Peromyscus difficilis (J. A. Allen, 1891)

Salame-Méndez, Arturo, Mancera-Jaime, Gustavo, Castro-Campillo, Alondra, Ávila-Valle, Zamira & Ramírez-Pulido, José, 2024, Intragonadal evaluation of sexual steroid hormones during three reproductive events in two species of Peromyscus (Rodentia: Cricetidae), Zoologia (e 23032) 41, pp. 1-23 : 8-13

Peromyscus difficilis ( Fig. 2B , Table S1). Proestrus:

the descending order was [E

2

], [T], [P

4

], and [A]; in the ∆

4

pathway, [A] decreased not significantly 1 pg/mg, from [P 4], but the 8.5 pg/mg increase in [T] from [A] was significant

(p = 0.02), and the following increase of 4.1 pg/mg in [E

2

]

from [ T] was not significant; [P 4] was 11.6 pg/mg extremely significantly lower (p = 7.3E –04) than [E

2

] and 7.5 pg/mg significantly lower (p = 0.04) than [T]; [A] was 12.6 pg/mg

extremely significantly lower (p = 1.2E –04) than [E 2]. Estrus heat: [E

2

] was the highest followed by [P

4

], [T], and [A]; in the biotransformation pathway, the reduction of 7.9 pg/mg

in [A] from [P 4] was very significant (p = 0.002), the slight increase of 1.3 pg/mg from [A] into [T] was not significant,

but there was conspicuous increase of 19 pg/mg in [E

2

]

from [ T] extremely significant (p = 6.6E –05); [P 4] was 12.4 pg/mg lower than [E

2

] and very significantly different (p =

0.002), 6.6 pg/mg higher than [ T] and also very significantly different (p = 0.001); [E 2] was also 20.3 pg/mg and extremely significantly higher than [A] (p = 7.6E –05). Metestrus: [P

4

]

scored the highest and was statistically different from other

[SSH]; in the ∆ 4 biotransformation, a decrease of 5 pg/mg from [P

4

] into [A] was significant (p = 0.02), but the slight increase of 0.3 in [T] from [A] and the decrease of 1.6 pg/mg

in [E 2] from [T] were not significant, respectively; [P 4] almost doubled (6.3 pg/mg) and was very significantly higher (p =

0.001) than [E

2

]. Diestrus: [E

2

] was the highest, followed by

[P 4], [T], and [A]; in the biotransformation pathway, there was a very significant (p = 0.003) decrease of 4.3 pg/mg from

[P

4

] into [A], a slight, not significant increase of 1.8 pg/mg in

[T] from [A], and an almost significant (p = 0.055) increase of 4.5 pg/mg in [E

2

] from [T]; [P

4

] was 2 pg/mg lower than

[E

2

] but not significantly different, but it was 2.5 pg/mg

significantly higher (p = 0.03) than [T], and [E 2] was 6.3 pg/ mg and significantly higher (p = 0.01) than [A]. Early gestation G1: [P

4

] was the highest, followed by [E

2

], [T], and [A];

in the biotransformation pathway, the collapse of almost seven times with a reduction of 22.2 pg/mg from [P

4

] into

[A] was extremely significant (p = 1.4E –07), followed by not significant slight increases of 1.2 pg/mg between the two androgens and of 4.4 ng /m in [E

2

] from [T], respectively; [P

4

]

almost tripled [E

2

] with an extremely significant difference

(p = 5.7E –05) of 16.6 pg/mg and was also about five times more concentrated than [T] with an extremely significant

(p = 3.5E –07) difference of 21 pg/mg; [E

2

] was also 5.6 pg/

mg significantly (p = 0.02) higher than [A]. Late gestation

G2: [E

2

] was the highest, followed by [T], [P

4

], and [A]; in the

∆

4

pathway, there was reduction of 0.3 pg/mg in [A] from

[P 4], an increase of 0.7 pg/mg in [T] from the latter, and an increase of 3.2 pg/mg in [E

2

], none of which were significant;

[P

4

] was 3.6 pg/mg significantly lower (p = 0.02) than [E

2

] and the estrogen was 3.9 pg/mg significantly higher (p = 0.03)

than [A]. Lactation: the concentration order was the same as in G2; there were only slight and no significant changes from the progestogens into androgens because [A] was only

0.2 pg/mg lower than [P

4

] and [T] was only 0.8 higher than the former, but the increase of 4.1 pg/mg in [E 2] from [T] was significant (p = 0.01); the estrogen was 4.7 pg/mg very significantly higher (p = 0.002) than [P 4] and 4.9 pg/mg very significantly higher (p = 0.001) than [A].

