Impact of various cryo-preservation steps on sperm rheotaxis and sperm kinematics in bull.

Semen cryopreservation is an important tool that has massively contributed to the progression of animal reproduction, especially in cattle. Nonetheless, a large part of the sperm population suffers from cryostress and loses fertility during the process. Although bovine semen cryopreservation is more advanced than any other species, there are still some missing links in the technology knowledge. The aim of the current study was to detect the effect of cryopreservation steps on sperm rheotaxis. Semen samples were collected from sex bulls and analyzed inside a microfluidic platform with CASA after each step of cryopreservation, including control, dilution with yolk citrate, cryoprotectant addition, and cooling or freezing. The results showed that positive rheotaxis % (PR) was not affected during cryopreservation. On the contrary, the sperm kinematics of the positive rheotactic sperm undergo significant changes, as velocity parameters (VCL, VSL, and VAP) were lower in both the cryoprotectant adding and cooling/freezing steps than in the control and yolk citrate dilution steps, while progression parameters (LIN and BCF) were higher in the cryoprotectant and cooling/freezing steps than in the control and yolk citrate dilution steps. Beside these results, an interesting phenomenon of sperm backward positive rheotaxis has been observed. The results of backward sperm rheotaxis samples revealed a significant decrease in PR%, while all sperm kinematics except BCF were significantly higher than normal rheotaxis samples. Based on these results, we conclude that positive rheotactic sperm cells are the elite of the sperm population; however, they still get some sublethal cryodamage, as shown by alterations in sperm kinematics. We also suggest that the sperm-positive rheotaxis mechanism is a mixture of an active and passive process rather than a passive physical one.

Autologous mesenchymal stem cell therapy for diabetic men with erectile dysfunction. Is it promising? A pilot study.

PURPOSE: to assess safety and efficacy of autologous mesenchymal bone marrow stem cell injection in penile cavernosal tissue for erectile dysfunction therapy in diabetic men. METHODS: The subjects of this study were diabetic men suffering erectile dysfunction, non-responding to maximum dose of oral PDE5I. Mesenchymal bone marrow stem cells were aspirated and injected after preparation in both corpora cavernosa at 3, 9 o'clock position. Erectile function was assessed by the International Index of Erectile Function and penile Doppler study, before and after 6 months after injection. RESULTS: 4 patients out of 10 achieve hard erection adequate for satisfactory coitus, and 2 patients achieved penile hardness with addition of pharmacological therapy with sildenafil 100mg. Peak systolic velocity increased significantly in 4 patients (2 arteriogenic and 2 mixed erectile dysfunction), from 12∼22 cm/s to 32∼69 cm/s. Variations in end-diastolic velocity increased substantially in 2 patients with venogenic insufficiency alone at follow-up from 4∼5 cm /s to -4∼-3 cm/s. CONCLUSIONS: Despite promising stem cell treatment efficacy for patients with erectile dysfunction, more clinical studies and researches are still warranted.

Sperm tendency to agglutinate in motile bundles in relation to sperm competition and fertility duration in chickens.

A unique sperm behavior was observed in Egyptian chickens. Sperm showed a tendency to agglutinate forming motile thread-like bundles. Sperm agglutination behavior, kinematics, and some morphometric measures were studied in relation to sperm competition and fertility duration in Sharkasi and Dandarawi chickens. Sperm tendency to agglutinate was assessed by examining sperm morphology using scanning electron microscopy, Acridine orange-stained semen smears using fluorescence microscopy, and recording videos of sperm under phase contrast microscope. Sperm velocity and morphometric measures were evaluated using image-J software. To assess sperm competition, Sharkasi and Dandarawi hens were artificially inseminated by semen pools possessing equal number of Sharaksi and Dandarawi sperm. Artificial insemination was repeated ten times. The eggs obtained were incubated, and the hatchlings were discriminated as descending from Sharkasi or Dandarawi fathers according to their phenotype. To assess the fertility duration, Sharkasi and Dandarawi hens were inseminated by semen collected from roosters of the same strain. Eggs were collected for a period of 28 days post-insemination and incubated. Sharkasi spermatozoa showed higher tendency to agglutinate forming longer and thicker motile bundles. No significant differences were observed in sperm curvilinear and straight line velocity and in sperm morphometric measures between Sharkasi and Dandarawi chickens. Sharkasi roosters fathered 81.6% and 67.7% of the hatchlings produced by Sharkasi and Dandarawi mothers, respectively. The fertility period in Sharkasi and Dandarawi was 22 and 14 days, respectively. We suggest that the differences seen in sperm competitiveness and fertility duration can be attributed to sperm agglutination behavior.

