Spermatogenic Waves and Expression of AR and ERs in Germ Cells of Podarcis sicula

Androgens are considered the main hormones that regulate spermatogenesis, but a lot of evidence confers to estrogen a key role in this process. In the testis of the seasonal breeder lizard Podarcis sicula we analyzed by means of in situ hybridization the expression of the androgen receptor (AR) and of the two types, α and β, of the estrogen receptors (ERs) throughout the three periods of the annual cycle: mating (spring-early summer), postmating-refractory (late summer), and autumnal recrudescence. The results show that during themating periodAR and ERs are expressed in all germ cells present in the tubules from spermatogonia to spermatozoa. During the postmating-refractory period, when only spermatogonia are present in the tubules, almost all express ERs and very few AR mRNA. During the autumnal recrudescence the localization of AR and ERs is the same of the mating period except for the population of the primary spermatocytes. The expression of the investigated receptors is peculiar in these cells that are positive in the middle-late pachytene stage of the meiotic prophase and negative in preleptotene. A possible functional role of the observed differences during spermatogenesis and in the spermatozoa is also discussed.


Introduction
The androgen receptor (AR) and the two types of the estrogen receptors (ER and ER) are members of the large superfamily of ligand-activated hormone receptors and act as ligand-inducible transcription factors [1,2].Even though the expression of these three types of steroid receptors is reported in the testis of many mammalian species [3][4][5][6][7][8][9] their role is not well defined in male reproductive system and even if the male tract is an example where these receptors are expressed [6,10] the reports about their localization are controversial [11].
More than 20 years ago in mammals, it was thought that AR was not expressed in male germ cells [12,13], but later some evidence showed the expression of AR in spermatogonia, spermatocytes, elongated spermatids, and spermatozoa [14][15][16].Regarding ERs, in all mouse germ cells only ER is detected [6,17]; in the human some authors reported a complete absence of ER [18,19] while some others suggested its expression in primary spermatocytes and spermatids and in the ejaculated sperm [4,[20][21][22].
Among lower vertebrates, in the amphibians that show a cystic structure of the testis, the estrogen guarantees in Rana esculenta the progression of spermatogenesis and release of spermatozoa [23][24][25] and in Triturus marmoratus immunoreactive AR, ER, and ER are reported in the germ cells [26].In the tubular testis of rooster, only spermatogonia and early spermatocytes express ER but do not express ER [27].In Trachemys scripta among reptiles, ER immunoreactivity was found in spermatogonia [28]; in Chelonia mydas AR and ER were found in germ cell at stage IV while ER was observed only in the somatic cells [29]; in the snake Zaocys dhumnades AR and ER were reported in spermatids [30].No data are available in reptiles about the localization of the transcripts of both androgen and estrogen receptors in the seminiferous epithelium.
Our experimental model is the lizard Podarcis sicula that, as many reptiles living in regions with marked circannual climatic variations, is a seasonal breeding species and its spermatogenic cycle is dependent on temperature and 2 International Journal of Zoology photoperiod [31].Furthermore in this lizard the testis shows a tubular structure like mammals.
In Podarcis the mating period occurs in spring when the intense spermatogenic activity in the testis gives rise to a lot of spermatozoa sent to the rete testis and epididymal channel to be then ejaculated.In the early summer the spermatogenesis is over and Podarcis enters in the status considered a condition of physiological hypophysectomy named refractoriness during which the lizards are unresponsive to endogenous or exogenous hormones [32].In the fall Podarcis shows a spermatogenic recrudescence considered the reminiscence of two reproductive events in the ancestor of this lizard living in the milder environments [31].Furthermore, in this lizard are known some data about the expression of androgen and estrogen receptors in the whole testis [33][34][35] and the pattern of sexual steroids hormones, that is, during the reproductive cycle [31].
The aim of this research was to obtain in Podarcis an overview on the expression of AR, ER, and ER in the seminiferous epithelium throughout the annual cycle.We will also try to assign a possible functional role to the difference in the ability of germ cells to transcribe the receptors during their progression in the spermatogenic events.For this purpose we analyzed the expression of these receptors in the testis by means of in situ hybridization with homologous probes already cloned by us [36][37][38].

