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Biology of Reproduction 61, 1356-1361 (1999)
© 1999 Society for the Study of Reproduction, Inc.

Articles

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

K.S. Sidhu2,a, K.E. Matea, F.C. Moliniab, and J.C. Rodgera

a Co-operative Research Centre for Conservation and Management of Marsupials, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia b Manaaki Whenua Landcare Research, Lincoln, Canterbury, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many eutherian species (for review see [1]) and one South American marsupial, Monodelphis domestica [2], capacitation and fertilization have been achieved in vitro in the absence of any oviducts-derived factor(s). However, in vitro fertilization has not been achieved for any Australian marsupial, suggesting that unknown factor(s) possibly derived from the oviduct may play a more critical, or even essential, role in preparing the sperm and possibly egg for fertilization (for review see [3, 4]). Recent in vitro and in vivo studies involving Australian marsupials support this suggestion [5–7].

As in most eutherian mammals [8, 9], marsupial spermatozoa are required to maintain their fertilizing capacity for extended periods within the female reproductive tract until the arrival of an egg. There is a considerable interval, varying from several hours (possums [phalangerids], kangaroos [macropods], and opossums [didelphids]) to several days (dasyurids), between mating and ovulation [10–14]. The caudal isthmal oviduct functions as a storage reservoir in most mammals (including marsupials), in which sperm are stored attached to the oviduct epithelial lining in specialized crypts or simply trapped in the secreted mucus [11–13, 15–21]. In the brushtail possum and tammar wallaby, sperm associate with the oviducal mucosa, which has deep folds, but there are no specialized crypts for sperm storage [19] as described in two other marsupials, the American opossum (Didelphis virginiana) and brown antechinus (Antechinus stuartii) [11, 12].

In most Australian marsupials, spermatozoa are released during spermiation with a thumbtack orientation and achieve a streamline orientation during epididymal transit (for review see [4]). These streamlined epididymal sperm again turn thumbtack in the oviduct as seen in situ in an Australian marsupial, Sminthopsis crassicauda [5], and in sperm flushed from the brushtail possum oviduct around the time of fertilization [6]. In marsupials, the sperm head has distinct dorsal and ventral aspects because of a different plane of nuclear flattening during spermiogenesis; this plane in marsupials is at 90° to that in eutherian mammals (for references on marsupial sperm see [22]). Current evidence suggests that marsupial spermatozoa associate with the zona pellucida via the acrosome, which is located on the dorsal surface of the sperm head (for review of the dorsi-ventral morphology of marsupial sperm see [22]), indicating that thumbtack orientation of spermatozoa may be important for fertilization in these species.

Several in vitro studies in eutherian species [19–21] and one study in marsupials [7] have demonstrated that spermatozoa attach to monolayers of cultured oviduct epithelial cells and that attachment results in functional changes in both spermatozoa and oviduct cells, which may be essential for fertilization. This study describes a model system of OEC monolayers and sperm coculture in the brushtail possum (Trichosurus vulpecula) that results in a high percentage of thumbtack sperm, a vital step in the development of in vitro capacitation and fertilization for Australian marsupials. This system will also be useful for examining the role of the oviduct and its products in marsupial development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Brushtail possums (Trichosurus vulpecula) were captured in box traps in the North Canterbury region and transported to the Landcare Research Animal Facility at Lincoln, New Zealand. The animals were housed individually in cages (1.0 m x 0.4 m x 0.55 m) with a removable nest box and fed a diet of cereal pellets supplemented with fruits, vegetables, and water ad libitum. Research on these animals was approved by the Animal Ethics Committee of Landcare Research under the Animal Protection (Codes of Ethical Conduct) Regulations 1987.

