Molluscan Research, 2015Vol. 35, No. 3, 161–172, http://dx.doi.org/10.1080/13235818.2015.1045055

Seasonal changes of progesterone and testosterone concentrations throughout gonadmaturation stages of the Mexican octopus, Octopus maya (Octopodidae: Octopus)

Omar Hernando Avila-Povedaa∗ , Rubén Cornelio Montes-Pérezb , Noussithé Kouetac , FranciscoBenítez-Villalobosd , Jorge Saúl Ramírez-Péreza , L.R. Jimenez-Gutierreza and Carlos Rosase

aFacultad de Ciencias del Mar (FACIMAR), Universidad Autónoma de Sinaloa (UAS), Mazatlán, Sinaloa, Mexico; bDepartamentode Reproducción Animal y Mejoramiento Genético, Laboratorio de Radioinmunoanálisis, Facultad de Medicina Veterinaria yZootecnia (FMVZ), Universidad Autónoma de Yucatán (UADY), Merida, Yucatan, Mexico; cLaboratoire de Biologie des Organismeset Ecosystèmes Aquatiques(BOREA), Institut de Biologie Fondamentale et Appliquée (IBFA), Université de Caen, France; dInstitutode Recursos, Universidad del Mar (UMAR), Puerto Ángel, Oaxaca, Mexico; eUnidad Multidisciplinaria de Docencia e Investigación(UMDI), Facultad de Ciencias, Universidad Nacional Autónoma de Mexico (UNAM), Puerto de Abrigo S/N. Sisal, Yucatán, Mexico

(Received 5 December 2013; final version received 22 March 2015 )

Variations in progesterone (P4) and testosterone (T) levels in the gonad of Octopus maya from Sisal in Yucatan State,Mexico, were investigated by radioimmunoassays and in relation to four gonad maturation stages (GMS) and to thereproductive cycle, as represented by two maturity indices (microscopic ‘MiMI’ and macroscopic ‘MaMI’). Accordingto the GMS and the maturity indices, the reproductive season of O. maya from Yucatan occurred from February to June.In females, P4 and T displayed the same pattern, with a tendency to increase at the same time, although on average, P4had seven-fold higher concentrations than T. In contrast, P4 and T in male gonads displayed a different pattern, whereT concentrations were relatively stable throughout all of the study months. In the female gonad P4 was lowest (close to0 pg g–1) during both developing (GMS-I) and maturing (GMS-II) stages, and increased (189 ± 53 pg g–1) approach-ing the mature stage (GMS-III) to a maximum value of 611 pg g–1. Concentrations of T in the male gonad were lowest(106 ± 9 pg g–1) during the maturing stage (GMS-II) and increased up to the mature stage (GMS-III), reaching a max-imum of 440 pg g–1. Pearson’s correlation (r) between hormones and maturity indices showed strong relationships forfemales (around 0.4 and − 0.7; p < 0.05), but there were negligible or weak relationships for males (0.2 and − 0.1;p > 0.05). Hormone correlations in females were inverse with MaMI and direct with MiMI. Our major findings showedthat gonadal P4 levels were elevated during GMS-III and GMS-IV (i.e. periods of vitellogenic oocytes), where the charac-teristic aspect is an ovary with very high oocyte diameters, with the primary follicle cells deeply infolded in the ooplasmfor yolk synthesis. These results suggested a synchrony between P4 and the process of folliculogenesis, and in turn,vitellogenesis.

Keywords: radioimmunoassay; sex steroids; reproductive cycle; maturity index; reproductive season; cephalopod

IntroductionSeveral reviews have found that sex steroids have a bio-logical effect on gonadal maturation in molluscs (Lafontand Mathieu 2007; Scott 2013); however, it is generallyagreed that the hormonal control of reproduction in inver-tebrates is not as well documented as in vertebrates (Gore2002; Okubo and Nagahama 2008), and even less so forcephalopod molluscs (Lehoux and Sandor 1970; Joosse1972; Lafont 2000; Lafont and Mathieu 2007; Ketataet al. 2008; Kikuyama and Tsutsui 2011; Huffard 2013).Hormones are major proximate factors mediating repro-duction (Chedrese 2009) and behaviour (Adkins-Regan2005), and have therefore contributed to the evolutionof the diverse mating systems and neurobiology seen invertebrates and invertebrates (Di Cristo 2013; Huffard2013).

