The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation

The present experiments compared the ability of pig oocytes matured either in vivo or in vitro to structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12-14 h after in vitro fertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher for in vivo than for in vitro matured (IVM) oocytes (P < 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN (P < 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% of in vivo matured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturation in vivo (up to germinal vesicle breakdown; GVBD) and then completed the process in vitro, displayed the same PN asymmetry as oocytes matured entirely in vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.     

Cryptorchidism in common eutherian mammals

Cryptorchidism is failure of one or both testes to descend into the scrotum. Primary fault lies in the testis. We provide a unifying cross-species interpretation of testis descent and urge the use of precise terminology. After differentiation, a testis is relocated to the scrotum in three sequential phases: abdominal translocation, holding a testis near the internal inguinal ring as the abdominal cavity expands away, along with slight downward migration; transinguinal migration, moving a cauda epididymidis and testis through the abdominal wall; and inguinoscrotal migration, moving a s.c. cauda epididymidis and testis to the bottom of the scrotum. The gubernaculum enlarges under stimulation of insulin-like peptide 3, to anchor the testis in place during gradual abdominal translocation. Concurrently, testosterone masculinizes the genitofemoral nerve. Cylindrical downward growth of the peritoneal lining into the gubernaculum forms the vaginal process, cremaster muscle(s) develop within the gubernaculum, and the cranial suspensory ligament regresses (testosterone not obligatory for latter). Transinguinal migration of a testis is rapid, apparently mediated by intra-abdominal pressure. Testosterone is not obligatory for correct inguinoscrotal migration of testes. However, normally testosterone stimulates growth of the vaginal process, secretion of calcitonin gene-related peptide by the genitofemoral nerve to provide directional guidance to the gubernaculum, and then regression of the gubernaculum and constriction of the inguinal canal. Cryptorchidism is more common in companion animals, pigs, or humans (2-12%) than in cattle or sheep (< or =1%). Laboratory animals rarely are cryptorchid. In respect to non-scrotal locations, abdominal testes predominate in cats, dogs, and horses. Inguinal testes predominate in rabbits, are common in horses, and occasionally are found in cats and dogs. S.c. testes are found in cattle, cats and dogs, but are most common in humans.     

Insights into the molecular basis of sperm-egg recognition in mammals

The zona pellucida surrounding the egg and pre-implantation embryo is required for in vivo fertility and early development. Explanatory models of sperm-egg recognition need to take into account the ability of sperm to bind to ovulated eggs, but not to two-cell embryos. For the last two decades, investigators have sought to identify an individual protein or carbohydrate side chain as the 'sperm receptor'. However, recent genetic data in mice are more consistent with the three-dimensional structure of the zona pellucida, rather than a single protein (or carbohydrate), determining sperm binding. The mouse and human zonae pellucidae contain three glycoproteins (ZP1, ZP2, ZP3) and, following fertilization, ZP2 is proteolytically cleaved. The replacement of endogenous mouse proteins with human ZP2, ZP3 or both does not alter taxon specificity of sperm binding or prevent fertility. Surprisingly, human ZP2 is not cleaved following fertilization and intact ZP2 correlates with persistent sperm binding to two-cell embryos. Taken together, these data support a model in which the cleavage status of ZP2 modulates the three-dimensional structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not (cleaved).     

Accumulation of perfluorooctane sulfonate in marine mammals

Perfluorooctane sulfonate (PFOS) is a perfluorinated molecule that has recently been identified in the sera of nonindustrially exposed humans. In this study, 247 tissue samples from 15 species of marine mammals collected from Florida, California, and Alaskan coastal waters; and northern Baltic Sea; the Arctic (Spitsbergen); and Sable Island in Canada were analyzed for PFOS. PFOS was detected in liver and blood of marine mammals from most locations including those from Arctic waters. The greatest concentrations of PFOS found in liver and blood were 1520 ng/g wet wt in a bottlenose dolphin from Sarasota Bay, FL, and 475 ng/mL in a ringed seal from the northern Baltic Sea (Bothnian Sea), respectively. No age-dependent increase in PFOS concentrations in marine mammals was observed in the samples analyzed. The occurrence of PFOS in marine mammals from the Arctic waters suggests widespread global distribution of PFOS including remote locations.     