Preparation and Growth of OEC Monolayers

Primary cultures of OEC monolayers were prepared according to the procedure of Sidhu et al. [7]. Briefly, oviducts were obtained from twelve female brushtail possums, and each received 15 IU eCG (i.m.) followed three days later with 4 mg of porcine LH (i.m.) [23]. Animals were restrained and then masked with CO2:O2 (3 L:1 L) and killed by a single intracardiac injection of sodium pentabarbiturate (125 mg/kg) 30 h after the LH injection, and tissues were processed within 30–45 min. Oviducts were dissected free of connective tissues, washed with PBS, and divided into two arbitrary halves, the isthmus (adjacent to the uterus) and the ampulla (adjacent to the ovary). The term "ampulla," which describes the swollen portion of the oviduct in eutherian mammals, does not accurately describe the same region of the brushtail possum oviduct, which is actually narrower in diameter than the isthmus part of the oviduct. However, the terms "ampulla" and "isthmus" will be used in this paper for clarity.

The oviductal epithelium was gently expelled from the isthmus and ampullary regions of the oviduct by squeezing with the back of sterilized forceps. Cell clumps were suspended in minimum essential medium with Earle's salts, L-glutamine, and nonessential amino acids (E-MEM; Trace Biosciences Pty. Ltd., Sydney, Australia) and dispersed gently by passing several times through a 26-gauge needle. The medium was supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 µg/ml streptomycin, and growth factors (10 ng/ml epidermal growth factor, 5 µg/ml insulin, 5 ng/ml transferrin, 50 IU/ml selenium), all from Sigma Chemical Company (St. Louis, MO). The cell suspensions in 450 µl E-MEM were cultured in 24-well tissue culture plates previously coated with 100 µl of 100% Matrigel (Collaborative Research Inc., Bedford, MA) diluted 1:1 with serum-free medium and maintained at 36°C in 5% CO2 and 95% humidified air. The medium (50%) was replaced with fresh medium on Day 5 of culture when about 60–70% confluence was achieved. Complete confluence was achieved after 7–10 days. As a control, tracheal epithelial cell monolayers from the same species were prepared in a similar way. Evidence for monolayer viability was vigorous ciliary activity. The purity of epithelial cells was assessed using indirect immunofluorescence analysis of epithelial cytokeratins [24].

OEC monolayer cultures prepared under the conditions described maintained some degree of differentiation for 7–10 days as evidenced by ciliary activity. The presence of FBS and growth factors was found to be beneficial for maintaining the differentiated state of these cells. The primary OEC cultures lose cilia quickly, within 5 days of culture, if grown from individual cells obtained after complete dispersion of OEC clumps. However, it was observed that if OEC monolayers were allowed to grow slowly from OEC clumps instead of individual OEC cells, the ciliary activity was maintained beyond 7–10 days. OEC monolayers appeared differentiated and survived beyond 10 days when grown on Matrigel-coated rather than uncoated wells of culture plates. Indirect immunofluorescence staining of monolayers using antibodies against cytokeratin demonstrated that > 90% of the cells were derived from epithelial cells.

Preparation of Spermatozoa and OEC Monolayer Coculture

Epididymal spermatozoa from two brushtail possum males were collected by back-flushing the cauda epididymidis through the vas deferens with 0.5 ml of medium, and sperm samples were pooled and washed by swim-up in E-MEM medium with the FBS replaced with polyvinyl alcohol (PVA, 1 mg/ml) as described previously [7] and diluted to 10 x 106/ml. PVA instead of FBS was used in the medium, because possum sperm agglutinate in the presence of 10% FBS. Cocultures of spermatozoa and OEC monolayers were established at a final sperm concentration of 2 x 106/ml. The E-MEM containing FBS was replaced in monolayers by E-MEM containing PVA 24 h before the coculture. Cocultures were incubated at 36°C in 5% CO2 and 95% humidified air.

Experimental Design

Washed epididymal brushtail possum spermatozoa were incubated under the following conditions: 1) with OEC from ampulla, 2) with OEC from isthmus, 3) with epithelial cells from possum trachea (control for the ciliated cells), 4) with Matrigel (control for the Matrigel used as basement material in the epithelial cell cultures), 5) in E-MEM in a plastic dish (control for the plastic culture plates and medium used in the epithelial cell cultures). All cultures were incubated at 36°C in 5% CO2 and 95% humidified air. Percentages of sperm attached, motility, sperm head-to-tail orientation, and acrosomal status were estimated at 2-h intervals for 0–6 h. The data represent means ± SEM from four replicates of sperm assessments.