*Corresponding author. Emails: oavila@uas.edu.mx; ohavilapo@conacyt.mx

Cephalopods, a group of more than 850 species,exhibit a diversity of reproductive strategies: semelparityand/or iteroparity, with or without parental care, and mat-ing offshore, in the open ocean, and in the deep sea (Nig-matullin and Laptikhovsky 1994; Laptikhovsky 1998,2013; Rocha et al. 2001; Hoving 2008). It is well knownthat the reproductive process (gametogenesis and sexualmaturation) in cephalopods is triggered by secretion ofan Octopus gonadotrophin-releasing hormone released bythe olfactory lobes and the subpedunculate area of thecentral nervous system, a system that is highly centralisedand consists of a ‘brain’ and the optic lobes (Young 1971;Di Cosmo and Di Cristo 1998; De Lisa et al. 2012).Octopus gonadotrophin-releasing hormone stimulates theoptic gland, which produces a gonadotrophin factor (notyet characterised) that induces gonadal steroidogenesis

© 2015 The Malacological Society of Australasia and the Society for the Study of Molluscan Diversity

162 O.H. Avila-Poveda et al.

and in turn, gonad maturation (Wells and Wells 1959,1969, 1972, 1975; O’Dor and Wells 1973, 1978; Wellset al. 1975; Di Cosmo and Di Cristo 1998; Iwakoshiet al. 2002; Iwakoshi-Ukena et al. 2004; Kanda et al.2006). The ovary and testis are sites of steroidogenesis(D’Aniello et al. 1996; Di Cosmo et al. 1998, 2001, 2002;Di Cristo et al. 2008, 2009; Osada and Treen 2013). Theinvolvement of olfaction in the control of reproductionhas been proposed (Di Cosmo and Polese 2014; Poleseet al. 2015).

Vertebrate-type sex steroids (e.g. progesterone [P4],testosterone [T] and 17β-oestradiol) have been reportedto occur in several organs of the reproductive systemof octopuses, both male and female. In male octopuses,these sex hormones occur in the testis, vas deferens, sem-inal vesicle, prostate and Needham’s sac, with the testisbeing the organ that contains the highest concentration ofT and the seminal vesicle possessing the highest concen-tration of P4 (D’Aniello et al. 1996). In female octopuses,P4 has been detected in the ovary, and the concentrationschange with ovarian maturation (Di Cosmo et al. 2001).In cephalopods, progestogens, such as P4, are involved infollicle cell maturation (Di Cosmo et al. 1998) and subse-quently in the regulation of egg yolk protein synthesis (DiCristo et al. 2008), but they may also play a role in thepre-activation of spermatozoa stored in the female tract(Tosti et al. 2001; De Lisa et al. 2013). Progesterone iselevated during periods of full vitellogenesis and loweredduring non-vitellogenic periods (Di Cosmo et al. 2001).Androgens, such as T, stimulate reproductive behavioursand spermatogenesis in males (D’Aniello et al. 1996).

Research into cephalopod sex steroid hormones (i.e.P4, T and/or 17β-oestradiol) has been limited to thefollowing species: Eledone cirrhosa (Lamarck, 1798)(Nikitina et al. 1977; Young 1992), Octopus mimusGould, 1852 (Olivares-Paz et al. 2003) and Octopus vul-garis Cuvier, 1797 (Wells and Wells 1959; D’Anielloet al. 1996; Di Cosmo et al. 2001, 2002; Di Cristo et al.2008).

