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Although it is known that the whitefish, an ancient salmonid, expresses three distinct gonadotropin-releasing hormone (GnRH) forms in the brain, it has been thought that the later-evolving salmonids (salmon and trout) had only two types of GnRH: GnRH2 and GnRH3. We now provide evidence for the expression of GnRH1 in the gonads of Atlantic salmon by rapid amplification of cDNA ends, real-time quantitative PCR and immunohistochemistry. We examined six different salmonid genomes and found that each assembly has one gene that likely encodes a viable GnRH1 prepropeptide. In contrast to both functional GnRH2 and GnRH3 paralogs, the GnRH1 homeolog can no longer express the hormone. Furthermore, the viable salmonid GnRH1 mRNA is composed of only three exons, rather than the four exons that build the GnRH2 and GnRH3 mRNAs. Transcribed gnrh1 is broadly expressed (in 17/18 tissues examined), with relative abundance highest in the ovaries. Expression of the gnrh2 and gnrh3 mRNAs is more restricted, primarily to the brain, and not in the gonads. The GnRH1 proximal promoter presents composite binding elements that predict interactions with complexes that contain diverse cell fate and differentiation transcription factors. We provide immunological evidence for GnRH1 peptide in the nucleus of 1-year-old type A spermatogonia and cortical alveoli oocytes. GnRH1 peptide was not detected during other germ cell or reproductive stages. GnRH1 activity in the salmonid gonad may occur only during early stages of development and play a key role in a regulatory network that controls mitotic and/or meiotic processes within the germ cell. [ABSTRACT FROM AUTHOR]

Nlrp2 encodes a protein of the oocyte subcortical maternal complex (SCMC), required for embryo development. We previously showed that loss of maternal Nlrp2 in mice causes subfertility, smaller litters with birth defects, and growth abnormalities in offspring, indicating that Nlrp2 is a maternal effect gene and that all embryos from Nlrp2-deficient females that were cultured in vitro arrested before the blastocysts stage. Here, we used time-lapse microscopy to examine the development of cultured embryos from superovulated Nlrp2-deficient and wild-type mice after in vivo and in vitro fertilization. Embryos from Nlrp2-deficient females had similar abnormal cleavage and fragmentation and arrested by blastocyst stage, irrespective of fertilization mode. This indicates that in vitro fertilization does not further perturb or improve the development of cultured embryos. We also transferred embryos from superovulated Nlrp2-deficient and wild-type females to wild-type recipients to investigate if the abnormal reproductive outcomes of Nlrp2-deficient females are primarily driven by oocyte dysfunction or if a suboptimal intra-uterine milieu is a necessary factor. Pregnancies with transferred embryos from Nlrp2-deficient females produced smaller litters, stillbirths, and offspring with birth defects and growth abnormalities. This indicates that the reproductive phenotype is oocyte-specific and is not rescued by development in a wild-type uterus. We further found abnormal DNA methylation at two maternally imprinted loci in the kidney of surviving young adult offspring, confirming persistent DNA methylation disturbances in surviving offspring. These findings have implications for fertility treatments for women with mutations in NLRP2 and other genes encoding SCMC proteins. [ABSTRACT FROM AUTHOR]

Background: Members of the cysteine-rich secretory proteins (CRISPS), antigen 5 (Ag5) and pathogenesis-related 1 (Pr-1) (CAP) superfamily of proteins are found across the bacterial, fungal, plant and animal kingdoms. Although many CAP superfamily proteins remain poorly characterized, over the past decade evidence has accumulated, which provides insights into the functional roles of these proteins in various processes, including fertilization, immune defence and subversion, pathogen virulence, venom toxicology and cancer biology.Objective and Rationale: The aim of this article is to summarize the current state of knowledge on CAP superfamily proteins in mammalian fertility, organismal homeostasis and disease pathogenesis.Search Methods: The scientific literature search was undertaken via PubMed database on all articles published prior to November 2019. Search terms were based on following keywords: 'CAP superfamily', 'CRISP', 'Cysteine-rich secretory proteins', 'Antigen 5', 'Pathogenesis-related 1', 'male fertility', 'CAP and CTL domain containing', 'CRISPLD1', 'CRISPLD2', 'bacterial SCP', 'ion channel regulator', 'CatSper', 'PI15', 'PI16', 'CLEC', 'PRY proteins', 'ASP proteins', 'spermatogenesis', 'epididymal maturation', 'capacitation' and 'snake CRISP'. In addition to that, reference lists of primary and review article were reviewed for additional relevant publications.Outcomes: In this review, we discuss the breadth of knowledge on CAP superfamily proteins with regards to their protein structure, biological functions and emerging significance in reproduction, health and disease. We discuss the evolution of CAP superfamily proteins from their otherwise unembellished prokaryotic predecessors into the multi-domain and neofunctionalized members found in eukaryotic organisms today. At least in part because of the rapid evolution of these proteins, many inconsistencies in nomenclature exist within the literature. As such, and in part through the use of a maximum likelihood phylogenetic analysis of the vertebrate CRISP subfamily, we have attempted to clarify this confusion, thus allowing for a comparison of orthologous protein function between species. This framework also allows the prediction of functional relevance between species based on sequence and structural conservation.Wider Implications: This review generates a picture of critical roles for CAP proteins in ion channel regulation, sterol and lipid binding and protease inhibition, and as ligands involved in the induction of multiple cellular processes. [ABSTRACT FROM AUTHOR]

