Unbound Sperm Research Articles

P-049 Identification of human spermatozoa with zona pellucida-binding ability using deep learning

Abstract Study question Can a deep learning model be used for identification of fertilization-competent human spermatozoa based on their distinctive morphological features? Summary answer Our results validated the clinical significance of image analysis of sperm morphology using deep learning to identify high-quality spermatozoa with zona pellucida (ZP)-binding ability. What is known already Sperm morphology is manually evaluated as part of the conventional semen analysis in clinical settings. However, the clinical predictive power of sperm morphology remains debatable due to its lack of association with the fertilization potential of spermatozoa. Human zona pellucida (ZP)-binding ability is a measurable, independent sperm parameter associated with in vitro fertilization (IVF) and pregnancy outcome. Supervised deep learning is an advanced branch to systematically identify the underlying relationship between the input images and designated output. A well-trained model with high generalization ability can generate plausible predictions for unseen images collected under the same condition as the training datasets. Study design, size, duration Human spermatozoa and oocytes were obtained with written consents from the donors attending our assisted reproduction program. Acrosome intact, ZP-bound spermatozoa were recovered after 30 min of gamete coincubation. Unbound spermatozoa were collected from clinical samples of spermatozoa with defective ZP-binding ability leading to fertilization failure following conventional insemination. Sperm images of infertile men (n = 165) with fertilization rates including 0-40% (low), 41-70% (intermediate) and 71-100% (high) following conventional insemination were collected for validation. Participants/materials, setting, methods Diff-Quik stained images of spermatozoa were processed by the K-means clustering algorithm to generate grey-scaled images of individual sperm heads. All sperm images of clinical samples were subjected an additional image conversion step using Cycle Generative Adversarial Network (CycleGAN) to ensure that their microenvironments are similar to those of the laboratory samples. A VGG 13 model was fine-tuned to classify a total of 1,083 images of ZP-bound and unbound spermatozoa over 50 epochs. Main results and the role of chance The newly fine-tuned VGG-13 model demonstrated excellent performance in classifying the ZP-bound and unbound spermatozoa, as reflected by the high sensitivity of 97.6%, specificity of 96.0%, and accuracy of 96.4%. It had a high discriminative power to distinguish between ZP-bound and unbound spermatozoa with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.992. Saliency maps indicated that the model focused on the sperm head and mid-pieces in all images, with a primary emphasis on the anterior regions (43.0%±5.0%). The model was further validated on a vast collection of over 40,000 sperm images collected from the aforementioned fertilization groups. Our results revealed that the high fertilization rate group had a significantly higher percentage of predicted ZP-binding ability than the low fertilization rate group, as determined by the model (16.8%±7.2 vs. 3.2%±1.4; p < 0.05). By analyzing sperm images from infertile men (n = 113) in the high and low fertilization rates groups, a clinical threshold of 4.8% was identified using ROS curve (AUC:0.970) and Youden’s index, which can be used to distinguish between ZP-bound and unbound spermatozoa of clinical samples with a specificity of 93.8% and a sensitivity of 90.9%. Limitations, reasons for caution Our deep learning model is only applicable to diff-quik stained images of spermatozoa captured at high magnification. Further investigation is needed to examine its classification performance on images captured by different platforms and settings, ensuring consistent and reliable prediction accuracy regardless of varying degrees of image quality. Wider implications of the findings Our newly fine-tuned model can be used to evaluate sperm fertilizing potential, irrespective of the parameters evaluated in conventional semen analysis and to identify normospermic patients with defective ZP-binding ability who may benefit from intracytoplasmic sperm injection (ICSI) to improve their fertilization outcome. Trial registration number not applicable

#338 : Insights into the Zona-Pellucida Mediated Selection of Fertilization-Competent Spermatozoa

