Structural features of the epididymis in a dasyurid marsupial (Antechinus stuartii)

Summary The ductus epididymidis of the marsupial mouse Antechinus stuartii was divided into caput, corpus, and caudal regions using several constant morphological landmarks. Tubule diameter and epithelial height increased gradually from caput to cauda. In contrast, the surface area of the lumen of the ductus epididymidis increased to a maximum in the distal caput region, but decreased markedly in the distal cauda in association with characteristic changes in lumen shape (from circular to slit-shaped) and epithelial height. Epithelial cells of the ductus epididymidis were generally similar in structure to those described in other mammalian species. Principal and basal cells were common throughout the epithelium. Clear and mitochondria-rich cells were also identified, but occurred less frequently. Regional variations in cell ultrastructure were observed only in principal cells. Numerous vesicular inclusions occurred in the apical cytoplasm of cells in caput segments, membrane-bounded, electron-dense bodies were common in distal corpus regions, and a brush border of microvilli characterized the luminal surface of principal cells in caudal segments. Sperm index increased in the proximal caput, declined to basal levels in the distal caput and proximal corpus, and then increased to a maximum in segment 9 of the distal corpus and remained at about this level throughout the cauda epididymidis. Nuclear rotation, loss of cytoplasmic droplets, and other sperm maturational changes were observed along the epididymis. Discarded cytoplasmic droplets collected in large masses interspersed between aggregates of spermatozoa throughout the distal regions of the duct. There was no evidence of phagocytosis by principal cells of cytoplasmic droplets. The epididymis of A. stuartii differs from that of other mammals. The unusual caudal region, which has little storage capacity for sperm, is an unusual adaptation in a species in which the male is known to be polygamous.     

Relationship between longevity of spermatozoa after insemination and the percentage of normal embryos in brown marsupial mice (Antechinus stuartii)

Twenty-six female brown marsupial mice in a laboratory colony were mated at intervals ranging from 1 to 20 days between coitus and ovulation. The numbers of corpora lutea and normal embryos were counted. A multiple regression model examined the parabolic relationship between the proportion of normal embryos and the time from coitus to ovulation. The proportion of normal embryos increased until a mean of 9.5 days and decreased thereafter. This relationship was independent of the year of breeding and the number of corpora lutea. After survival of spermatozoa for up to 13 days in the female reproductive tract, the fertility levels of females was 88-92%. Low fertility levels after 13 days appeared to be due to a decrease in the number of spermatozoa. Reproductive tracts from 7 females killed after insemination and examined histologically showed many spermatozoa in the isthmus of the oviduct and the uterus at 5 days post coitum; spermatozoa confined to the isthmus between 6 and 13 days; and few spermatozoa in the isthmus at 14 days after copulation. A comparison between the fertility levels in the females which had been inseminated once and a further 17 females which had been inseminated 2 or 3 times suggested that spermatozoa from 2nd and 3rd inseminations can contribute spermatozoa for fertilization. In these females fertility levels did not decline with time after the first mating.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

Phase delay of the natural photoperiod alters reproductive timing in the marsupial Antechinus stuartii

The dasyurid marsupial Antechinus stuartii (20–40 g) has a brief, highly synchronized mating period in spring followed by complete male mortality. Analysis of breeding times in nature suggests that the rate of change of photoperiod is the main Zeitgeber for reproductive timing in this species. To test this hypothesis the natural photoperiod was phase delayed by two months, one experiment beginning in late summer, and the other in the autumn, before the spring breeding period.
The reproductive cycle of males and females exposed to the delay in summer was synchronously delayed by two months, coincident with the exact duration of the delay. Animals exposed to the two month phase delay in autumn also exhibited a delay in reproductive timing, but it was less synchronized than either the control or the other experimental group. Reproduction in control animals exposed to natural photoperiod was synchronous with that of a wild population monitored simultaneously. It is therefore likely that the rate of change of photoperiod is the dominant Zeitgeber for the reproductive cycle in this species.     

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

Size breeds success: multiple paternity, multivariate selection and male semelparity in a small marsupial, Antechinus stuartii

Mating in the marsupial genus Antechinus is a synchronous annual event that is characterized by monoestry in females and abrupt postmating mortality in males. Male semelparity (multiple copulations during a single breeding season per lifetime) is often assumed to occur as a consequence of the intense mating effort expended by males in the rut, but the forces selecting for this remain elusive. Here, we investigate selection in male brown antechinus, Antechinus stuartii, and test two hypotheses for the evolution of semelparity: intermale competition and sperm competition. If intermale competition drives semelparity, we predicted that males would be under strong selection for large body size. If sperm competition is important, we predicted that selection would be strongest on scrotal size, a surrogate for testes volume. Using microsatellite markers, we found that 92% of females in free-living conditions mated with multiple males, producing litters of eight that had up to four fathers. These observations confirm the potential for sperm competition. Using selection analysis, we then found paternity success in 119 males to be related most strongly to body mass and scrotal size, thus providing support for both hypotheses. Large males presumably experience increased paternity success by gaining more matings or prolonged copulations via mate guarding, while large testes may allow increased sperm investment per copulation. Increased levels of free corticosteroid hormones in males facilitate the extreme mating effort during the short period of rut, but lead to immune suppression and consequently to the phenomenon of postmating mortality.     

