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J.D. Konvalina and S.E. Trauth
22001188 SOUTHEASTERN NATURALIST 1V7o(3l.) :1572,1 N–5o3. 03
Seasonal Variation of Testicular Tissue in Northern Rough
Greensnakes, Opheodrys a. aestivus, from Alabama
John D. Konvalina1,* and Stanley E. Trauth2
Abstract - Gamete production is a fundamental component of the reproductive cycle of an
organism. Studies dedicated to the testicular cycle in mammals and birds vastly outnumbers
those discussing the process in reptiles. To help increase the availability of such knowledge
for reptiles, we histologically examined the testicular cycle of Opheodrys a. aestivus
(Northern Rough Greensnake) from populations in Alabama. We measured seminiferous
tubule diameter and seminiferous tubule epithelial height from 30 specimens. The individuals
in our sample exhibited small seminiferous tubule diameters in spring followed by
increases in summer. By October, the lumen was mostly empty of sperm because they had
migrated to the vas deferens for winter storage. Seminiferous tubule epithelial height was
significantly correlated with seminiferous tubule diameter. Using AIC model selection, we
compared both additive and interactive models to determine if either seminiferous tubule
diameter or season influenced seminiferous tubule epithelial height. We found that only
seminiferous tubule diameter was a significant predictor of seminiferous tubule epithelial
height. Like other temperate snakes, Northern Rough Greensnakes in Alabama have postnuptial
spermatogenesis where sperm are produced in the summer following spring mating.
Future studies of this species need to investigate the testicular cycle in other parts of its
geographic distribution to see if this monthly pattern is consistent.
Introduction
The reproductive cycle of an organism can be divided into 4 stages: production
of gametes, mating, oviposition, and birth. Gamete production is the most understudied
and fundamental of these components. Books have been dedicated to the
study of the testicular cycle in mammals and birds (Ewing et al. 1980, Russell et
al. 1990), but only 3 reviews cover the process in reptiles (Gribbins 2011; Gribbins
and Rheubert 2011, 2014).
In some snakes, such as Thamnophis s. sirtalis L. (Eastern Gartersnake), sperm
are stored over winter in the vas deferens (Clesson et al. 2002), whereas in others
like Carphophis vermis (Kennicott) (Western Wormsnake), sperm are retained
within the ductus deferens year-round (Aldridge and Metter 1973). In Zaocys dhumnades
(Cantor) (Chinese Ratsnake), sperm are stored in the vas deferens and also
the epididymis (Liang et al. 2011). Comparing testis volume to the total mass of the
organism can be used to decipher seasonal allocation of resources between somatic
growth and reproduction (Moshiri et al. 2014).
1Department of Biology, University of Central Florida, 4000 Central Florida Boulevard,
Orlando, FL 32816. 2Department of Biological Sciences, Arkansas State University, PO
Box 599, State University, AR 72467. *Corresponding author - jkonvalina@knights.ucf.edu.
Manuscript Editor: John Placyk
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Changes in season trigger the different testicular cycle phases, with environmental
temperatures playing a pivotal role in initiating spermatogenesis (Hawley
and Aleksiuk 1976). We can divide the testicular cycle in squamates into 4 phases:
maximum activity (all stages of spermatogenesis are active), regression (cells start
to die), quiescence (cessation of spermatogenesis), and recrudescence (reinitiation
of spermatogenesis) (Hernández-Gallegos et al. 2014). Seasonal differences in
testicular stages are noted by measuring interstitial cell nuclear diameters, seminiferous
tubule diameters, and epithelial heights. Differences in season have been
found in colubrids (Goldberg and Parker 1975), elapids (Shine 1977), homalopsids
(Jadhav and Padgaonkar 2011) and viperids (Gribbins et al. 2008).
