brain research 1608 (2015) 66–74
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Research Report
Biostimulation and nursing modify mating-induced c-FOS immunoreactivity in the female rabbit forebrain Gabriela Gonza´lez-Mariscaln, Cipatli Garcı´a Dalma´n, Angeles Jime´nez Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala, Apdo. Postal 62, Tlaxcala 90000, Mexico
ar t ic l e in f o
abs tra ct
Article history:
Mating in rabbits lasts only 3–5 s but profoundly changes the female's physiology and
Accepted 15 February 2015
behavior (e.g., inhibition of scent-marking and ambulation, changes in EEG, and release of
Available online 23 February 2015
GnRH). The behavioral responsiveness to copulation is reduced in lactating rabbits, relative
Keywords:
to estrous does, but is enhanced after suppressing a single nursing bout (“biostimulation”).
Mating
Little is known about the mechanisms mediating the differential responsiveness to mating
c-FOS
among estrous, lactating, and biostimulated rabbits. To begin addressing this issue we
Preoptic area
quantified the number of c-FOS-immunoreactive (IR) cells in the preoptic area (POA),
Nursing
dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), infundibular
Lactational anestrus
nucleus (INF), paraventricular nucleus (PVN), supraoptic nucleus (SON), and lateral septum (LS) in mated and unmated does from the above three reproductive conditions. Mating increased c-FOS-IR cells in the POA and PVN relative to unmated estrous does. Biostimulation increased c-FOS-IR cells in the PVN, relative to lactating does, regardless of mating. Lactation reduced the responsiveness of the LS and INF to copulation but increased it in the DMH. No differences were found in the VMH. Conclusions: a) copulation activates forebrain nuclei that regulate scent-marking (POA), ovulation (INF), and post-coital oxytocin release (PVN); b) lactation and suppression of one nursing bout modulate the magnitude of such changes. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
Copulation in rabbits is a brief event, lasting between 3–5 s, during which the male displays a single mount followed immediately by intromission and ejaculation (Contreras and Beyer, 1979). Despite its brevity, copulation induces in doe rabbits n
Corresponding author.Fax: þ52 248 48 15476. E-mail address: [email protected] (G. González-Mariscal).
http://dx.doi.org/10.1016/j.brainres.2015.02.033 0006-8993/& 2015 Elsevier B.V. All rights reserved.
a series of specific behavioral and neuroendocrine changes, such as: a) the inhibition of scent-marking and ambulation in an open field (González-Mariscal et al., 1997); b) the appearance of slow waves in the EEG of the cerebral cortex, hypothalamus, and hippocampus (Sawyer and Kawakami, 1959); c) the activation of the noradrenergic system in the brain stem (Caba et al., 2000a,
10 133748 9 119731 8 115723 7 107736 a
n ¼ 20 does, except on day 10, when the biostimulated group (n ¼10) was not given kits to suckle.
6 95727 5 93737 4 78725 3 61729 2 56718 1 44722 Grams/day (mean7SD)
Days
Table 1 – Milk output across lactation days 1–11a.
2000b) which, in turn, provokes the release of gonadotrophinreleasing hormone (GnRH) (Yang et al., 1996) and the massive secretion of luteinizing hormone (LH) (Ramírez and Beyer, 1988). These behavioral and neuroendocrine consequences of mating occur only if rabbits are in estrus, i.e., if they have an adequate concentration of estrogens in blood (Hilliard and Eaton, 1971), if their caloric intake is sufficient (Brecchia et al., 2006), and if they are housed under a photoperiod of around 12–14 h of light/day (Hudson et al., 1994). Lactating rabbits, by contrast, are in a state of lactational anestrus during which sexual receptivity and scent-marking are markedly reduced (Beyer and Rivaud, 1969; García Dalmán and González-Mariscal, 2012; González-Mariscal et al., 1990) and, even if copulation is achieved, ovulation does not occur (Ramírez and Beyer, 1988). Little is known about the neuroendocrine mechanisms mediating lactational anestrus although a participation of progesterone in this regard can be safely excluded as this hormone is absent in nursing does (González-Mariscal et al., 1997). Therefore, suckling stimulation – and its associated neuroendocrine consequences – seems the obvious candidate for maintaining the inhibition of estrus throughout lactation. Yet, unlike most mammals, rabbits nurse their litters only once per day, for around three min, throughout lactation (González-Mariscal et al., 1997, 2013; Drewett et al., 1982; Zarrow et al., 1965). Despite this low frequency and duration of suckling the sensory stimulation received at each nursing bout, plus the neuroendocrine cascade that follows, is sufficient and necessary to induce a state of anestrus (García Dalmán and González-Mariscal, 2012). Accordingly, a number of studies have shown that canceling a single nursing episode in early lactation (usually between days 8 and 10) leads to the restoration of estrus within 24–48 h and to the consequent pregnancy following natural or artificial insemination (Alvariño et al., 1998; Castellini et al., 1998; Theau-Clément and Mercier, 1999). This phenomenon has been termed “biostimulation” and it is reliably used worldwide to accelerate rabbit production (Theau-Clément et al., 1998). Yet, the factors underlying the effectiveness of the “biostimulation” method to restore estrus have been little explored. To our knowledge only the concentrations of prolactin and estradiol in blood have been compared between lactating and “biostimulated” does in early lactation; the latter show higher concentrations of prolactin and estradiol at the time of insemination, relative to control lactating does in which nursing was not interrupted (Ubilla et al., 2000). Studies using in situ hybridization (Caba et al., 2000a, 2000b), RT-PCR (Reyna-Neyra et al., 2000), and immunocytochemistry (Caba et al., 2000b) to c-FOS have found that, in estrous rabbits, mating activates specific brain areas, like the preoptic region, several brainstem-noradrenergic nuclei, the ventrolateral hypothalamus, and the encapsulated portion of the bed nucleus of the stria terminalis, some of which may be related to the behavioral and neuroendocrine consequences of copulation described above. However, we ignore if the enhanced sexual receptivity and probability of ovulation found in biostimulated does and the decreased reactivity seen in lactating rabbits is associated with concomitant differences in the responsiveness of specific forebrain nuclei to mating. To begin investigating this issue we quantified the number of c-FOS immunoreactive cells in the forebrain of mated and unmated does from the above three reproductive states.
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2.