Acuariid, capillariid and hymenolepidid parasites of the dasyurid marsupial Antechinus stuartii Macleay, 1841, from southeastern Australia.

Stammerinema suffodiax sp. n. is described from the dasyurid marsupial Antechinus stuartii. It differs from S. soricis, the only other member of the genus, in lacking lateral body spines, as well as in the spicules and characters of the anterior extremity. As well as redescribing Synhimantus australiensis (Johnston and Mawson, 1952) now collected from the stomach of A. stuartii, a new capillariid, Capillaria rickardi from the stomach and two cestodes Hymenolepis aklei sp. n. and H. bradleyi sp. n. are described from the same host.     

Seasonal morphometry of the vomeronasal organ in the marsupial mouse,Antechinus subtropicus

The vomeronasal system consists of a peripheral organ and the connected central neuronal networks. The central connections are sexually dimorphic in rodents, and in some species, parameters of the vomeronasal organ (VNO) vary with sex, hormonal exposure, body size and seasonality. The VNO of the dasyurid marsupial mouse, Antechinus subtropicus is presumed to be functional. The unusual life history (male semelparity) is marked by distinct seasonality with differences in hormonal environments both between males and females, and in males at different time points. Body size parameters (e.g., length, weight) display sexual dimorphism and, in males, a pronounced weight gain before breeding is followed by a rapid decline during the single, short reproductive season. VNO morphometry was investigated in male and female A. subtropicus to identify possible life cycle associated activity. The overall length of the VNO is positively correlated with the size of the animal. The amount of sensory epithelium exhibits a negative correlation, decreasing with increasing size of the animal. The effects of sex and breeding condition are not obvious, although they do suggest that sensory vomeronasal epithelium mass declines in the breeding period. The VNO may be more important in A. subtropicus before breeding when it may participate in synchronising reproduction and in the development of the male stress response. J. Morphol. 277:1517–1530, 2016. © 2016 Wiley Periodicals, Inc.     

Phylogeography and environmental correlates of a cap on reproduction: teat number in a small marsupial, Antechinus agilis

Natural selection should optimize litter size in response to the distribution and abundance of resources during breeding. In semelparous, litter-bearing antechinuses, teat number limits litter size. Consequently adaptation has been inferred in explaining intraspecific, geographic variability in teat number for several Antechinus spp. The phylogeography of teat number variation and associated genetic divergence were assessed in A. agilis using nine microsatellites and mitochondrial cytochrome b sequence data. Six-teat Otway Range animals were divergent in microsatellite allele identity and frequencies: samples from three Otway six-teat sites demonstrated significantly greater similarity genetically to those from six-teat animals approximately 250 km to the west, than to nearby Otway 10-teat samples, or to the six-teat animals at Wilsons Promontory. Gene flow between Otway phenotypes appears to have been limited for sufficient time to enable different microsatellite alleles to evolve. Nonetheless, nuclear genetic evidence suggested only incomplete reproductive isolation, and mitochondrial DNA (mtDNA) haplotypes showed no association with teat number. Other populations across the range were no more genetically differentiated from one another than expected from geographic separation. Principal components and distance-based redundancy analyses found an association between environmental variables and geographic distribution of A. agilis teat number - six-teat animals inhabit more temperate forests, whilst those with more teats experience greater seasonality. The apparent restricted breeding between phenotypically distinct animals, together with phylogenetically separate groups of six-teat animals in different locations with similar environments, are consistent with the hypothesis that adaptation to different habitats drives teat number variation in A. agilis.     