One example of seasonal differences is in the testicular cycle of Agkistrodon piscivorus
(Lacépède) (Northern Cottonmouth). It begins in April with recrudescence,
followed by peak sperm production in July and August, and ending with the testes
regressing in the winter months (Johnson et al. 1982). Aldridge et al. (1990) also
noted that in Opheodrys a. aestivus L. (Northern Rough Greensnake), spermatogenesis
peaks in July. However, all their specimens were collected from Arkansas, so
their data may not represent characteristics of the testicular cycle in other parts of the
expansive range of this species. Rough Greensnakes occur widely in the eastern and
southeastern United States, from southern New Jersey and Indiana south along the
East Coast to Florida and west to central Texas, eastern Kansas and central Oklahoma
(Trauth et al. 2004). We addressed this concern in our study by examining seasonal
variation in the testicular cycle in Alabama populations of Northern Rough Greensnake.
We hypothesized that both season and seminiferous tubule diameter would
have a significant effect on seminiferous tubule epithelial height. Thus, we expected
to find significant differences in seminiferous tubule epithelial height among seasons
and among different seminiferous tubule diameters. We also hypothesized that
Northern Rough Greensnakes from Alabama would exhibit post-nuptial spermatogenesis,
corroborating what Aldridge et al. (1990) found in Arkansas.
Methods
We obtained 30 Northern Rough Greensnakes collected in multiple Alabama
counties over a period of 18 years (1957–1975) from the Auburn University Museum
(AUM) herpetological collection (Table 1). We dissected the testes from each
specimen and dehydrated them in a graded series of increasing ethanol solutions
(50–100%) and embedded them in paraffin following the methods of Presnell and
Schreibman (1997). We serially sectioned the Paraffin-tissue blocks into ribbons
10 μm in thickness using a rotary microtome and affixed the ribbons, as they were
floating on a 2% formalin solution, to microscope slides by using Haupt’s adhesive.
We stained slides with hematoxylin and eosin for general cytology. We used
a Nikon Eclipse 600 epi-fluorescent light microscope with a Nikon DXM 1200C
digital camera (Nikon Instruments Inc., Melville, NY, USA) for photomicroscopy.
For each specimen, we randomly selected 20 seminiferous tubules and measured
seminiferous tubule diameter and seminiferous tubule epithelial height. Seminiferous
tubule diameter was defined as the lumen plus the epithelium at the widest point
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of the tubule. We averaged the 20 measurements to give each individual an average
seminiferous tubule diameter and average seminiferous tubule epithelial height.
We used a mixed model to test for significant differences in seminiferous tubule
diameter and seminiferous tubule epithelial height among samples from different
months. Then, we used a post hoc test (extension of Tukey for mixed models) to
identify the significant differences among months. Due to small monthly sample
sizes, we collapsed the months into 3 seasons: spring (April and May, n = 18),
summer (June, n = 5) and fall (September and October, n = 7). ). Each season was
normally distributed, but variances were not equal among seasons. Therefore, we
performed a non-parametric Kruskal-Wallis test followed by a Pairwise Wilcoxon
rank sum test to identify differences in seminiferous tubule diameter and seminiferous
tubule epithelial height among seasons. Next, we used a Pearson correlation
test to look for a correlation between seminiferous tubule diameter and seminiferous
tubule epithelial height.
Table 1. Opheodrys a. aestivus (Northern Rough Greensnake) specimens from Alabama used for light
microscopy. All sections taken were of the testis and prepared by Stanley E. Trauth.
Month Date Alabama county
April 9-Apr-59 Lee
April 10-Apr-63 Russell
April 29-Apr-66 Calhoun
April 24-Apr-67 Lee
April 13-Apr-68 Dale
April 18-Apr-68 Butler
April 04-Apr-70 Lee
April 13-Apr-73 Baldwin
May 19-May-43 Lee
May 4-May-57 Walker
May May-61 Lee
May May-61 Lee
May 12-May-68 Cherokee
May 19-May-68 Lee
May 4-May-69 Marshall
May 19-May-69 Barbour
May 10-May-75 Dallas
May 12-May-75 Randolph
June 25-Jun-49 Lee
June 22-Jun-57 Cleburne
June 22-Jun-57 Cleburne
June 12-Jun-65 Mobile
June 6-Jun-66 Morgan
September 30-Sep-67 Lauderdale
September 2-Sep-70 Covington
September 27-Sep-70 Elmore
October 12-Oct-61 Shelby
October 1-Oct-67 Barbour
October 12-Oct-67 Russell
October 8-Oct-68 Macon
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Finally, we tested 4 linear models to determine what factors influenced seminiferous
tubule epithelial height. The first model was seminiferous tubule epithelial
height as function of seminiferous tubule diameter, the second was seminiferous
tubule epithelial height as function of season, the third was seminiferous tubule
epithelial height as a function of seminiferous tubule diameter plus season, and the
final model was seminiferous tubule epithelial height as a function of seminiferous
tubule diameter plus season plus an interaction between those 2 terms. We used AIC
to determine which model best fit the data. We performed all statistical analyses at
a 5% significance level using the statistical programming software R (R Development
Core Team 2014).