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Results
All estrous does responded to mating by showing lordosis to the three mounts received from the male. A similar sexual receptivity response was observed in lactating and biostimulated rabbits. In these two groups milk production increased steadily across lactation days 1–11 (or 1–10 in biostimulated does): Table 1. Abundant c-FOS-IR cells were visualized in the POA as individual dark dots over a clear background in the six experimental groups. A significant interaction between mating and reproductive condition was found (F5,29 ¼161.928; po0.006), as well as a specific effect of copulation (F5,29 ¼12.618; po0.001), though not of reproductive condition (F5,29 ¼ 1.048; po0.365). Indeed, copulation markedly increased the number of c-FOS-IR cells in the POA of estrous rabbits; approximately an eight-fold increase, with respect to unmated estrous females (po0.0001; Fig. 1). Lactation, however, blunted the effect of mating as the number of c-FOS-IR cells was very similar between the two groups of biostimulated does (i.e., mated and unmated). Yet, these animals showed around five times more c-FOS-IR cells than unmated estrous rabbits.
Fig. 1 – Upper panels: photographs of c-FOS-IR cells visualized in the POA of estrous unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the POA of female rabbits from the six experimental groups. A significant interaction between mating and reproductive condition was found (F5,29 ¼ 161.928; po0.006), as well as a specific effect of copulation (F5,29 ¼ 12.618; po0.001), though not of reproductive condition (F5,29 ¼1.048; po0.365). Mating increased the number of c-FOS-IR by approximately eightfold. apo0.0001.
In the PVN (Fig. 2) we also observed a significant interaction between mating and reproductive condition (F5,29 ¼ 23.914; po0.039) as well as a specific effect of reproductive condition (F5,29 ¼6.288; po0.006), though not of mating (F5,29 ¼0.072; po0.790). Indeed, mating significantly increased the number of c-FOS-IR cells only in estrous does (po0.0001). By contrast, biostimulated and lactating does responded to mating with a significant decrease in the number of c-FOS-IR cells (po0.0001 and po0.047, respectively). In addition, biostimulation per se doubled the number of c-FOS-IR cells with respect to regular lactating does (po0.02). In the SON no significant interactions between mating and reproductive condition were found (Fig. 3; F5,29 ¼ 10.019; po0.087) or INF (Fig. 4; F5,29 ¼6.358; po0.128). However, in both regions significant overall effects of mating (SON: F5,29 ¼ 16.01; po0.0001; INF: F5,29 ¼ 25.804; po0.0001) and reproductive condition (SON: F5,29 ¼9.659; po0.001; INF: F5,29 ¼ 10.27; po0.001) occurred. In both the SON (Fig. 3) and the INF (Fig. 4) mating markedly reduced the number of c-FOS-IR cells in lactating
Fig. 2 – Upper panels: photographs of c-FOS-IR cells visualized in the PVN of biostimulated unmated or mated rabbits. Calibration bar¼200 lm. Lower panel: number of c-FOS-IR cells quantified in the PVN of female rabbits from the six experimental groups. A significant interaction between mating and reproductive condition (F5,29 ¼ 23.914; po0.039) as well as a specific effect of reproductive condition (F5,29 ¼6.288; po0.006) though not of mating (F5,29 ¼ 0.072; po0.790) was found. Mating significantly increased the number of c-FOS-IR cells only in estrous does but reduced it in biostimulated and lactating does. Biostimulation per se doubled the number of c-FOS-IR cells with respect to regular lactating does (po0.02). apo0.0001, dpo0.05.
brain research 1608 (2015) 66–74
Fig. 3 – Upper panels: photographs of c-FOS-IR cells visualized in the SON of biostimulated unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the SON of female rabbits from the six experimental groups. No significant interactions between mating and reproductive condition were found (F5,29 ¼ 10.019; po0.087) but significant overall effects of mating (F5,29 ¼ 16.01; po0.0001) and reproductive condition (F5,29 ¼ 9.659; po0.001) were evident. Mating reduced the number of c-FOS-IR cells only in lactating and in biostimulated rabbits (apo0.0001; cpo0.005). Suppressing a nursing episode increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulated-unmated vs lactating-unmated does; po0.0001).
and in biostimulated rabbits but copulation had no effect in estrous does. In both SON and INF the suppression of a nursing episode on lactation day 10 increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulated-unmated vs lactating-unmated does; po0.0001). A significant interaction between reproductive condition and mating was evident in the DMH (F5,29 ¼35.01; po0.027), as well as overall effects of each of these factors alone (mating: F5,29 ¼ 46.62; po0.0001; reproductive condition: F5,29 ¼ 3.739; po0.037). In biostimulated and in lactating does, but not in estrous rabbits, the DMH was highly responsive to mating: the number of c-FOS-IR cells increased by more than sevenfold after copulation (Fig. 5).
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Fig. 4 – Upper panels: photographs of c-FOS-IR cells visualized in the INF of biostimulated unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the INF of female rabbits from the six experimental groups. No significant interactions between mating and reproductive condition were found (F5,29 ¼ 6.358; po0.128) but significant overall effects of mating (F5,29 ¼25.804; po0.0001) and reproductive condition (F5,29 ¼ 10.27; po0.001) occurred. Mating markedly reduced the number of c-FOS-IR cells in lactating and in biostimulated rabbits but it provoked the reverse effect in estrous does. Suppression of a nursing episode on lactation day 10 increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulatedunmated vs lactating-unmated does). apo0.0001; bpo0.002.
In the LS no significant interaction between mating and reproductive condition (F5,29 ¼ 8.7; po0.098) nor specific effects of mating (F5,29 ¼ 1.049; po0.315) were evident (Fig. 6). Thus, copulation did not significantly modify the number of c-FOS-IR cells in either estrous or biostimulated does (po0.261 and po0.095, respectively) but provoked a significant decline in the lactating group (po0.002). Moreover, each of the four groups of nursing does (i.e., biostimulated and lactating, mated or not) showed significantly fewer c-FOS-IR cells in the LS than did the estrous groups (po0.001), coinciding with a significant overall effect of reproductive condition (F5,29 ¼ 224.357; po0.0001). The VMH did not respond to either mating or biostimulation; the six experimental groups showed similar and low numbers of c-FOS-IR cells (Fig. 6).