Effects of photoperiod and endogenous control on timing of reproduction in the marsupial genus Antechinus

The timing of reproduction was investigated and compared in natural pop ulations and laboratory colonies of three species of dasyurid marsupials, Antechinus stuartii, A. swainsonii and A. flavipes. In the natural populations, reproductive events occurred only during the austral winter (July-September), and began at almost exactly the same time each year. The mating period lasted–3 weeks, and coincided with the onset of pouch changes (oestrus) in females. In males, spermatozoa appeared in the urine up to five weeks before the first matings occurred, but sexual activity tended to be suppressed during this time by female aggression. In the laboratory colonies, most individuals were paired (females with males) and housed under 12L: 12D or ambient light. Irrespective of the light regime, all first-year animals entered reproductive condition at the same time or only slightly earlier than those in the natural populations. However, in females housed for two years under 12L: 12D, oestrus occurred–5 weeks earlier, on average, than in field individuals, and–5 weeks earlier than in equivalent individuals housed under ambient light. Males again achieved reproductive condition before females, and may have stimulated and helped to synchronize oestrus. These findings showed that the timing of reproduction is controlled by an endogenous circannual rhythm, which is probably entrained by some component of the photoperiod. The timing mechanism ensures that all young are nursed and weaned in spring and summer when food is most predictably abundant, and hence maximizes individual reproductive success.     

Influence of daylength on the initiation of the breeding season of the marsupial possum, Trichosurus vulpecula

In Queensland, possums in the wild and in captivity first give birth during March and continue to give birth throughout the year until November. In this study the effect of short daylengths on the initiation of breeding activity was examined. One male and 4 female possums were transferred from an outside enclosure into a light control room (10 h light, 14 h dark) on 22 November. A control group of possums was housed in outside enclosures. The possums held in the light room gave birth 81.2 +/- 14.7 days (s.e.m.) after being placed in the short-day photoperiod. The control group gave birth 133.8 +/- 9.8 days after 22 November. This result suggests that photoperiod plays a role in the initiation of the breeding season of the brushtail possum.     

Influence of melatonin on the initiation of the breeding season of the marsupial bandicoot, Isoodon macrourus

Melatonin implants were administered to 6 female bandicoots during the months of May and July. These animals, together with 6 control bandicoots were housed in large outside enclosures with mature males. Births were observed in the 6 control animals from 26 July to 2 September, but no births were observed in the 6 bandicoots with melatonin implants. These results would suggest that photoperiod, which is known to influence melatonin concentrations, may be a factor in the initiation of births in the bandicoot. However, the gradual build-up of births would suggest that other factors such as temperature and rainfall may also have some influence.     

Short term, repeated exposure to rams during the transition into the breeding season improves the synchrony of mating in the breeding season

The ram effect is widely used in Mediterranean breeds of sheep but its use in temperate genotypes is restricted by breed seasonality. However, ewes from these highly seasonal genotypes are sensitive to stimulation by rams close to the onset of the natural breeding season. In this study we developed a pre-mating protocol of repeated, short-term exposure to rams (fence-line contact or vasectomised rams) beginning during late anoestrus and continuing into the breeding season. We hypothesised that this pre-mating protocol would synchronise the distribution of mating of North of England Mule ewes during the breeding season above that observed in ewes isolated from rams prior to mating. Ram-exposed ewes were given contact with rams (Experiment 1: fence-line; FR, n = 94 and Experiment 2: vasectomised rams; VR; n = 103) for 24 h on Days 0 (10 September), 17 and 34 of the experiment. Control ewes (Experiment 1; FC, n = 98 and Experiment 2; VC; n = 106) remained isolated from rams prior to mating. In Experiment 2, a subset of VR (n = 35) and VC ewes (n = 35) were blood sampled twice weekly to monitor their pre-mating progesterone profiles. At mating, harnessed entire rams were introduced, 17 or 16 days after the last ram exposure (Experiments 1 and 2) and raddle marks were recorded daily. The median time from ram introduction to mating was reduced in ewes given both fence-line and vasectomised ram contact (P < 0.001), leading to a more compact distribution of mating and lambing (At least P < 0.01). In the blood sampled VR ewes, there was a progressive decline in the number of days from ram exposure to the onset of dioestrus (at least P < 0.05). This observation indicates that the cycles in VR ewes became increasingly synchronised over the pre-mating period, a pattern not evident in VC ewes. In conclusion, repeated, short-term exposure of ewes to rams during the transition into the breeding season is an effective method of synchronising the distribution of mating during the breeding season.     

Some effects of breeding season and castration on the prostate and epididymis of the brushtail possum, Trichosurus vulpecula

