B. Fernandez-Fuertes

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150–200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x105EVs/mL, 1.5x105EVs/mL or 7.5x104EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7–9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

Asynchronous embryo transfer as a tool to understand embryo–uterine interaction in cattle: is a large conceptus a good thing?

The aim was to examine the effect of embryo-uterine synchrony on conceptus elongation and pregnancy rate in cattle. In Study 1, crossbred beef heifers each received 10 Day-7 in vitro-produced blastocysts on either Day 5, 7 or 9 after oestrus. A proportion of Day 5 recipients were supplemented with progesterone, via a progesterone-releasing intravaginal device from Days 3-5 plus either 750IU equine chorionic gonadotrophin or 3000IU human chorionic gonadotrophin on Day 3. At embryo age Day 14, all heifers were slaughtered and the uterus was flushed. Fewer recipients yielded conceptuses (P<0.05) and fewer conceptuses were recovered (P<0.05) following transfer on Day 5 compared with Day 7 or 9. Supplementation with progesterone resulted in short cycles in approximately 50% of recipients. Mean conceptus length was greater (P<0.05) following transfer to an advanced uterus. In Study 2, overall pregnancy rate following the fresh transfer of a single in vitro-produced blastocyst was 43.5% (2065/4749). Transfer of a Day 7 embryo to a synchronous Day-7 uterus resulted in a pregnancy rate of 47.3%. Transfer to a Day-5 (40.8%) or a Day-8 (41.3%) uterus moderately impacted pregnancy rate (P<0.01) while transfer to a uterus 2 days in advance (Day-9, 24.4%) or 3 days behind (Day-4, 27.0%) reduced (P<0.001) pregnancy rate compared with synchronous transfers. In conclusion, transfer of an embryo into an advanced uterus results in an acceleration of conceptus development, but does not result in greater pregnancy rates.

Cauda Epididymis-Specific Beta-Defensin 126 Promotes Sperm Motility but Not Fertilizing Ability in Cattle

ABSTRACT Bovine beta-defensin 126 (BBD126) exhibits preferential expression for the cauda epididymis of males, where it is absorbed onto the tail and postacrosomal region of the sperm. The aim of this study was to examine the role of BBD126 in bull sperm function. Fresh and frozen-thawed semen were incubated in the presence of different capacitating agents as well as with phosphatidylinositol-specific phospholipase C. These treatments, which have been successful in releasing beta-defensin 126 from macaque sperm, proved to be ineffective in bull sperm. This finding suggests that the protein behaves in a different manner in the bovine. The lack of success in removing BBD126 led us to use corpus epididymis sperm, a model in which the protein is not present, to study its functional role. Corpus sperm were incubated with cauda epididymal fluid (CEF) in the absence or presence of BBD126 antibody or with recombinant BBD126 (rBBD126). Confocal microscopy revealed that rBBD126 binds to corpus sperm with the same pattern observed for BBD126 in cauda sperm, whereas an aberrant binding pattern is observed when sperm are subject to CEF incubation. Addition of CEF increased motility as well as the number of corpus sperm migrating through cervical mucus from estrus cows. However, it decreased the ability of sperm to fertilize in vitro matured oocytes. The presence of the antibody failed to abrogate these effects. Furthermore, when rBBD126 was added in the absence of other factors and proteins from the CEF, an increase in motility was also observed and no negative effects in fertility were seen. These results suggest that BBD126 plays a key role in the acquisition of sperm motility in the epididymis.

Sperm-Coating Beta-Defensin 126 Is a Dissociation-Resistant Dimer Produced by Epididymal Epithelium in the Bovine Reproductive Tract