Fluctuations in the concentration of each SSH through estrous cycle, pregnancy, and overall lactation

The ANOVA ( Fig. 3 , Table S2) for each [SSH] also resulted in several significant differences in its concentrations between the stages of EC, the two parts of pregnancy, and overall lactation in adult females of P. melanotis ( Fig. 3A ) and P.difficilis ( Fig. 3B ). In these analyses, the significant differences in the concentration of the same SSH were also arranged, according to three intervals for the respective p-values, in order to emphasize its physiological increase or decrease in a particular reproductive stage or event: significant differences fluctuated from 0.01 to 0.05; very significant differences fluctuated from 0.001 to 0.009; extremely significant differences fluctuated from four to eight ceros after a period and before any digit as 1.1E –04 to 3.2E –08.

Peromyscus melanotis ( Fig. 3A , Table S2). Overall arrangement from most to least [P 4] was G1> Est> Met> Pro> Die> Lac> G2. The maximum peak of [P 4] after fecundation and initial development of products in early gestation (G1) was extremely significantly higher than most reproductive stages of a complete estrous cycle (CEC: Pro p = 3.7E –04; Met p = 8.4E –05; Die p = 5.5E –05), as well as than late gestation (G2, p = 6.1E –06) and overall lactation (Lac p = 1.5E –06), being also significantly higher (p = 0.02) than its other conspicuous peak in estrus. Except for proestrus, this second maximum [P 4] at estrus was also statistically significant and conspicuously higher than everything else (Met p = 0.03; Die p = 0.008; G1, p = 0.02; G2 p = 0.001; Lac p = 3.8E –04). In metestrus, [P 4] was also statistically higher than in diestrus (p = 0.04), late gestation G2 (p = 0.005), and lactation (p = 0.002).

Androstenedione [A] was arranged as Die> Met> G2> Lac> Est> Pro> G1. The lowest [A] in G1 was statistically different from metestrus (p = 0.002), diestrus (p = 0.014), late gestation G2 (p = 0.02), and lactation (p = 0.03); [A] in metestrus was statistically higher than in lactation (p = 0.04) and proestrus (p = 0.02). Thus, the maximum [A] reached at diestrus in the CEC was not statistically different from the maximum during G 2 in a successful EC (SEC); however, after fecundation, the minimum [A] in early gestation G1 was significantly lower than in G2 and lactation. In the CEC, the increase of [A] from proestrus to diestrus only had significant differences between proestrus and metestrus,

A

B

whereas in the SEC, the collapse of [A] from estrus to early gestation G1 was statistically significant, as was the [A] increase from G1 to G2.

The order of [ T] was Pro> Die> Est> G2> Lac> Met> G1. The lowest [ T] in early gestation G1 was statistically different from everything else (Pro p = 3.5E –04, Est p = 4.9E –04, Die p = 0.01, G2 p = 4.9E –04, Lac p = 8.2E –04), except for the second lowest [ T] in metestrus; the latter also was very significantly segregated from the maximum [ T] in proestrus (p =0 0.002) and estrus (p = 0.008), whereas it was significantly different from late gestation G2 (p = 0.01), and overall lactation (p = 0.02). The two collapses of [ T] in metestrus and G1 were not significantly different from each other. The maximum [ T] in proestrus was significantly higher in either a CEC or a SEC; indeed, the only other [ T] peak in G2 was statistically lower. In the CEC, the fall of [ T] is interrupted by its statistically significant increase in diestrus, whereas during the SEC, both the collapse from estrus to G1, and its increase towards G2 were statistically significant .

Estradiol [E 2] was arranged as Est> Pro> Lac> G2> Die> G1> Met. The maximum [E 2] peak in estrus differed extremely significantly from most [ SSH] (Met p = 6.0E –06, G1 p = 1.6E –06, G2 p = 7.4E –05, Lac p = 6.5E –05) and very significantly from diestrus (p = 0.004), being almost significantly different from proestrus (p = 0.054). The minimum [E 2] in metestrus was significantly different from that in proestrus (p = 0.03) and very significantly different from that in both late gestation G2 (p = 0.002) and lactation (p = 0.003), whereas the next minimum in early gestation G1 was significantly different from that in proestrus (p = 0.04) and lactation (p = 0.01) and very significantly different from that in G2 (p = 0.007); [E 2] in metestrus and G1 were not statistically different from each other nor from that in diestrus. The maximum [E 2] in the estrus of either a CEC or a SEC was statistically the highest. On the other hand, both the [E 2] in late gestation G2 and lactation were significantly the lowest. In a CEC, both the sudden increase of [E 2] from proestrus to estrus and the abrupt collapse in metestrus were statistically significant. In the SEC followed by pregnancy and lactation, the collapse of [E 2] into G1 was statistically significant, as was its increase in G2 and lactation .

