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J C Rodger

Publications and source records attributed to J C Rodger.

At least 19 recordsLinked to original sources
PubMed2004

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).

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

Animals↗
PubMed2003

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

Animals↗
PubMed2003

Effects of repeated superovulation and surgical oocyte collection on ovarian response and natural breeding ability of the tammar wallaby (Macropus eugenii).

The aim of this study was to assess the response of a marsupial, the tammar wallaby (Macropus eugenii) to repeated superovulation and surgical oocyte collection and monitor any effects on subsequent natural breeding ability. Animals (n = 5 per group) were superovulated once, twice or three times with pig FSH (pFSH; 6 mg administered twice per day for 4 days) followed by 4 mg pig LH (pLH). There was an interval of either 5-6 weeks (n = 9) or 12 weeks (n = 1) between the first and second superovulation and 13-17 weeks (n = 5) between the second and third superovulation. Oocytes were collected surgically after each treatment. Serum was collected at the time of each treatment to monitor the formation of anti-pFSH and anti-pLH antibodies. Animals were allowed to mate naturally in the season following superovulation treatment(s). There was no significant difference between groups in the number of large follicles (2-5 mm diameter, mean +/- standard error) produced in response to the first (21.2 +/- 4.3), second (18.0 +/- 6.5) or third (29.0 +/- 4.9) superovulation treatment. Eggs were recovered from approximately 80% of follicles that were flushed during laparotomy. There were significant concentrations of anti-pFSH and anti-pLH antibodies (P < 0.05) detected in previously superovulated animals at the time of the second superovulation but not at the time of the third superovulation. The anti-gonadotrophin antibodies present at the time of repeated superovulation did not cause a significant decrease in average number of follicles. All animals produced pouch young in the breeding seasons after repeated superovulation. Combined with other reproductive technologies, repeated superovulation has the potential to increase the production of offspring from rare or valuable marsupials in captivity.

Animals↗
PubMed2000

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination.

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.

Animals↗
PubMed2000

Sperm binding and penetration of the zona pellucida in vitro but not sperm-egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.

Animals↗
PubMed1999

Ultrastructural observations on in vivo fertilisation in the brushtail possum, Trichosurus vulpecula, following PMSG/LH superovulation and artificial insemination.

Information on the dynamics of gamete interaction in marsupials is very limited and not available for any species from the major Australian Order Diprotodontia which includes most of the more familiar animals such as kangaroos, possums and the koala. This study addressed this deficiency by examining the ultrastructure of in vivo fertilised eggs from common brushtail possums (Trichosurus vulpecula). Females were superovulated by treatment with 15 IU PMSG and then 4 mg porcine LH 3 days later, and inseminations were performed 910-13 h after LH) using epididymal spermatozoa. Between 33 and 39 h after LH injection females were killed, reproductive tracts excised and the oviduct ampulla segment flushed for eggs. Three of the six eggs examined were fertilised as judged by the presence of sperm remnants in the cytoplasm. On the basis of these eggs it was found that sperm penetration left a large hole in the zona pellucida (ZP), suggesting that sperm zona penetration occurs primarily by the enzymatic action of acrosomal enzymes. Sperm lying within the perivitelline space were lacking both an outer acrosomal membrane and the associated acrosomal contents, while both these structures were found on sperm embedded within the mucoid layer, which is consistent with induction of the acrosome reaction by binding to the ZP. Once inside the egg cytoplasm, the sperm head travelled only a short distance before chromatin decondensation occurred. Fertilised eggs showed signs of cytoplasmic activation including cytoskeleton association with apparently dividing mitochondria and prominent rough endoplasmic reticulum. Unfertilised eggs appeared to be undergoing degenerative changes and lacked any evidence of activation. This study was demonstrated that superovulation and laparoscopic intravaginal artificial insemination provide a system through which perifertilisation events in the possum and other monovular Australian marsupials can be examined experimentally.

Animals↗
PubMed1999

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the 'scoop'. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

Acrosome↗
PubMed1999

Secretory proteins from the female reproductive tract of the brushtail possum (Trichosurus vulpecula): binding to sperm and effects on sperm survival in vitro.