New insights into sperm rheotaxis, agglutination and bundle formation in Sharkasi chickens based on an in vitro study.

Fertility in birds is dependent on their ability to store adequate populations of viable sperm for extended durations in sperm storage tubules (SSTs). The exact mechanisms by which sperm enter, reside, and egress from the SSTs are still controversial. Sharkasi chicken sperm showed a high tendency to agglutinate, forming motile thread-like bundles comprising many cells. Since it is difficult to observe sperm motility and behavior inside the opaque oviduct, we employed a microfluidic device with a microchannel cross-section resembling close to that of sperm glands allowing for the study of sperm agglutination and motility behavior. This study discusses how sperm bundles are formed, how they move, and what role they may have in extending sperm residency inside the SSTs. We investigated sperm velocity and rheotaxis behavior when a fluid flow was generated inside a microfluidic channel by hydrostatic pressure (flow velocity = 33 µm/s). Spermatozoa tended to swim against the flow (positive rheotaxis) and sperm bundles had significantly lower velocity compared to lonesome sperm. Sperm bundles were observed to swim in a spiral-like motion and to grow in length and thickness as more lonesome sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Scanning and transmission electron microscopy revealed that sperm bundles were supported by a copious dense substance. The findings show the distinct motility of Sharkasi chicken sperm, as well as sperm's capacity to agglutinate and form motile bundles, which provides a better understanding of long-term sperm storage in the SSTs.

Seminal cyclooxygenase relationship with oxidative stress in infertile oligoasthenoteratozoospermic men with varicocele.

This study aimed to assess the relation of seminal cyclooxygenase COX-1, COX-2 with oxidative stress in infertile oligoasthenoteratozoospermic (OAT) men with varicocele (Vx). In all, 128 men were allocated into fertile men, fertile men with Vx, infertile OAT men without Vx and infertile OAT men with Vx. They were subjected to history taking, clinical examination and semen analysis. Also, seminal COX-1, COX-2, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated. Mean levels of seminal COX-1, COX-2 were over-expressed, the mean level of seminal MDA was significantly increased, and the mean level of seminal GPx was significantly decreased in infertile OAT men with Vx compared with other groups. Seminal COX-1 and COX-2 were over-expressed in cases with Vx grade III compared with Vx grades I, II cases and in cases with bilateral Vx compared with unilateral Vx. There was significant negative correlation between seminal COX-1 and COX-2 with sperm concentration, sperm motility, sperm normal morphology, seminal GPx and significant positive correlation with seminal MDA. It is concluded that seminal COX-1 and COX-2 are over-expressed in infertile OAT men with Vx compared with fertile men with/without and infertile OAT men without Vx being associated with oxidative stress, Vx grade and Vx laterality.

Development of computer-assisted sperm analysis plugin for analyzing sperm motion in microfluidic environments using Image-J.

We modified a previously reported computer-assisted sperm analysis (CASA) plugin for Image-J to enable analyzing motion of sperm cells in microfluidic environments. Microfluidics is increasingly being used in sperm-related applications such as sperm selection, IVF, and sperm motion behavior. Current CASA systems are not capable of analyzing motion of sperm cells in microfluidic devices where both sperm cells and the liquid itself are constantly moving, contrary to the conventional situation of sperm cells moving in a stationary liquid. We resolved this deficiency in the modified plugin reported here and built an image processing pipeline to enhance object detection, which increased CASA accuracy considerably. More importantly, particle tracking was improved and modified to accommodate sperm cells going out of focus for short periods during swimming on the same track. This last feature is particularly important in microfluidics where height of the microchannel is larger than that of CASA custom chambers to avoid channel blockage; this increased height causes sperm cells to frequently come in and out of focus. New parameters were introduced to allow studying new aspects of sperm motion behavior such as rheotaxis and wall tracking. The new plugin was able to detect and analyze motion of human, bull, and chicken sperm. A preliminary study using this tool agreed well with previously reported studies on rheotaxis and wall tracking behavior of sperm.

Characterization of rheotaxis of bull sperm using microfluidics.