Background and Aims: Human zona pellucida (ZP) selectively interacts with high-quality spermatozoa characterized by superior morphology and normal chromatin structure to achieve fertilization. Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. Method: Human spermatozoa and oocytes were obtained with written consents from the donors attending our assisted reproduction program. Acrosome intact, ZP-bound and unbound spermatozoa were recovered using the modified spermatozoa-ZP coincubation assays. ZP-binding protein expression on ZP-bound and unbound spermatozoa were examined by immunofluorescence staining. And the genetic integrity and epigenetic landscapes were evaluated by TUNEL assay, COMET assay, acridine orange staining, chromomycin A3, and immunofluorescence staining for 5-methylcytosine. Results: The time course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. ZP-bound spermatozoa had a higher percentage of positive HSPA2 (85.8% vs. 45.9%, p<0.05) and SPACA3 signals (86.3% vs. 30.5%, p<0.05) than the unbound ones. The signal intensities of both markers were stronger in the ZP-bound spermatozoa than the unbound ones. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound ones. Conclusion: Our results validated the functional role of spermatozoa-ZP interaction in providing diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

P-103 Three-dimensional oviductal organoid model to study spermatozoa-oviduct interaction in humans

Abstract Study question Is it possible to establish an in vitro model that recapitulating human oviduct to investigate sperm-oviduct interaction Summary answer Three-dimensional (3D) oviductal organoid model was established from either primary human oviductal tissues or immortalized human OE-E6/67 cell line, based on our protocol. What is known already The sperm reservoir in oviduct is formed to maintain the fertilizing capacity of spermatozoa until ovulation by the binding of sperm to the epithelial lining of the oviduct, which is also significant to select sperm with normal chromatin structure and superior fertilization ability. The regulation and mechanism of sperm reservoir formation in human is still unknown, mainly due to the ethical issue and technical difficulty in obtaining enough human oviduct. We previously demonstrated that sperm fucosyltransferase-5 (sFUT5), a carbohydrate-binding protein on spermatozoa, is responsible for spermatozoa-oviduct interaction in humans. Study design, size, duration Human oviductal epithelial cell line immortalized by HPV 16 E6/E7 open reading frame by retroviral expression system was employed to establish the co-culture model. Fresh human samples (human spermatozoa,N = 30; 1-2 cm primary oviductal tissue,N = 15) were collected in hospital with signed consent. Fluorescent pre-labelled sperm were co-cultured with organoids for 2 hours. At least three replicates were conducted for each assay. Participants/materials, setting, methods Morphological characterization and markers expressions of the organoid were determined by immunohistochemistry and confocal microscopy analysis. The sperm binding capacity of the oviductal organoid was studied by co-culture system. Sperm chromatin structure of the organoid-bound sperm was assessed by flow cytometry-based TUNEL assay. Affinity chromatography followed by nano-liquid chromatography–mass spectrometry was used to identify the sFUT5-binding proteins on ovidutal epithelial cells (OECs) and their expressions on OECs were validated by immunostaining/flow cytometry. Main results and the role of chance The organoid exhibited a hollow structure surrounded by a single layer of OECs. Immunohistochemistry analysis revealed the high expressions of epithelial marker E-cadherin (basolateral membrane), secretory marker pair-box 8 (PAX8) and proliferative marker Ki67. Expression of ciliated cell marker, acetylated tubulin (AcTUB) can also be detected. The modification of culture environment successfully reversed the OEC organoids polarity as demonstrated by immunostaining using antibodies against ZO-1 (apical marker) and E-cadherin (basolateral marker), making the organoid accessible during the co-culture. These apical-out OEC organoids have significantly higher sperm binding capacities than their basal-out counterparts. Furthermore, OEC organoid-bound spermatozoa possessed more intact DNA than the unbound spermatozoa. The results indicate the relevance of using OEC organoid-spermatozoa co-culture model as a tool to assess spermatozoa-oviduct interaction in humans. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was then identified as a potential sFUT5-interacting protein on OECs. This result was further supported by flow cytometry and immunofluorescent staining. 3-D oviductal organoid model demonstrates profound potential for in vitro investigation of human oviduct physiology. Limitations, reasons for caution Our 3D model may not fully represent the entire cellular complexity and tubular architecture of the oviductal epithelium. Further investigation on the sperm morphology and DNA integrity and analysis on organoid transcriptome will enhance our understanding on oviduct in human reproduction. Wider implications of the findings The results from co-culture may give a clue to future study on whether oviductal organoid can be applied clinically for functional sperm selection in in vitro fertilization. Our apical-out organoid model may also be applied to study tubal ectopic pregnency. Trial registration number not applicable

The role of spermatozoa-zona pellucida interaction in selecting fertilization-competent spermatozoa in humans.