Sperm Assessments

Aliquots (50 µl) of unattached sperm were drawn after vigorous pipetting 5 times from the same well at 0, 2, 4, and 6 h of incubation each time and were assessed for sperm concentration, motility, viability, acrosome status, and sperm head-to-tail orientation by standard procedures [25]. Briefly, the acrosome status of the unattached spermatozoa was evaluated after fixation in paraformaldehyde and staining with Bryan's stain, an equal combination of 0.1% (w:v) each of fast green FCF, eosin Y, and flavianic acid (all Sigma Chemical Company) in 1% acetic acid. The percentage of motile sperm was assessed subjectively (the assessor was blind to treatments) using an inverted microscope with Hoffman modulation contrast optics. Sperm progressive motility was assessed subjectively using an arbitrary scale (1–5, with 1 lowest and 5 highest). Sperm viability was assessed using a live/dead fertilight sperm viability kit according to the instructions of the supplier (Molecular Probes Inc., Eugene, OR). Sperm with damaged membranes (dead) emitted red fluorescence, and sperm with intact membranes (live) emitted green fluorescence, with emission maxima of 516 nm and 617 nm, respectively. Vigorous pipetting did not affect sperm viability.

The percentage of sperm attached to monolayers was calculated from counts of the number of unattached spermatozoa in an aliquot using a hemocytometer and the formula:

where n may be 2, 4, 6.

Corrections required in data were made to compensate for the sperm aliquot removed at each time point for sperm assessments before calculating the percentage of sperm attached using the formula above.

Statistical Analysis

Means (n = 4) of sperm concentration, motility, and acrosome status during coculture for each time point and treatment were calculated and compared using ANOVA (SigmaStat program version 2 for Windows; Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Attachment of Sperm to the Monolayer

Similar numbers of brushtail possum epididymal sperm attached to OEC monolayers derived from both the isthmus and ampulla regions of the oviducts (Fig. 1). At 2 h of coculture, a large percentage of sperm were attached to OEC monolayers (ampulla, 60.1 ± 4.7%; 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%). There were fewer sperm attached to the tracheal epithelial cell monolayer (1.2 ± 0.2%; P < 0.01) and Matrigel (10.2 ± 2.5%; P < 0.01) after 6 h compared to those attached to ampulla and isthmus OEC monolayers (37.9 ± 7.2% and 44.6 ± 2.2%, respectively), and none on the plastic surface. Fewer sperm were released from the ampullary and isthmal OEC monolayers compared to controls (P < 0.05). Sperm were not evenly distributed on monolayers: they attached via the apical end of the sperm head and were predominantly in contact with the nonciliated secretory cells of the epithelium (Fig. 2, a and b). Sperm attached to the Matrigel mainly by the lateral surface of the head.



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  		FIG. 1. Percentages of sperm attachment during coculture with OEC monolayers derived from ampulla and isthmal parts of the oviduct and with controls (tracheal epithelial cell, Matrigel, and plastic) during different periods of coculture. Values are mean ± SEM, n = 4. Circles, ampulla; squares, isthmus; triangles, trachea; inverted triangles, Matrigel; diamonds, plastic



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  		FIG. 2. Brushtail possum OEC grown as monolayers and during coculture with sperm. a) OEC confluent monolayers at 7 days of culture. Scale bar = 44.9 µm. b) OEC monolayer with sperm attachment. Scale bar = 100 µm.