The mechanism of sperm-oocyte fusion in mammals

Sperm-oocyte fusion is one of the most impressive events in sexual reproduction, and the elucidation of its molecular mechanism has fascinated researchers for a long time. Because of the limitation of materials and difficulties in analyzing membrane protein-protein interactions, many attempts have failed to reach this goal. Recent studies involving gene targeting have clearly demonstrated the various molecules that are involved in sperm-oocyte binding and fusion. Sperm ADAMs (family of proteins with a disintegrin and metalloprotease domain), including fertilin alpha, fertilin beta and cyritestin, have been investigated and found to be important for binding rather than for fusion and painstaking studies have raised suspicions that their putative receptors, oocyte integrins, are necessary for the sperm-oocyte interaction. Recently, several studies have focused the spotlight on CD9 and glycosylphosphatidylinositol (GPI)-anchored proteins on oocytes, and epididymal protein DE on sperm, as candidate molecules involved in sperm-oocyte fusion. Lack of, or interference with the function of, these proteins can disrupt the sperm-oocyte fusion without changing the binding. In this review we highlight the candidate molecules involved in the sperm-oocyte interaction suggested from the recent progress in this research field.     

Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement

In mammals, only a few spermatozoa arrive at the fertilization site. During the last step in the journey to the egg, apart from their self-propulsion, spermatozoa may be assisted by oviduct movement and/or a guidance mechanism. The proportion of rabbit spermatozoa that arrive at the fertilization site was determined under in vivo conditions, in which either the ovulation products (secreting chemoattractants) and/or the oviduct movement (causing the displacement of the oviductal fluid) was inhibited. When only one of these components was inhibited, sperm transport to the fertilization site was partially reduced. However, when both the ovulation products and the oviduct movement were inhibited, almost no spermatozoa arrived at the fertilization site. The results suggest that spermatozoa are transported to and retained at the fertilization site by the combined action of a chemical guidance and the oviduct movement. A working model is proposed to explain how these two mechanisms may operate to transport spermatozoa to the fertilization site, probably as an evolutionary adaptation to maximize the chance of fertilizing an egg.     

Cytoskeleton localization in the sperm head prior to fertilization

Three major cytoskeletal proteins, actin, tubulin and spectrin, are present in the head of mammalian spermatozoa. Although cytoskeletal proteins are implicated in the regulation of capacitation and the acrosome reaction (AR), their exact role remains poorly understood. The aim of this study was to compare the distribution of the sperm head cytoskeleton before and after the AR in spermatozoa representing a range of acrosome size and shape. Spermatozoa from the human and three rodents (rat, hamster and grey squirrel) were fixed before and after the AR in appropriate medium in vitro. Indirect immunofluorescent localization of cytoskeletal proteins was undertaken with antibodies recognizing actin, spectrin and alpha-tubulin. Preparations were counterstained with propidium iodide and examined by epifluorescent and confocal microscopy. Our results clearly demonstrated changes in localization of cytoskeleton during the AR, mainly in the apical acrosome with further changes to the equatorial segment and post-acrosomal regions. The pattern of cytoskeletal proteins in the sperm head of all the species was similar in respect to various sub-compartments. These observations indicated that the sperm head cortical cytoskeleton exhibits significant changes during the AR and, therefore, support the image of cytoskeletal proteins as highly dynamic structures participating actively in processes prior to fertilization.     

Early history of in vitro fertilization

Although in vitro fertilization (IVF) is used widely for a variety of purposes, it is often not appreciated how this technology was developed. A large number of experiments beginning in 1878 contributed to the first successful reports of IVF over 75 years later. The discovery of sperm capacitation in 1951 was central to the development of IVF technology, and it was rapidly followed by the first convincing reports of IVF in several species. The ability to fertilize oocytes in vitro has allowed major advances to be made into understanding the mechanisms involved in fertilization and early development, and IVF now supports reproductive biotechnology in animals and in humans. This article is a historical review of key experiments that helped to provide the basis for present day IVF procedures, placed into context with current practice.     

Modules for cloning-free chromatin tagging in Saccharomyces cerevisae

We describe a straightforward two-step PCR-based method to insert arrays of lac or tet operators (lacO or tetO) at specific loci in the budding yeast genome. The method entails insertion of a marker generated by PCR with classical long primers recognizing the locus of interest, followed by the replacement of this marker by a linearized plasmid bearing an array of lacI- or tetR-binding motifs. Using this technique, loci located either in the yeast genome or on yeast artificial chromosomes can be efficiently tagged. We provide a set of plasmids with different markers for cloning-free integration of lacO or tetO repeats into the yeast genome.     

