Steroid hormones and chondrichthyan reproduction: physiological functions, scientific research, and implications for conservation

RIA

Assay based on antigen-antibody complex and radioactively labelled antigens (Goldsmith, 1975). This immunoassay methodology was first carried out in chondrichthyans by Sumpter & Dodd (1979) using the plasma of S. canicula. Since then, RIA have analysed an average of 3.36 (S.D. ± 1.31) steroid hormones per paper (Fig. 3) with hormone recoveries ranging from 17% to 111% (Table 2). Minimum and maximum number of specimens were 2–248 for males (Tricas, Maruska & Rasmussen, 2000; Sulikowski et al., 2016) and 1–248 for females (Rasmussen & Murru, 1992; Tricas, Maruska & Rasmussen, 2000). From the reviewed studies, only 61.7% specified the polyclonal antibodies used. A total of 28 species of chondrichthyans have been analysed through RIA, including 17 sharks, 10 rays and one chimaera (Table 2). This biochemical assay was used in 76.3% (n = 45) of the reviewed studies, making RIA the most widely used technique for the study of steroid hormones in these group of cartilaginous fishes (Fig. 2A).

EIA/ELISA

Biochemical assay techniques focused on the detection and quantification of a wide range of substances including antibodies, allergens, proteins, peptides, hormones, etc. (Vashist & Luong, 2018). These biochemistry assays were first used to describe steroid hormones in the plasma of the spotted ray Torpedo marmorata (Prisco et al., 2008), and subsequently in the Atlantic sharpnose shark Rhizoprionodon terranovae (Hoffmayer et al., 2010). Since then, it has been used for the study of captive endangered species by analysing semen of the whale shark R. typus (Nozu et al., 2015) and plasma of the reef manta ray M. alfredi (Nozu et al., 2017) and zebra shark Stegostoma fasciatum (Nozu et al., 2018). The studies that have applied EIA/ELISA detected an average of 2.62 (S.D. ± 1.06) steroid hormones per paper with recoveries of 85–108% (Fig. 3). Minimum and maximum number of specimens were 2–81 for males (Nozu et al., 2017; Wyffels et al., 2019) and 3–94 for females (Nozu et al., 2015; Mylniczenko et al., 2019). All papers with EIA/ELISA as the main analytical method mentioned the antibodies used and constitute 13.6% (n = 8) of the reviewed literature.

TRFIA

This non-radioactive immunoassay involves the use of Europium labelled antibodies and antigens for the detection of substances along with a fluorometer for the quantification through means of lanthanide fluorescence (Diamandis, 1988). In chondrichthyans it was used by Matsumoto et al. (2019) for the analysis of T and P4 in plasma of a captive whale shark. The monitoring for 20 years of this young male allowed to describe the effect of T and P4 on the sexual maturation of this endangered species, since an increase of T and P4 was related to the morphogenesis and functioning of the claspers. The antibodies used in this article were not specified. This study constitutes 1.7% of the reviewed papers.

PC/TLC-GC

Physical techniques for the separation of components from a specific solution trough a stationary and a moving phase, which is paper and a liquid solvent in PC and silica plates interacting with liquids or gas in TLC and GC, respectively (Bhawani et al., 2010). PC was first reported for steroid hormones in chondrichthyans by Simpson, Wright & Gottfried (1963) and Simpson, Wright & Hunt (1964) in semen of S. canicula. In the study of Di Prisco, Vellano & Chieffi (1967), the analysis of plasma of T. marmorata allowed the detection of eight sex steroid by using TLC-GC (Table 2). The studies that have required this technique have analysed an average of 3.36 (S.D. ± 1.68) steroids per paper (Fig. 3). Recoveries of steroid hormones ranged from 34% to 65% depending on the author (Table 2). Minimum and maximum number of specimens were 5–40 for males (Simpson, Wright & Hunt, 1964; Gottfried & Chieffi, 1967) and 38 for females (Di Prisco, Vellano & Chieffi, 1967). These methodologies were mostly used during the sixties and constitute 6.8% (n = 4) of the reviewed studies.