Octopus maya Voss & Solís-Ramírez, 1966, knownas the red octopus and/or Mexican four-eyed octo-pus, is one of the most fished species and represents70% of the octopus resource, comprising, in additionto O. maya, Octopus vulgaris Cuvier, 1797 and Octo-pus hubbsorum Berry, 1953 in Mexican national fish-ing (CONAPESCA 2011). Octopus maya is an endemicspecies from the Campeche Bank (i.e. the continentalshelf of the Yucatan Peninsula, comprising the statesCampeche, Yucatan and Quintana Roo) and inhabits ben-thic habitats down to 58 m (Solís-Ramírez and Chávez1986). Due to the considerable demand for red octopusas an important source of meat for local people (Santos-Valencia and Medina-Martinez 2003; Salas et al. 2006)and for export (CONAPESCA 2011), the ExperimentalCephalopod Production Unit (EPHAPU) at the Unidad

Multidisciplinaria de Docencia e Investigación, Univer-sidad Nacional Autónoma de Mexico (UMDI-UNAM) inMexico has successfully raised captive-born red octopus.Cconsiderable effort has also improved our knowledge ofthe reproductive traits of this species (Rosas et al. 2014).

Aspects of O. maya’s reproductive anatomy andreproductive cycle have been reported, but what littleinformation is known has been restricted to conferenceproceedings and grey literature. This information focuseson gonad emergence, gonad maturation and develop-ment of the reproductive system during early life stages(Avila-Poveda et al. 2009a; Calva et al. 2009; Calva-Fierro 2010), descriptions of gonad maturation stagesduring adult life history (Santos-Valencia et al. 2006;Avila-Poveda et al. 2009b; Rosas et al. 2012; Avila-Poveda 2014), and the reproductive season, which showstwo reproductive peaks (transferring eggs) per year inCampeche, the first between June and July and the secondbetween November and December (Solís-Ramírez 1967,Santos-Valencia and Re-Regis 2003; Santos-Valencia andDel-Río-Rodríguez 2006; Santos-Valencia et al. 2006),whereas a single reproductive season occurs from Febru-ary to June in Yucatan (Avila-Poveda 2014). Recently, ithas also been suggested that gonad maturation of femaleO. maya is related to sex steroid levels (Avila-Povedaet al. 2009c). Therefore, variations in the hormonal con-centrations in O. maya during gonad maturation couldhave potential applications in aquaculture, improvingcaptive breeding practices and studies of the life cycle ofthis species, as well as providing a better understandingof endocrine control in O. maya.

The aims of this study were: (1) to quantify seasonalchanges of P4 and T in the gonad of female and male octo-puses throughout a sampling period of 10 months, and(2) to examine the relationship of steroid hormone con-centrations (of P4 and T) with events of the reproductivecycle, represented by maturity indices (microscopic andmacroscopic) in the red octopus, O. maya.

Materials and methodsSample collectionEach month from September 2007 to June 2008, an aver-age of 32 O. maya adults with body weight > 250 gwere caught off the coast of Sisal, Yucatan (21°9′55′′N,90°1′50′′W) (Fig. 1). Only red octopuses > 250 g inbody weight were included in this study, because cap-tive females of O. maya above this size lay fertile eggs(Romero-Soberanis 2007; Rosas et al. 2014; Rosas, pers.obs.). Individuals were caught at depths ranging from 5to 10 m, using the local method of fishing-adrift, knownas ‘gareteo’, a canoe equipped with an artisanal fish-ing line attached to a 7- to 8-m-long bamboo rod witha crab (Callinectes ornatus, Menippe mercenaria or Lib-inia sp) tied on as bait (Solís-Ramírez 1967). The line

Sex steroids in Octopus maya 163

Figure 1. Map of the Campeche Bank (area until contour of200-m isobaths), and Yucatan Peninsula (shaded grey), Mex-ico, showing the location of Octopus maya capture site (blacksquare) off the coast of Sisal.

is left drifting and carried away by the wind. All octo-puses were transported to the UMDI-UNAM laboratoryin a 120-L circular tank, filled with seawater. In the lab-oratory, each individual was separated for 2 days insidea continuous-flow 80-L tank with aerated seawater andwithout food to avoid any interference by feeding on thephysiological condition of the animals (Rosas et al. 2011,2014). One piece of PVC tube (10 cm in diameter) peroctopus was placed in each tank as a refuge.