Relationship between paternal involvement and child malnutrition in a rural area of Vietnam

BACKGROUND: Malnutrition is a public health problem in Vietnam. Child health and the status of women have been targets for various health programs in the country. In general, reports in the literature suggest that care is positively correlated with positive nutritional status of children. In the household, the father is considered a resource for care. However, the role of paternal care in health programs has not received the attention it deserves. OBJECTIVE: To identify associations between the involvement of fathers in child care and housework and the nutritional status of children under 3 years of age. METHODS: This cross-sectional study was based on a random sample of 547 children under 3 years of age from intact families and their biological parents. The main outcome variable was child nutrition. Predictor variables represented two domains of father's involvement. Multivariable general linear modeling and multivariable logistic regression modeling were performed with the use of a combination of stepwise and hierarchical approaches in data analysis. RESULTS: The overall prevalence of underweight among children was 19.1%, and the prevalence of stunting was 14.4%. Children whose fathers did not bring them to a medical facility for immunization were about 1.7 times more likely to be underweight and stunted than those whose fathers did bring them for immunization after child's age, household economic status, and mother's education were controlled for. Father's involvement in housework was not found to be related to the prevalence of malnutrition. CONCLUSIONS: Paternal involvement in child immunization should be encouraged by health-care providers who manage immunization programs.     

Cytogenetic damage in preimplantation mouse embryos generated after paternal and parental gamma-irradiation and the influence of vitamin C

Cytogenetic damage expressed as micronuclei (MN) in 4-8-cell embryos generated after irradiation of male or male and female mice in the absence and presence of vitamin C was investigated. Male NMRI mice were whole body exposed to 4 Gy gamma-rays and mated with non-irradiated superovulated female mice in 6 successive weeks after irradiation in a weekly interval. In experiments involving irradiation of both male and female mice, irradiated male mice for 6 weeks post irradiation were mated with female mice irradiated after induction of superovulation. Effect of 100 mg/kg vitamin C (ascorbic acid) on the frequency of MN was also studied. Pregnant animals were euthanized and embryos flushed from the oviducts and fixed on slides. The rate of MN observed in embryos generated from irradiated male compared with control group dramatically increased (P<0.01). Frequency of MN in this group decreased dramatically after vitamin C treatment (P<0.01). Frequency of MN in embryos generated by mating both male and female irradiated mice was higher than that observed for those embryos generated by irradiated male mice alone. However, a considerable modifying effect of vitamin C was observed for this group too (P<0.05). Results indicate that irradiation of gonads during spermatogenesis and preovulatory stage oocytes may lead to unstable chromosomal aberrations and probably stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos expressed as MN. The way vitamin C reduces clastogenic effects of radiation on germ cells leading to reduced frequency of MN in pre-embryos might be due to its antioxidation and radical scavenging properties.     

Organotin compounds in the liver tissue of marine mammals from the Polish coast of the Baltic Sea

Butyltins (BTs) and phenyltins (PhTs) were determined in the livers of marine mammals found by-caught or stranded along the Polish coast of the Baltic Sea. During the investigation an original analytical method was developed. Butyltin compounds were detected in all the liver samples, whereas phenyltins were not detected in any of the samples. The total concentrations of BTs ranged from 43.9 to 7698 ng(Sn) x g(-1) dry weight. Age-related trends to accumulate BTs in immature porpoises were found. At the same time there were no male-female differences in BTs concentrations observed. No statistically significant spatial distribution differences were found between the locations corresponding to the open Baltic Sea waters and inside the Gulf of Gdańsk, which is characterized by high maritime activity. In comparison to butyltin levels in marine mammals from other geographic regions, the samples analyzed indicate a significant degree of tributyltin pollution along the Polish coast of the Baltic Sea. On the basis of a literature review, higher BT levels are usually found in waters close to highly industrialized areas, such as Japan, Hong Kong, and the United States.     

黑龙江省甜菜优化施肥技术研究

在黑龙江省黑土和白浆土甜菜主产区,采用3×3优化施肥设计开展甜菜优化施肥研究.试验结果表明,黑龙江省甜菜高产施肥氮和钾的适宜用量为90 - 135kg/hm2,磷的适宜用量为80 - 120kg/hm2,氮磷钾的适宜比例为N∶P2O5∶K2O=1.11∶1∶1.12.不施氮肥平均减产33.8％,不施磷肥平均减产21.1％,不施钾肥平均减产21.6％.从产量和含糖率综合考虑,最佳处理为N2P2K2,即N 90kg/hm2、P2O80kg/hm2、K2090 kg/hm2,较对照平均增产31.9％,含糖率平均增加1.08个百分点.建立了三种不同土壤的甜菜高产施肥模型.     