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Fig. 5 – Upper panels: photographs of c-FOS-IR cells visualized in the DMH of biostimulated unmated or mated rabbits. Calibration bar¼200 lm. Lower panel: number of cFOS-IR cells quantified in the DMH of female rabbits from the six experimental groups. A significant interaction between reproductive condition and mating was evident (F5,29 ¼35.01; po0.027), as well as overall effects of each of these factors alone (mating: F5,29 ¼ 46.62; po0.0001; reproductive condition: F5,29 ¼3.739; po0.037). In biostimulated and in lactating does, but not in estrous rabbits, mating increased the number of c-FOS-IR cells by more than seven-fold. a po0.0001.
3.
Discussion
The present results show that copulation in doe rabbits selectively stimulates the expression of the c-FOS protein in specific forebrain nuclei and not in others. Some of these results agree with earlier findings from us (Reyna-Neyra et al., 2000) and others (Caba et al., 2000b), such as the large activation of the POA. This, in turn, coincides with the fact that: a) GnRH-producing neurons, in rabbits, are located precisely in that region (Foster and Younglai, 1991); b) an increased number of double-labeled cFOS/GnRH cells is observed in the preoptic region (specifically, the anteroventral periventricular nucleus) at 90 min post-copula (Caba et al., 2000b); c) the perfusion of certain agents (like norepinephrine and some progestins) through “push-pull” cannulae placed in the POA stimulates the release of GnRH in unmated estrogen-treated does (Ramírez and Beyer, 1988). The present results extend such findings by showing that the responsiveness to mating differs among estrous, biostimulated,
Fig. 6 – Upper panel: number of c-FOS-IR cells quantified in the LS of female rabbits from the six experimental groups. No significant interaction between mating and reproductive condition (F5,29 ¼ 8.7; po0.098) nor specific effects of mating (F5,29 ¼1.049; po0.315) were evident. Copulation provoked a significant decline in the lactating group. bpo0.002. Lower panel: Number of c-FOS-IR cells quantified in the VMH of female rabbits from the six experimental groups. No effects of mating or reproductive condition were observed.
and lactating does. Thus, mating had practically no effect on the number of c-FOS-IR cells in the POA of nursing rabbits agreeing with their state of anestrus (García Dalmán and GonzálezMariscal, 2012). By contrast, copulation provoked, in the POA, a much larger increase in the number of c-FOS-IR cells in estrous than in biostimulated does. This finding coincides with the fact that our non-lactating does were in a state of estrus, as expected of rabbits kept under a long photoperiod, with an adequate food supply (Brecchia et al., 2006; Hudson et al., 1994). Although biostimulation increases fertility above that seen in estrous rabbits (Theau-Clément et al., 1998) the lack of an effect of mating on the number of c-FOS-IR cells may indicate that, although the “general” reactivity of the POA to mating (as determined by the c-FOS protein) is smaller in biostimulated rabbits, the responsiveness of specific subnuclei directly involved in triggering ovulation may be larger. Further studies in particular subnuclei of the POA, using double-label immunocytochemistry to c-FOS and specific peptides promoting ovulation are warranted to explore such possibility. Of particular interest are: a) neuropeptide Y, which is co-released with GnRH in the
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rabbit hypothalamus in vivo (Pau et al., 1989) and promotes the release of GnRH and pituitary gonadotropins in vitro (Khorram et al., 1988) and b) kisspeptin. In addition the finding that the number of c-FOS-IR cells in the POA did not differ between biostimulated-unmated and lactating-unmated does coincides with the recent report that the number of PER1 protein-IR cells is not significantly reduced in the POA of does deprived of nursing for 24 h (Meza et al., 2015). Moreover, the present finding contributes to the understanding of the ways by which the biostimulation method promotes fertility without abolishing nursing behavior, as the POA is crucial for both ovulation (Foster and Younglai, 1991; Caba et al., 2000b) and maternal care (Cruz and Beyer, 1972; González-Mariscal et al., 2009b) in rabbits. The time of nursing used in the present study (10:00 h) is different from the one at which lactating does spontaneously suckle the litter under laboratory conditions, namely: 03:52 h (González-Mariscal et al., 2013). Yet, we have found that nursing the kits at 10:00 h triggers a robust c-FOS response in the LS, PVN, and SON (González-Mariscal et al., 2009b). Moreover, the PER1 protein shows a rhythm that shifts in parallel to the time of nursing in both the POA and the LS (Meza et al., 2015). These findings indicate that the brain's responsiveness to suckling can be effectively measured by such methods at different times of day. In the PVN a clear effect of biostimulation was evident: in unmated does this group showed significantly more c-FOS-IR cells than (unmated) lactating or estrous animals. To our knowledge this is the first evidence showing that the restoration of estrus, provoked by suppressing a single nursing bout, is associated with an increase in the activity of a specific brain region. Although we ignore the phenotype of the c-FOS-IR cells counted it is tempting to speculate that many of them are oxytocinergic as the PVN of rabbits is rich in oxytocin (OT)producing neurons, especially during lactation (Caba et al., 1996). The secretion of this peptide is stimulated by copulation (Todd and Lightman, 1986) and by nursing (Fuchs et al., 1984) so it seems puzzling that (regular) lactating mated females, which received both of those stimuli, showed fewer c-FOS-IR cells than estrous does, which were only mated. This issue needs to be further explored by using double-immunostaining to c-FOS and OT to determine if the proportion of double-stained neurons is larger in lactating-mated rabbits than in estrous ones. Both groups of estrous rabbits (i.e., mated and unmated) showed practically the same number of c-FOS-IR cells in the LS, a finding strongly suggesting that such region is probably not related with the perception of sensory stimulation received during copulation nor with the release of GnRH. Mated lactating and mated biostimulated does showed similar counts of cFOS-IR cells which, in turn, were about three-fold lower than those seen in estrous does. In agreement with our earlier study (González-Mariscal et al., 2009b) c-FOS immunoreactivity in the LS was stimulated by suckling as unmated lactating rabbits showed significantly more c-FOS-IR cells than did unmated biostimulated does, which missed a nursing bout. These results agree with a recent publication showing that canceling a single nursing episode reduces (by approximately 50%) the number of PER1 protein-IR cells in the dorsal and ventral septum (Meza et al., 2015). In mated animals the difference between lactating and biostimulated does disappeared suggesting that mating can blunt the responsiveness of the LS to suckling.