We study rheotaxis of bull sperm inside microchannels to characterize the effects of flow and wall shape on sperm swimming behavior. We found that a large percentage of sperm cells, 80 to 84%, exhibited positive rheotaxis (sperm cells swimming against the flow) within flow velocities of 33 to 134 µm s(-1). Sperm cells were also found to reverse their swimming direction when the liquid flow direction was reversed. Time taken by sperm cells to reverse their swimming direction was inversely proportional to the flow velocity. Sperm behavior was significantly affected by the sperm position with respect to the channel wall. Sperm cells close to the channel wall moved upstream faster than sperm cells moving along the channel centerline. Shear stress, which is an indicator of velocity distribution, was found to play an important role in regulating rheotactic behavior of sperm cells. Side pockets were added to some microchannels to mimic storage sites in mucosal folds and pockets in the fallopian tube of the female reproductive system and sperm interaction with these pockets was monitored. We found that sperm cells tend to follow channel walls and enter these pockets without any chemical binding, which further confirms the wall tracking behavior of mammalian sperm cells. Our results confirm that sperm rheotaxis is a strong mechanism for guiding sperm cells towards the oocyte along the female genital tract.

Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

Effect of dinoprost and cloprostenol on serum nitric oxide and corpus luteum blood flow during luteolysis in ewes.

In this study we compared the effect of dinoprost and cloprostenol on changes of corpus luteum blood flow during luteolysis. Ten nonlactating cyclic ewes were synchronized with double PGF2α injections 11 days apart. At Day 10, the animals were classified into 2 groups and received the third dose of PGF2α after confirmation of the presence of a mature CL. The first group received (12.5 mg/im) dinoprost and the second group received (250 µg/im) cloprostenol. A color Doppler ultrasound scan was performed by the same operator according to the following timeline: 0, 0.5, 1, 2, 4, 6, 12, and 24 hours, then every 24 hours until Day 4). The size, morphology, and blood flow of the CL was evaluated during the regression. The results showed that regression of the CL did not differ between the dinoprost and cloprostenol groups. There was no significant effect on diameter of the CL in both groups, though the size of the CL decreased gradually and slowly. Pretreatment progesterone concentration did not differ between groups. The results showed that the nitric oxide level was significantly increased within half an hour after the dinoprost treatment, and was significantly decreased in the cloprostenol group after half an hour. The blood velocity was increased significantly half an hour after the dinoprost treatment and it was decreased in the cloprostenol-treated group. In conclusion, both cloprostenol and dinoprost affect CL by controlling the nitric oxide level and blood supply of the CL via different mechanisms to induce luteolysis.

Development of 3D hydrogel culture systems with on-demand cell separation.

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations.

Cavernous antioxidant effect of green tea, epigallocatechin-3-gallate with/without sildenafil citrate intake in aged diabetic rats.

This study aimed to assess the cavernous antioxidant effect of green tea (GT), epigallocatechin-3-gallate (EGCG) with/without sildenafil citrate intake in aged diabetic rats. One hundred and four aged male white albino rat were divided into controls that received ordinary chow, streptozotocin (STZ)-induced aged diabetic rats, STZ-induced diabetic rats on infused green tea, induced diabetic rats on epigallocatechin-3-gallate and STZ-induced diabetic rats on sildenafil citrate added to EGCG. After 8 weeks, dissected cavernous tissues were assessed for gene expression of eNOS, cavernous malondialdehyde (MDA), glutathione peroxidase (GPx), cyclic guanosine monophosphate (cGMP), and serum testosterone (T). STZ-induced diabetic rats on GT demonstrated significant increase in cavernous eNOS, cGMP, GPx and significant decrease in cavernous MDA compared with diabetic rats. Diabetic rats on EGCG demonstrated significant increase in cavernous eNOS, cGMP, GPx and significant decrease in cavernous MDA compared with diabetic rats or diabetic rats on GT. Diabetic rats on EGCG added to sildenafil showed significant increase in cavernous eNOS, cGMP and significant decrease in cavernous MDA compared with other groups. Serum T demonstrated nonsignificant difference between the investigated groups. It is concluded that GT and EGCG have significant cavernous antioxidant effects that are increased if sildenafil is added.

Effect of testosterone and frequent low-dose sildenafil/tadalafil on cavernous tissue oxidative stress of aged diabetic rats.

This study aimed to assess the effect of testosterone (T) administration and chronic low-dose sildenafil/tadalafil on cavernous tissue oxidative stress (OS) of aged diabetic rats. In all, 140 Sprague-Dawley aged rats were subdivided into the following: controls; streptozotocin (STZ)-induced diabetic rats; diabetic rats injected with T every 4 weeks; diabetic rats on sildenafil orally daily; diabetic rats on T and daily sildenafil; diabetic rats on tadalafil orally every other day; diabetic rats on T and tadalafil; diabetic rats on alternate sildenafil/tadalafil; and diabetic rats on alternate sildenafil/tadalafil with T. After 12 weeks, the rats were euthanised where in dissected cavernous tissues malondialdehyde (MAD), glutathione peroxidase (GPx) and cGMP (cyclic guanosine monophosphate) were estimated. Compared with controls, aged diabetic rats demonstrated significant increase in cavernous tissue MDA and significant decrease in GPx and cGMP where diabetic rats injected with T had marked improvement of these parameters. Diabetic rats on sildenafil, tadalafil or alternate sildenafil/tadalafil demonstrated significant increased cavernous tissue GPx, cGMP and decreased cavernous MDA that was further improved when supplemented with T. It is concluded that frequent low-dose use of sildenafil and/or tadalafil supplemented with T has a marked impact on ameliorating cavernous OS in aged diabetic rats.