Previous studies have demonstrated that co-culture of brushtail possum epididymal spermatozoa with oviduct epithelial cell monolayers prolongs sperm survival and results in the re-orientation of the sperm head and tail to the T-shape (thumbtack) configuration. Transformation of sperm to thumbtack orientation is believed to be associated with marsupial sperm capacitation. Here we report that incubation in oviduct-conditioned media also significantly prolongs sperm survival and results in the transformation of sperm to the thumbtack orientation. The major objective of the current study was to examine the proteins present in the conditioned media, to determine whether any of these proteins specifically bound to sperm and the relationship between these proteins and sperm survival and thumbtack orientation. Co-culturing brushtail possum sperm with biotin-labeled proteins in conditioned media (CM) from ampulla, isthmus and uterine explants demonstrated strong binding of two proteins of molecular mass 230 and 61 kD and weak binding of nine proteins of molecular mass 200, 180, 120, 140, 55, 52, 48, 34, 30 kD to sperm within 30 min. The binding of the 61-kD protein from the conditioned media appeared specific as increasing concentrations of non-labeled oviduct proteins, but not serum proteins, inhibited the binding of labeled proteins. The binding of oviduct and uterine proteins in the conditioned media significantly prolonged sperm survival and percentage motility and also transformed a large number of sperm to a thumbtack orientation. The implication of binding of these proteins is discussed in the context of sperm survival and capacitation in this species.

Animals↗
PubMed1999

Induction of thumbtack sperm during coculture with oviduct epithelial cell monolayers in a marsupial, the brushtail possum (Trichosurus vulpecula).

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.

Animals↗
PubMed1998

Influence of lead selection and population on automated measurement of QT dispersion.

BACKGROUND: The study of QT dispersion (QTd) is of increasing clinical interest, but there are very few data in large healthy populations. Furthermore, there is still discussion on the extent to which QTd reflects dispersion of measurement. This study addresses these problems. METHODS AND RESULTS: Twelve-lead ECGs recorded on 1501 apparently healthy adults and 1784 healthy neonates, infants, and children were used to derive normal limits of QTd and QT intervals by use of a fully automated approach. No age gradient or sex differences in QTd were seen and it was found that an upper limit of 50 ms was highly specific. Three-orthogonal-lead ECGs (n=1220) from the Common Standards for Quantitative Electrocardiography database were used to generate derived 12-lead ECGs, which had a significant increase in QTd of 10.1+/-13.1 ms compared with the original orthogonal-lead ECG but a mean difference of only 1.63+/-12.2 ms compared with the original 12-lead ECGs. In a population of 361 patients with old myocardial infarction, there was a statistically significant increase in mean QTd compared with that of the adult normal group (32.7+/-10.0 versus 24.53+/-8.2 ms; P<0. 0001). An estimate of computer measurement error was also obtained by creating 2 sets of 1220 ECGs from the original set of 1220. The mean error (difference in QTd on a paired basis) was found to be 0. 28+/-9.7 ms. CONCLUSIONS: These data indicate that QTd is age and sex independent, has a highly specific upper normal limit of 50 ms, is significantly lower in the 3-orthogonal-lead than in the 12-lead ECG, and is longer in patients with a previous myocardial infarction than in normal subjects.

Adult↗
PubMed1998

Characterisation of fibrous sheath and midpiece fibre network polypeptides of marsupial spermatozoa with a monoclonal antibody.

In this study cytoskeletal antigens common to brushtail possum and tammar wallaby spermatozoa were characterised using a monoclonal antibody (PSA-10). Using indirect immunofluorescence, the PSA-10 antibody detected antigens predominantly associated with the midpiece and principal piece of mature, permeabilised marsupial spermatozoa. The principal piece determinant, shared by a variety of other species, was found to arise in the marsupial testis. Midpiece localisation of the PSA-10 epitope was detected only in marsupial spermatozoa and shown to arise in the epididymis. Immunogold labelling demonstrated that the PSA-10 antigens were predominantly associated with the fibrous sheath and midpiece fibre network of both possum and wallaby spermatozoa. Western blotting suggested that two major possum and wallaby sperm polypeptides of 158 and 182 kDa were associated with the midpiece fibre network, a cytoskeletal structure unique to marsupial spermatozoa. A 32 kDa polypeptide was associated with the principal piece fibre network and/or fibrous sheath. The finding that these marsupial sperm cytoskeletal proteins share a common linear epitope suggests that they share some sequence similarity. The midpiece fibre network of marsupial sperm, like the fibrous sheath, has been proposed to have a structural role in providing passive stiffening for the flagellum (Harding et al., 1975, 1979; Olsen, 1975). The PSA-10 monoclonal antibody may provide a tool for comparative studies of mammalian sperm cytoskeletal proteins, particularly the marsupial midpiece fibre network. It may also allow the formation of this unique marsupial cytoskeletal structure, and its fate during the fertilisation process, to be followed by immunological means.