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In the INF the highest number of c-FOS-IR cells was observed in unmated biostimulated animals followed by unmated lactating ones. These findings are consistent with the activity of as yet unidentified secretory neurons involved in the regulation of lactation (Tucker, 1994). Moreover, these results indicate that the INF is one of the brain structures whose activity is clearly increased following the suppression of one nursing bout and which may be related to the facilitation of ovulation and fertility through biostimulation (Bonanno et al., 2002; Theau-Clément et al., 1998). The impact of reproductive condition on the number of c-FOS-IR cells following mating was especially clear in the SON while estrous mated rabbits showed an insignificant increase in c-FOS-IR cells relative to their unmated counterparts in both groups of lactating does mating provoked a reduction in such parameter. Moreover, in unmated females, biostimulation markedly increased the number of such cells above the levels seen in lactating or estrous rabbits. Many of such cells could be oxytocinergic or vasopressinergic neurons that are far more abundant in lactating than in estrous rabbits (Caba et al., 1996). Copulation did not impact the number of c-FOS-IR cells in the DMH of estrous rabbits, a finding in agreement with the idea that this structure does not have a known role in ovulation or sexual behavior. However, mated lactating and mated biostimulated does showed two to three times more c-FOS-IR cells than estrous animals. Because: a) the DMH plays an important role in the regulation of feeding (Yang et al., 2001) and b) nursing rabbits increase their food intake across lactation (González-Mariscal et al., 1994, 2009a) we believe that the larger number of c-FOS-IR cells observed in the DMH of lactating mated does is related with such phenomena. Yet, the mechanisms by which the condition of lactation increases the responsiveness to mating (relative to the state of estrus) remain to be determined. In contrast with the above regions, no significant differences were seen among the six experimental groups in the VMH. This is a puzzling finding as this region is rich in estradiol receptor alpha (Caba et al., 2003) and estradiol implants in the VMH stimulate sexual receptivity in ovariectomized rabbits (Melo et al., 2008). This result apparently also contradicts our earlier finding that mating promotes an increased expression of the c-FOS gene in the hypothalamus of estrous does, as determined by RT-PCR (Reyna-Neyra et al., 2000). These differences may be due to the lack of fine neuroanatomical resolution associated with the RT-PCR method and/or the activation of a specific neuronal phenotype, not identified in the present study, by mating. Indeed, a marked increase in the number of double-labelled c-FOS/ GnRH cells was found at 90 min post-coitus in the ventrolateral hypothalamus of estrous does (Caba et al., 2000b). On the other hand, the lack of differences between estrous-mated and biostimulated-mated does may be related with the fact that our mother–litter separation lasted only 24 h. Indeed, an interval of 48 h is necessary to provoke a massive increase in the concentration of estradiol in plasma (Ubilla et al., 2000) and longer mother–litter separations have been reported to provoke larger increases in fertility (Alvariño et al., 1998). However, the 24 h separation is usually preferred in rabbit farms as it both enhances fertility and allows the survival of the 10-day-old kits. Finally, we must consider that lactational anestrus is a complex phenomenon revealed through several
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physiological and behavioral indicators. We have found, for instance, that: a) the magnitude of estrus inhibition is dependent on the number of suckling young and b) three behaviors characteristic of estrous does (i.e., ambulation, scent-marking, and lordosis) have different thresholds of inhibition by suckling (García Dalmán and GonzálezMariscal, 2012). Accordingly, a 24-hr doe–litter separation may not be enough for the “state of estrus” to be reflected in terms of c-FOS-IR cells in the VMH. Clearly, more work is needed to unravel the ways by which nursing inhibits and biostimulation promotes estrus and fertility. In summary, our work has found that the reproductive state of female rabbits determines the responsiveness of specific forebrain regions to mating. This information can be a starting point to begin investigating the neuroendocrine signals and neuronal pathways involved in enhancing (biostimulation) or decreasing (lactation) the reactivity to copulatory stimulation in a reflex ovulator.
4.
Experimental procedure
4.1.
Animals
New Zealand white adult (3.5–4.5 kg body weight) virgin rabbits bred in our colony were used. They were housed inside a colony under controlled light (14 h light; 10 h dark; lights on at 07:00 h) and natural temperature (13–25 1C) conditions. They were provided with water and rabbit pellets (Conejina, Purina™) ad libitum. Throughout this work animal care adhered to the Law for the Protection of Animals (Mexico).
4.2.
Experimental groups
4.2.1.
Estrous rabbits
Each female was placed within a round wire mesh arena (1 m in diameter 80 cm height) and a sexually experienced male was then introduced. They were allowed to copulate three times while an observer determined whether lordosis occurred (or not) following each mount from the male. After this test the female was returned to her home cage (estrous mated group; n ¼5). Estrous females in the unmated group
(n ¼5) were placed in the arena alone for about 5 min after which they were returned to their individual wire mesh cages (52 cm long 42 cm wide 41 cm high).
4.2.2.
Lactating rabbits
They were mated as above but were housed in individual maternal cages (90 cm long 60 cm wide 40 cm high) within which a wooden nest box (50 cm long 30 cm wide 32 cm high) was placed to allow the construction of the maternal nest, as previously described (González-Mariscal et al., 1994). From pregnancy day 20 until parturition 300 g of straw were provided daily as material for nest-building. Starting on pregnancy day 30, females were spot-checked across the day to determine the approximate time of delivery. At parturition mothers were left undisturbed for 5–8 h after which kits were removed, litter size was adjusted to eight, and kept (away from the mother) inside a box containing paper shavings, under a mild heat source. On each day when nursing was recorded (see below) litters were weighed and introduced into the nest box at 10:00 h. The mother was allowed to nurse the kits after which they were removed and weighed again; the difference in their body weight (with respect to the one shown before nursing) was taken as a measure of milk output. This method for determining milk yield, reliably used by us in earlier studies (González-Mariscal et al., 1994, 2009b), was repeated for nine consecutive days. On lactation day 10 only half of the does were provided with their litters and allowed to nurse (lactating groups; n¼ 10) while the other half was not given the kits (biostimulated groups; n¼ 10). On the following day all mothers were allowed to nurse at the same time as before (i.e., 10:00 h) and half of them were mated immediately after nursing as described above; the other half was not mated. This strategy produced four experimental groups: lactatingmated (n¼ 5), lactating unmated (n¼5), biostimulated-mated (n¼5), and biostimulated-unmated (n¼ 5). The following diagram summarizes the experimental design:
4.3.