Animals.
For this study were used males of lizard Podarcis sicula whose spermatogenic cycle in natural or experimental conditions is well known [31].
Adult males (about 7.5-8 cm snout-vent) of lizard Podarcis sicula were captured near Naples (Italy) during the mating period (spring-early summer) [ = 10], the postmatingrefractory period (late summer) [ = 10], and the recrudescence (fall) [ = 10].They were kept in terraria at natural temperature and photoperiod and fed ad libitum with larvae of Tenebrio molitor.The animals were killed by decapitation after anaesthesia on ice and the testes, immediately excised, were processed for histological and biomolecular analyses.Authorization to capture the animals for experimental treatments was granted by the Italian Ministry of the Environment (auth.SCN/2D/2000/9213).

Histology.
Both testes of each animal were fixed in Bouin's fluid [39], alcohol-dehydrated, and paraffin-embedded.Sections 7 m in thickness were obtained with Reichert-Jung 2030 microtome.Some histological sections were stained with Mallory's trichrome modified by Galgano [39]; other sections were processed by in situ hybridization (ISH).The results were examined at Nikon-MicroPhot-Fxa microscope.

In Situ Hybridization (ISH).
The ISH was performed by using homologous digoxigenin (DIG) labelled cDNA fragments of ER, ER, or AR, as already reported for the liver and epididymis [37,38,40].Briefly, the dewaxed sections were treated with proteinase K (10 g/mL) at 50 ∘ C for 10 min.The probes were used at a concentration of 80 ng/100 L in hybridization buffer overnight at 50 ∘ C in a moist chamber.The slides were washed with formamide 50% and SSC 2x for 30 min, formamide 50% and SSC 1x for 30 min, and formamide 50% and SSC 0.5x for 15 min, washed in 2x SSC for 3 min and in NTP (Tris-HCl 0.1 M pH 7.5; NaCl 0.15 M), and then incubated in 2% blocking solution (Roche Diagnostics, Mannheim, Germany) in maleic acid buffer for 1 h.The sections were kept overnight at 4 ∘ C with alkaline phosphatase-conjugated sheep anti-DIG antibody (Roche Diagnostics) (1 : 2500) in blocking solution and rinsed in NTP buffer for 30 min and in NTM buffer (Tris-HCl 100 mM pH 9.5, MgCl 50 mM, NaCl 100 mM) for 30 min.Finally, the sections were kept in the dark at room temperature in the colour detection substrate solution BCIP/NBT (nitroblue tetrazolium and 5-bromo-4-chloro-3  -indolyphosphate, Roche) in NTM until appearance of the reaction.
Control sections were obtained by omitting incubation with the probes.Some other sections were pretreated with DNase to exclude cross-link with genomic DNA.

Histology.
In the males of lizard Podarcis sicula the reproductive annual cycle starts with the mating period lasting from spring to early summer, followed by the postreproductive-refractory period and by the autumnal recrudescence.
Although the structure of the testis during this cycle is known, a concise description of the histological features of the seminiferous tubules around the year is necessary for the evaluation of the results concerning the purpose of this research.In the mating period a great amount of sperms fills the lumen of the tubules where all stages of the spermatogenesis are detectable as spermatogonia (spg), primary spermatocytes (spcI), secondary spermatocytes (spcII), round and elongated spermatids (spd), and spermatozoa (spz) (Figure 1(a)).The spg are located on the basal membrane; above these, many spcI in different stages of the meiotic prophase from preleptotene, pachytene, diplotene until diakinesis are easily recognizable according to the aspect of the chromatin.The spcII are located upper and show a smaller diameter.Toward the lumen several round spermatids show the presence of the acrosomal bubble (Figures 1(b) and 1(c)).Such organization of the seminiferous epithelium is not uniform on the whole surface of the tubule because the development of the germ cells from spc to spz proceeds in a spiral as reported in other reptiles and in mammals [41,42].
At the end of mating period (late July) the spermatogenesis is over and the seminiferous epithelium gradually reduces in thickness up to only spg and Sertoli cells remain (Figure 2(a)).Sometimes few residual cells are still evident in the restricted lumen of the tubules.At this stage the refractoriness sets in.
In the fall the refractoriness slows down and the spermatogenesis resumes (autumnal recrudescence) until the production of few spermatozoa (Figure 3(a)) that are useless since the couplings do not occur in this period.At the beginning of the new year spermatogenesis starts again to culminate in spring with the onset of a new mating period (data not shown).

