Testis asymmetry, in which the testes in an individual differ in size, has recently been reported in koalas (Phascolarctos cinereus) in the Mount Lofty Ranges population of South Australia. We describe the morphology and histology of both testes from affected individuals in this population (n=56) and the parameters of koalas with normal-sized testes based on age and breeding season (n=56). Morphologic measurements included testis weight, length, width, and volume; histologic parameters included seminiferous tubule diameter, seminiferous epithelial height, and seminiferous tubule (interstitial tissue ratio and presence or absence of spermatozoa). Of the 56 koalas with intraindividual variation in testes size, 47 were classified as asymmetric and nine as microtestes. For koalas with asymmetric testes, all morphologic parameters were significantly decreased in the smaller testes compared with normal-sized testes, but for the histologic parameters, only seminiferous tubule diameter was significantly less. Histopathologic examination of the asymmetric testes showed 38 with normal parenchyma histologically indistinguishable between intraindividual testes, four with degeneration and atrophy, and three with hypoplasia, whereas examination of microtestes showed degeneration and atrophy in seven, hypoplasia in one, and aplasia in one. No association of testis size difference with Chlamydia pecorum infection was found in a subset of animals. For the 56 koalas with normal-sized testes, morphologic parameters were found to increase with age, and juvenile and young adults were found to have smaller seminiferous tubule diameters than adults. No differences were found between testes of koalas in the breeding and nonbreeding season. Overall, these findings indicate that testis asymmetry in koalas from the Mount Lofty Ranges population is common but not associated with decreased function, except where testis malformations such as hypoplasia or aplasia occur or when parenchyma has been disrupted by acquired disease.
Mild asymmetry in testis size has been reported in humans (Vaganée et al. 2018) and some species of mammals (Yu 1998). Marked testis asymmetry occurring in mammalian populations at high prevalence appears uncommon and has only been reported in the European bison (Bison bonasus), with a high proportion of males having 10–30% difference in testis size (Krasińska et al. 2009).
In the Mount Lofty Ranges koala (Phascolarctos cinereus) population of South Australia (SA), a recent necropsy study found that marked testes asymmetry occurred in 6 of 56 (11%) males (Speight et al. 2018). In all of these cases, both testes were present, but one was 30–50% smaller than the other, with histopathology of four cases showing changes consistent with atrophy (Speight et al. 2018). Testis asymmetry has not been reported in other koala populations, and the morphologic and histologic differences occurring in individuals with asymmetric testes have yet to be described in detail.
The cause of testis asymmetry in koalas may or may not be associated with previously reported infectious or developmental testicular diseases. Chlamydia pecorum infection has been found to cause abnormalities in the male reproductive tract, with orchitis, epididymitis, and testis fibrosis documented for koalas from Queensland (Deif 2011; Johnston et al. 2015). In the Mount Lofty Ranges koala population in SA, incidence of orchitis appears to be low, with no cases reported in a histopathologic study of 50 males (Fabijan et al. 2020) and only 5% in another study of 40 males (Speight et al. 2016). However, one koala in the previous report of testis asymmetry in Mount Lofty Ranges koalas showed interstitial orchitis in the smaller testis (Speight et al. 2018), which may have indicated chlamydial infection. The developmental abnormality testis aplasia has previously been reported in Kangaroo Island (Seymour et al. 2001; Montgomery 2002; Cristescu et al. 2009; Fabijan et al. 2019) and Eyre Peninsula koala populations in SA, as well as in the French Island koala population in Victoria, Australia (Seymour et al. 2001; Montgomery 2002). Incidence of testis aplasia was found to be positively correlated with high inbreeding coefficients in these populations, suggestive of inbreeding depression (Seymour et al. 2001). The Mount Lofty Ranges koalas share common origins with these populations (Robinson 1978), so they may also be more predisposed to testis aplasia.
Histologic structure of the normal koala testis has previously been described in a Queensland-based study of the germ cell changes that occur during the various stages of the cycle of seminiferous epithelium (Oishi et al. 2013). Also, in the surrounding Sertoli cells, large crystalloid inclusions of unknown function have been found (Harding et al. 1982; Kerr et al. 1987). A previous study of testis morphology in New South Wales koalas included an investigation as to whether changes occur during the breeding season, but no seasonal differences in testis volume were found (Cleva et al. 1994). Studies of the effects of age on koala testis are limited, with Kerr et al. (1987) reporting that juvenile koalas from Victoria have seminiferous tubules composed of “nonlumenated cords” containing mostly Sertoli cells, few spermatogonia and primary spermatocytes, whereas in adults, full spermatogenesis and spermatozoa were present.