Motility and Viability of Sperm During Coculture

There was a decline in motility of attached and unattached spermatozoa in all treatments during incubation (Fig. 3). After 6 h of coculture with ampullary and isthmal OEC, the percentage motility of both attached and unattached spermatozoa was 40–50%, which decreased to 35% at 24 h. In controls, i.e., tracheal monolayer, Matrigel, and plastic, the motility of the attached spermatozoa declined to < 25% by 6 h, and all sperm were immotile by 24 h. However, the motility of the unattached spermatozoa in the controls (tracheal monolayer, Matrigel, and plastic) was approximately 35% at 6 h, and all were immotile by 24 h of incubation. Sperm progressive motility decreased during coculture over time, but there was no significant difference between different treatments (progressive motility in all treatments at 0 h was 3.5 ± 0.0, and at 6 h it varied from 1.8 ± 0.3 to 2.3 ± 0.3). A decline (P < 0.001) in the percentage of viability of unattached sperm was observed during coculture in all groups (Fig. 4). At 6 h, more than 60–70% sperm were viable in all groups; however, only 35% of sperm were motile at this time, indicating that sperm may become quiescent before they die.



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  		FIG. 3. Percentage motility of top) attached and bottom) unattached sperm during coculture with OEC monolayers derived from ampulla (circles) and isthmus (squares), and with trachea epithelial cell (triangles), Matrigel (inverted triangles), and plastic (diamonds) controls. Values are means ± SEM, n = 4



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  		FIG. 4. Percentage of unattached sperm that were viable during coculture with OEC monolayers derived from ampulla (circles) and isthmus (squares), and with trachea epithelial cell (triangles), Matrigel (inverted triangles), and plastic (diamonds). Values are means ± SEM, n = 4

Capacitation-Like Changes to Sperm

Coculture of sperm with OEC monolayers induced transformation from streamlined orientation of sperm head and tail to thumbtack orientation in 60% of spermatozoa by 2 h of coculture compared to 25% in controls (Fig. 5). After 2 h of coculture, there was no significant increase in the number of thumbtack sperm in any treatment (Fig. 5). After 2 h of coculture, thumbtack spermatozoa continued to attach to monolayers, where they alternated between streamlined and thumbtack orientation while still attached to epithelial cells; however, this could not be quantitated. Brushtail possum spermatozoa did not show any acrosomal modifications during coculture.



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  		FIG. 5. Percentage of unattached sperm that were thumbtack during coculture with OEC monolayers derived from ampulla (circles) and isthmus (squares) and with trachea epithelial cell (triangles), Matrigel (inverted triangles), and plastic (diamonds). Values are means ± SEM, n = 4


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes a procedure to obtain confluent layers of brushtail possum OEC monolayers within 7–10 days based on techniques developed in eutherian mammals [18, 20–21] and another marsupial, the tammar wallaby [7]. Matrigel was found to help maintain the differentiated state of the epithelial cells for 7–10 days in brushtail possum. This observation is consistent with previous studies, in which Matrigel improved polarization and differentiation in cultured mammary epithelial cells [26], human endothelial cells [27], and bovine and equine OEC [24, 28].

Brushtail possum spermatozoa attached to both isthmus- and ampulla-derived OEC monolayers during coculture, but in lower numbers than observed in the tammar wallaby [7]. Although spermatozoa also attached to Matrigel, tracheal epithelial cells, and plastic, the attachment to OEC monolayers was specifically via the apical sperm-head surface. Sperm were unevenly distributed on the monolayer, suggesting that they attached only to differentiated and polarized cells or perhaps to specific cell types in the monolayer. By 6 h of coculture, a large number of sperm remained attached to the ampulla and isthmus monolayers (37.9 ± 7.2% and 44.6 ± 2.2%, respectively) compared to those in controls (tracheal epithelial cell monolayer, 1.2 ± 0.2%; Matrigel, 10.2 ± 2.5%; plastic, none). Chian and Sirard [20] also reported that in the bull more than 50% of sperm remained attached to OEC monolayers. Recently, Nickel et al., [19] studying sperm transport after artificial insemination in the brushtail possum, have reported association of spermatozoa with the luminal epithelium as well as with the luminal epithelial folds (crypts) of the oviduct. The prolonged attachment of large numbers of possum spermatozoa to OEC cells may therefore have some functional relevance. Some of the proposed biological roles of sperm attachment include an additional block to polyspermy, protection of sperm against phagocytosis, prolongation of motility and fertility, conservation of sperm energy by protecting against the stimulatory effects of uterine and ampullary fluids, and mediation of sperm capacitation and acrosome reaction [9, 20–22, 28–33].

