In this lizard, testes have a typical tubular organization that changes during the annual cycle determining six different conditions [17,18,19,20]. During the breeding season (May–June), the seminiferous epithelium is thick, germ cells are in all stages of differentiation, and it is rich in spermatozoa ready to be ejaculated. In the summer stasis (July–August), tubules regress and the epithelium becomes thin and is composed only of spermatogonia and Sertoli cells. In early (September) and mid-autumn (October–November), a recovery occurs: spermatogenesis is resumed, spermatocytes I (early autumn) are produced, and then all the germ cells, including few spermatozoa (mid-autumn). The latter, however, are not used for reproduction; this spermatogenic recovery is considered the reminiscence of two reproductive events once present in the ancestor of this lizard, living in a milder environment [20]. This morphological organization of the seminiferous tubules is maintained during the winter stasis, while during spring recovery there is the complete activation of spermatogenesis, and the tubules are rich in spermatozoa ready to be ejaculated (Figure 2). Photoperiod and temperature are the most important players in the regulation of spermatogenesis [15,19,21,22].

P. siculus represents one of the main animal models used to study the intricate process of spermatogenesis since the 1950s, as evidenced by the studies of Galgano and D’Amore [17]. Over time, an abundance of studies has documented spermatogenesis control mechanisms in P. siculus, many of which helped to elucidate the spermatogenic process in other vertebrates that share a similar testicular organization, including mammals. It has been shown that the process is regulated by a network of interaction between several factors of both systemic and testicular origin [20]; among these, the pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) [23,24,25,26,27,28], D-aspartic amino acid (D-Asp) [29,30], beta-endorphin [31,32], retinoic acid [33], and steroidogenic enzymes [27,34,35] deserve to be mentioned. Numerous studies have demonstrated the presence in P. siculus testis of these molecules with their corresponding receptors both in the germline and in somatic cells, suggesting their involvement in the control of testicular activities [24,25,26]. In detail, qPCR experiments revealed a cyclical pattern of PACAP and VIP mRNA levels during the different phases of the reproductive cycle, with the lowest mRNA expression levels recorded during the summer and winter stasis, when spermatogenesis is blocked; the highest transcript levels were detected during the reproductive period, characterized by maximum testicular activity; finally, intermediate expression levels were found in the recovery phases, when spermatogenesis is reactivated [24,25,26]. These data, therefore, highlight that the seasonal changes in PACAP and VIP mRNA levels are directly linked to changes in P. siculus testis activity. Furthermore, in situ hybridization and immunocytochemical investigations have shown that PACAP, VIP, and their receptors are widely distributed in the Podarcis testis throughout the reproductive cycle and localized in both germ cells and somatic cells [24,25,26,27,28,34]. Taken together, all these data suggest that PACAP and VIP could act as local control factors of spermatogenesis and spermiogenesis in P. siculus.

In addition to neuropeptides, the presence in the testis of P. siculus of beta-endorphin (β-EP), an endogenous opioid peptide involved in the control of reproductive processes in vertebrates, has also been demonstrated [31,32,36,37]. Immunohistochemistry demonstrates a seasonal variation in β-EP positivity, with a strong signal, especially in the interstitial cells of sexually quiescent lizards (December) [31].

In another study, it was shown that an additional molecule such as vitamin A and its main biologically active derivative, retinoic acid (RA), is involved in the control of lizard spermatogenesis [33]. Indeed, quantitative PCR analysis showed a different expression of its receptors (RAR α and RAR β) during the different phases of the reproductive cycle. In the reproductive period (May), only RAR β is expressed, while in the post-reproductive phase (August) only the transcript for RAR α is present [38].

More recently, through biochemical and molecular investigations in the testis of P. siculus, the expression of the Vasa gene, which encodes a protein involved in different aspects of germ cell development in many animals [39], has been demonstrated in three significant moments of the reproductive cycle: reproductive period, summer stasis, and autumn recovery [40,41]. Using immunohistochemical investigations, the authors also demonstrated the presence of the Vasa protein in germ cells, from spermatogonia to spermatids, but not in spermatozoa [41].

A major role in the control of P. siculus spermatogenesis is also played by steroidogenic enzymes, which represent the key to managing the local action of sex hormones on spermatogenesis, whose localization and levels have been studied (StAR, 3β-HSD, 17β-HSD, P450 aromatase, and 5α-Red) [28,35]. The presence and localization of these enzymes in the P. siculus testis throughout the reproductive cycle have been established by immunocytochemistry; this analysis showed that, in lizard testis, as occurs in mammals [42,43,44], sex hormones are produced by both germ cells and somatic cells [28,35].

The balance between cytochrome P450 aromatase expression and sex hormone levels is really interesting: the highest levels of P450 aromatase, the enzyme responsible for estrogen biosynthesis, and 17β-estradiol were recorded during the summer and winter stasis when the hormone is involved in blocking the spermatogenesis [15,28,45]; conversely, the highest level of testosterone and the concurrently low level of P450 aromatase were detected during the recovery period and, in particular, the reproductive period, when testosterone is responsible for the massive production of spermatozoa and the development and maintenance of secondary sexual characteristics [19,28,45].