We describe the morphology and histology of the testes of the Mount Lofty Ranges koala population in SA. Our study aimed to determine 1) the normal testicular morphologic and histologic parameters, including changes associated with breeding season and age, and 2) the morphology and histopathology of abnormal testes that occur in this population.
MATERIALS AND METHODS
Formalin-fixed left and right testes from 112 male koalas from the Mount Lofty Ranges population that were necropsied between 2012 and 2019, following death or euthanasia on welfare grounds for trauma or illness, were obtained for this study from an archived collection of 275 male koalas. For this study, “abnormal testes” (n=56) were considered those from individuals in which marked testes size differences had been recorded at necropsy. Any koalas with testicular lesions associated with trauma such as bruising or hemorrhage were excluded from the study. “Normal testes” (n=56) were of similar size to the contralateral testis of the same animal, lacked any gross lesions, and were from koalas with a good body condition score (>3.5) at necropsy (scale 1–5; Blanshard and Bodley 2008). A pairwise comparison of the morphologic and histologic measurements for the left and right testis of koalas in the normal group showed no significant differences (P>0.05).
Koalas were aged by using the tooth wear classification (TWC; Martin and Handasyde 1990) and grouped into three age groups based on the stage of sexual maturity. These were juvenile (TWC I, <2 yr old); young adult (TWC II–III, 2–4 yr old); and adult (TWC IV+, 5+ yr old). For the normal cohort, breeding season status was determined by using the date of euthanasia, with those euthanized between September and February classified as being in the breeding season and those between March and August in the nonbreeding season (Martin and Handasyde 1990). All animal use was approved by the University of Adelaide Animal Ethics Committee (S-2013-198 and S-2016-169) in conjunction with Department for Environment and Water Scientific Research approval (Y26054).
Four morphologic parameters were determined for the formalin-fixed testes and epididymides: testis weight, testis length, testis width, and epididymal weight. Using the maximum length (L) and width (W) of the testes, total testicular volume (TTV) was calculated with the equation TTV=0.524×L×W2 (Allen et al. 2010). The testis mass percentage of body weight was recorded as relative testis mass (RTM).
For histologic examination, two parasagittal incisions were made in each testis to produce a central section, and the remaining outer pieces of testis each trimmed tangentially to produce tissue representing each pole of the testis (Kamstock et al. 2011). Tissues were routinely processed for histology, with paraffin blocks sectioned at 5 µm and slides stained with H&E.
Histologic analyses included determination of seminiferous tubule diameter (in micrometers) and seminiferous epithelial height (in micrometers) by using LabSens™ Software (Olympus, Shinjuku-ku, Tokyo, Japan) and a semiquantitative assessment of the area of the seminiferous tubules within the testes, representative of spermatozoa producing tissue, as well as the intertubular area of the interstitial tissue, occupied predominantly by testosterone-producing Leydig cells. The presence or absence of sperm in seminiferous tubules was also recorded.
For histologic measurements, each testis section was divided into quadrants, and a region in each quadrant was randomly selected to be photographed with 10× objective magnification on a light microscope. Within each image, five seminiferous tubules were randomly selected for measurement; to correct for plane of sectioning, the shortest diameter of seminiferous tubules was measured. Epithelial height was determined in the same five tubules by measuring the epithelium from the seminiferous tubule basement membrane to the apical surface. For each testis, 20 to 30 tubule diameters and epithelial height measurements were determined, as well as four to six assessments of the proportion of tubules to interstitial tissue. The averages were then calculated for each individual. In the abnormal testes, in addition to histologic measurements, histopathologic changes were also described.
Dry cotton swabs (Copan Italia SpA, Brescia, Italy) of the conjunctiva and urethra were available for a subset of 75 males (38 with normal testes and 37 with abnormal testes) that had been collected at necropsy and stored at –80 C. DNA extraction was performed by using the Qiagen DNAEasy Kit (Qiagen, Hilden, Germany), following the manufacturer's protocol. Chlamydia pecorum detection was performed on a pooled sample for each koala with quantitative PCR (qPCR; Marsh et al. 2011) by using forward (5′-CCAAGCATAATCGTAACAA-3′) and reverse (5′-CGAAGCAAGATTCTTGTC-3′) primers that targeted the C. pecorum outer membrane protein gene (Hulse et al. 2018). Each reaction was run in triplicate by using 2.5 µL of Power SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.5 µL of each primer, and 1.5 µL of DNA template (1/50) to create a volume of 5 µL in each reaction. The qPCR was performed by using the following conditions: denaturation for 10 min at 95 C, 40 cycles of 15 s at 95 C, and 60 s at 60 C. For sample confirmation, a melt curve analysis was run for 15 s at 95 C, 15 s at 60 C, and 15 s at 95 C. For quality control, β-actin qPCR was performed as described earlier (Shojima et al. 2013) to determine the presence of koala genomic DNA, with samples that were negative for β-actin removed.