Marine and freshwater spawning environments present fish sperm with unique challenges, but for both, gametes often signal prior to contact via biochemical interactions through maternally-derived compounds (i.e. eggs and ovarian fluid; OF). For example, when OF is incorporated into the fertilization environment, sperm have been observed to exhibit changes in swimming trajectories (e.g. motility and velocity), yet it remains unclear whether the presence of this OF consistently improves sperm performance. The objectives of this study were to determine the overall effect of OF on sperm performance using meta-analysis. Published literature was searched for studies comparing sperm motility and/or velocity in the presence and absence of OF. For each study, the log response ratios (lnRR) was calculated, in which positive values indicate improved performance in the presence of OF. For motility, the overall effect size was non-significant (lnRR = 0.09, CL = −0.06, 0.24), whereas velocity was positively affected by OF (lnRR = 0.10; CL = 0.04, 0.17). When segregated by environment, for freshwater species there was a significant positive effect of the OF on velocity (lnRR = 0.18, CL = 0.07, 0.29), which translated to an increase in velocity of 20%. In contrast, no effect was detected for velocity in marine species (lnRR = −0.01, CL = −0.02, 0.01). Overall, there is evidence that OF improves sperm performance, although spawning environment and/or taxonomic factors are likely to moderate these sperm-OF interactions. Together, these results further our understanding of natural reproductive processes governing sperm performance, mating systems, and fertilization dynamics. [ABSTRACT FROM AUTHOR]

Symsagittifera roscoffensis is a small marine worm found in the intertidal zone of sandy beaches around the European shores of the Atlantic. S. roscoffensis is a member of the Acoelomorpha, a group of flatworms formerly classified with the Platyhelminthes, but now recognized as Xenacoelomorpha, a separate phylum of disputed affinity. We have used electron microscopy to examine the process of spermiogenesis (the final stage of spermatogenesis) in S. roscoffensis, by which spermatids form highly elongated spermatozoa. Their nuclei are long and thread-like, running most of the cell's length, and during the process, a pair of flagella are fully incorporated into the cell body. Two previously undescribed interorganelle contact sites form at different stages of spermiogenesis. Strikingly, there is an extensive nucleus–plasma membrane contact site. Golgi-derived granules containing electron-dense filaments line up along the spermatid plasma membrane, undergo a conformational change, and donate material that forms a perinuclear layer that cements this contact site. We also show in spermatids at an earlier stage that the same granules are associated with microtubules, presumably for traffic along the elongating cell. We identify a second spermiogenesis-specific contact site where sheaths engulfing each internalizing flagellum contact the nuclear envelope. [ABSTRACT FROM AUTHOR]

Fertilization is a multistep process during which two terminally differentiated haploid cells, an egg and a sperm, combine to produce a totipotent diploid zygote. In the early 1950s, it became possible to fertilize mammalian eggs in vitro and study the sequence of cellular and molecular events leading to embryo development. Despite all the achievements of assisted reproduction in the last four decades, remarkably little is known about the molecular aspects of human conception. Current fertility research in animal models is casting more light on the complexity of the process all our lives start with. This review article provides an update on the investigation of mammalian fertilization and highlights the practical implications of scientific discoveries in the context of human reproduction and reproductive medicine. [ABSTRACT FROM AUTHOR]