Three-dimensional in vitro tri-culture platform to investigate effects of crosstalk between mesenchymal stem cells, osteoblasts, and adipocytes.

The bone marrow niche for mesenchymal stem cells (MSCs) contains different amounts of bone and fat that vary with age and certain pathologies. How this dynamic niche environment may affect their differentiation potential and/or healing properties for clinical applications remains unknown, largely due to the lack of physiologically relevant in vitro models. We developed an enabling platform to isolate and study effects of signaling interactions between tissue-scale, laminated hydrogel modules of multiple cell types in tandem. We applied this platform to co- and tri-culture of primary human MSCs, osteoblasts, and adipocytes over 18 days in vitro. Each cell type was analyzed separately with quantitative polymerase chain reaction (qPCR) and histochemistry for several mesenchymal lineage markers. Distinct expression dynamics for osteogenic, adipogenic, chondrogenic, and myogenic transcriptional regulators resulted within each cell type depending on its culture setting. Incorporating this data into multivariate models produced latent identifiers of each emergent cell type dependent on its co- or tri-culture setting. Histological staining showed sustained triglyceride storage in adipocytes regardless of culture condition, but transient alkaline phosphatase activity in both osteoblasts and MSCs. Taken together, our results suggest novel emergent phenotypes for MSCs, osteoblasts, and adipocytes in bone marrow that are dependent on and result in part from paracrine interactions with their neighboring cell types.

Development of nano- and microscale chondroitin sulfate particles for controlled growth factor delivery.

Size scale plays an important role in the release properties and cellular presentation of drug delivery vehicles. Because negatively charged chondroitin sulfate (CS) is capable of electrostatically sequestering positively charged growth factors, CS-derived nanoscale micelles and microscale spheroids were synthesized as potential growth factor carriers to enhance differentiation of stem cells. Particles were characterized for morphology, size distribution, surface charge and cytocompatibility, as well as release of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α). CS micelles were spherical and negatively charged with a bimodal distribution of 324.1±8.5 and 73.2±4.4 nm diameters, and CS microspheres possessed a rounded morphology and a diameter of 4.3±0.93 µm. Positively charged TGF-ß1 demonstrated minimal release after loading in CS microspheres, while negatively charged TNF-α exhibited substantial release over the first 15 h, suggesting that TGF-ß1 electrostatically complexed with CS. The micelles and microparticles were found to be cytocompatible at moderate concentrations with marrow stromal cell monolayers and within embryonic stem cell embryoid bodies. These synthesis techniques, which allow the formation of CS-based carriers over a variety of nano- and microscale sizes, offer versatility for tailored release of positively charged growth factors and controlled CS presentation for a variety of stem cell-based applications in tissue engineering and regenerative medicine.

Long-term spatially defined coculture within three-dimensional photopatterned hydrogels.

Spatially controlled coculture in three-dimensional environments that appropriately mimic in vivo tissue architecture is a highly desirable goal in basic scientific studies of stem cell physiological processes (e.g., proliferation, matrix production, and tissue repair) and in enhancing the development of novel stem-cell-based clinical therapies for a variety of ailments. This study describes a novel fabrication system for photopatterning and assembling cell-laden oligo(polyethylene glycol)-fumarate:poly(ethylene glycol)-diacrylate hydrogels with high spatial fidelity and thickness using a controlled, inert nitrogen environment without the need for expensive precision equipment. Cross-linking was performed using Irgacure-2959 photoinitiator and 365-nm light (∼7 mW/cm²) to form gels ranging from 0.9 to 3 mm in width. Employing a nitrogen environment increased gel thickness up to 240%, generating gels > 1 mm thick before swelling. This technique was further applied for spatially controlled patterning of primary tendon/ligament fibroblasts and marrow stromal cells in a single 1.5-mm-thick laminated hydrogel construct. Cells encapsulated using this technique maintained viability over 14 days in culture. This system potentially enables better understanding of paracrine effects on a range of stem cell functions and therefore may be useful as an in vitro model system for a wide array of regenerative medicine applications.