Animals↗
PubMed1998

Further observations of the ovarian response of the tammar wallaby (Macropus eugenii) to exogenous gonadotrophins: an improved method for superovulation using FSH/LH.

This study reports the development of an improved superovulation protocol in the monovulatory tammar wallaby, Macropus eugenii. Treatment with pregnant mare's serum gonadotrophin (PMSG; 10-20 IU) inhibited follicle development in the corpus luteum (CL)-bearing ovary and only 2-3 eggs per female could be recovered after ovulation induction with gonadotrophin releasing hormone (GnRH; 3 x 30 microg at 3-h intervals) or porcine luteinizing hormone (LH; 4, 5 or 8 mg) 3 days after PMSG priming. Treatment with porcine FSH (8 x 6 mg at 12-h intervals for four consecutive days) was found to override this inhibition and resulted in the recovery of 7-13 eggs per female after ovulation induction with porcine LH (4 mg on day 5). For these animals, there was no difference in numbers of developing follicles, ovulation sites and eggs recovered between the CL- and non-CL-bearing ovaries. This FSH/LH protocol was effective in both cycling and non-cycling females, and multiple ovulation occurred from about 36 h after LH treatment. After LH treatment, eggs were recovered from the oviduct at 36-50 h. At 51-57 h, 12-25% of eggs were recovered from the uterus, and by 75 h all eggs were recovered from the uterus. It is concluded that the described FSH/LH protocol used results in higher ovulation success than the PMSG/GnRH method.

Animals↗
PubMed1998

Manipulation of the fertility of marsupials for conservation of endangered species and control of over-abundant populations.

Marsupials present a dichotomy in population management; the numbers of many Australian marsupial species have declined due to loss of habitat, competition from introduced herbivores and predation by introduced carnivores, but other species have become locally overabundant in Australia or are introduced pests in New Zealand. The manipulation of reproduction offers the means to increase or decrease productivity; however, considerable fundamental research is required before reproductive technologies can be applied to marsupials. Marsupials differ from eutherian mammals in several aspects of their reproduction including sex differentiation, gamete function and endocrinology, as well as in the relative lengths of gestation and lactation. Although these differences present unique problems in the application of reproductive technologies to marsupials, they also present unique opportunities for marsupial-specific fertility control. This paper summarises the assisted breeding technologies currently being applied to marsupials including superovulation, artificial insemination, in vitro fertilization and gene banking; unique marsupial targets for contraceptive intervention including gamete production, sperm capacitation, gamete surface antigens and embryonic development; and some options for the delivery of contraceptive vaccines to marsupial populations.

Animals↗
PubMed1998

Successful fertilization after superovulation and laparoscopic intrauterine insemination of the brushtail possum, Trichosurus vulpecula, and tammar wallaby, Macropus eugenii.

Fertilization has been achieved in superovulated brushtail possums and tammar wallabies after laparoscopic intrauterine artificial insemination. Various superovulation protocols and insemination times were examined but a maximum of 2-5 eggs including 1-2 embryos per possum were recovered. The female possums were superovulated by treatment with 15 iu pregnant mares' serum gonadotrophin and then either GnRH (4 x 50 micrograms, at intervals of 90 min) or 4 mg LH, 3 days later. Inseminations were performed within 6 h before or 4-10 h after (pregnant mares' serum gonadotrophin-GnRH group only) the expected onset of ovulation using epididymal spermatozoa. Superovulation in wallabies was achieved by treatment with FSH (8 x 6 mg, at intervals of 12 h for 4 days) followed by 4 mg LH on day 5. Inseminations were performed 4-6 h before the expected onset of ovulation using ejaculated spermatozoa, which resulted in the recovery of 7-8 eggs including 3-4 embryos per female. All embryos recovered were from possums and wallabies examined 1-2 days after insemination and included fertilized eggs, two-cell and four-cell embryos. Motile spermatozoa were recovered from the oviducts and uteri but only immotile spermatozoa were found in the vaginal complex. Five to thirty per cent of spermatozoa recovered from the oviducts of possums examined 2-6 h after insemination had thumbtack morphology, which is thought to be correlated with capacitation. Although embryo yields per female were low, this study has established that intrauterine artificial insemination after superovulation is a feasible assisted breeding strategy for marsupials with implications for species conservation and population control.

