmark of sperm maturation in rats.


Assuntos
Epididimo , Glicopeptídeos/metabolismo , Espermatozoides/metabolismo , Proteínas ADAM/metabolismo , Animais , Cromatografia de Afinidade , Ontologia Genética , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Proteoma , Ratos , Ratos Wistar
12.
Cell Tissue Res ; 363(1): 279-287, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26239910

RESUMO

It is hard to fathom that one of the most highly differentiated cells in the body, the spermatozoon, spends over half of its developmental life without the capacity for nuclear protein biosynthesis. This is even more incredible when considering that protein synthesis is switched off long before the sperm is mature. As such, in order to obtain full functionality, spermatozoa rely on post-translational modifications (PTM) of existing proteins. Many PTM have been shown to play a role in the development of a sperm cell. These include phosphorylation and glycosylation events that occur both in the epididymis and during capacitation. In addition, several other PTM such as disulfide cross-linking, ubiquitination, acetylation and methylation all play a role to both develop and enable a spermatozoon to achieve its final destiny.


Assuntos
Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo , Animais , Cromatina/química , Cromatina/genética , Epigênese Genética , Humanos , Masculino , Proteoma/análise , Proteoma/genética , Proteômica , Capacitação Espermática , Espermatozoides/química , Espermatozoides/citologia
13.
Reproduction ; 152(6): 665-672, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27624483

RESUMO

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm-egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.


Assuntos
Sistemas CRISPR-Cas , Fertilidade/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Fertilização/fisiologia , Imunoglobulinas/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Fosforilação , Domínios Proteicos , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo
14.
Reprod Fertil Dev ; 28(1-2): 1-10, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27062870

RESUMO

Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including the development of effective antioxidant formulations, are becoming increasingly urgent.


Assuntos
Apoptose , Dano ao DNA , Infertilidade Masculina/etiologia , Peroxidação de Lipídeos , Modelos Biológicos , Estresse Oxidativo , Espermatozoides/metabolismo , Animais , Quebras de DNA , DNA Glicosilases/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Fosforilação , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Capacitação Espermática , Interações Espermatozoide-Óvulo , Espermatozoides/enzimologia , Espermatozoides/patologia , Tirosina/metabolismo
15.
J Cell Physiol ; 230(2): 449-63, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25078272

RESUMO

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.


Assuntos
Acrossomo/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Animais , Imunoprecipitação/métodos , Masculino , Proteômica/métodos , Receptores de Superfície Celular , Capacitação Espermática , Suínos
16.
Biol Reprod ; 92(1): 11, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25411390

RESUMO

The maturation of spermatozoa throughout the epididymal environment occurs in the complete absence of nuclear protein biosynthesis. As such, these cells rely heavily on posttranslational modifications of existing proteins in order to obtain the potential for fertilization. We have used an OxiCat approach to label both free and oxidized cysteine residues in rat sperm proteins and compared the ratio of reduced:oxidized peptides as these cells undergo epididymal transit. In all, 20 peptides, corresponding to 15 proteins, underwent a change in their redox status. Included in this list were A-kinase anchoring protein 4 and fatty acid-binding protein 9. Both of these proteins undergo intradisulfide bonding, leading to reduced solubility and, in the case of the latter, is likely to cause a loss of protein function. Interestingly, two glycolytic enzymes, hexokinase-1 and lactate dehydrogenase, also display increased cysteine oxidation during epididymal transit, which may be involved in the regulation of the enzyme activities.


Assuntos
Epididimo/fisiologia , Proteínas de Plasma Seminal/análise , Proteínas de Plasma Seminal/metabolismo , Maturação do Esperma , Compostos de Sulfidrila/análise , Animais , Masculino , Microscopia de Fluorescência , Oxirredução , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Espermatozoides/química , Espermatozoides/metabolismo , Compostos de Sulfidrila/metabolismo
17.
Biol Reprod ; 92(4): 108, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25673561

RESUMO

Lipid peroxidation products such as the naturally occurring aldehyde 4-hydroxynonenal (4-HNE) are known to be cytotoxic toward different cell types, including spermatozoa. In order to understand this at the molecular level, we have employed a proteomic approach to characterize direct 4-HNE adducts on human spermatozoa. Several proteins were identified to be of particular interest, including aldehyde labeling of histone methyltransferase and dynein heavy chain. In addition, we found that 4-HNE bound to part of the activation segment, cysteine residue 199, of protein kinase A (PKA). Interestingly, at low levels, addition of 4-HNE had a stimulatory effect on PKA. However, this did not correlate to increased phosphotyrosine levels during capacitation. This data explains the link between reactive oxygen species and sperm toxicity. Given that epigenetic regulation is likely affected in oxidative-stressed spermatozoa, this data show that spermatozoa appear to shut down under these conditions before reaching the egg.


Assuntos
Aldeídos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dineínas/metabolismo , Epigênese Genética , Humanos , Técnicas In Vitro , Masculino , Fosforilação , Fosfotirosina/metabolismo , Proteômica , Capacitação Espermática/efeitos dos fármacos
18.
FASEB J ; 28(8): 3480-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24790034

RESUMO

Importin α proteins function as adaptors to connect a cargo protein and importin ß1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and ß1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and ß1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Espermatogênese/fisiologia , alfa Carioferinas/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Compartimento Celular , DNA Helicases/química , Masculino , Meiose , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Estágio Paquíteno , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína , Proteômica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Espermátides/metabolismo , Espermátides/ultraestrutura , Fatores de Transcrição/química , alfa Carioferinas/análise , beta Carioferinas/análise , beta Carioferinas/fisiologia
19.
Int J Mol Sci ; 16(10): 24732-50, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26501274

RESUMO

Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.


Assuntos
Sistemas CRISPR-Cas/genética , Espermatogênese/fisiologia , Animais , Masculino , Camundongos , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatogênese/genética
20.
Biochim Biophys Acta ; 1833(3): 731-42, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23159777

RESUMO

Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin α2, α3 and α4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (cNLS) for importin α binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin α2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin α together with importin ß1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin αs, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin α binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin αs.


Assuntos
Proteoma/análise , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Imunoprecipitação , Masculino , Meiose/fisiologia , Sinais de Localização Nuclear , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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