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The time-course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.

P–064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer: The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings: In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number Not applicable

P-064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number not applicable

Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead's surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis)

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67–96.56%) compared to unbound (range 8.70–21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo.

Oviductal epithelial cells selected boar sperm according to their functional characteristics

The interaction of oviductal epithelial cells (OECs) with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization (IVF), tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF: (i) W30 group: spermatozoa were incubated with oocytes in the absence of OEC; (ii) NB group: after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and (iii) B group: after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates (NB: 19.6%, B: 94.9%, and W30: 62.9%; P < 0.001) and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups (NB: 58.7%, B: 2.5%, and W30: 4.5%; P < 0.01). A similar trend was observed in phosphatidylserine translocation (NB: 93.7%, B: 5.7%, and W30: 44.2%; P < 0.01). These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

149 DEVELOPMENT OF AN IN VITRO BIOLUMINESCENT SPERM BINDING ASSAY: PRELIMINARY DATA

Quantum dot technology has enabled researchers to incorporate the intrinsic properties of such nanoparticles into physiological exploration. Previous work from our laboratory has demonstrated that quantum dots can be incorporated into spermatozoa without deleterious effects to physiological parameters such as motility, viability, and fertilizing potential (Feugang et al. 2012). However, the journey of spermatozoa within the female reproductive tract is met with many physicochemical obstacles and checkpoints that include the binding of spermatozoa to utero-oviducal epithelial cells. Moreover, the binding ability/affinity of quantum dot-labelled spermatozoa has not been tested and therefore, the objective of this study is to test the binding semblance of quantum dot-labelled spermatozoa to uterine epithelial cells compared to normal sperm, and the subsequent use of the technology to develop a bioluminescent sperm binding assay. Porcine uterine epithelial (PUE) cells were seeded into 96-well clear-bottomed plates (20 000 cells/well) and allowed to grow to 95% confluency. Motile spermatozoa were selected from fresh pooled semen of fertile boars and labelled with quantum dot nanoparticles to form quantum sperm, as previously described (Feugang et al. 2012). Final concentrations of 107 labelled (QD+) and non-labelled (QD–) spermatozoa were added to monolayers of PUE cells and co-incubated in PBS/polyvinylpyrrolidone (PVP) at 37°C, 5% CO2. The control consisted of PUE cells alone in the PBS/PVP medium. Each treatment was performed in triplicate and experiments were repeated 3 times. After 1 h of co-incubation, the supernatant from each well was transferred to the adjacent three wells. The co-incubated wells containing expected PUE-sperm binding were then washed 3 times with PBS/PVP to eliminate any unbound sperm. PUE-quantum sperm (QD+) and PUE-non-labelled sperm (QD–) complexes were verified using bright field microscopy, followed by measurement of photonic emission from each well (GloMax Multi Detection System, Promega, Madison, WI, USA). Data was analysed by ANOVA with the threshold of significance fixed at P &lt; 0.05. There were no visual differences in binding patterns between QD+ and QD–, which appeared similar under the microscope. However, the photonic signals (relative luminescent units; RLU) from QD+ wells were significantly higher than both the control and QD– wells (2534.84 ± 639.91 v. 542.46 ± 639.91 and 806.48 ± 639.91 RLU; P &lt; 0.05). Supernatants collected from the QD+ wells, representing unbound quantum sperm, had the highest photonic emissions when compared to all other wells, with or without spermatozoa (19 948.23 ± 639.91 RLU; P &lt; 0.05). Results demonstrate that quantum dot nanoparticles can be incorporated into boar spermatozoa without affecting their binding affinity to uterine epithelial cells, and their subsequent use in a biophotonic sperm binding assay. Further optimization and experimentations are ongoing to establish whether bioluminescent quantum sperm could serve to develop sensitive in vitro binding assays to better characterise sperm viability. Support was provided by U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) grant number 58-6402-3-0120