Sacrifice, perfusion, and immunocytochemistry
All females were killed on postpartum day 11 one hour after mating or nursing (or equivalent time of day in the unmated
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group). They were anesthetized with an overdose of sodium pentobartibal and perfused transcardially with saline and 4% para-formaldehyde. Brains were cryoprotected in sucrose (10%, 20%, 30%) and sectioned (40 mm) coronally from the rostral border of the preoptic area to the mammillary bodies. Every single section was collected but only one of every six was reacted with the monoclonal antibody against the c-FOS protein or was stained with cresyl violet for visualization of neuroanatomical structures. Free-floating sections were washed three times in phosphate buffer (PB) 0.1 M, pH 7.4 to remove excess aldehydes and then exposed for 10 min to 0.05% hydrogen peroxide to eliminate endogenous peroxidase activity. After preincubation for 1 h in 1% normal donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA) sections were incubated for 48 h at 4 1C in the monoclonal antibody sc-52 (Santa Cruz Biotechnology), diluted at 1:5000 with 0.3% Triton X-100 (Sigma, St. Louis, MO) in 0.1 M PB. This antibody has been reliably used by us (González-Mariscal et al., 2009b) and others (Caba et al., 2000b; Toledo et al., 2005) to visualize the c-FOS protein in the adult female rabbit brain. Sections were then incubated sequentially in biotinylated donkey antigoat serum IgG (1:200; Vector laboratories, Burlingame, CA) plus avidin–biotin horseradish peroxidase, HRP, complex (1:250; Vector Laboratories). HRP label was visualized using 0.05% diaminobenzidine (Polysciences, Warrington, PA) in 0.1 M PB as the chromogen with 0.01% hydrogen peroxide as substrate. Sections were mounted onto gelatin-subbed slides, dehydrated, and cleared in Hemo-De and then cover-slipped with Permount. Control sections were processed omitting the primary antibody.
slices counted (in our case one of every six); c) the proportion of the height of the slice that will be counted (in our case the middle third); d) the proportion of grid squares on which c-FOSIR cells will be counted (in our case one of every four). We placed a grid with squares of defined size (89 mm2); factor a/p in the formula below) over the specific structure and counted the number of intersections over the area analyzed (number of points). We obtained the distance between the sections analyzed (T) by multiplying the thickness of each slice (40 mm) by the inverse of the slice sampling factor (we selected one slice out of every six). Thus, T¼ 40 E6¼240 mm. We then estimated the volume of each of the structures specified above with the formula \widehatV¼(T) (a/p) (number of points). Next, we counted the number of c-FOS-IR cells (Q) in the selected slices (i.e., one of every six; slice sampling fraction; ssf¼ 1/6), choosing an area sampling fraction (asf) of one fourth (i.e., we counted cFOS-IR cells in one of every four squares of the grid placed over the corresponding slice) and a height sampling fraction (hsf) of one third (i.e., we counted the middle third of the selected 40 mm thick slices). According to the optical fractionator method [34] these factors were introduced into the equation \widehatNV ¼Q 1/asf 1/ssf 1/hsf to estimate the number of particles (\widehatNV) per unit volume in a given structure. This number was then multiplied by the volume of the corresponding brain structure (determined by the Cavalieri method described above) to obtain the total number of c-FOS-IR cells within that structure. Consequently, the c-FOS counts made in all animals in the brain structures selected was not biased as the probability of a cell being counted was not influenced by the size of the region analyzed.
4.4.
4.5.
Quantification of c-FOS-IR cells
Sections were examined under bright-field illumination in an Olympus CH2 microscope and camera lucida drawings were made of all sections. The distribution of c-FOS-IR cells was determined in reference to the rabbit stereotaxic atlas of Girgis and Shih-Chang (1981). The quantification of c-FOS-IR cells per brain region was done manually, by an observer blind to the condition of the animal, using three-dimensional stereology. We adhered to the criteria of Howard and Reed (1988), as described in an earlier publication from us performed in the rabbit brain (González-Mariscal et al., 2009b). The advantages of this method are: a) every cell (or object) within a given structure has the same chance of being selected and counted; b) brain structures are counted in their entirety, thus minimizing the possibility that large structures are sampled more than smaller ones. The nomenclature used and the delineation of the brain structures analyzed was the one adopted in earlier studies from our group (Caba et al., 2003; González-Mariscal et al., 2009b), specifically: preoptic area (POA), paraventricular nucleus (PVN), supraoptic nucleus (SON), lateral septum (LS), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), and infundibular nucleus (INF). We first estimated the volume \widehatV of each of the above structures by the method of Cavalieri (Gundersen and Jensen, 1987) using Nissl-stained sections. This method takes into account the following factors, which are determined a priori by the investigator and are kept constant for all animals and all brain regions analyzed: a) the thickness of the slices (in our case 40 mm); b) the proportion of
Statistical analysis
A 2-way ANOVA was performed among the six experimental groups, in every brain region analyzed, to determine: a) the interaction between reproductive condition and mating and b) the impact of each factor alone on the number of c-FOS-IR cells. To compare between two specific groups a Student's Ttest was performed.
Acknowledgments This work was supported by CONACYT (National Council of Science and Technology, Mexico) Grant #128625 to GGM.