All data were tested for normality and homogeneity by using SPSS version 26 (IBM Corporation, Armonk, New York, USA), with extreme outliers removed. Data in the normal cohort were compared based on breeding season and age, and data of the abnormal cohort were compared pairwise between normal and abnormal testes of individuals. Variables that were normally distributed were analyzed by using a one-way analysis of variance and post hoc t-tests to determine differences between groups by using the Levene test to determine equality of variances. Variables that were not normally distributed were analyzed by using nonparametric tests, including Kruskal-Wallis and post hoc Mann-Whitney U-test, to determine differences between groups. A chi-square association test was used to determine the association between sperm production and age, breeding season and normal and abnormal testes, and between C. pecorum status and normal or abnormal testes. Results were considered statistically significant at P≤0.05, and all data are presented as mean±SD.
Of 56 koalas classified as having normal testes, 33 were obtained in the breeding season and 15 in the nonbreeding season (eight unknown seasons). No significant differences were found for any of the morphologic or histologic parameters based on breeding season status (P≥0.05). Based on TWC, there were five juvenile koalas, 29 young adults, 21 adults, and one with age no recorded. The mean testis weight of the adult Mount Lofty Ranges koalas was 3.07±0.62 g, volume was 3.42±0.79 cm2 (n=13), the seminiferous tubule diameter was 221±20 µm, and the overall seminiferous tubule area was 82±7.3% (n=21). Significant differences were found between each of the age groups by using analysis of variance or Kruskal-Wallis tests for all of the morphologic measurements, except RTM (see Table 1).
Juvenile koalas had significantly lower testes weight (t-test, t=–4.429, df=19, P<0.001), epididymal weight (Mann-Whitney U-test, Z=–2.502, P=0.012), testis length (t-test, t=–3.385, df=19, P=0.003), and testis width (t-test, t=–5.788, df=18, P<0.001) compared with the young adults, as well as a lower testis volume (Mann-Whitney U-test, Z=–2.613, P=0.009). Juvenile koalas also had significantly lower testes weight (t-test, t=–7.249, df=14, P<0.001), epididymal weight (Mann-Whitney U-test, Z=–2.635, P=0.008), testis length (ttest, t=–5.432, df=14, P<0.001), and testis width (t-test, t=–9.984, df=14, P<0.001) than the adult group, as well as a lower testis volume (Mann-Whitney U-test, Z=–2.625, P=0.009). The young adult group, compared with adults, had significantly lower testis weight (t-test, t=–3.331, df=29, P=0.002), epididymal weight (Mann-Whitney U-test, Z=–2.858, P=0.004), testis length (t-test, t=–2.946, df=29, P=0.006) and width (t-test, t=–3.577, df=28, P=0.001), as well as testis volume (Mann-Whitney U-test, Z=–3.063, P=0.002). The RTM was similar between the juvenile and young adult groups, but both were significantly lower than those of the adult group (t-test, t=–2.661, df=14, P=0.019; and t-test, t=–2.110, df=28, P=0.044, respectively).
Based on histologic parameters, the diameter of seminiferous tubules was significantly lower in juvenile koalas compared with both young adult (t-test, t=–4.811, df=32, P<0.001) and adult (t-test, t=–6.256, df=24, P<0.001) koalas. For seminiferous epithelial height, there were no significant differences found between the young adult and adult groups; autolysis in some tissue sections prevented measurement of this parameter in some samples (see Table 1). No epithelial measurements were obtained in juvenile koalas, due to the lack of lumina in seminiferous tubules (explained soon). Seminiferous tubule to interstitial tissue ratio was significantly lower in the young adults than adults (Mann-Whitney U-test, Z=–3.206, P=0.001); however, the juvenile group showed no significant differences. The presence of spermatozoa was significantly associated with age (χ2 test, χ2=12.34, df=1, P<0.001), with only 14/29 of young adults having spermatozoa present compared with 20/21 adults and complete absence of spermatozoa in juveniles.