Before dissecting, each octopus was cooled by immer-sion in seawater at 5 °C for 3 min (derived from Roperand Sweeney 1983) to induce narcotisation to enablehumane killing (Andrews et al. 2013) in considerationof ethical protocols (Mather and Anderson 2007) and theanimals’ welfare during manipulations (Moltschaniwskyjet al. 2007). Soon after narcotisation, the organismswere dissected and two weights ( ± 0.001 g) were takenper octopus: (1) RCW–reproductive complex weight:identified as accessory glands without gonad, i.e. sper-matophoric complex, Needham’s sac and vas deferens inmales, and oviducts and oviducal glands in females; and(2) GW–gonad weight. From the dissected gonads, onepart was frozen in liquid nitrogen and stored at − 40 °Cfor hormonal assays.

Hormonal radioimmunoassaysConcentrations of P4 and T were quantified in gonadextracts by using the corresponding antibody andtracer ligand-I125 taken from commercially availablesolid-phase 125Iodine radioimmunoassay (DSL-3900 andDSL-4000; Diagnostic Systems Laboratories

®, Inc. Web-

ster, TX, USA). The extraction of sex steroids from

gonads (twice with 15 mL of diethyl-ether each time)and the preparation of gonad extracts (re-dissolved inmethanol and phosphate buffer solution) were performedas described by Avila-Poveda et al. (2013). For eachhormone, nine standards, serially diluted from 10 ngmL–1 in 0.1 M phosphate-buffered saline at pH 7.0, weremade in the laboratory and run in each assay. Eachdetermination of P4 and T was carried out in dupli-cate. For both hormones, precision (intra-assay) was 13%and reproducibility (inter-assay) was < 14%. The preci-sion/reproducibility ratio was 75% for P4 and 96% for T,which indicates a high stability of the system (Rodbard,1974). Sensitivities (the smallest amount of hormone sig-nificantly different from zero at the 95% confidence limit)were registered as 17 pg g–1 for P4, and 1 pg g–1 for T.

Reproductive statusThe characterisation of the reproductive status (par-ticularly, periodicity of reproductive cycle and pre-paredness to transfer gametes) throughout the gonadmaturation stages (GMS), was represented by two repro-ductive maturity indices: (1) macroscopic maturity index:MaMI = RCW/(RCW + GW); after Hayashi (1970) andGuerra (1975), where the functional maturation infemales corresponds to the lowest MaMI value and inmales to the highest MaMI value; and (2) microscopicmaturity index: MiMI = ∑

(ni × si) / N (statisticallya weighted mean, Zar 2010) applied in several Classesof invertebrates like Cephalopoda (Ortiz 2013), Bivalvia(Meneghetti et al. 2004), Echinoidea (Yoshida 1952),Holothuroidea (Sewell 1992; Despalatovic et al. 2004;Benítez-Villalobos et al. 2013; Kazanidis et al. 2014);where ni is the number of octopuses at each gonad mat-uration stage, si is the numerical score attributed to thatstage, and N is the total number of octopuses collected permonth. Hence, each MiMI value indicates the respectiveperiodicity of each GMS throughout the sampling period.Four GMS were recognised for both sexes (see Table1): GMS-I—developing, GMS-II—maturing, GMS-III—mature and GMS-IV—transferring. These were basedon the most obvious histological characteristics for thestages used in other octopuses (Ishiyama et al. 1999;Khallahi 2001; Gonçalves et al. 2002; Rodríguez-Rúaet al. 2005; Idrissi et al. 2006; ICES 2010; MEDITS2012; López-Peraza et al. 2013; de Lima et al. 2014).