In Situ Hybridization with ER𝛼, ER𝛽, or AR Probes.
In the seminiferous tubules during the mating period almost all the germinal cells, from spg to spz, are positive for AR (Figure 1(d)), ER (Figure 1(e)), or ER (Figure 1(f)) mRNA.Occasionally few preleptotene spcI were negative.
In the postmating-refractory period the spg are poorly positive to AR mRNA (Figure 2 The ISH performed on control sections by omitting the incubation with AR, ER, or ER probes was negative in all the different stages of the reproductive cycle (for all we reported one picture, Figure 1(g)).The sections pretreated with DNAse showed the same features of the untreated ones (data not shown).

Discussion
Our research analyzed in parallel for the first time the expression of AR, ER, and ER by means of in situ hybridization  6 International Journal of Zoology with homologous probes in the seminiferous epithelium of the lizard Podarcis sicula during the reproductive annual cycle.The present data indicate that the arrangement of the reproductive events in a seasonal breeding species makes it easier to correlate the seasonal histological changes with biomolecular approaches.In particular the results show that all male differentiating germ cells are able to express androgen and/or estrogen receptors and suggest that the almost constant presence of AR and ERs mRNA often observed may be related to the continuous hormonal occurrence throughout the cycle even if at different concentration.In fact in Podarcis the plasma level of testosterone (T) reaches the maximum during the mating period and falls down in the postmating period while its endotesticular profile shows always significant levels.The plasma levels of estradiol 17- (E 2 ) are instead consistent with endotesticular titers, rising at the end of the mating and remaining discrete until the autumnal recrudescence [31,43].Furthermore our findings show that in Podarcis during the mating period the expression of the AR, ER, and ER mRNA occurs at the same time in spg, spcI, spcII, spd, and spz.The significance of the simultaneous expression of the androgen and estrogen receptors during the spermatogenesis has yet to be clarified in Podarcis as in other vertebrates, but some data showed in mammals a possible interplay between them.In hamster multiple consensus sequences in the promoter region of AR recognize ER or AR itself as transcription factors [44].In mouse mammary cells the interaction E 2 /ER inhibits transcriptional activity of DHT/AR complex, in vitro [45].Physical AR-ER interactions, in mammalian twohybrid systems, arise between the C-terminal ER ligandbinding domain and the N-terminal AR transactivational domain or with the full-length of AR, while ER does not interact with AR [46].Moreover E 2 itself can bind weakly to the AR [47,48] and can activate an androgen-responsive element [49,50].In addition, the two types of the estrogen receptors may form homodimers or heterodimers which have different affinities with the estrogen-responsive element [51] and, in the uterine cells, ER may negatively regulate ER [52].
In Podarcis at the end of the mating period, almost exclusively spg and Seroli cells remain in the seminiferous tubules and very few spg express AR-mRNA while most of them are positive for ERs.In this period the plasma levels of E 2 are at zenith in this species [31] and in vitro E 2 autoregulates ER-mRNA and downregulates AR-mRNA [34].Therefore it is possible to hypothesize that the expression of AR in some spermatogonia may distinguish ones that will come in meiosis.
In vertebrates the spermatogenesis is guaranteed by a series of mitotic and meiotic waves that in Podarcis takes place twice in a year, the first in spring and the second in the fall.In these two spermatogenic events we recorded some differences in the expression of the investigated receptors in particular at level of spz and on the population of spcI.Concerning the spermatozoa we found the expression of AR, ER, and ER in the mating period unlike in the fall.In some mammals [53,54] the terminal differentiation of spermatids and their release from the seminiferous epithelium are AR dependent and in AR knockout mice [55] the development of spermatozoa is impaired.On the other hand the estrogens, through their receptors, guarantee sperm function and release and prevent spermatid death [14,24,25,[56][57][58][59].Since in Podarcis the spz produced during the autumnal recrudescence do not pass in the epididymis [31,37], the expression of the three receptors in the mating period may characterize the spz that will be ejaculated.
The other difference in the two annual spermatogenic events of Podarcis concerns the population of spcI.The large amount of spcI in middle or late pachytene stage, present in the mating period, expresses mRNA for all the three receptors whereas the few preleptotene spcI are negative.On the contrary, the amount of spcI in pachytene stage is much smaller during the autumnal recrudescence and remains positive to the AR or ERs probes while the large amount of preleptotene remains negative.Taken together these findings suggest that the spcI cannot express the receptors at the early stage of meiotic prophase and acquire such ability later.In reptiles the pachytene is highly synthetic and represents the longest stage of prophase as in mammals [41,42] and the conversion from middle to late pachytene spermatocytes is critical for the maturation of rat germ cells in vitro [60].
In synthesis in Podarcis during the mating period the great amount of spcI expressing the three receptors may be likely due to the high frequency in the succession of spermatogenic waves in this period.In the same way the presence in the autumnal recrudescence of many negative spcI and of very few positive pachytene cells may be consequence of a lower frequency of the spermatogenic waves in this period.Furthermore the almost complete lack of expression observed almost always in preleptotene during the autumnal recrudescence and only occasionally during the mating period may represent a sort of interwave resting stage.
Therefore in Podarcis, while the timing of the reproductive cycle is given by the seasonal changes as it is known, the speed and frequency of the spermatogenic waves in the mating period or in the autumnal recrudescence may be set by the amount of spcI expressing AR, ER, and ER-mRNA.
It would be interesting to check if the role ascribed to the spcI in Podarcis can be verified in other reptiles or in higher vertebrates showing two or more mating periods in the year.