r e f e r e nc e s
Alvarin˜o, J.M.R., Del Arco, J.A., Bueno, A., 1998. Effect of mother– litter separation on reproductive performance of lactating rabbit females inseminated on day 4 or 11 post-partum. World Rabbit Sci. 6, 191–194. Beyer, C., Rivaud, N., 1969. Sexual behavior in pregnant and lactating domestic rabbits. Physiol. Behav. 4, 753–757. Bonanno, A., Di Grigoli, A., Alabiso, M., Boiti, C., 2002. Parity and number of repeated doe–litter separation treatments affect differently the reproductive performances of lactating does. World Rabbit Sci. 10, 63–70. Brecchia, G., Bonanno, A., Galeati, G., Federici, C., Maranesi, M., Gobbetti, A., Zerani, M., Boiti, C., 2006. Hormonal and
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metabolic adaptation to fasting: effects on the hypothalamicpituitary-ovarian axis and reproductive performance of rabbit does. Domest. Anim. Endocrinol. 31, 105–122. Caba, M., Bao, J., Pau, K.Y.F., Spies, H.G., 2000a. Molecular activation of noradrenergic neurons in the rabbit brainstem after coitus. Mol. Brain Res. 77, 222–231. Caba, M., Pau, K.Y.F., Beyer, C., Gonzalez, A., Silver, R., Spies, H.G., 2000b. Coitus-induced activation of c-fos and gonadotropinreleasing hormone in hypothalamic neurons in female rabbits. Mol. Brain Res. 78, 69–79. Caba, M., Silver, R., Gonza´lez-Mariscal, G., Jime´nez, A., Beyer, C., 1996. Oxytocin and vasopressin immunoreactivity in rabbit hypothalamus during oestrus, late pregnancy, and postpartum. Brain Res. 720, 7–16. Caba, M., Beyer, C., Gonza´lez-Mariscal, G., Morrell, J.I., 2003. Immunocytochemical detection of estrogen receptor alpha in the female rabbit forebrain: topography and regulation by estradiol. Neuroendocrinology 77, 208–222. Castellini, C., Canali, C., Boiti, C., 1998. Effect of mother–litter separation for 24 h, by closing the nestbox or change of cage, on rabbit doe reproduction performance. World Rabbit Sci. 6, 199–203. Contreras, J.L., Beyer, C., 1979. A polygraphic analysis of mounting an ejaculation in the New Zealand white rabbit. Physiol. Behav. 23, 939–943. Cruz, M.L., Beyer, C., 1972. Effect of septal lesions on maternal behavior and lactation in the rabbit. Physiol. Behav. 9, 361–365. Drewett, R.F., Kendrick, K.M., Sanders, D.J., Trew, A.M., 1982. A quantitative anaylysis of the feeding behavior of suckling rabbits. Dev. Psychobiol. 15, 25–32. Foster, W.G., Younglai, E.V., 1991. An immunohistochemical study of the GnRH neuron morphology and topography in the adult female rabbit hypothalamus. Am. J. Anat. 191, 293–300. Fuchs, A.R., Cubile, L., Dawood, M.Y., Jørgensen, F.S., 1984. Release of oxytocin and prolactin by suckling rabbits throughout lactation. Endocrinology 114, 462–469. Garcı´a Dalma´n, C., Gonza´lez-Mariscal, G., 2012. Major role of suckling stimulation for inhibition of oestrous behaviors in lactating rabbits: acute and chronic effects. Horm. Behav. 61, 108–113. Girgis, M., Shigh Chang, W., 1981. In: A New Stereotaxic Atlas of the Rabbit Brain. Warren Green Inc., St Louis Missouri, USA. Gonza´lez-Mariscal, G., Albonetti, M.E., Cuamatzi, E., Beyer, C., 1997. Transitory inhibition of scent-marking by copulation in male and female rabbits. Anim. Behav. 53, 323–333. Gonza´lez-Mariscal, G., Dı´az-Sa´nchez, Melo, A.I., Beyer, C., Rosenblatt, J.S., 1994. Maternal behavior in New Zealand white rabbits: quantification of somatic events, motor patterns and steroid plasma levels. Physiol. Behav. 55, 1081–1089. Gonza´lez-Mariscal, G., Gallegos, J.A., Sierra-Ramı´rez, A., Garza Flores, J., 2009a. Impact of concurrent pregnancy and lactation on maternal nest-building, estradiol and progesterone concentrations in rabbits. World Rabbit Sci. 17, 145–152. Gonza´lez-Mariscal, G., Jime´nez, A., Chirino, R., Beyer, C., 2009b. Motherhood and nursing stimulate c-FOS expression in the rabbit forebrain. Behav. Neurosci. 123, 731–739. Gonza´lez-Mariscal, G., Melo, A.I., Zavala, A., Beyer, C., 1990. Variations in chin-marking behavior of New Zealand female rabbits throughout the whole reproductive cycle. Physiol. Behav. 48, 361–365. Gonza´lez-Mariscal, G., Lemus, A.C., Vega-Gonza´lez, A., AguilarRoblero, A., 2013. Litter size determines circadian periodicity of nursing in rabbits. Chronobiol. Int. 30, 711–718. Gundersen, H.J.G., Jensen, E.B., 1987. The efficiency of systematic sampling in stereology and its prediction. J. Microsc. 147, 229–263. Hilliard, J., Eaton, L.W.M., 1971. Estradiol-17β, progesterone and 20α-hydroxypregn-4-en-3-one in rabbit ovarian venous
plasma. II. From mating through implantation. Endocrinology 89, 522–527. Howard, C.V., Reed, M.G., 1998. In: Unbiased Stereology. SpringerVerlag, New York. Hudson, R., Melo, A.I., Gonza´lez-Mariscal, G., 1994. Effect of photoperiod and exogenous melatonin on correlates of oestrus in the domestic rabbit. J. Comp. Physiol. A 175, 573–580. Khorram, O., Pau, K.Y.F., Spies, H.G., 1988. Release of hypothalamic neuropeptide Y and effects of exogenous NPY on the release of hypothalamic GnRH and pituitary gonadotropins in intact and ovariectomized does in vitro. Peptides 9, 411–417. Melo, A.I., Chirino, R., Jime´nez, A., Cuamatzi, E., Beyer, C., Gonza´lez-Mariscal, G., 2008. Effect of forebrain implants of testosterone or estradiol on scent-marking and sexual behavior in male and female rabbits. Horm. Behav. 54, 676–683. Meza, E., Aguirre, J., Waliszewski, S., Caba, M., 2015. Suckling induces a daily rhythm in the preoptic area and lateral septum but not in the bed nucleus of the stria terminalis in lactating rabbit does. Eur. J. Neurosci. 41, 196–204, http://dx. doi.org/10.1111/ejn.12776. Pau, K.Y.F., Khorram, O., Kaynard, A.H., Spies, H.G., 1989. Simultaneous induction of neuropeptide Y and gonadotropinreleasing hormone release in the rabbit hypothalamus. Neuroendocrinology 49, 197–201. Ramı´rez, V.D., Beyer, C., 1988. The ovarian cycle of the rabbit: its neuroendocrine control. In: Knobil, E., Neill, J. (Eds.), The Physiology of Reproduction, Vol 2. Raven Press, New York, pp. 1873. Reyna-Neyra, A., Camacho-Arroyo, I., Cerbo´n, M.A., Gonza´lezMariscal, G., 2000. Mating modifies c-fos expression in the brain of male and female rabbits. Neurosci. Lett. 284, 1–4. Sawyer, C.H., Kawakami, M., 1959. Characteristics of behavioral and electroencephalographic after-reactions to copulation and vaginal stimulation in the female rabbit. Endocrinology 65, 622–630. Theau-Cle´ment, M., Castellini, C., Maertens, L., Boiti, C., 1998. Biostimulations applied to rabbit reproduction: theory and practice. World Rabbit Sci. 6, 179–184. Theau-Cle´ment, M., Mercier, P., 1999. Effect of a doe–litter separation on rabbit doe reproductive performance and growth of the young. World Rabbit Sci. 7, 177–179. Todd, K., Lightman, S.L., 1986. Oxytocin release during coitus in male and female rabbits: effect of opiate receptor blockade with naloxone. Psychoneuroendocrinology 11, 367–371. Toledo, R., Aguilar-Roblero, R., Canchola, E., Caba, M., 2005. Circadian and photic-induced expression of Fos protein in the suprachiasmatic nucleus of the rabbit. Biol. Rhythm Res. 36, 47–55. Tucker, H.A., 1994. Lactation and its hormonal control. In: 2nd ed In: Knobil, E., Neill, J. (Eds.), The Physiology of Reproduction, Vol 2. Raven Press, New York, pp. 1065. Ubilla, E., Rebollar, P.G., Pazo, D., Esquifino, A., Alvarin˜o, J.M.R., 2000. Effects of doe–litter separation on endocrinological and productivity variables in lactating rabbits. Livest. Prod. Sci. 67, 67–74. Yang, L., Scott, K.A., Hyun, J., Tamashiro, K.L., Tray, N., Moran, T. H., Bi, S., 2001. Role of dorsomedial hypothalamic neuropeptide Y in modulating food intake and energy balance. J. Neurosci. 29, 179–190. Yang, S.P., Pau, K.Y.F., Hess, D.L., Spies, H.G., 1996. Sexual dimorphism in secretion of hypothalamic gonadotropinreleasing hormone and norepinephrine after coitus in rabbits. Endocrinology 137, 2683–2693. Zarrow, M.X., Denenberg, V.H., Anderson, C.O., 1965. Rabbit: frequency of suckling in the pup. Science 150, 1835–1836.