The object of this study was to investigate if testis germ cell transplantation (TGCT) into a heterologous recipient would result in donor-origin spermatogenesis in the dromedary camel. First, we investigated a workable protocol for TGCT in camels, including donor cell isolation, enrichment by density gradient centrifugation (Percoll and Bovicoll), rete testis injection and microsatellite detection of donor and recipient genotypes. Second, the effects of three doses of Dolichos biflorus agglutinin (DBA), a glycoprotein that specifically binds to gonocytes or Type A spermatogonia, on testis germ cell depletion were investigated by direct injection into the rete testis of a male camel. Seven recipients were prepared with DBA treatment, two males were castrated at 4 weeks for depletion assessment and the remaining five received donor cells 4–6 weeks after treatment. On average, ~17 million cells were isolated per gram of testis tissue, with 19.5 ± 1.9% DBA-positive (DBA+) cells. Percoll centrifugation yielded a 1.5-fold increase in DBA+ cells while Bovicoll centrifugation produced a 2.5-fold increase from the input cells of 18.6 ± 2.1% DBA+ cells. Semen was collected from the recipients 13–20 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In two of the five recipients, all semen samples were shown to be positive for donor-derived cells. These results demonstrate for the first time that: (1) heterologous testicular germ cell transplantation in camels is feasible and the recipients are able to produce spermatozoa of donor origin and (2) DBA can be used effectively to deplete endogenous stem cells. Using stem cell technology, a group of surrogate males that produce donor sperm were created for the first time in the dromedary camel. This technology has the potential to disseminate a favoured genotype in species where artificial insemination is not feasible due to the lack of effective semen freezing protocols. The treatment of the surrogate male's testis with a protein, DBA lectin, is critical for the success of this process. [ABSTRACT FROM AUTHOR]

Much progress has been made regarding our understanding of aromatase regulation, estrogen synthesis partitioning and communication between the germinal and somatic compartments of the differentiating gonad. We now know that most of the enzymatic and signaling apparatus required for steroidogenesis is endogenously expressed within germ cells. However, less is known about the expression and localization of steroidogenic components within mature spermatozoa. We have assembled a sperm library presenting 197,015 putative transcripts. Co-expression clustering analysis revealed that 6687 genes were present at higher levels in sperm in comparison to fifteen other salmon tissue libraries. The sperm transcriptome is highly complex containing the highest proportion of unannotated genes (45%) of the tissues analyzed. Our analysis of highly expressed genes in late-stage sperm revealed dedication to tasks involving chromatin remodeling, flagellogenesis and proteolysis. In addition, using various different embedding and microscopic techniques, we examined the morphology of salmon spermatozoa and characterized expression and localization of several estrogenic regulatory and signaling proteins by immunohistochemistry. We provide evidence for the endogenous synthesis and localization of aromatase (CYP19A and CYP19B1) and potential mediators of estrogen [i.e., ER-alpha and soluble adenylyl cyclase (sAC)] or phosphate (i.e., CREB and FOXL2A) signaling. Partitioning of select transcripts that encode AR-beta, FSH and the LH receptor, but not AR-alpha, LH or the FSH receptor, further points to localized specificity of function in the steroidogenic circuitry of the sperm cell. These results open new avenues of investigation to further our understanding of the intra- and intercellular regulatory processes that guide sperm development and biology. [ABSTRACT FROM AUTHOR]

Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes' maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR ("before IVM" group), or first in vitro matured and then if classified as BCB+ passed to molecular analyses ("after IVM" group). As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes' successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte's achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes. [ABSTRACT FROM AUTHOR]

The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (1VF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa-ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were coincubated for 15 min. A significant increase in spermatozoa-ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa. [ABSTRACT FROM AUTHOR]

Abstract: The study assesses the possibility to estimate the potential fertility of post-thawed antelope (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi) epididymal sperm using homologous and heterologous IVF and the functioning of cattle IVF system to produce antelope embryos. Cauda epididymal sperm were collected from the antelope and cryopreserved under field conditions. In vitro matured domestic cow, blesbok and springbok oocytes were co-incubated in modified-Tyrode Lactate (m-TL) IVF media with springbok, impala and blesbok sperm for heterologous IVF and springbok and blesbok sperm for homologous IVF. A group of presumptive zygotes from each treatment were examined for sperm penetration and male pronuclear formation after 18h and the remainder were cultured and evaluated for embryo cleavage 22h later. The study shows that Modified Tyrode Lactate in vitro fertilization media supports survivability, capacitation and hyperactivation of springbok, impala and blesbok sperm. Springbok, impala and blesbok post-thawed epididymal spermatozoa are capable of fertilizing domestic cow oocytes under conditions that support domestic cattle IVF. Penetration, male pronuclear formation and embryo cleavage did not differ (p >0.05) between cow oocytes inseminated with sperm from springbok, impala or blesbok however these parameters were higher (p <0.05) for oocytes inseminated with bull sperm. Modified Tyrode Lactate IVF media supported homologous fertilization and embryo development in springbok and blesbok however did not support blastocyst development. These findings suggest that cattle provide a useful model for evaluating springbok, impala and blesbok post-thawed cauda epididymal sperm functionality. Domestic cattle embryo culture conditions need to be modified to promote blastosyst development in these antelope species. Such research provides an important tool in assisted reproductive technology development when high biological value material is utilized for wild species recovery plans. [Copyright &y& Elsevier]