Statistical analysisData were expressed as means ± standard error (SE).Differences among monthly values of each hormone level(P4 and T) and of the macroscopic maturity index (MaMI)for each sex were tested by one-way analysis of variance(ANOVA), and the homogeneous groups were estab-lished according to the post hoc Tukey HSD test (honestly

164 O.H. Avila-Poveda et al.

Table 1. Gonad maturation stages in Octopus maya.

Stage Characteristics

I – Developing Corresponds to the formation of earlyoocytes or early spermatocytes(i.e. non-mature gametes, includinggerminal epithelium).

II – Maturing Comprises the formation of secondaryoocytes surrounded by the folliclecells (oocyte II) but without yolkor secondary spermatocytes andspermatids.

III – Mature Corresponds to the formation of matureova with the primary follicle cellsdeeply infolded in the ooplasmrealising yolk synthesis (oocytes III)or lumen of the seminiferous tubulesfilled with spermatozoa.

IV – Transferring Comprises mature ova of large sizeand completely filled with yolkgranules (oocyte IV), which begin tobe transferred into accessory glandsfrom where the spawning will takeplace. Males show empty spacesin the lumen of the seminiferoustubules indicating that spermatozoahave been expelled from the testis toaccessory glands.

significant difference), and indicated in each graph bydifferent letters. Before ANOVA, assumption tests werecarried out to determine the homogeneity of variancesfor each hormone. Pearson correlation and simple linearregression were used to relate the levels of each hor-mone (P4 and T) to each maturity index (macroscopic andmicroscopic) in the population of O. maya (Zar 2010).Statistical analysis was carried out with STATISTICA 7.Statistical significance was accepted at p < 0.05.

ResultsConcentrations of P4 and TIn female octopuses, P4 concentrations ranged from 1 to611 pg g–1, whereas T concentrations ranged from 1 to80 pg g–1. The two steroids P4 and T displayed the samepattern between them throughout all of the months of thestudy, with a tendency to increase together at the sametime, with P4 having a seven-fold higher concentration,on average, than levels of T. Statistical analysis revealedthat P4 and T concentrations peaked in February andJune (p > 0.05 for these 2 months) (Fig. 2). The relativesynchrony between variation patterns of P4 and T concen-trations during the reproductive cycle was confirmed byPearson’s r correlation (r = 0.831 and p < 0.05).

In male octopuses, the variation pattern of the twosteroids was quite different between them and both steroidconcentrations were significantly higher than reported forfemales (p < 0.05) through all of the months of the study.

P4 concentrations increased from 304 to 1,146 pg g–1,reaching the highest value in February (p < 0.05). T con-centrations were relatively stable throughout the monthsof the study, ranging from 75 to 159 pg g–1, except duringApril (440 pg g–1) when the highest value was recorded(p < 0.05) (Fig. 2).

Maturity indicesThe MaMI showed different fluctuation patterns betweensexes, being more pronounced in females than in males(Fig. 3). Female octopuses had values between 0.24 and0.52, where the lowest values correlated with the repro-ductive season during February and May-June. Malesshowed MaMI values between 0.39 and 0.52, being high-est in the reproductive season during March to June(p > 0.05). The statistical analyses showed significantdifferences between sexes (p < 0.05).

The MiMI was more pronounced in females than inmales (Fig. 3). Females were observed to be develop-ing (GMS-I) and maturing (GMS-II) from Septemberto December 2007, mature (GMS-III) during January,March and May 2008, and transferring (GMS-IV) dur-ing February 2008. Throughout the sampling period, themales were close to maturing (GMS-II), but coming fromdeveloping (GMS-I) during September and October 2008and heading towards mature (GMS-III) during March2008.

Progesterone and testosterone relationships withmaturity indicesFigure 4 shows the P4 and T relationships with respect tomaturity indices in each sex. Progesterone in the femalegonad was lowest (close to zero pg g–1) during the devel-oping (GMS-I) and maturing (GMS-II) stages, and wasincreasing (189 ± 53 SE pg g–1) to maturation (GMS-III)with a maximum value (611 pg g–1) when transferring(GMS-IV) took place during February 2008. Testos-terone in the female gonad showed the same trend, butat concentrations that were much lower than P4.