The motility of brushtail possum sperm was significantly higher after coculture on OEC monolayers than on controls (Matrigel, plastic, and tracheal epithelium), indicating a specific effect of the oviduct on sperm motility. This specific effect could be through physical interactions with the oviducal epithelial cells. It is also possible that specific secretory products of oviductal cells may add to this effect. Alterations in sperm motility characteristics are believed to be indicative of either metabolic, structural, or membrane changes in sperm [30]. Sperm viability was 60–70% in all the treatment groups at 6 h. Beneficial effects of OEC monolayers and explants have been reported for bovine spermatozoa [29, 30].

More than 60% of spermatozoa cocultured with ampulla and isthmus OEC for 2 h exhibited thumbtack orientation and high motility, characteristics that are considered an indicator of sperm capacitation in marsupials [6, 7]. Highly motile thumbtack sperm have also been observed in the oviduct in situ in an Australian marsupial, Sminthopsis crassicauda [5], and in sperm flushed from the brushtail possum oviduct around the time of fertilization [6], indicating that thumbtack orientation of spermatozoa may be important for fertilization in these species. We have observed possum spermatozoa attached to oviduct monolayers with their tails oscillating around a fulcrum at the implantation fossa, alternating between streamline and thumbtack orientation, indicating that it is a reversible transformation. Since in the present study we have observed a re-orientation of the sperm head in 25% of control sperm, thumbtack orientation alone is probably not an absolute indication of sperm capacitation. In controls, sperm were unable to bind or penetrate the zona pellucida of brushtail possum eggs (unpublished observation). Thus it appears that it is the combination of head re-orientation and other as yet unknown biochemical and physiological changes that constitutes the events of sperm capacitation in this species. Unlike bovine, equine, ram, buffalo, and tammar wallaby spermatozoa [7, 32, 34–37], which undergo acrosome modifications or acrosome reaction during coculture with OEC monolayers or oviduct environment, brushtail possum sperm do not show any acrosome modifications detectable at the light microscope level during coculture. Electron microscope studies of cocultured sperm and their interactions with OEC monolayers are planned to examine any ultrastructural changes occurring in the sperm.

Although possum sperm attach to OEC monolayers in lower numbers than in other species, the secretory products of the OEC may interact with sperm to induce the capacitation-like changes (thumbtack sperm) and to maintain the higher sperm viability and motility. Oviduct culture is thus a promising in vitro model for studying cell-to-cell interactions and autocrine, paracrine, and endocrine regulation of such events as sperm capacitation and the influence of the oviductal environment on these events during in vitro culture. Such a system will contribute to our understanding of sperm capacitation, fertilization, and manipulation of fertility in marsupials [38].


   ACKNOWLEDGMENTS
 
The staff at the Landcare Research Animal Facility at Lincoln, New Zealand, is thankfully acknowledged for the help in the procurement and care of the possums. Dr. John Clulow and Dr. David Kay, Department of Biological Sciences, University of Newcastle, Callaghan, NSW, are acknowledged for reviewing the manuscript and providing useful comments.


   FOOTNOTES
 
1 This study was funded by the Possum Biocontrol Program of MAF Policy, New Zealand, and the Australian Government's Co-operative Research Centre Program. Back

2 Correspondence: Kuldip S. Sidhu, Marsupial CRC, Department of Biological Sciences, Macquarie University, Sydney NSW 2109, Australia. FAX: 61 2 9850 9686; ksidhu{at}possum.bio.mq.edu.au Back

Accepted: June 25, 1999.

Received: October 26, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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K S Sidhu, K E Mate, T Gunasekera, D Veal, L Hetherington, M A Baker, R J Aitken, and J C Rodger
A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)
Reproduction, January 1, 2004; 127(1): 95 - 103.
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