Abstract: In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality. [Copyright &y& Elsevier]

Abstract: Ethanol is a known modulator of neural stem cell development, but the consequences of ethanol toxicity on the cell fate decisions of spermatogonial stem cells (SSCs) is poorly understood. Using an in vivo treatment and stem cell transplantation approach, we investigated the effects of acute ethanol exposure on formation of the growing adult SSC population in neonatal and pre-pubertal mice. Treatment with a single dose of ethanol disrupted SSC homeostasis in vivo evidenced by a significant reduction (7-fold) of stem cell colonization efficiency in the testes of recipient mice following transplantation. Ethanol treatment also increased the rate of apoptosis in adult differentiating germ cells in situ. Gene expression analysis indicates that ethanol exposure has transient and long-term effects on the expression of GDNF and VEGF family molecules and supports the hypothesis that the niche microenvironment for SSCs is sensitive to ethanol toxicity during pre-pubertaland adult life. [Copyright &y& Elsevier]

A tetraspanin family protein, CD9, has not previously been identified in sperm cells. Here, we characterize sperm CD9 in the mouse, including its unique localization in sperm, appearance during spermatogenesis, and behavior and fate during mouse fertilization. In sperm, CD9 is an inner acrosomal membrane-associated protein, not a plasma membrane-associated protein. Its molecular weight is approximately 24 kDa throughout its processing, from testicular germ cells to acrosome-reacted sperm. A temporal difference was found between mRNA and protein expression; CD9 mRNA was detected in the stages from spermatogonia through round spermatids showing the strongest levels in midpachytene spermatocytes. CD9 protein was detected in the cytoplasm throughout the stages from spermatogonia to spermatocytes. While CD9 was weakly expressed in the spermatids from step 1 through step 14, the signals became clearly positive at the marginal region of the anterior acrosome in elongated spermatids. After the acrosome reaction, the majority of sperm CD9 was retained in the inner acrosomal membrane, but some quantity of CD9 was found on the plasma membrane covering the equatorial segment as detected by immunogold electron microscopy using anti-CD9 antibody. CD9 was maintained on the sperm head after reaching the perivitelline space of CD9-deficient eggs that were recovered after natural mating with wild males. Thus, this study characterizes CD9 in sperm development and fertilization. [ABSTRACT FROM AUTHOR]

It is accepted that recombination errors during human female meiotic prophase have some influence on the origin of trisomy 21. A total of 335 oocytes from four euploid fetuses were analysed by immunofluorescence and fluorescence in-situ hybridization in order to assess the recombination nodules along chromosome 21. Results based on the analysis of recombination points on the bivalent 21 during human female meiosis showed that both number [none (3.70%), one (79.01%) and two (17.29%)] and distribution (always positioned interstitially on the q-arm) are different in males, ensuring that the two homologues more efficiently remain together until anaphase I. Therefore, the mainly maternal origin of trisomy 21 appears not be linked to the first stages of oocyte development during fetal life, and this leads to the suggestion that the influence of environmental factors on the segregation of chromosome 21 homologues in later meiotic stages could have a significant role in the predominant maternal origin of trisomy 21. [ABSTRACT FROM AUTHOR]

Abstract: We have carried out a study on the influence of prolonged cold storage (5°C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5°C and stored for 24, 96 and 192h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37°C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192h at 5°C (P <0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192h. [Copyright &y& Elsevier]

Abstract: The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology. [Copyright &y& Elsevier]