LC-MS/MS

This method combines chromatography and mass spectrometry for the separation and quantification of a wide range of substances including most organic compounds (large proteins, pollutants, drugs, and hormones; Wudy et al., 2018). It was recently used by Lyons & Wynne-Edwards (2019) for the simultaneous analysis of 13 steroid hormones in plasma and histotroph samples of the round stingray U. halleri. While some of these steroids were below the limit of quantification for selected samples, this was the study with the highest number of detected hormones overall. However, this paper did not report recoveries of the extracted steroids from the tissues of the 55 female specimens analysed. This technique was used only in this study and constitutes 1.7% of the reviewed papers.

The above methodologies used for the detection and quantification of steroid hormones were applied to six biological matrices: blood (plasma or serum), semen, yolk, histotroph, gonads and muscle. Most studies were performed using plasma (69.5%; n = 41 papers) or serum (13.6%; n = 8 papers), with three studies including semen (5.1%; n = 3 papers) and seven papers involving the analysis of other matrices (11.9%; Fig. 2B). In the case of blood, the original volume per sample ranged between 0.5 and 20 mL, while the volume for analysis was 25–1,000 µL (Table 2). Other tissues such as muscle or gonad required approximately 3–5 g of the original sample for hormone extraction (Prohaska et al., 2013a) and 19 g in the case of semen (Gottfried & Chieffi, 1967). In the majority of the studies, the obtention of samples in these quantities was met through the sacrifice of specimens, or through the interaction with captive elasmobranchs.

The sample mass required for the extraction of hormones in tissues like blood, gonad, histotroph and yolk usually entailed the use of a lethal methodology in 69.0% (n = 40) of the analyses (Fig. 2B). Most blood studies included sample collection carried out through cardiac, caudal or dorsal venepuncture (Table 2). Studies that did not required a lethal methodology (31.0%; n = 18) were considered invasive due to the capture or manipulation needed for the collection of samples. In addition to extraction kits, the main methods for hormones analyses in chondrichthyans were solvent/ether extractions using diethyl ether, ethyl acetate, and petroleum ether; although other substances such as chloroform: methanol, dichloromethane, ethyl acetate/cyclohexane, and benzene ether were also used (Table S1).

Some of the first papers that described steroid hormones in tissues other than blood were carried out via PC/TLC-GC by Simpson, Wright & Gottfried (1963) and Simpson, Wright & Hunt (1964) and Gottfried & Chieffi (1967) using semen of Squalus acanthias and S. stellaris, respectively. More than two decades later, analyses of histotroph, gonads and plasma from S. canicula and T. marmorata were performed via RIA (Fasano et al., 1992; Garnier, Sourdaine & Jégou, 1999). Analyses of steroid hormones from yolk were only studied in S. tiburo by RIA (Manire et al., 2004). The use of skeletal muscle was first analysed by Barnett et al. (2009) and further by Prohaska et al. (2013a), both via RIA. The latter suggested the use of muscle as an alternative for the study of protected elasmobranchs, since a correlation between steroid concentrations in plasma and muscle was observed (Hammerschlag & Sulikowski, 2011; Prohaska et al., 2013a).

All the analysed steroids used for reproductive assessment according to each biological matrix are detailed in Table 2. The average number of steroid hormones analysed per study that used plasma was 3.3 (S.D. ± 1.7); 4.2 (S.D. ± 1.2) for serum; 5.3 (S.D. ± 3.2) for gonad; 1.3 (S.D. ± 0.6) for semen; 3.0 for muscle; and 7.0 (S.D. ± 4.2) for histotroph. To date, most of the studies have focused on the analysis of several tissues using immunoassay-based methods such as RIA or ELISA/EIA and therefore the antigen-antibody complex interaction (Fig. 2A). Overall, the results from these analyses of the six biological matrices in several species are the current basis of the endocrinological knowledge of chondrichthyans in terms of reproductive biology. This scientific effort has allowed the detection and quantification of a wide diversity of steroid hormones and furthered the knowledge of their role in the reproduction of these cartilaginous fishes.