ABSTRACT Beta-defensins are innate immune molecules, often described as antimicrobial peptides because of their bactericidal activity and are now known to have diverse additional functions, including cell signaling, chemoattraction, immunoregulation, and reproduction. In humans and primates, beta-defensin 126 has been shown to regulate the ability of sperm to swim through cervical mucus and to protect sperm from attack by the female immune system during transit toward the oviduct. Bovine beta-defensin 126 (BBD126) is the ortholog of human defensin 126, and computational analysis here revealed significant conservation between BBD126 and other mammalian orthologs at the N-terminus, although extensive sequence differences were detected at the C-terminus, implying possible species-specific roles for this beta-defensin in reproduction. We had previously demonstrated preferential expression of this and related beta-defensin genes in the bovine male reproductive tract, but no studies of bovine beta-defensin proteins have been performed to date. Here, we analyzed BBD126 protein using a monoclonal antibody (a-BBD126) generated against a 14 amino acid peptide sequence from the secreted fragment of BBD126. The specificity of a-BBD126 was validated by testing against the native form of the peptide recovered from bovine caudal epididymal fluid and recombinant BBD126 generated using a prokaryotic expression system. Western blot analysis of the native and recombinant forms showed that BBD126 exists as a dimer that was highly resistant to standard methods of dissociation. Immunohistochemical staining using a-BBD126 demonstrated BBD126 protein expression by epithelial cells of the caudal epididymis and vas deferens from both mature and immature bulls. BBD126 could also be seen (by confocal microscopy) to coat caudal sperm, with staining concentrated on the tail of the sperm cells. This study is the first to demonstrate beta-defensin 126 protein expression in the bovine reproductive tract and on bull sperm. Its dissociation-resistant dimeric structure is likely to have important functional implications for the role of BBD126 in bovine reproduction.

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

Profiling bovine blastocyst microRNAs using deep sequencing

MicroRNAs (miRNAs) are known to control several reproductive functions, including oocyte maturation, implantation and early embryonic development. Recent advances in deep sequencing have allowed the analysis of all miRNAs of a sample. However, when working with embryos, due to the low RNA content, miRNA profiling is challenging because of the relatively large amount of total RNA required for library preparation protocols. In the present study we compared three different procedures for RNA extraction and prepared libraries using pools of 30 bovine blastocysts. In total, 14 of the 15 most abundantly expressed miRNAs were common to all three procedures. Furthermore, using miRDeep discovery and annotation software (Max Delbrück Center), we identified 1363 miRNA sequences, of which bta-miR-10b and bta-miR-378 were the most abundant. Most of the 179 genes identified as experimentally validated (86.6%) or predicted targets (13.4%) were associated with cancer canonical pathways. We conclude that reliable analysis of bovine blastocyst miRNAs can be achieved using the procedures described herein. The repeatability of the results across different procedures and independent replicates, as well as their consistency with results obtained in other species, support the biological relevance of these miRNAs and of the gene pathways they modulate in early embryogenesis.

Subfertility in bulls carrying a nonsense mutation in transmembrane protein 95 is due to failure to interact with the oocyte vestments

Abstract In a recent genome-wide association study, 40 Fleckvieh bulls with exceptionally poor fertility were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95) encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, only 1.7% of inseminations resulted in pregnancies. The aim of this study was to examine the effect of this mutation in TMEM95 on bovine sperm function in vitro. Sperm from homozygous (mt/mt) males had lower in vitro fertility than sperm from wild-type (wt/wt) or heterozygous (wt/mt) bulls (P < 0.01). In addition, early embryo division was affected in the mt/mt group (P < 0.01). This translated into a lower (P < 0.01) blastocyst rate at day 8. Fluorescent staining revealed that TMEM95 is lost after the acrosome reaction. This led us to hypothesize that TMEM95 might be involved in events that lead to sperm-oocyte interaction. After fertilization, a lower number (P < 0.01) of sperm from mt/mt bulls bound to the zona pellucida (ZP). Sperm from mt/mt bulls were also less able to penetrate oocytes with no ZP (P< 0.01). However, when sperm from these animals were injected into mouse oocytes, they could decondense as successfully as sperm from wt/wt bulls. No differences between genotypes were observed in the ability of sperm to retain motility in an ex vivo oviduct, or in the percentage of sperm exhibiting markers for capacitation and acrosomal reaction. These results suggest that fertilization failure in mt/mt bulls is due to the inability of their sperm to interact with the oocyte vestments. Summary Sentence Bulls homozygous for a nonsense mutation in TMEM95 exhibit extremely poor fertility in vivo and in vitro. This is due to the inability of sperm from these animals to interact with the vestments of the oocyte.

Do differences in the endometrial transcriptome between uterine horns ipsilateral and contralateral to the corpus luteum influence conceptus growth to day 14 in cattle

Abstract Embryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n = 5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n = 9) or contralateral to the CL (n = 8) or into both horns (i.e., bilateral, n = 9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P < 0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean ± SE 2.85 ± 0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14. Summary Sentence Differences in endometrial gene expression exist between the ipsilateral and contralateral uterine horns in cattle, but conceptus growth to day 14 is not different between the horns.