The seminiferous tubules of the juvenile koala testes lacked lumina, with the epithelium consisting mainly of large Sertoli cells and some germ cells at various stages of meiosis I (Fig. 1). In the young adults, there were germ cells in meiosis I or meiosis II, with some having completed spermatogenesis with late spermatids and spermatozoa present, and the latter having the characteristic hook-shaped head as previously reported (Temple-Smith and Taggart 1990; Breed et al. 2001). Adult koalas similarly exhibited all stages of spermatogenesis.
Of the 56 koalas found to have abnormal testes, 47 (84%) were classified as asymmetric, whereby both testes were fully formed but of different size (Fig. 2a). Within this group, there were two juveniles, 23 young adults, and 22 adults. The remaining nine koalas (16%) were classified as having “microtestes” in which one (n=8) or both testes (n=1) were very small (Fig. 2b), with one juvenile, five young adults, and three adults in this group. The overall prevalence of testis asymmetry and microtestes in the Mount Lofty Ranges population was 17% and 3%, respectively, based on the total of 275 necropsy records of male koalas from which the koala samples in this study were selected. Of 22 koalas with available data, the left testis was smaller in 64% of cases (χ2 test, χ2=1.63, df=1, P=0.2); no cases of cryptorchidism were observed.
In the asymmetric testes group, the smaller testes differed in weight by 27.7±13.5% from the normal-sized testes, weighing significantly less (paired t-test, t=–7.744, df=29, P<0.001). Based on the other morphologic parameters (Table 2), the smaller testes also showed significantly reduced testis length (paired ttest, t=–6.741, df=32, P<0.001), width (paired t-test, t=–7.366, df=36, P<0.001), and volume (paired t-test, t=–8.434, df=31, P<0.001) compared with the normal testes. Measurements of normal-sized testes from the asymmetric testes group were statistically comparable (P>0.05) to those of koalas in the normal cohort. The proportion of koalas in each age group in the asymmetric group was also comparable with that of the normal cohort.
For the histologic parameters, only seminiferous tubule diameter was significantly lower in the asymmetric testes group (paired t-test, t=–2.311, df=41, P=0.026). Also, histopathologic examination found that 38 had smaller testes that were histologically indistinguishable from the normal testes. However, four koalas showed unilateral testicular degeneration and seminiferous tubule atrophy, and three koalas had testis hypoplasia. Degenerative changes included coagulative necrosis, hemorrhage, tubular loss, and fibrosis probably secondary to ischemic or inflammatory etiologies. Testes classified as hypoplastic showed a lower number of seminiferous tubules, with smaller diameter, lower seminiferous epithelial height, a lower density of Sertoli cells, and variation in lobular structure. In the nine koalas with microtestes, histopathologic abnormalities included unilateral testicular degeneration or necrosis or both, replacement fibrosis, and seminiferous tubule atrophy (n=7), hypoplasia (n=1; Fig. 3), and aplasia (n=1), with the latter individual showing no testicular tissue and an atrophic epididymis. For cases of testicular degeneration, changes were consistent with an ischemic event or chronic inflammation leading to parenchymal destruction and replacement fibrosis.
There was no significant association between C. pecorum infection status for koalas that had normal testes (11/38 positive; 29%) and abnormal testes (18/37 positive, 49%; χ2 test, χ2=3.06, df=1, P=0.08). Of the 18 koalas that were positive for C. pecorum in the abnormal group, 14 had asymmetric but histologically indistinguishable testes, one had unilateral testicular degeneration, two hypoplasia, and one aplasia.
In 47 koalas from the Mount Lofty Ranges of SA, we found marked testis asymmetry (mean 28% size difference); however, only 9% of the smaller testes showed histopathologic changes. Conversely, in nine koalas with microtestes, degeneration and atrophy, hypoplasia, or aplasia were evident in all cases. This suggests that testis asymmetry is a common syndrome in this population, but unlikely to affect overall fertility, and that testis malformations are rare.
The mean testis weight of adult Mount Lofty Ranges koalas (3.07±0.62 g) was similar to weights reported by Taggart et al. (1998) and Montgomery (2002) in koalas from two other SA populations: Kangaroo Island (3.21±0.25 g), and the Eyre Peninsula (3.27±0.16 g). Also, the testes volume of the adult koalas in this study (3.42±0.79 cm2) was similar to that found in Queensland koalas by Allen et al. (2010), despite the body mass differences between southern and northern koalas (Lee and Martin 1988). The adult seminiferous tubule diameter (221±20 µm) was similar to that previously reported for adult Queensland koalas by Oishi et al. (2013), but the overall seminiferous tubule area at 82±7.3% was considerably higher than the 67.3±1.9% previously found in Queensland koalas (Oishi et al. 2013). This might reflect differences in the method of area determination in the two studies or possibly population-based differences.