Figure 1 :Figure 2 :
Figure 1: Histology and mRNA distribution of AR and ERs in the testis during the mating period.(a, b, c) Mallory's trichrome staining: (a) all stages of germ cells differentiation: spg, spcI, spcII, spd, and spz are evident in a seminiferous tubule; (b) spcI in preleptotene stage (arrows), the red nucleolus is still evident (magnification of (a)); (c) spcI in pachytene stage (arrows), note the condensation of chromatin to constitute chromosomes.The asterisk marks the round spermatids (magnification of (a)).(d, e, f) ISH with homologous probes: (d) expression of AR (the square indicates the spcI in pachytene stage); in (e) expression of ER; (f) expression of ER; (g) ISH-negative control section by omitting the probes.The bar is 30 m.
(b)) but are strongly positive to ER (Figure 2(c)) or ER probes (Figure 2(d)).In the months of the autumnal recrudescence AR (Figure 3(b)), ER (Figure 3(c)), and ER (Figure 3(d)) were expressed in almost all spg, spcII, and spd as in the mating period, while a lot of spcI in preleptotene were negative and constitute a large circular ring between the spg and spcII (Figures 3(b), 3(c), and 3(d)).The few spcI in pachytene stage, detectable in this area, were positive for the three receptors.No reaction was observed in the spermatozoa.The responsiveness of the spermatozoa during the mating period and autumnal recrudescence was displayed in Figure 4.

Figure 3 :
Figure 3: Histology and mRNA distribution of AR and ERs in the testis during the autumnal recrudescence.(a) Mallory's trichrome staining: all stages of germ cells differentiation from spg to spz are evident in the seminiferous epithelium as in the mating period.(b, c, d) ISH with homologous probes: intense positivity to AR (b), ER (c), or ER (d) probes of the spg, on the basis of the tubules, and of spcII near the lumen ( * ); the spcI in pre-leptotene constitute a large negative circular ring where only few pachitene cells (arrows) are slightly positive.The bar is 30 m.

Figure 4 :
Figure 4: Spermatozoa in the seminiferous epithelium.(a) Mallory's trichrome stained section.(b, c, d, e) ISH with homologous probes: spermatozoa positive to AR (b), ER, (c) and ER (d) in the mating period; spermatozoa negative in the autumnal recrudescence (e) (one for all is depicted ER).The bar is 30 m.