Results
There were significant differences in seminiferous tubule diameter among
months (χ2 = 289.64, df = 4, P < 0.001). Seminiferous tubule diameter was significantly
different between April and June (z = 3.15, P = 0.014), April and September
(z = 3.97, P less than 0.001), May and June (z = -4.87, P less than 0.001), May and September (z =
5.41, P less than 0.001), and May and October (z = 4.06, P less than 0.001). All other month-tomonth
comparisons were not significant (Fig. 1).
There were significant differences in seminiferous tubule epithelial height
among months (χ2 = 118.63, df = 4, P < 0.001). Epithelial height was significantly
Figure 1. (Left) Average seminiferous tubule diameter by month for Opheodrys a. aestivus
(Northern Rough Greensnake) from Alabama. The letters above the boxes represent significance
levels; if 2 months share at least one letter, they are not significantly different,
and if 2 months do not share any letters, they are significantly different. (Right) Average
seminiferous tubule epithelial height by month for Northern Rough Greensnakes from Alabama.
The symbol above the box for May represents that it is significantly different from
June. The black horizontal line within each box represents the median value. The bottom
and top of each box represents the 1st and 3rd quartiles, respectively. The “whiskers” above
and below the box show the maximum and minimum values. The numbers below the boxes
represent sample size (i.e., number of snakes per month).
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different between May and June (z = -2.84, P = 0.04). All other month-to-month
comparisons were not significant (Fig. 1).
Mean seminiferous tubule diameter was significantly different among seasons
(Kruskal-Wallis χ2 = 13.3, df = 2, P = 0.001). Both fall (P = 0.003) and summer
(P = 0.01) had significantly greater seminiferous tubule diameters than spring
(Table 2, Fig. 2). No significant differences in epithelial height were found among
seasons (Kruskal-Wallis χ2 =5.23, df = 2, P = 0.07; Table 2, Fig. 2). There was a
positive correlation between seminiferous tubule epithelial height and seminiferous
tubule diameter (r = 0.64, P < 0.001; Fig. 3). Model 1 (seminiferous tubule
epithelial height ~ seminiferous tubule diameter) was the best model according to
AIC (Table 3). The summary (Table 4) and plot (Fig. 4) of model 1 show that seminiferous
tubule diameter is a significant factor in predicting seminiferous tubule
epithelial height. Season was not included in this model and, therefore, was not a
significant factor in predicting seminiferous tubule epithelial height.
Figure 2. Average seminiferous tubule diameter (left) and average seminiferous tubule
epithelial height (right) by season for Opheodrys a. aestivus (Northern Rough Greensnake)
from Alabama. The asterix above the box for spring tubule diameter represents that it is
significantly different from summer and fall. Open circles above the boxplots are outliers.
Boxplot description is the same as that in Figure 1.
Table 2. Mean seminiferous tubule diameter and seminiferous tubule epithelial height by season from
Opheodrys a. aestivus (Northern Rough Greensnake) testes from Alabama. 95% confidence intervals
are listed in brackets beside each mean.
Season n Mean tubule diameter Mean epithelial height
Spring 18 114.47 μm [102.58, 126.36] 28.95 μm [25.25, 32.65]
Summer 5 155.93 μm [137.29, 174.57] 38.24 μm [26.34, 50.13]
Fall 7 161.67 μm [139.68, 183.67] 34.72 μm [29.41, 40.04]
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Figure 3. Correlation between seminiferous tubule epithelial height and seminiferous tubule
diameter for Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. The correlation
coefficient is 0.64 with a P value of < 0.001.