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Research Report
Biostimulation and nursing modify mating-induced c-FOS immunoreactivity in the female rabbit forebrain Gabriela Gonza´lez-Mariscaln, Cipatli Garcı´a Dalma´n, Angeles Jime´nez Centro de Investigación en Reproducción Animal, CINVESTAV-Universidad Autónoma de Tlaxcala, Apdo. Postal 62, Tlaxcala 90000, Mexico
ar t ic l e in f o
abs tra ct
Article history:
Mating in rabbits lasts only 3–5 s but profoundly changes the female's physiology and
Accepted 15 February 2015
behavior (e.g., inhibition of scent-marking and ambulation, changes in EEG, and release of
Available online 23 February 2015
GnRH). The behavioral responsiveness to copulation is reduced in lactating rabbits, relative
Keywords:
to estrous does, but is enhanced after suppressing a single nursing bout (“biostimulation”).
Mating
Little is known about the mechanisms mediating the differential responsiveness to mating
c-FOS
among estrous, lactating, and biostimulated rabbits. To begin addressing this issue we
Preoptic area
quantified the number of c-FOS-immunoreactive (IR) cells in the preoptic area (POA),
Nursing
dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), infundibular
Lactational anestrus
nucleus (INF), paraventricular nucleus (PVN), supraoptic nucleus (SON), and lateral septum (LS) in mated and unmated does from the above three reproductive conditions. Mating increased c-FOS-IR cells in the POA and PVN relative to unmated estrous does. Biostimulation increased c-FOS-IR cells in the PVN, relative to lactating does, regardless of mating. Lactation reduced the responsiveness of the LS and INF to copulation but increased it in the DMH. No differences were found in the VMH. Conclusions: a) copulation activates forebrain nuclei that regulate scent-marking (POA), ovulation (INF), and post-coital oxytocin release (PVN); b) lactation and suppression of one nursing bout modulate the magnitude of such changes. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
Copulation in rabbits is a brief event, lasting between 3–5 s, during which the male displays a single mount followed immediately by intromission and ejaculation (Contreras and Beyer, 1979). Despite its brevity, copulation induces in doe rabbits n
Corresponding author.Fax: þ52 248 48 15476. E-mail address: [email protected] (G. González-Mariscal).
http://dx.doi.org/10.1016/j.brainres.2015.02.033 0006-8993/& 2015 Elsevier B.V. All rights reserved.
a series of specific behavioral and neuroendocrine changes, such as: a) the inhibition of scent-marking and ambulation in an open field (González-Mariscal et al., 1997); b) the appearance of slow waves in the EEG of the cerebral cortex, hypothalamus, and hippocampus (Sawyer and Kawakami, 1959); c) the activation of the noradrenergic system in the brain stem (Caba et al., 2000a,
10 133748 9 119731 8 115723 7 107736 a
n ¼ 20 does, except on day 10, when the biostimulated group (n ¼10) was not given kits to suckle.
6 95727 5 93737 4 78725 3 61729 2 56718 1 44722 Grams/day (mean7SD)
Days
Table 1 – Milk output across lactation days 1–11a.