Animals↗
PubMed1998

Sperm-oviduct epithelial cell monolayer co-culture: an in vitro model of sperm-female tract interactions in a marsupial, the tammar wallaby (Macropus eugenii).

Oviduct epithelial cell (OEC) monolayers were prepared from the isthmic and ampullary parts of the oviducts of FSH-primed tammar wallabies. Co-culture experiments found that 50-60% of wallaby spermatozoa attached immediately to OEC monolayers, tracheal cell monolayer controls, and the surface of culture dishes with and without Matrigel coating. Spermatozoa were considered to be attached if they remained on the culture surface after rapidly pipetting the co-culture medium five times. The percentages of attached and unattached spermatozoa were calculated from the number of spermatozoa recovered in the agitated supernatant. After 2 h co-culture the percentage of attached spermatozoa rose to 60-80%. After 6 h co-culture the number of spermatozoa attached to OEC monolayers derived from the oviductal isthmus remained high and only a small percentage were recovered in the agitated supernatant (unattached spermatozoa 3.85 +/- 0.76%, P = 0.67). However, after 6 h co-culture of spermatozoa with OEC monolayers derived from the ampulla and with the controls the percentage of attached spermatozoa declined significantly (unattached spermatozoa: ampullary monolayer 23.08 +/- 4.80%, P < 0.01; tracheal monolayer 23.23 +/- 5.18%, P < 0.01; Matrigel 27.23 +/- 7.76%, P < 0.01; plastic surface 28.19 +/- 5.30%, P < 0.01). After 6 h co-culture with ampullary and isthmic OEC monolayers, the percentage motility of both attached and unattached spermatozoa was maintained at 64.00 +/- 1.90% and 56.66 +/- 3.18% and 62.00 +/- 3.11% and 52.00 +/- 2.43%, respectively, and was then maintained at > or = 35% after 24 h incubation. In the controls, that is, tracheal monolayer and Matrigel, the motility of attached spermatozoa declined rapidly to 48.66 +/- 2.15% and 33.63 +/- 8.66%, respectively, at 6 h, and all spermatozoa were immotile after 24 h incubation. However, the motility of unattached spermatozoa in the controls (tracheal monolayer and Matrigel) was maintained at 57.33 +/- 3.00% and 34.54 +/- 9.27%, respectively, until 6 h and then declined rapidly, and all spermatozoa were immotile after 24 h incubation. Co-culture of wallaby spermatozoa with OEC monolayers also induced acrosomal modifications that were followed by acrosomal loss. At 6 h incubation 38.92 +/- 3.98% of spermatozoa on ampullary OEC monolayers and 36.50 +/- 3.81% spermatozoa on isthmic OEC monolayers had shed their acrosome. Acrosomal loss during co-culture with both isthmic and ampullary OEC monolayers was significantly (P < 0.01) greater than that observed on tracheal epithelial monolayer (24.42 +/- 1.90%, P < 0.01), Matrigel (20.70 +/- 2.71%, P < 0.01) and plastic (15.54 +/- 2.49%, P < 0.01). Co-culturing spermatozoa with OEC monolayers also induced a transformation from streamlined orientation of sperm head and tail to T-shaped (thumbtack) orientation in a small number (10-15%) of motile spermatozoa after 6 h incubation (data not shown). The significance of these results in relation to the role of the oviduct in sperm capacitation is discussed.

Acrosome Reaction↗
PubMed1997

Posttesticular development of spermatozoa of the tammar wallaby (Macropus eugenii).

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.

Acrosome↗
PubMed1997

Spermiogenesis and spermiation in a marsupial, the tammar wallaby (Macropus eugenii).

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis: the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm-egg fusion site proposed for Monodelphis domestica (Taggart et al. 1993). The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principle piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.

Acrosome↗