Peromyscus difficilis ( Fig. 3B , Table S2). The order for [P 4] was G1> Est> Met> Die> G2> Pro> Lac. Clearly, the maximum [P 4] in early gestation G1 was statistically the highest and extremely significantly different from anything else (Pro p = 2.0E –05, Est p = 1.1E –04, Met p = 8.9E –05, Die p = 1.6E –05, G2 p = 6.8E –06, Lac p = 3.2E –08); the other but lower peak of [P

4

] in estrus was also statistically higher than everything else (Die p = 0.02, G2 p = 0.002, Lac p = 4.2E –05), except for metestrus; the three minimum concentrations of [P 4] were also statistically different from the higher concentration in metestrus (Lac p = 3.4E –04, Pro p = 0.02, G2 p = 0.009), whereas the higher concentration in diestrus also differed from lactation (p = 8.0E –04) and G2 (p = 0.03). Therefore, the maximum [P 4] in G1 during a SEC was statistically higher than the maximum [P 4] in the estrus-metestrus of the CEC, while the lowest [P 4] in the proestrus was not different from the minimum [P 4] in G2 and lactation. In the CEC, the increase of [P 4] from proestrus to estrus is statistically significant, whereas in the SEC interrupted by pregnancy, both the sharp increase of [P 4] from estrus to G1 and its abrupt collapse from G2 to G1 were statistically significant.

In [A], the order was Met> G2> Lac> Pro> Die> Est> G1. Only the lowest [A] in early gestation G1 differed statistically from all other reproductive stages or events (Pro p = 0.04, Met p = 0.008, Die p = 0.003, G2 p = 0.004, Lac p = 7.5E –04), except for estrus. Therefore, the concentration of this anabolic androgen remained almost constant around 6–9 pg/mg; the two slight peaks during metestrus in the CEC and late gestation G2 of the SEC, respectively, were similar to each other. In the CEC, due to the dispersion of data, there were no significant gaps between lower (e.g., Pro, Est, Die) and higher (e.g., Met) [A]. However, in the SEC, both the decrease of [A] from estrus to G1 and the increase from G1 to G2 were statistically significant.

The arrangement for [T] was Pro> Met> G2> Die> Lac> Est> G1. The maximum [T] in proestrus differed from all others (Pro p = 0.03, Met p = 0.04, G1 p = 0.005, Lac p = 0.02), except for [T] in late gestation G2, and was almost significant for [T] in diestrus (p = 0.052), whereas the minimum [T] in early gestation G1 also differed from [T] in metestrus (p = 0.009), diestrus, and lactation (p = 0.02 both). The fluctuating [T] showed three peaks: the highest [T] in proestrus was statistically significant in the CEC and the SEC; the second peak in metestrus and the third one in G2 were similar to each other. In a CEC, only the fall of [T] from proestrus to estrus was statistically significant. The collapse of [T] at G 1 in the SEC was statistically significant, whereas both its decrease from estrus to G1 and its increase from G1 to G2 were statistically not significant.

Estradiol [E 2] was arranged as Est> Pro> Die> Lac> G2> G1> Met. The maximum [E 2] peak in estrus was significantly higher than everything else (Pro p = 0.05, Met p = 1.7E –05, Die p = 0.003, G1 p = 3.6E –04, G2 p = 0.001, Lac p = 1.7E –004), and the next highest [E 2] peak in proestrus was also statistically different from the others (Met p = 3.2E –05, Die p = 0.03, G1 p = 0.002, G2 p = 0.005, Lac p = 0.003); the minimum [E 2] peak in metestrus was statistically lower than everything else (Die p = 0.01, G2 and Lac p = 0.005), except for early gestation G1. Once again, the maximum [E

2

] reached at estrus, was clearly statistically defined and higher than the peak in G2-Lac after fecundation. In the CEC, the two increases of [E

2

] in Pro-Est and Met-Die were statistically significant. Also, the two abrupt collapses of [E 2] from estrus to metestrus in the CEC and from estrus to G 1 in the SEC were statistically significant, respectively.

Figures

Figure 2. Production and biotransformation profiles in four selected SSH of the ∆4 pathway by reproductive event in two species ofPeromyscus. Mean intraovarian concentrations of sexual steroid hormones ([SSH]: progesterone, P4; androstenedione, A, testosterone, T; estradiol, E2) were assessed in free-living, adult females ofP.melanotis (A) andP.difficilis (B), during estrous cycle (PRO, proestrus; EST, estrus; MET, metestrus; DIE, diestrus), pregnancy (early gestation, G1; late gestation, G2), and overall lactation. Bars depict mean [SSH] and vertical whiskers the respective standard errors;arrows signal which [SSH] had significant differences with others; when more than one [SSH] was statistically different in ANOVA analyzes, horizontal lines were added and arrows below and above them signal the respective [SSH]; number of asterisks indicate degree of significance in p-values (see Methods, Results, and the ANOVA in Table S1): * = significant; ** = very significant; *** = extremely significant; the plus sign (+) shows an almost statistically significant instance. Note that scales of plots differ.

Figure 3. Fluctuations of each intraovarian [SSH] in the ∆4 pathway throughout three reproductive events in two species of Peromyscus. Mean concentrations of sexual steroid hormones, [SSH], were obtained from estrous cycle, pregnancy and lactation in free-living, adult females of P. melanotis (A) and P. difficilis (B). Symbology as in Fig. 2. Note that scales differ; complete ANOVA information is available in Table S2.