Isolation, culture, and characterization of equine oviduct epithelial cells in vitro

A reorientation of the sperm head so that it is perpendicular to the sperm tail (i.e., T-shape or thumbtack) is considered an indicator of sperm capacitation in the Australian marsupial the brushtail possum (Trichosurus vulpecula). This study describes a method of oviduct epithelial cell monolayer and sperm coculture in the brushtail possum to obtain a high percentage of thumbtack sperm. The oviduct epithelial cell (OEC) monolayers were prepared in vitro from the isthmal and ampullary segments of eCG- and LH-primed brushtail possum oviducts. Coculture experiments demonstrated that cauda epididymidal sperm from the brushtail possum attached equally to the OEC monolayers derived from the isthmal and ampullary segments of the oviduct. After 2 h of coculture, a large number of sperm attached to OEC monolayers (ampulla, 60.1+/-4.7% and isthmus, 63.1+/-5.7%) as well as to controls (tracheal epithelial cell monolayer, 46.2+/-3.7%; Matrigel, 57.4+/-7.7%; plastic, 29.2+/-3.2%). After 6 h, fewer sperm were attached to tracheal epithelial cell monolayers (1.2+/-0.2%; P<0.01) and Matrigel (10.2+/-2.5%; P<0.01), compared to those attached to ampullary and isthmal OEC monolayers (37.9+/-7.2% and 44.6+/-2.2%, respectively), and none were attached to the plastic surface. Fewer sperm were released from the ampullary and isthmal OEC monolayers compared to those from controls (P<0.05). At 6 h of coculture with ampullary and isthmal OEC, the percentage motility of both attached and unattached spermatozoa was maintained at 40-50%, which was higher (P<0.05) than in controls. Progressive motility of unattached sperm was maintained at about 2 (on an arbitrary scale of 1-5) and was not different among treatments until 6 h. More than 60-70% sperm were viable at 6 h of coculture in all the treatments. Coculture of brushtail possum epididymal sperm with OEC monolayers transformed 60% of motile streamlined spermatozoa to thumbtack orientation at 2 h compared to approximately 25% in controls. No acrosomal modifications were induced in spermatozoa in any of the treatments. This study has demonstrated a role of the oviduct in transforming a large number of sperm from a streamlined to thumbtack orientation, which may have relevance in sperm capacitation and fertilization in this species.

Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Induction of Thumbtack Sperm During Coculture with Oviduct Epithelial Cell Monolayers in a Marsupial, the Brushtail Possum (Trichosurus vulpecula)1

Sidhu

Mate

Molinia

et al. 1999

“…Unless otherwise noted, all reagents were supplied by Sigma Chemical Company (St. Louis, MO). Monolayers of OEC were derived from the oviductal isthmus of two mares in the preovulatory stage of the cycle [24]. Briefly, oviducts were opened longitudinally and epithelial cells were mechanically dissociated from the underlying stroma.…”

Section: General Methodsmentioning

confidence: 99%

Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro

Dobrinski

Suárez

Ball

1996

Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and at 0.5, 2, 4, and 6 h of incubation by ratio image analysis of fluorescent images captured at 405 nm and 490 nm. At each time point, [Ca2+]i was lower in motile spermatozoa attached to OEC than in free-swimming spermatozoa. [Ca2+]i in spermatozoa attached to Matrigel was lower than in free-swimming spermatozoa and was comparable to [Ca2+]i in spermatozoa attached to OEC only at 0.5 h incubation. Beyond 0.5 h of incubation, [Ca2+]i was higher in spermatozoa attached to Matrigel than in spermatozoa attached to OEC. These results indicate that spermatozoa with low [Ca2+]i might preferentially attach to OEC and to Matrigel, but that [Ca2+]i is maintained at this basal level only in spermatozoa attached to OEC. The reduced [Ca2+]i in spermatozoa associated with OEC may prevent premature capacitation and acrosomal exocytosis of spermatozoa stored in the isthmic sperm reservoir.

“…With regard to the quantification of sperm-oviduct binding, we were aware that standardization is the main challenge when using equine oviduct explants, due to the invaginated and irregular oviductal surface, which is very different from the flattened surface of an oviduct monolayer [66,67]. To achieve standardization in the oviduct explant model, we decided that the ideal sperm concentration needed to be below the sperm- 6 spermatozoa/ml), and this was set in noncapacitating conditions at 2 3 10 6 spermatozoa/ml.…”

Section: Leemans Et Almentioning

confidence: 99%

Oviduct Binding and Elevated Environmental pH Induce Protein Tyrosine Phosphorylation in Stallion Spermatozoa1

Leemans

Gadella

Šoštarić

et al. 2014

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 3 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 timedependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviductbound 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 (pH i ) 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 pH i , presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pH i 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.