The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum. Because of their stage specificity and perinuclear rather than spherical distribution, these clustered Balbiani body mitochondria may have been missed previously. The Balbiani body also contains Trailer hitch, a widely conserved member of a protein complex that associates with endoplasmic reticulum/Golgi-like vesicles and transports specific RNAs during Drosophila oogenesis. Our results provide evidence that mouse oocytes develop using molecular and developmental mechanisms widely conserved throughout the animal kingdom. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to test the hypothesis that bulls have significant intra-individual differences in the hydraulic conductivity (L p) and permeability coefficient for glycerol (P s) of the sperm cell membrane. The permeability parameters were determined at 22, 10, and 0°C of sperm from 7 Holstein Frisian artificial insemination (AI) bulls, using four ejaculates per bull. A stopped-flow approach was applied to provide temporal resolution sufficient to measure rapid cell volume changes under anisosmotic conditions in the absence or presence of glycerol. This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute L p in the absence glycerol, and L p in the presence of glycerol () and P s. Averaged over all bulls, L p in the absence of glycerol was 0.28±0.01, 0.15±0.01 and 0.10±0.01μmmin−1 atm−1 (mean±SD) at 22, 10 and 0°C, respectively, yielding an Arrhenius activation energy (E a) of 7.39kcal/mol. The average value at 22°C, was 3.8 times lower than L p in the absence of glycerol (P <0.05). , P s, and the reflection coefficient (σ) at 22°C were 0.073±0.015μmmin−1 atm−1, 0.80±0.33×10−3 cmmin−1, and 0.92±0.10 (mean±SD), respectively. Subsequent experiments were performed at 10 and 0°C. Activation energies for and P s were 10.08 and 8.77kcal/mol, respectively. The significant differences between individual bulls in L p and P s indicate that individual males may require individual adjustments of the cooling protocol. Application of these data in a theoretical model to simulate the osmotic events during freezing resulted in predicted optimal cooling rates in the range of published empirical values. [Copyright &y& Elsevier]

Abstract: Intracytoplasmic sperm injection (ICSI) in pigs is a technique with potential application in diverse fields of animal production and biomedicine. Even though there are some cases of live offspring resulting from this technique, its yield is still quite low compared to other species. The aim of this study was to evaluate different factors affecting the ICSI performance. This was done by studying (1) the sequence of culture media for the oocytes after injection; (2) modifications in the in vitro maturation system (IVM) through meiotic inhibitors such as roscovitine, and changes in the IVM time; (3) oocyte activation through injection of inositol triphosphate (InsP3) together with the sperm. In vitro matured oocytes were employed. All the ICSI experiments were performed with fresh ejaculated semen. Results showed that porcine ICSI zygotes give an improved proportion of two-cell embryos using the sequence IVF medium-embryo culture medium (NCSU-23) rather than transferring directly to NCSU-23. Pronuclear formation ability was not affected by prematuration, but a faster embryo development was observed in roscovitine treated oocytes. In relation to IVM times, oocytes matured for 36h can achieve better fertilization percentages than oocytes matured for 44h. These results were independent of the roscovitine treatment. Finally, no influence on embryo development was observed until the blastocyst stage with the use of the InsP3 as an exogenous activating factor. [Copyright &y& Elsevier]

Abstract: Proteins implicated in the “SNARE hypothesis” for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p &#60;0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility. [Copyright &y& Elsevier]

Abstract: The Signal transducers and activators of transcription (Stat) family of proteins plays diverse roles during differentiation in many tissues. Stat3 is an essential mammalian gene, critical during embryonic development. In mammals, Stat3 is differentially distributed in the cytoplasm of mature oocytes and in preimplantation embryos suggesting that Stat3 may be involved in determination of polarity. Here, we report that Stat3 protein is expressed in the cytoplasm of oocytes from primordial, primary and secondary follicles in the adult ovary and in developing acrosomes of round spermatids in the adult testis. Stat3 is also expressed in gonocytes, prospermatogonia, oogonia and oocytes of embryonic and neonatal gonads. [Copyright &y& Elsevier]

Degradation of proteins mediated by ubiquitin–proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation. [Copyright &y& Elsevier]

To review and critique the current English literature describing the effects of antisperm antibodies (ASA) on mammalian fertility.A comprehensive English language literature was searched using Medline and by hand-searching. Emphasis was placed on clinically relevant articles.Results from the studies were extrapolated and the effects of ASA on fertility described.Antisperm antibodies may interfere with fertility. Not all ASA cause infertility. Current tests cannot differentiate the infertility-related ASA from those that do not interfere with infertility, because the antigenic specificities of these ASA are not known. The antigens which the infertility-related ASA must be characterized to allow an accurate detection and proper treatment for couples with ASA. [Copyright &y& Elsevier]

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