Testosterone

High concentrations of this androgen have been related to follicle development and to the maintenance of spermatozoa along with E2 in females (Manire, Rasmussen & Gross, 1999; Tricas, Maruska & Rasmussen, 2000). Additionally, an increase of T was observed during the mating seasons of several elasmobranchs, suggesting that this steroid could play an important role in their reproductive ethology (Rasmussen & Gruber, 1993; Manire et al., 1995; Garnier, Sourdaine & Jégou, 1999; Mull, Lowe & Young, 2010). In males, several studies described an increase of this androgen during spermatogenesis and sperm transportation, which could indicate a role in the maturation and motility of gametes (Heupel, Whittier & Bennett, 1999; Sulikowski, Tsang & Howell, 2005). Additionally, T could be an anabolic agent for the growth of testes, reproductive ducts and claspers (Sulikowski, Tsang & Howell, 2005; Lyons & Wynne-Edwards, 2019). In contrast to other vertebrates, knowledge regarding the functions of T in the body or in the development of gonads is still scarce (Awruch, 2013). This androgen has been quantified in six biological matrices using the five analytical methods described in this paper. A total of 52 papers have included T in their analyses, which constitutes 88.1% of the reviewed literature (Table 2).

5α-dihydrotestosterone

In chondrichthyans, DHT has been suggested as an important steroid for spermatogenesis along with T as a possible precursor of E2 (Manire & Rasmussen, 1997; Tricas, Maruska & Rasmussen, 2000; Awruch, 2013). However, there is no evidence regarding an aromatisation of DHT into oestrogens such as E2, so this hypothesis is unlikely. In terms of steroid synthesis control, Tricas, Maruska & Rasmussen (2000) suggested that DHT could provide an inhibitory feedback in the production of gonadotropin-releasing hormone (GnRH), based on their observations in the ray Hypanus sabinus. Additionally, an increase of DHT and T was observed during ovulation and later during histotroph nourishment. This could suggest that the action of DHT is related to the sexual differentiation in batoids as observed in other vertebrates (Tricas, Maruska & Rasmussen, 2000; Lyons & Wynne-Edwards, 2019). In viviparous sharks such as S. tiburo, DHT was related to reproductive behaviour, as the highest concentration of this steroid occurred during preovulation and mating stages (Manire et al., 1995). DHT was detected and quantified in blood, gonads and histotroph of elasmobranchs using RIA and ELISA (Table 2). There are no detections of DHT in holocephans to date. From the reviewed literature, a total of 13 papers (22.0%) have included this steroid in their analyses (Table 2).

11-ketotestosterone

In female elasmobranchs, 11-KT may play a role in the synthesis of oestrogens and ovarian follicle development, although most of the research has been focused on males (Manire, Rasmussen & Gross, 1999). In this regard, a higher concentration of 11-KT could favour the sexual development of juvenile males, with an increase during the peak of sperm storage influencing sperm maturation and preparation for mating (Garnier, Sourdaine & Jégou, 1999; Mull, Lowe & Young, 2008). This androgen showed a negative correlation with photoperiod in specimens of U. halleri with levels of 11-KT decreasing with daylight. The effect of light on the synthesis of some steroid hormones such as 11-KT could explain differences in sexual segregation regarding depth and habitat use for males, although research in this topic remains scarce. In holocephans, levels of 11-KT were 300% higher during pre-parturition stages, which suggest that this steroid could be related not only to parturition, but to sexual behaviour favouring the next reproductive cycle (Barnett et al., 2009). In general, it is likely that 11-KT is involved in the sexual maturity and mating behaviour of male chondrichthyans, although more research is needed in order to understand the exact role in their development and further reproduction. 11-KT has been detected and quantified in blood, gonads and muscle of chondrichthyans using RIA (Table 2). From the reviewed literature, a total of six papers (10.2%) have included 11-KT in their analyses (Table 2).

Androstenedione

In batoids, A4 was higher during the greater fertile period and early gestation of Rhinoptera bonasus, while there were no differences in A4 along with E2 in the analysed males (Sheldon et al., 2018). A similar increase was observed in early and mid-term females just before the appearance of claspers in embryos of the round stingray U. halleri, which could suggest an intermediary role for sex differentiation along with 17-OHP (Lyons & Wynne-Edwards, 2019). In contrast, the concentrations detected in S. canicula demonstrated an increase before a peak in sperm reserves, and A4 was consistently detected in the testis as one of the principal steroids along with T and P4 (Garnier, Sourdaine & Jégou, 1999). A4 has been quantified in plasma and testis of S. canicula and in the plasma of both sexes of R. bonasus using RIA, as well as in the histotroph of U. halleri (Table 2). Three papers (5.1%) included A4 in their analyses (Table 2).