In an attempt to understand the mechanism(s) responsible for the reported marked seasonal increase in prostatic, but not epididymal, weight in T. vulpecula, a number of parameters were measured in tissues from mature, entire males sampled within and outside of the breeding season and from castrates. Conditions for the measurement of cytosol androgen receptors were also established. The weight of both the prostate and the epididymis was significantly elevated in the breeding season but the relative increase in prostate weight was considerably greater. The increase in prostatic weight was associated with a decrease in DNA: g tissue and an increase in protein: DNA and RNA: DNA ratios, each indicative of cellular hypertrophy and/or accumulation of secretory product. In the epididymis there were no significant seasonal changes in RNA: DNA, protein: DNA or DNA: g tissue ratios. Low-capacity, high-affinity binding was demonstrated in the epididymal and prostatic cytosols and values for the equilibrium association constants and receptor concentrations were within the range reported for androgen receptors in eutherian species. The temperature sensitivity of the binding, steroid specificity and slow dissociation in the cold indicated that in both tissues cystosol receptor and not androgen-binding or serum-binding protein(s) were being measured. In prostatic, but not epididymal, cytosol a low level of progesterone binding was observed and was masked by triamcinolone acetonide. When expressed in terms of tissue DNA, cytosol androgen receptor level in the prostate only was elevated in the breeding season. Prostatic tissue showed a low level of 5 alpha-reductase in vitro which was not influenced by season. However, both tissues showed a high concentration of 5 alpha-dihydrotestosterone and in the prostate, where seasonal effects were measured, the concentration was higher in the breeding season. This indicates that although 5 alpha-dihydrotestosterone is the likely active androgen in the prostate it may be formed elsewhere. Part of the explanation for the increased growth of the prostate in the breeding season appears to be a change in receptor concentration coupled with elevated tissue androgen level.     

The effects of cortisol and testosterone on renal function in male Antechinus stuartii (Marsupialia)

Seasonal changes in the physiology of Antechinus stuartii result in complete male mortality after mating. The most important endocrine changes in males are large rises in plasma testosterone and cortisol concentrations. Glomerular filtration rate (GFR) in males declines coincident with high plasma testosterone and cortisol. In the present study GFRs were measured in males captured in May (when endogenous plasma testosterone and cortisol levels are low) and given depot injections of either saline, testosterone-only, cortisol-only or testosterone plus cortisol at doses designed to mimic plasma levels during the mating period. GFR decreased significantly with testosterone injection, independent of cortisol treatment. Urinary concentrations of sodium and chloride, and osmolality decreased significantly with cortisol treatment, although the addition of testosterone reversed the effect. Total urinary excretion of electrolytes was similar between groups. Plasma potassium levels significantly increased in testosterone plus cortisol treated males. Plasma sodium levels significantly increased and plasma chloride significantly decreased in all groups treated with cortisol. Water consumption significantly increased in all cortisol-treated males and food consumption significantly increased in all testosterone-treated males. The seasonal renal functional changes observed in A. stuartii were mimicked by testosterone administration. Accepted: 23 January 1998     

Social and foraging components of the home range in Antechinus stuartii (Dasyuridae: Marsupialia)

The home range of Antechinus stuartii has two components. A small area (0.94 ha for males, 0.38 ha for females) is used for foraging, while a much greater social range (mean for males in a given year may exeed 5 ha; the mean for females never exceeded 3 ha) encompasses communal nests, which are used by both males and females. The foraging range of females is stable throughout the year. The foraging range may not coincide at all, or only partially coincide with her social range. By contrast, male foraging ranges drift through the year, and generally lie within their social range. Intersexual differences reflect the different selection pressures on the sexes. Females require access to resources to sustain a costly lactation, while males require a knowledge of many possible sites for mating and early resolution of agonistic interactions with other males with which they cohabit during mating.     

Effect of glutathione depletion on antioxidant enzymes in the epididymis, seminal vesicles, and liver and on spermatozoa motility in the aging brown Norway rat

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.     

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.     

Anatomic distribution of lectin-binding sites in mouse testis and epididymis

Testis and epididymis of sexually mature mice were studied histochemically using 25 fluorescein-isothiocyanate-labeled lectins. Several lectin-specific binding patterns were recognized. Thus, HAA, HPA, GSA-I, and UEA-II reacted only with spermatozoa. PNA, GSA-II, SBA, VVA, BPA, RCA-I, and RCA-II reacted with spermatozoa and spermatocytes. WGA, PEA, LCA, and MPA reacted with spermatogonia, spermatocytes, and spermatozoa in increasing order of intensity. ConA, Suc. ConA, LAA, STA, LTA, LPA, PHA-E, PHA-L, UEA-I, and LBA reacted with all spermatogenic cells with equal intensity. In the epididymis, 12 lectins reacted uniformly with the epithelial cells lining all segments of this organ. One lectin (VVA) did not react with epididymal lining cells. The remaining 12 lectins reacted in a specific manner with portions of the head, body, or tail, thus selectively outlining different portions of the epididymis. RCA-I and RCA-II selectively accentuated the so-called halo cells of the epididymis. These findings provide a detailed map of lectin-binding sites in the mouse testis and epididymis and show that certain lectins can be used as specific markers for spermatogenic cells and segments of the epididymis.