Testosterone of the male gonad was lowest (106 ± 9SE pg g–1) during the maturing stage (GMS-II), andincreased toward the mature stage (GMS-III) in March toa maximum value of 440 pg g–1. Progesterone of the malegonad showed a concentration of 505 ± 42 SE pg g–1

during the maturing stage (GMS-II), but with an abruptpeak in February 2008 (1146 pg g–1), before the onset ofthe mature stage (Fig. 4).

Table 2 presents the correlation (r) and determination(R2) coefficients and the simple linear regression betweenhormones and indices. Pearson’s correlation (r) showedstrong relationships for females (around 0.4 and − 0.7;p < 0.05); however, there was a negligible or weak rela-tionship for males (0.2 and − 0.1; p > 0.05). Hence,

Sex steroids in Octopus maya 165

Figure 2. Monthly variations in progesterone (P4) and testosterone (T) concentrations (pg g–1) in the gonads of females and malesof Octopus maya. Each value represents a mean + SE (n, the sample sizes are each value in brackets). Different letters on each figureindicate significant differences between values at p < 0.05.

hormone correlations in females were inverse with MaMIand direct with MiMI. Coefficients of determination (R2)were greater for female models than for males, whichindicated that the percentage of variability in each hor-mone could be explained by each index in females. Theonly model in which males had a higher coefficient ofdetermination (0.46; p < 0.05) was the MiMI model.

Finally, the correlations for those models in whichfemale octopuses showed a significant effect were: (1)concentration of P4 decreased with increasing value ofthe MaMI, indicating an inverse correlation; (2) concen-tration of P4 increased with increasing value of the MiMI,indicating a direct correlation; (3). concentration of Tdecreased with increasing value of the MaMI, indicat-ing an inverse correlation; and, (4) concentration of Tincreased with increasing value of the MiMI, indicatinga direct correlation (Table 2).

DiscussionThe study of sex steroids and the regulatory mechanismsof biochemical and physiological processes involvedin reproductive and other biological functions is an

interesting field that needs to be explored in detail ininvertebrates to identify any similarities and differenceswith vertebrates (e.g. Joosse 1972; Sandor 1980; Lafont2000; Lafont and Mathieu 2007; Kikuyama and Tsut-sui 2011). There is evidence that sex steroid hormonesare involved in the regulation of the reproductive cyclein many marine invertebrates, such as crustaceans (VanBeek and De Loof 1988), polychaetes (García-Alonsoet al. 2006), echinoderms (Schoenmakers and Dieleman1981; Xu and Barker 1990; Unuma et al. 1999; Wassonet al. 2000; Barbaglio et al. 2007), and various mol-luscs, such as scallops (Matsumoto et al. 1997; Wang andCroll 2004, 2006), mussels (Reis-Henriques and Coimbra1990), clams (Gauthier-Clerc et al. 2006), oysters (Mat-sumoto et al. 1997) and cephalopods (Di Cosmo et al.2001; Di Cristo et al. 2008).

The present study is the first that has established alink between the sex steroid levels (P4 and T) and thereproductive cycle in the cephalopod O. maya. This sug-gests that seasonal variations of P4 and T may play animportant role as endogenous modulators of gonad mat-uration and, in turn, the reproductive cycle in both maleand female O. maya.

166 O.H. Avila-Poveda et al.

Figure 3. Monthly variations in macroscopic and microscopic maturity indices (MaMI and MiMI, respectively) of females andmales of Octopus maya. Values in MaMI are mean + SE, while those in MiMI are absolute values. Levels (grey areas) of MiMI are:Stage I—developing; Stage II—maturing; Stage III—mature; Stage IV—transferring. Different letters indicate significant differencesbetween values at p < 0.05.