11-ketoandrostenedione

This steroid was detected in serum of S. tiburo using RIA and appears only in the study of Manire, Rasmussen & Gross (1999). It is possible that an increase in 11KA4 plays a role in sperm storage and in the maintenance of pregnancy in viviparous species, as the concentrations rise from mating until prior to parturition. In males, the function of 11KA4 is unclear, as levels appear to be similar throughout the annual study periods in mature individuals. Further research involving different maturity stages could provide the first insights regarding the role of 11KA4 in most chondrichthyans. The paper of Manire, Rasmussen & Gross (1999) constitutes 1.7% of the reviewed literature.

5α-androstane-3α,17β-diol

This hormone was quantified throughout the year in plasma and testis of S. canicula with significantly higher concentrations during winter, just prior to an increase in steroid hormones such as E2, E1, DHT,11-KT and a peak in sperm reserves (Garnier, Sourdaine & Jégou, 1999). While 3α-diol may play a role in the preparation of spermatogenesis, the metabolism of this steroid and its effects on the reproduction of chondrichthyans remain unclear. The paper of Garnier, Sourdaine & Jégou (1999) used RIA as the analytical method and constitutes 1.7% of the reviewed literature.

Progesterone

In females, this progestogen was suggested to act as an antagonist of E2 and consequently decrease the synthesis of vitellogenin in the liver (Tsang & Callard, 1987; Tricas, Maruska & Rasmussen, 2000; Prisco et al., 2008; Mull, Lowe & Young, 2010). An increase of P4 along with 11KA4 could be related to the beginning and maintenance of pregnancy in viviparous species since the concentration of these hormones drop prior to parturition (Manire, Rasmussen & Gross, 1999; Mull, Lowe & Young, 2010). In contrast, it is possible that a decrease in P4 is related to the regulation of encapsulation and oviposition since low levels of P4 were detected after ovulation in oviparous species (Koob, Tsang & Callard, 1986; Sulikowski, Tsang & Howell, 2004). The function of P4 in males remains unclear, with different studies showing contrasting results (Awruch, 2013). However, it is probable that P4 is linked to the synthesis of T and other steroid hormones, which could partially be related to sexual maturation (Simpson, Wright & Gottfried, 1963; Rasmussen & Gruber, 1993). P4 has been quantified in five biological matrices using the five analytical methods described in this paper. A total of 44 papers have included P4 in their analyses, which constitutes 74.6% of the reviewed literature (Table 2).

17-hydroxyprogesterone

This hormone was detected throughout the complete reproductive cycle of S. canicula with no significant differences among the analysed months (Garnier, Sourdaine & Jégou, 1999). In contrast, hormones such as A4 and 17-OHP could be related to sex differentiation in male embryos of U. halleri (Lyons & Wynne-Edwards, 2019). This progestogen has been quantified in plasma and testis of S. canicula and in the plasma and histotroph of U. halleri using RIA and LC-MS/MS, respectively (Table 2). Two papers (3.4%) included 17-OHP in their analyses (Table 2).

Dihydroprogesterone

The study of Manire, Rasmussen & Gross (1999) demonstrated that levels of DHP significantly increased during maturation in females of S. tiburo, but decreased before spermatogenesis in adult males along with an increase in E2 and 11-KT levels. This could be related to the synthesis of E2 and 11-KT, although this process remains unclear. The analysis of DHP was made in serum of this carcharhinid using RIA. The paper of Manire, Rasmussen & Gross (1999) constitutes 1.7% of the reviewed literature.

17-hydroxypregnenolone

The study of Gottfried & Chieffi (1967) was the only one to include 17P5 by analysing semen of S. stellaris using TLC-GC. However, this study was purely technical, focusing on hormone detection without any biological inferences. 17P5 may have receptors in the brain of elasmobranchs or may be involved in the synthesis of other hormones such as 17-OHP (Diotel et al., 2011). However, there is no evidence to date to support the functions of 17P5 in chondrichthyans. The paper of Gottfried & Chieffi (1967) constitutes 1.7% of the reviewed literature.