Docking onto chromatin via the Saccharomyces cerevisiae Rad9 Tudor domain

An integrated cellular response to DNA damage is essential for the maintenance of genome integrity. Recently, post-translational modifications to histone proteins have been implicated in DNA damage responses involving the Rad9 family of checkpoint proteins. In budding yeast, methylation of histone H3 on lysine 79 (H3-K79me) has been shown to be required for efficient checkpoint signalling and Rad9 localization on chromatin. Here, we have used a rad9 Tudor mutant allele and cells mutated for Dot1, the H3-K79 methylase, to analyse the epistatic relationship between RAD9 and DOT1 genes regarding the DNA damage resistance and checkpoint activation pathways. Our results show that RAD9 is epistatic to DOT1 and suggest that it acts downstream of the Dot1 methylase in the damage resistance and checkpoint response. We have also found that the Tudor domain of Rad9 is necessary for in vitro binding to H3-K79me as well as Rad9 focal accumulation in response to DNA damage in vivo. In summary, our study demonstrates that the interaction between Rad9, via its Tudor domain, and methylated H3-K79 is required at two different steps of the DNA damage response, an early step corresponding to checkpoint activation, and a late step corresponding to DNA repair. The study further shows that the function of this interaction is cell cycle-regulated; the role in checkpoint activation is restricted to the G(1) phase and its role in DNA repair is restricted to G(2).     

Increased disruption of sperm plasma membrane at sperm immobilization promotes dissociation of perinuclear theca from sperm chromatin after intracytoplasmic sperm injection in pigs

The effects of sperm-immobilization methods on decondensation of sperm chromatin and retention of subacrosomal sperm perinuclear theca (SAR-PT) after intracytoplasmic sperm injection (ICSI) were examined in pigs. Sperm membrane damage caused by different immobilization methods by rubbing with a micropipette without piezo pulses (R), or with a low (L) or high (H) intensity of piezo pulses while rubbing, was assessed by the time required for staining of sperm heads with eosin Y solution. The average time for staining of sperm heads immobilized by the R, L or H treatments was 76, 41 or 26 s, respectively. The fertilization rate following ICSI was increased by sperm immobilization by piezo pulses compared with R, but increased intensity of pulses from L to H did not cause further improvements (29, 48 and 47%, respectively). An immunofluorescence study revealed that H immobilization promoted the dissociation of SAR-PT from sperm chromatin compared with L and R, and it increased the frequency of male pronuclear formation in which chromatin appeared uniformly decondensed. With in vitro fertilization (IVF), SAR-PT disassembled coordinately with sperm chromatin decondensation and it was not detectable around male pronuclei. This was different from most of the oocytes after ICSI in which remnants SAR-PT were detected adjacent to male pronuclei. We concluded that increased damage on the sperm plasma membrane at immobilization improved fertilization rates and decondensation of sperm chromatin after ICSI due to the accelerated dissociation of SAR-PT from the sperm nucleus. Also, the behavior of SAR-PT after ICSI was different from that observed in oocytes after IVF.     

Roles of stress response-related signaling and its contribution to the toxicity of zearalenone in mammals

Zearalenone (ZEA) is a mycotoxin frequently found in cereal crops and cereal-derived foodstuffs worldwide. It affects plant productivity, and is also a serious hazard to humans and animals if being exposed to food/feed contaminated by ZEA. Studies over the last decade have shown that the toxicity of ZEA in animals is mainly mediated by the various stress responses, such as endoplasmic reticulum (ER) stress, oxidative stress, and others. Accumulating evidence shows that oxidative stress and ER stress signaling are actively implicated in and contributes to the pathophysiology of various diseases. Biochemically, the deleterious effects of ZEA are associated with apoptosis, DNA damage, and lipid peroxidation by regulating the expression of genes implicated in these biological processes. Despite these findings, the underlying mechanisms responsible for these alterations remain unclear. This review summarized the characteristics, metabolism, toxicity and the deleterious effects of ZEA exposure in various tissues of animals. Stress response signaling implicated in the toxicity as well as potential therapeutic options with the ability to reduce the deleterious effects of ZEA in animals were highlighted and discussed.