Regarding P4, our results showed fluctuations thatwere in agreement with the reproductive cycle, similarto the results observed in O. vulgaris Cuvier, 1797 (DiCosmo et al. 2001). In O. vulgaris, P4 is involved in fol-licle cell maturation, which covers the oocyte (Di Cosmoet al. 1998), and subsequently in the regulation of eggyolk synthesis (Di Cristo et al. 2008). In O. maya, theincrease of P4 during vitellogenesis (stages III and IV)seems to suggest its involvement in yolk synthesis, anaspect that is also sustained by a decrease when vitelloge-nesis is completed, similar to what has been demonstratedin O. vulgaris (Di Cosmo et al. 2001).

Regarding T, there have been no studies suggestingits role in reproduction in female cephalopods. However,there is evidence in our study for transient increases ofT at the maturing stage (GMS-II) and during the transfer-ring stage (GMS-IV), as shown previously for some othermolluscs such as the clams Mya arenaria Linnaeus, 1758(Gauthier-Clerc et al. 2006) and Sinonovacula constricta(Lamarck, 1818) (Yan et al. 2011), and in the asteroidAsterias vulgaris Linnaeus, 1758 (current name acceptedas Asterias rubens) (Hines et al. 1992). This indicates thatT in females could also play a role in processes related tooocyte development in the female reproductive cycle.

Olivares-Paz et al. (2003) indicated that both P4 and Tshow the same pattern of change in testis maturation formale Octopus mimus, where both steroids are low whenspermatozoa are being expelled from the testis (here,GMS-IV) and increase at the onset of the maturing stage(here, GMS-II), reaching a peak during spermatogenesis(here, GMS-III). In male O. maya, levels of P4 and T didnot apparently change in relations to the stages of gonadmaturation during their reproductive cycle; however, weobserved a peak in testis P4 levels before the mature stage(GMS-III), which is concomitant with the active maturestage of females. In cephalopods, androgens such as Tstimulate reproductive behaviours and spermatogenesisin males (D’Aniello et al. 1996), but can also play a rolein the pre-activation of spermatozoa stored in the femaletract (Tosti et al. 2001; De Lisa et al. 2013).

The concentrations of both P4 and T in gonads ofO. maya throughout the annual reproductive cycle wereslightly higher in males than in females. This suggeststhat the role of these steroids is not the same in malesand females and involves the regulation of sex-specificprocesses occurring in the gonads, particularly aroundthe reproductive season. Previous data suggest that P4could be the main sex steroid hormone that modulates the

Sex steroids in Octopus maya 167

Figure 4. Reproductive cycle represented by maturity indices (MaMI—macroscopic maturity index; MiMI—microscopic maturityindex), and monthly means of progesterone (P4) and testosterone (T) concentrations, from September 2007 to June 2008, at Sisal,Yucatan, Mexico. The grey area indicates the main reproductive season, which covered the mature (Stage III) and transferring (StageIV) stages. Levels of MiMI are: Stage I—developing; Stage II—maturing; Stage III—mature; Stage IV—transferring. Different letterson each figure indicate significant differences between values at p < 0.05.

168 O.H. Avila-Poveda et al.

Table 2. Parameter values of the regression by least squares between hormones (progesterone [P4] and testosterone [T]) andthe macroscopic and microscopic maturity indices (MaMI and MiMI, respectively) per each sex.

Sex n r R2 Equations p-value

Females 62 − 0.7206 0.5193 P4 = 898.3942 – 2020.3225*(MaMI) 0.0000Females 55 0.4643 0.2156 P4 = − 207.7783 + 140.814*(MiMI) 0.0004Females 67 − 0.6880 0.4733 T = 93.3269 – 203.2997*(MaMI) 0.0000Females 61 0.4360 0.1901 T = − 22.3192 + 15.0848*(MiMI) 0.0004Males 34 − 0.1796 0.0323 P4 = 833.0311 – 652.2115*(MaMI) 0.3095Males 20 0.6838 0.4676 P4 = 250.8865 + 159.3049*(MiMI) 0.0009Males 39 0.2536 0.0643 T = 39.5863 + 159.4806*(MaMI) 0.1193Males 25 − 0.1320 0.0174 T = 114.2523 – 6.2078*(MiMI) 0.5230

Note: n—sample size; r—Pearson correlation coefficient; R2—coefficient of determination. Relationship significant at valuesp < 0.05.

endocrine role during gonad maturation and reproductivestrategy for both males and females of O. maya.