Estrone

This steroid was at higher levels before the gestation period of T. marmorata, with no detections observed during the pregnancy of this species (Di Prisco, Vellano & Chieffi, 1967). In contrast, levels of E1 and androstenedione (A4) were only detected in the histotroph of early and mid-term pregnant females of U. halleri (Lyons & Wynne-Edwards, 2019). This could suggest a transfer of energy and substances for the development of these species; however, evidence about these mechanisms remains scarce. This oestrogen has been quantified in plasma and histotroph of U. halleri, and in the plasma of T. marmorata using LC-MS/MS and TLC-GC, respectively (Table 2), but has not been investigated in holocephans. From the reviewed literature, four papers (6.8%) have included E1 in their analyses (Table 2).

17β-oestradiol

In chondrichthyan females, E2 has been linked to the synthesis of vitellogenin in the liver (Prisco et al., 2008), follicle development in the ovary (Koob, Tsang & Callard, 1986; Heupel, Whittier & Bennett, 1999; Manire, Rasmussen & Gross, 1999; Henningsen et al., 2008), development and functions of the oviducal gland including the storage of spermatozoa along with T (Koob, Tsang & Callard, 1986; Tsang & Callard, 1987; Manire et al., 1995; Tricas, Maruska & Rasmussen, 2000; Gelsleichter & Evans, 2012; Awruch, 2013), and the synthesis of protein for the capsules of oviparous species (Dodd & Goddard, 1961). An increase of E2 has been related to the secretion of histotroph in the uterus of batoids (Snelson et al., 1997; Tricas, Maruska & Rasmussen, 2000; Lyons & Wynne-Edwards, 2019), as well as to the formation of the placental connection in some viviparous sharks (Manire et al., 1995). It has been proposed that E2 is linked to the presence of relaxin and therefore involved in the preparation for parturition (Tsang & Callard, 1987; Koob, Laffan & Callard, 1984). In recent years, it was suggested that E2 and T could play an important role in sexual differentiation of batoids such as U. halleri (Lyons & Wynne-Edwards, 2019). In males, an increase of E2 was related to spermatogenesis (Tricas, Maruska & Rasmussen, 2000; Sulikowski, Tsang & Howell, 2004) and the development of gonads along with other steroids such as P4, T and DHT (Gelsleichter et al., 2002). This oestrogen has been quantified in six biological matrices using the analytical methods described in this review except for PC/TLC-GC. From the reviewed literature, a total of 49 papers (83.0%) have included E2 in their analyses (Table 2).

Estriol

In female batoids, E3 was more abundant in immature individuals compared to mature individuals of T. marmorata (Di Prisco, Vellano & Chieffi, 1967). In a recent study, this steroid was postulated to influence sex differentiation along with E2, as it was detected in the early development of female-only litters of U. halleri (Lyons & Wynne-Edwards, 2019). This oestrogen was quantified in the plasma and histotroph of U. halleri and in plasma of T. marmorata using LC-MS/MS and TLC-GC, respectively. From the reviewed literature, only two papers (3.4%) have included E3 in their analyses (Table 2).

Cortisone

This glucocorticoid was analysed by Di Prisco, Vellano & Chieffi (1967) using plasma of T. marmorata and TLC-GC. The concentrations of E where consistently lower than F, with a decrease of these steroids from immaturity to gestation in the analysed females. In contrast, the concentrations of CORT increased. The study of Lyons & Wynne-Edwards (2019) did detect the hormone, yet it indicated concentrations below the limit of quantification in plasma, along with a lack of detections in the histotroph of U. halleri (Table 2). Two papers (3.4%) using LC-MS/MS and TLC-GC included E in their reproductive analyses (Table 2).

Corticosterone

An increase in CORT levels was linked to sexual development and mating in males of elasmobranch species such as Negaprion brevirostris, S. tiburo and H. sabinus (Rasmussen & Gruber, 1993; Manire et al., 2007). In females of S. tiburo and T. marmorata, CORT was detected during mating, sperm storage and early gestation (Di Prisco, Vellano & Chieffi, 1967; Snelson et al., 1997; Manire et al., 2007). However, this pattern was not observed in females of H. sabinus where CORT levels were low during the entire study period (Snelson et al., 1997), but were elevated in late gestation and parturition in the study of Manire et al. (2007). Although CORT was detected in the plasma and histotroph of U. halleri, the concentrations were below the limit of quantification, so no biological inferences were provided (Lyons & Wynne-Edwards, 2019). CORT has been quantified in blood and histotroph of several elasmobranch species using RIA, TLC-GC or LC-MS/MS, but it has not been investigated in holocephans. From the reviewed literature, five papers (8.5%) have included CORT in their analyses (Table 2).