245 EFFECT OF INCUBATION IN OVIDUCTAL EPITHELIAL CELLS AND CO-CULTURE WITH OOCYTES IN THE PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF BOAR SPERM

The oviductal epithelial cells (OEC) play an important role in the process of sperm capacitation and the acquisition of fertilizing capacity. One of the most important changes that can be observed in spermatozoa is the increase in protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm after incubation in oviductal cells in the presence of oocytes. Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on a petri dish. Sperm cells treated by a discontinuous gradient of isotonic Percoll were added to the OEC and incubated for 30 min. After 30 min, the spermatozoa bound and unbound were separated and co-cultured with oocytes for 18 h. Five different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al., 2001 Biol. Reprod. 65, 784–792): ejaculated control, sperm unbound OEC after 30 min, sperm unbound after 30 min with oocytes, sperm bound after 30 min with oocytes, and sperm bound after 30 min and detached after culture with oocytes. Five patterns were determined according to their surface distribution: nonphosphorylated spermatozoa (pattern 1), head, tail, or both phosphorylated (pattern 2), equatorial segment phosphorylated (pattern 3), equatorial segment and head, tail, or both phosphorylated (pattern 4), and phosphorylation of the tail alone and in combination with other areas of the sperm (pattern 5). Sperm without any treatment showed lower tyrosine phosphorylation levels (87.04%; P &lt; 0.05). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment and head, tail, or both phosphorylated (47.71%; P &lt; 0.05), and this pattern was associated with a high level of capacitation. The same pattern was reduced when unbound sperm were co-cultured with oocytes for 18 h (25.67%; P &lt; 0.05), and was even lower in sperm bound with oocytes. The study of phosphorylation of the tail (pattern 5) showed no significant differences between different samples. We concluded that sperm-bound OEC have lower levels of phosphorylation, indicating a selective function in the sperm-OEC interaction. In addition, phosphorylation of the tails appears to be related more to the hyperactivation of sperm than to the different culture conditions.

244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 109 sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P &lt; 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.

Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida

To determine if Raman microspectroscopy (RMS) can distinguish sperm bound to the human zona pellucida (ZP) from those unbound sperm. Paired experiments to compare Raman scanning features of ZP-bound and unbound sperm. Public hospital-based clinical assisted reproduction center. Sperm samples from ten fertile donors were used in this study. None. Sperm-ZP binding, ZP-induced acrosome reaction, and scanning intensity of various regions of sperm. The RMS found two slightly low-intensity regions (800-900 and 3,200-4,000 cm(-1)) shifted to high-intensity grade at the acrosome region of the ZP-bound sperm compared with unbound sperm. Moreover, principal component analysis and statistical analysis showed that the RMS can distinguish the ZP-bound sperm from the unbound sperm. RMS scanning of single live sperm could be used to distinguish ZP-bound sperm from unbound sperm. Thus, RMS may be a useful tool to detect normal functional sperm and to select sperm for intracytoplasmic sperm injection.