Tosti et al. (2001) determined that O. vulgaris sper-matozoa are activated by P4 stimulation, that spermatozoatransferred toward the female distal oviduct after matingare stored in a pre-activated state, and then P4 plays arole in the activation of spermatozoa stored in the femaledistal oviduct. Di Cosmo et al. (1998) characterised a P4receptor in the reproductive system of female O. vulgaris(in particular, localised in the nuclei of the follicular cellsthat embedded it in the oocyte, in the proximal portion ofthe oviduct and in the outer region of the oviducal gland),specifically showing that a P4 receptor has biochemi-cal and immunohistochemical characteristics resemblingthose of P4 receptors in vertebrates; hence, these resultscould indicate the site of action.

Our major findings were that gonadal P4 levels areelevated during GMS-III and GMS-IV (i.e. periods ofvitellogenic oocytes), with the characteristic aspect beingan ovary with very high oocyte diameter increases, andprimary follicle cells that are deeply embedded with yolksynthesis. Also, the gonadal P4 levels were lower dur-ing GMS-I and GMS-II (i.e. periods of non-vitellogenicoocytes), with a characteristic aspect of an ovary withoocytes that are beginning to establish primary folliclecells. These findings agree with what is known aboutother species of octopus such as O. vulgaris (Di Cosmoet al. 1998, 2001; Di Cristo et al. 2008). Our results sug-gested a synchrony between P4 and the process of follicu-logenesis, and in turn, vitellogenesis, as well as a concor-dance between P4 and T, prominent in February–March(reproductive season), for this population of O. mayafrom Sisal, Yucatan, Mexico. Our study corroboratesthe main reproductive season (February–June) for thisspecies on the coasts of Sisal, Yucatan, and provides somedata on their hormones and reproductive physiology.

AcknowledgementsThis research was partially funded by PAPIIT project num-ber IN212012 and CONACYT CB: 2010–01/150810 to C

Rosas. OH Avila-Poveda receives a CONACYT scholarshipand a family grant and the results presented here are part ofhis PhD Dissertation. Thanks are due to the team Pulpo atSisal for their professional gear handling, but we are espe-cially grateful to Arturo George-Zamora and Felipe Briceñofor their direct assistance in octopus sampling and laboratorywork. The radioimmunoassay procedures were performed in theRadioimmunoassay laboratory of the Veterinary Medicine andZootechnics School, Yucatan University (UADY-FMVZ), inaccordance with the standard operating procedures given by theNational Commission of Nuclear Safety and Safeguard of Mex-ico (CNSNS, acronym in Spanish, http://www.cnsns.gob.mx/).The critical appraisals of reviewers Anna Di Cosmo, GianlucaPolese and Kathrin Bolstad, and of one anonymous reviewer,are greatly appreciated. OH Avila-Poveda and LR Jimenez-Gutierrez were commissioned from CÁTEDRAS-CONACYT(project No. 2137/FACIMAR-UAS).

Disclosure statementNo potential conflict of interest was reported by the authors.

ORCIDOmar Hernando Avila-Poveda

http://orcid.org/0000-0002-4411-0602Rubén Cornelio Montes-Pérez

http://orcid.org/0000-0003-4251-7342Noussithé Koueta

http://orcid.org/0000-0003-1791-2930Francisco Benítez-Villalobos

http://orcid.org/0000-0002-0951-7757Jorge Saúl Ramírez-Pérez

http://orcid.org/0000-0002-3994-424XL.R. Jimenez-Gutierrez

http://orcid.org/0000-0001-6805-4134Carlos Rosas

http://orcid.org/0000-0002-1301-7368

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