Cortisol

In terms of the reproductive biology of batoids, Lyons & Wynne-Edwards (2019) observed that F was only present in post-ovulatory females and one early term female of U. halleri. In another study by Di Prisco, Vellano & Chieffi (1967), F was detected during the whole reproductive cycle of T. marmorata in concentrations that were permanently higher than E. This glucocorticoid has been analysed in the plasma of T. marmorata and U. halleri using TLC-GC and LC-MS/MS, respectively, since the concentrations of these hormones were below the limit of quantification in the histotroph of U. halleri. Two papers (3.4%) included F in their analyses (Table 2).

11-deoxycortisol

Although S was recently detected during pregnancy in plasma and histotroph of U. halleri, the observed concentrations were below the limit of quantification, hence limiting our ability to draw conclusions regarding its function. The paper of Lyons & Wynne-Edwards (2019) using LC-MS/MS constitutes 1.7% of the reviewed literature.

11-Deoxycorticosterone

In elasmobranchs, DOC was detected during different maturity stages in T. marmorata females, which could indicate its role in the synthesis of CORT and the regulation of energy and stress (Di Prisco, Vellano & Chieffi, 1967). In the case of male sharks, DOC was suggested as a protein-bound component of seminal plasma in S. acanthias, yet its effect on sperm motility or other influence on semen remains unknown (Simpson, Wright & Hunt, 1964). DOC has been detected in semen of S. acanthias and plasma of T. marmorata using PC and TLC-GC, respectively (Table 2). Three papers included DOC in their analysis, which constitutes 5.1% of the reviewed literature.

11-dehydrocorticosterone

In chondrichthyans, 11-DHC was first detected in late term females of the round stingray U. halleri by Lyons & Wynne-Edwards (2019). However, the concentrations were found below the limit of quantification. It is possible that the presence of 11-DHC could be related to the conversion of this steroid to F or CORT by enzymes such as 11β-hydroxysteroid dehydrogenase (Pelletier, 2010). Nevertheless, the scientific research related to this topic is scarce and there is no evidence of its effects on chondrichthyan metabolism and reproduction. 11-DHC was detected using LC-MS/MS in the paper of Lyons & Wynne-Edwards (2019) which constitutes 1.7% of the reviewed literature.

General models illustrating the function of steroid hormones in male and female chondrichthyans are provided in Figs. 5 and 6. These theoretical diagrams summarise the observations made to date in all the analysed species and can highly differ between taxa. In this regard, considerations for each reproductive strategy, embryonic nutrition and species should be cautiously considered since research in most of the hormones is currently insufficient. A brief summary of the effects of steroid hormones during each biological phenomenon are mentioned below. Further details are also reviewed in Maruska & Gelsleichter (2011), Gelsleichter & Evans (2012) and Awruch (2013).

Overall, male chondrichthyan steroidogenesis is initiated in the testis by the action of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) produced in the pituitary gland on the Sertoli and Leydig cells (Fig. 5; Callard et al., 1989). During sexual maturation, some steroids such as T, P4, 11-KT and CORT play a role as anabolic agents promoting the mitosis and further development of testis, reproductive ducts, seminal glands and claspers (Rasmussen & Gruber, 1993; Manire, Rasmussen & Gross, 1999; Sulikowski, Tsang & Howell, 2005; Manire et al., 2007; Lyons & Wynne-Edwards, 2019). In mature individuals, an increase of T, DHT and oestrogens such as E2 will favour spermatogenesis, while the particular function of androgens such as T are related to sperm motility and transportation throughout the paired reproductive ducts (Manire & Rasmussen, 1997; Heupel, Whittier & Bennett, 1999; Tricas, Maruska & Rasmussen, 2000; Sulikowski, Tsang & Howell, 2004, 2005). The synthesis of seminal plasma will involve the presence of DOC as a component of this substance (Simpson, Wright & Hunt, 1964), where A4 and 3α-diol occur during the peak of sperm reserves prior the reproductive season (Garnier, Sourdaine & Jégou, 1999). The presence of A4 and 3α-diol could be a precursor for an increase in T, DHT and CORT that, in turn, will promote mating behaviours and the further copulation with females (Fig. 5; Rasmussen & Gruber, 1993; Manire et al., 1995; Garnier, Sourdaine & Jégou, 1999; Manire et al., 2007; Mull, Lowe & Young, 2010).