236 PROTEIN TYROSINE PHOSPHORYLATION IN BOAR SPERM DURING CO-CULTURE WITH OVIDUCTAL EPITHELIAL CELLS

Essentials steps of the capacitation are coordinated in the caudal isthmus of the oviduct, where sperm are stored in intimate contact with the epithelium. A crucial event involved in capacitation is the protein tyrosine phosphorylation. The aim of this study was to characterise the different protein tyrosine phosphorylation patterns in boar sperm after the co-culture with oviducal epithelial cells (POEC). Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on Petri dishes. Sperm (with and without treatment by discontinuous gradient of isotonic Percoll) were added and co-incubated for 1 h. Six different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al. 2001 Biol. Reprod. 65, 784–792): ejaculated control, ejaculated bound and unbound POEC, sperm washed through Percoll, and sperm Percoll-washed bound and unbound POEC. Four patterns were determined according to their surface distribution: nonphosphorylated spermatozoa, head and/or tail phosphorylated, equatorial segment phosphorylated, equatorial segment, and head and/or tail phosphorylated. Sperm without any treatment showed lower tyrosine phosphorylation levels than sperm washed through Percoll (66.90% v. 1.90%; P &lt; 0.01). The most common tyrosine phosphorylation distribution in sperm adhesion to POEC was located in equatorial segment (57.78%; P &lt; 0.01). A significant difference regarding head phosphorylation (P &lt; 0.01) was found between sperm bound to oviductal epithelium and unbound sperm (0.00% v. 4.75%). Binding to POEC occurred mainly in sperm with nonphosphorylated heads and tails, whereas higher proportions of equatorial segment and head and/or flagellum phosphorylated cells were found in unbound population (50.25% v. 83.75%; P &lt; 0.01). The data of this study showed differences in the sperm-POEC binding in relation to protein tyrosine phosphorylation patterns, indicating a selective function in the sperm-oviductal epithelial cells interaction. Supported by Fundación Séneca 08752/PI/08.

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology

During spermiogenesis, a plasma membrane remodelling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. Enrichment of Tygerberg normal spermatozoa in HA-bound versus semen sperm fractions was postulated. Semen was placed on the uncoated A side and HA-coated B side of a semen chamber. After 15 min, the HA binding score (proportion of HA-bound motile spermatozoa) was assessed on the B side, and unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology of A and B sides was evaluated by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal spermatozoa was higher in HA-bound versus semen spermatozoa ( n = 63 subjects) with a 3.04-fold improvement (95% confidence limits: 1.9–4.7) in 37 teratozoospermic men, comparable with a 4.2-fold enrichment in zona pellucida-bound spermatozoa reported earlier by the Tygerberg group. The morphology scores of three investigators were different but related, indicating that the variations reflect individual-to-individual differences in the perception of shape normality. The selection power of HA and zona pellucida for normal spermatozoa are similar. The sperm biomarkers of creatine phosphokinase (reflecting retained cytoplasm in arrested maturity spermatozoa) and chaperone protein HspA2 (heat shock protein) were proportional with sperm HA binding. As HA binding reflects sperm maturity and function, the combination of Tygerberg morphology and HA binding is likely to improve male infertility management.

Preliminary study of embryo development following assessment of male and female gametes

Gamete characteristics are likely to contribute to embryo development. In this preliminary study, early parameters were assessed in embryos generated following microinjection of hyaluronan-bound and unbound spermatozoa into 176 sibling oocytes, which had themselves been assessed using a polarizing light microscope imaging system. The fertilization and early cleavage rates, and day 3 embryo quality did not differ when oocytes displaying similar characteristics were inseminated with either bound or unbound sperm. Regardless of the binding characteristics of the sperm, early embryos displaying good and poor quality derived from oocytes displaying visible spindles and similar characteristics. These results indicate that early embryo developmental characteristics were independent from the hyaluronic acid binding capacity of the sperm when oocytes displayed a visible spindle.

Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies

To test a newly invented intracytoplasmic sperm injection (ICSI) sperm selection method based on sperm hyaluronic acid (HA) binding. Comparison of chromosomal disomy and diploidy frequencies in sperm arising from semen and in HA-bound sperm. Academic andrology laboratory. Men presenting for semen analysis. Washed sperm fractions of 32 semen samples were applied to Petri dishes or glass slides coated with immobilized HA. The unbound sperm were rinsed gently, and the HA-bound sperm were removed with an ICSI pipette. The control sperm population was the unselected sperm. Both HA-selected and unselected sperm were treated with fluorescence in situ hybridization with centromeric probes for the X, Y, and 17 chromosomes. Chromosomal disomy and diploidy frequencies. In the HA-bound sperm (495-2,079 per man, 41,670 in all) compared with unselected sperm (4,770 per man, 162,210 in all), the chromosomal disomy frequencies were reduced to 0.16% from 0.52%, diploidy to 0.09% from 0.51%, and sex chromosome disomy to 0.05% from 0.27% (a 5.4-fold reduction vs. 4-fold respective increase in ICSI offspring). The HA sperm selection method for ICSI, which is based on a relationship between sperm receptors for zona pellucida and HA, will likely reduce the potential genetic complications and adverse public health effects of ICSI.