Table 3. AIC table comparing linear models examining the factors contributing to seminiferous tubule
epithelial height in Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama.(EH = epithelial
height, TD = tubule diameter, and S = season).
Models ΔAICc df Weight
EH ~ TD 0.0 3 0.89
EH ~ TD + S 4.2 5 0.11
EH ~ TD * S 10.3 7 0.01
EH ~ S 13.0 4 0.001
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Table 4. Summary of best model (seminiferous epithelial height ~ seminiferous tubule diameter)
for explaining the factors that affect seminiferous tubule epithelial height in Opheodrys a. aestivus
(Northern Rough Greensnake) from Alabama.
Estimate Standard error t value P value
Intercept 8.37 5.54 1.51 0.14
Tubule diameter 0.18 0.04 4.37 less than 0.001
Figure 4. Plot of best model (seminiferous tubule epithelial height ~ seminiferous tubule
diameter) for explaining the factors that affect seminiferous tubule epithelial height in
Opheodrys a. aestivus (Northern Rough Greensnake) from Alabama. Shaded areas represent
95% confidence intervals.
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Discussion
Seminiferous tubule diameter was small in April and May and then increased
significantly in June. Seminiferous tubule diameter was not significantly different
in September or October. By October, the lumen was mostly empty of sperm since
they migrated to the vas deferens for winter storage. Seminiferous tubule epithelial
height followed a similar pattern as tubule diameter, with small values in May, a
significant increase in June, followed by little change in diameter in September and
October. These similar patterns were reflected by a positive correlation between
seminiferous tubule epithelial height and seminiferous tubule diameter.
Seminiferous tubule diameter increased significantly from spring to summer
and stayed significantly greater in fall. Contrastingly, there were no significant
differences in epithelial height among seasons. This contrasts with what Konvalina
et al. (2018) found while examining the testicular cycle of Northern Rough
Greensnakes in Arkansas. They found that both seminiferous tubule diameter and
seminiferous tubule epithelial height began with small values in spring, increased
in summer, and decreased in fall. The lack of significant decrease in the fall in the
Alabama specimens may be due to uneven sample sizes across the seasons. Our
sample was heavily favored toward spring (n = 18), whereas summer (n = 5) and
fall (n = 7) had few samples. More fall samples might yield a significant decrease
in tubule diameter and epithelial height from the peak in summer. Specifically,
adding November samples could accomplish this due to the testis entering the
quiescence phase.
Tubule diameter, but not season, was a significant predictor of epithelial
height. This result contrasts with the findings of Konvalina et al. (2018), who
found that both season and tubule diameter significantly affected epithelial height
in Arkansas specimens of Northern Rough Greensnake. However, both studies
found a peak in tubule diameter in summer, which correlates with peak sperm
production. Setting aside the uneven sample-size issues, our results suggest that
the seasonal variation in seminiferous tubule diameter and epithelial height is
less pronounced in southern populations of Northern Rough Greensnakes. Reproductive
physiology studies examining the impact of temperature on variation
of testicular tissue in this species could provide insight on such geographic
differences and also on the seasonal variation (or lack thereof) that climate
change-induced warmer temperatures would produce. Saint Girons (1982) found
that the only climatic factor that affects spermatogenesis in male snakes is temperature,
where cold winters stop spermatogenesis. Future climate models predict
warmer winters (Liu et al. 2017), which may lead to longer periods of sperm production
over the course of a year. An extended testicular cycle is possible as long
as the snakes can stay active and delay hibernation.
If the ecology of the Northern Rough Greensnake is similar to that of the colubrid
T. s. parietalis (Say) (Red-sided Gartersnake), then photoperiod likely has little to no
impact on the recrudescence of the testes (Hawley and Aleksiuk 1976). To test whether
only environmental temperature affects the initiation of spermatogenesis, future
studies should investigate the testicular cycle at the fringes of the Northern Rough
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Greensnake’s range, the tip of the Florida peninsula in the south and southern New
Jersey in the north, to see if these patterns are consistent across all populations.
Acknowledgments
We thank V. Rolland for assistance with R software and statistical analyses. We also
thank J. Bouldin and T. McKay for help with revising and editing this manuscript. All
specimens were acquired from the Auburn University Museum of Natural History.
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