4 SUBFERTILITY IN BULLS CARRYING A NONSENSE MUTATION IN TMEM95 IS DUE TO FAILURE TO PENETRATE THE ZONA PELLUCIDA

In a recent genome-wide association study, male reproductive ability was assessed for 7962 Fleckvieh sires (Pausch et al. 2014 PLoS Genet. 10, e1004044). Forty bulls in the population with exceptionally poor reproductive performance were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95)-encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, out of 35,671 inseminations with these bulls, only 1.7% resulted in pregnancies. In addition, none of the bulls with normal reproductive performance was homozygous for this mutation. The aim of this study was to examine the effect of this nonsense mutation in TMEM95 on bovine sperm function in vitro. In all experiments, data were assessed for normality of distribution and analysed using a one-way ANOVA. The model included the main effects of treatment, replicate and their interactions. In Experiment 1, the fertilizing ability of sperm, assessed as oocyte cleavage rate, from 5 homozygous (mt/mt), 3 heterozygous (wt/mt), and 2 wildtype (wt/wt) Fleckvieh bulls was assessed. Oocytes fertilised with sperm from mt/mt males had a lower cleavage rate at 48h post-fertilization than oocytes fertilised with sperm from wt/wt or wt/mt bulls (26±3%, 72±2%, and 79±3%, respectively; P<0.01). In addition, the kinetics of cleavage were slower in the mt/mt group as evidenced by a lower proportion of embryos beyond the 2-cell stage at 48h (32±12.6%, 90±3.6%, and 87±0.4%, respectively, P<0.01). This translated into a lower blastocyst rate at Day 8 in the mt/mt group in comparison with wt/mt and wt/wt groups (2±1%, 27±3%, and 40±2%, respectively, P<0.01). In Experiment 2, fluorescent staining of oocytes 3h after fertilization revealed a lower number of sperm from mt/mt bulls bound to the zona pellucida (ZP) in comparison with sperm from wt/wt or wt/mt individuals (3±1.0, 11±2.3, and 24±9.4, respectively; P<0.05). In order to further study the potential role of TMEM95 in sperm-ZP interaction, in Experiment 3 sperm from the 3 groups were used to fertilize ZP-free oocytes. Consistent with the previous results, sperm from mt/mt bulls were less able to penetrate oocytes with no ZP than sperm from wt/wt or wt/mt bulls (6±2%, 44±8%, and 68±14%, respectively; P<0.05). In conclusion, these results suggest that TMEM95 is involved in events that take place before ZP binding but are required for sperm to interact normally with the oocyte vestments and, ultimately, to achieve fertilization. Further study of the possible role of this protein in the initiation of sperm capacitation and hypermotility, as well as in early embryo development, will shed more light regarding the crucial role of TMEM95 in sperm function.

Infinity sperm storage: The gift that keeps on giving

© 2017 Wiley Periodicals, Inc. & M Female Drosophila retain sperm for up to two weeks post-copulation in a network of storage organs includng the bursa, the seminal receptacle, and the spermathecae. This image shows the seminal receptacle of a Drosophila melanogaster female, 2-hrs post-copulation between a LHM (wild-type) female with a male transgenic for fl uorescently tagged Protamine B (green), and -Tubulin (blue), whose sperm can be visualized within the female. This ‘infi nity’ structure of the seminal receptacle resembles the tubular coil that allows for the storage of very long sperm relative to female body size –up to 20 times the length of the female in some Drosophila species. These unusually long sperm carry essential seminal proteins from the male, which are gradually released from the gamete during their storage in the female (Adams and Wolfner. 2007. J. Insect Physiol. 53:319). These seminal proteins change female behavior, reducing the inclination to mate with other males; altering female feeding, immunity, longevity; and inducing relaxation of the oviduct for copulatory-induced ovulation (Mattei et al. 2015. PNAS. 112:8475). After these male-derived proteins modify her physiology, the female takes control and actively regulates the release of sperm from internal stores in time with ovulation. Gaining insight into the reproductive behavior of Drosophila provides a basic understanding of factors that regulate male and female reproductive success. This model could be translatable to population control of species that impact human health, including mosquitoes, which are vectors for diseases such as malaria, dengue fever, and Zika.