Sexual maturation and reproductive mechanisms in mature females are influenced by the presence of LH and FSH (Fig. 6; Maruska & Gelsleichter, 2011; Awruch, 2013). This promotes the synthesis of P4, T and E2 at the beginning of ovarian follicle development by its production in the granulosa and theca cells (Gelsleichter & Evans, 2012). During sexual maturation and the beginning of each reproductive cycle in mature specimens, the effect of E2 on the liver favours the synthesis of vitellogenin, which is essential for oocyte development (Prisco et al., 2008). During this process, the effects of DOC and 11-DHC are linked to the synthesis of CORT and the regulation of energy and stress (Di Prisco, Vellano & Chieffi, 1967; Pelletier, 2010; Lyons & Wynne-Edwards, 2019). In this regard, it is likely that specific steroid hormones such as 1α-hydroxycorticosterone play an important role during all reproductive phenomena; however, their functions in reproductive processes are still unclear (Iki et al., 2020). Follicle development in preparation for the beginning of the mating seasons or reproductive cycles is favoured by an increase in T, E2 and 11-KT, while the development of reproductive ducts and the oviducal gland will be related to such steroids and to the action of 11-KT (Koob, Tsang & Callard, 1986; Heupel, Whittier & Bennett, 1999; Manire, Rasmussen & Gross, 1999; Tricas, Maruska & Rasmussen, 2000; Henningsen et al., 2008).

After mating, the action of CORT and 11-KA4 allows sperm storage in preparation for conception and synthesis of a tertiary membrane. This structure becomes the egg case in oviparous species or a protective membrane in viviparous embryos (Hamlett, 2011). A rise in P4, E1 and A4 occurs during the preparation for the passing of fertilised oocytes from the oviducal gland to the uterus (Di Prisco, Vellano & Chieffi, 1967; Koob, Tsang & Callard, 1986; Sulikowski, Tsang & Howell, 2004; Sheldon et al., 2018). During pregnancy, this increase in P4 acts as a negative feedback for the synthesis of E2, resulting in a reduction of the vitellogenin synthesis (Tsang & Callard, 1987; Tricas, Maruska & Rasmussen, 2000; Prisco et al., 2008; Mull, Lowe & Young, 2010). As previously mentioned, all these effects would change according to the species. For instance, species showing oophagy or adelphophagy could present different routes since unfertilised or fertilised oocytes are provided for the nutrition of embryos (Compagno, Dando & Fowler, 2005; Hamlett, 2011). Bloodstream levels of P4, S and 11KA4 favour the conditions for the maintenance of embryos, with the presence of CORT, E1 and A4 playing a role in the potential regulation of energy and stress (Fig. 6; Di Prisco, Vellano & Chieffi, 1967; Manire, Rasmussen & Gross, 1999; Sheldon et al., 2018; Lyons & Wynne-Edwards, 2019).

In the case of histotrophic batoids, a rise in E2 affects the ciliated cells in the uterus for the synthesis of the histotroph (Snelson et al., 1997; Tricas, Maruska & Rasmussen, 2000; Lyons & Wynne-Edwards, 2019), while in placental species this E2 rise is linked to the consumption of the yolk and the start of a placental connection (Manire et al., 1995). Specific steroids involving androgens and oestrogens such as DHT, A4, 17-OHP, E3 and E2 will play a role in the sex differentiation of embryos (Tricas, Maruska & Rasmussen, 2000; Lyons & Wynne-Edwards, 2019), with high levels of CORT present during the late gestation and prior to parturition (Manire et al., 2007). In the last phases of the reproductive cycle, a drop in P4 will affect the original conditions for the maintenance of embryos or the start of oviposition (Koob, Tsang & Callard, 1986; Manire, Rasmussen & Gross, 1999; Sulikowski, Tsang & Howell, 2004; Mull, Lowe & Young, 2010). The absence of P4 allows the recommencement of E2 synthesis, which plays a role in the production of relaxin for parturition (Koob, Laffan & Callard, 1984; Tsang & Callard, 1987). The oviposition or the birth of the pups likely stimulates an increase in DHT levels that could act as a negative feedback for the synthesis of gonadotropin-releasing hormone (Fig. 6; Tricas, Maruska & Rasmussen, 2000).