The similar testis morphology between breeding and nonbreeding seasons is consistent with findings in captive New South Wales koalas (Cleva et al. 1994). Other marsupial species such as the tammar wallaby (Macropus eugenii; Inns 1982) and southern hairy-nosed wombat (Lasiorhinus latifrons; Taggart et al. 2005) also lack seasonal differences in the testes or epididymal weights, despite being seasonal breeders. However, Allen et al. (2010) found seasonal differences in several reproductive parameters in Queensland koalas, including that testicular volume peaked in spring, which could suggest an increase in testicular activity at the onset of the breeding season in that population.
In the Mount Lofty Ranges koalas, we found that testes morphologic measures increased with age, as well as seminiferous tubule diameter, which increased at the onset of sexual maturity and may reflect the development of tubule lumina at this time. Associated with this was an increase in RTM, with juveniles having an RTM of 0.038%, young adults 0.051%, and adults 0.060%, similar to that reported by Temple-Smith and Taggart (1990) and Taggart et al. (1998). Koalas have a lower RTM than many other marsupial species such as the brushtail possum (Trichosurus vulpecula) with 0.25% and the western grey kangaroo (Macropus fuliginosus) with 0.15% (Tyndale-Biscoe and Renfree 1987; Taggart et al. 1998). High RTM suggests postcopulatory intermale sperm competition and the possibility of a polyandrous mating system (Kenagy and Trombulak 1986; Simmons and Fitzpatrick 2012; Ramm and Schärer 2014), with the low RTM in koalas suggesting low levels of intermale sperm competition and the possibility of a monogamous mating system. This conclusion is supported by the finding that male koalas rarely exhibit physical aggression toward each other, with female choice possibly being a key factor in mate selection (Ellis and Bercovitch 2011).
We found testis size asymmetry to be the most common testis abnormality in the Mount Lofty Ranges koalas; however, histopathologic findings indicated that in most cases, the smaller testis was histologically normal and indistinguishable from that of the typical-sized testis, suggesting unimpaired sperm production capacity. High prevalence of testis asymmetry appears uncommon in other mammals, and its occurrence in these koalas suggests either a population-based syndrome that could be related to abnormal testicular development in juveniles or an adult-onset acquired disease.
The latter is supported by the finding that four koalas with asymmetric testes, and seven with microtestes, had unilateral testicular degeneration and atrophy with pathologic changes including coagulative necrosis, hemorrhage, tubular loss, and fibrosis. These may have been due to ischemic (e.g., torsion, trauma, localized thromboembolic disease, and neoplasia) or inflammatory etiologies, but the underlying cause needs further investigation, as no association with chlamydial infection was identified, and orchitis was not observed.
In relation to an underlying abnormality of testicular development, hypoplasia occurred in the testes of four individuals (asymmetric group n=3; microtestes group n=1). We found only one koala with testis aplasia or agenesis, in which there was no testis tissue present and the epididymis was hypoplastic or atrophic. Testicular aplasia has previously been reported at much higher prevalence in koalas from Kangaroo Island (13%), as well as Eyre Peninsula (24%), where it has been linked to low genetic diversity (Seymour et al. 2001). Whether testis asymmetry could also be associated with low genetic diversity in the Mount Lofty Ranges koalas is unknown, but a recent study has identified downregulated expression of several reproductive development genes, including an androgen receptor in this population, compared with that found in Queensland koalas, using lymph node tissue (Tarlinton et al. 2021). This could potentially be linked with testis developmental abnormalities but needs further investigation by using testis tissue from affected individuals to understand the unilateral nature of the condition.
In summary, our study of koalas from the Mount Lofty Ranges population in SA found significant changes occurring in normal testes as koalas increase in age, associated with increasing sexual maturity, but no differences in testis parameters between the breeding and nonbreeding season. Testis abnormalities included a relatively common syndrome of testis size asymmetry with histologically normal testes, which has rarely been reported in other mammals. Testis malformations including degeneration, atrophy, hypoplasia, and aplasia occurred more rarely. Further investigation of these abnormalities is needed to determine the potential pathogeneses as either inherited or acquired and to contribute to the overall understanding of the health status of this southern koala population.
The authors thank Cleland Wildlife Park, the Adelaide Koala and Wildlife Hospital, and Adelaide Koala Rescue; as well as Adrian Hines and other staff in the Veterinary Diagnostic Laboratory at the University of Adelaide.