Kinetics of protein tyrosine phosphorylation in sperm selected by binding to homologous and heterologous oviductal explants: how specific is the regulation by the oviduct?

Essential steps of the capacitation process take place in the oviductal isthmus. A crucial step in the process of capacitation is the phosphorylation of membrane proteins. The aims of this work were (1) to study the effect of dog sperm binding to oviductal epithelium on tyrosine phosphorylation and (2) to investigate the specificity of regulation of molecular changes by the oviduct of different species by comparing the numbers of canine sperm bound to heterologous (porcine) and homologous epithelium, and the kinetics of tyrosine phosphorylation. Semen was collected from four healthy dogs and washed through a Percoll gradient. Explants, small pieces of epithelium, were cut from porcine and estrous bitch oviducts. During 6 h of coincubation in Tyrode medium, the numbers of bound sperm were counted by microvideographic observation, and the state of tyrosine phosphorylation was determined immunocytochemically after 3, 30, 90, 180 and 360 min. Canine sperm bound in similar numbers to homologous and heterologous explants. Increasing tyrosine phosphorylation of tail proteins and subsequent phosphorylation of sperm head proteins were observed. Binding occurred mainly in sperm with non-phosphorylated heads (∼2% phosphorylated), while higher proportions of head-phosphorylated cells were found in unbound populations (∼40–60%; P<0.05). The head phosphorylation progressed significantly during incubation in unbound spermatozoa ( P<0.05), while it was suppressed in bound suspensions. The rate of tyrosine phosphorylation of sperm tail proteins was higher in cells bound to explants than in unbound cells or in those incubated in control medium. There were no significant differences with respect to the kinetics of tyrosine phosphorylation between the two coincubation systems. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. Tyrosine phosphorylation of sperm head proteins and capacitation are delayed in spermatozoa in close contact with oviductal epithelium. This mechanism appears to be species-independent, as sperm bound similarly to pig and dog oviduct explants, and similar phosphorylation kinetics were observed in both types of tissue.

Regulation of capacitation of canine spermatozoa during co-culture with heterologous oviductal epithelial cells.

Progress of essential steps of the capacitation is coordinated in the oviductal isthmus, where sperm are stored in close contact with the epithelium. A crucial capacitational event is the phosphorylation of sperm membrane proteins. Regulation of the tyrosine phosphorylation by the oviduct has not been examined in dog sperm yet. The aim of this work was to study the effect of dog sperm binding to porcine oviductal epithelium on capacitation-induced cellular and molecular changes. Epithelial cells were stripped from the oviducts of post-puberal sows and cultured for 5-7 days at 39 degrees C and 5% CO2 on Biomatrix-covered Chamber slides. Sperm washed through Percoll was co-incubated with the oviductal epithelium cell cultures in a bicarbonate Tyrode's medium. During co-incubation, sperm membrane changes, the state of tyrosine phosphorylation and motility were determined after 3, 30, 90, 180, 240 and 360 min. Significant increases in the percentage of capacitated and dead cells were observed in unbound sperm, while bound sperm remained uncapacitated, live and motile. An increasing tyrosine phosphorylation of tail proteins in bound, unbound and control sperm suspensions and a subsequent phosphorylation of head proteins in unbound and control sperm suspensions were observed. A significant difference regarding head phosphorylation (p < 0.05) was found between sperm bound to oviductal epithelium and unbound sperm. Binding occurred mainly in sperm with non- phosphorylated heads, while higher proportions of phosphorylated cells were found in unbound populations. The head phosphorylation progressed significantly during incubation in unbound spermatozoa (p < 0.05); however, it was suppressed in population of sperm attached to oviductal epithelium. Significant correlations between motility parameters related to hyperactivation and tail phosphorylation were found in unbound sperm. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. It can be concluded that tyrosine phosphorylation of head membrane proteins and capacitation are delayed in canine spermatozoa being in closed contact with oviductal epithelium.