2000b) which, in turn, provokes the release of gonadotrophinreleasing hormone (GnRH) (Yang et al., 1996) and the massive secretion of luteinizing hormone (LH) (Ramírez and Beyer, 1988). These behavioral and neuroendocrine consequences of mating occur only if rabbits are in estrus, i.e., if they have an adequate concentration of estrogens in blood (Hilliard and Eaton, 1971), if their caloric intake is sufficient (Brecchia et al., 2006), and if they are housed under a photoperiod of around 12–14 h of light/day (Hudson et al., 1994). Lactating rabbits, by contrast, are in a state of lactational anestrus during which sexual receptivity and scent-marking are markedly reduced (Beyer and Rivaud, 1969; García Dalmán and González-Mariscal, 2012; González-Mariscal et al., 1990) and, even if copulation is achieved, ovulation does not occur (Ramírez and Beyer, 1988). Little is known about the neuroendocrine mechanisms mediating lactational anestrus although a participation of progesterone in this regard can be safely excluded as this hormone is absent in nursing does (González-Mariscal et al., 1997). Therefore, suckling stimulation – and its associated neuroendocrine consequences – seems the obvious candidate for maintaining the inhibition of estrus throughout lactation. Yet, unlike most mammals, rabbits nurse their litters only once per day, for around three min, throughout lactation (González-Mariscal et al., 1997, 2013; Drewett et al., 1982; Zarrow et al., 1965). Despite this low frequency and duration of suckling the sensory stimulation received at each nursing bout, plus the neuroendocrine cascade that follows, is sufficient and necessary to induce a state of anestrus (García Dalmán and González-Mariscal, 2012). Accordingly, a number of studies have shown that canceling a single nursing episode in early lactation (usually between days 8 and 10) leads to the restoration of estrus within 24–48 h and to the consequent pregnancy following natural or artificial insemination (Alvariño et al., 1998; Castellini et al., 1998; Theau-Clément and Mercier, 1999). This phenomenon has been termed “biostimulation” and it is reliably used worldwide to accelerate rabbit production (Theau-Clément et al., 1998). Yet, the factors underlying the effectiveness of the “biostimulation” method to restore estrus have been little explored. To our knowledge only the concentrations of prolactin and estradiol in blood have been compared between lactating and “biostimulated” does in early lactation; the latter show higher concentrations of prolactin and estradiol at the time of insemination, relative to control lactating does in which nursing was not interrupted (Ubilla et al., 2000). Studies using in situ hybridization (Caba et al., 2000a, 2000b), RT-PCR (Reyna-Neyra et al., 2000), and immunocytochemistry (Caba et al., 2000b) to c-FOS have found that, in estrous rabbits, mating activates specific brain areas, like the preoptic region, several brainstem-noradrenergic nuclei, the ventrolateral hypothalamus, and the encapsulated portion of the bed nucleus of the stria terminalis, some of which may be related to the behavioral and neuroendocrine consequences of copulation described above. However, we ignore if the enhanced sexual receptivity and probability of ovulation found in biostimulated does and the decreased reactivity seen in lactating rabbits is associated with concomitant differences in the responsiveness of specific forebrain nuclei to mating. To begin investigating this issue we quantified the number of c-FOS immunoreactive cells in the forebrain of mated and unmated does from the above three reproductive states.
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67
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2.
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Results
All estrous does responded to mating by showing lordosis to the three mounts received from the male. A similar sexual receptivity response was observed in lactating and biostimulated rabbits. In these two groups milk production increased steadily across lactation days 1–11 (or 1–10 in biostimulated does): Table 1. Abundant c-FOS-IR cells were visualized in the POA as individual dark dots over a clear background in the six experimental groups. A significant interaction between mating and reproductive condition was found (F5,29 ¼161.928; po0.006), as well as a specific effect of copulation (F5,29 ¼12.618; po0.001), though not of reproductive condition (F5,29 ¼ 1.048; po0.365). Indeed, copulation markedly increased the number of c-FOS-IR cells in the POA of estrous rabbits; approximately an eight-fold increase, with respect to unmated estrous females (po0.0001; Fig. 1). Lactation, however, blunted the effect of mating as the number of c-FOS-IR cells was very similar between the two groups of biostimulated does (i.e., mated and unmated). Yet, these animals showed around five times more c-FOS-IR cells than unmated estrous rabbits.
Fig. 1 – Upper panels: photographs of c-FOS-IR cells visualized in the POA of estrous unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the POA of female rabbits from the six experimental groups. A significant interaction between mating and reproductive condition was found (F5,29 ¼ 161.928; po0.006), as well as a specific effect of copulation (F5,29 ¼ 12.618; po0.001), though not of reproductive condition (F5,29 ¼1.048; po0.365). Mating increased the number of c-FOS-IR by approximately eightfold. apo0.0001.
In the PVN (Fig. 2) we also observed a significant interaction between mating and reproductive condition (F5,29 ¼ 23.914; po0.039) as well as a specific effect of reproductive condition (F5,29 ¼6.288; po0.006), though not of mating (F5,29 ¼0.072; po0.790). Indeed, mating significantly increased the number of c-FOS-IR cells only in estrous does (po0.0001). By contrast, biostimulated and lactating does responded to mating with a significant decrease in the number of c-FOS-IR cells (po0.0001 and po0.047, respectively). In addition, biostimulation per se doubled the number of c-FOS-IR cells with respect to regular lactating does (po0.02). In the SON no significant interactions between mating and reproductive condition were found (Fig. 3; F5,29 ¼ 10.019; po0.087) or INF (Fig. 4; F5,29 ¼6.358; po0.128). However, in both regions significant overall effects of mating (SON: F5,29 ¼ 16.01; po0.0001; INF: F5,29 ¼ 25.804; po0.0001) and reproductive condition (SON: F5,29 ¼9.659; po0.001; INF: F5,29 ¼ 10.27; po0.001) occurred. In both the SON (Fig. 3) and the INF (Fig. 4) mating markedly reduced the number of c-FOS-IR cells in lactating
Fig. 2 – Upper panels: photographs of c-FOS-IR cells visualized in the PVN of biostimulated unmated or mated rabbits. Calibration bar¼200 lm. Lower panel: number of c-FOS-IR cells quantified in the PVN of female rabbits from the six experimental groups. A significant interaction between mating and reproductive condition (F5,29 ¼ 23.914; po0.039) as well as a specific effect of reproductive condition (F5,29 ¼6.288; po0.006) though not of mating (F5,29 ¼ 0.072; po0.790) was found. Mating significantly increased the number of c-FOS-IR cells only in estrous does but reduced it in biostimulated and lactating does. Biostimulation per se doubled the number of c-FOS-IR cells with respect to regular lactating does (po0.02). apo0.0001, dpo0.05.
brain research 1608 (2015) 66–74
Fig. 3 – Upper panels: photographs of c-FOS-IR cells visualized in the SON of biostimulated unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the SON of female rabbits from the six experimental groups. No significant interactions between mating and reproductive condition were found (F5,29 ¼ 10.019; po0.087) but significant overall effects of mating (F5,29 ¼ 16.01; po0.0001) and reproductive condition (F5,29 ¼ 9.659; po0.001) were evident. Mating reduced the number of c-FOS-IR cells only in lactating and in biostimulated rabbits (apo0.0001; cpo0.005). Suppressing a nursing episode increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulated-unmated vs lactating-unmated does; po0.0001).
and in biostimulated rabbits but copulation had no effect in estrous does. In both SON and INF the suppression of a nursing episode on lactation day 10 increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulated-unmated vs lactating-unmated does; po0.0001). A significant interaction between reproductive condition and mating was evident in the DMH (F5,29 ¼35.01; po0.027), as well as overall effects of each of these factors alone (mating: F5,29 ¼ 46.62; po0.0001; reproductive condition: F5,29 ¼ 3.739; po0.037). In biostimulated and in lactating does, but not in estrous rabbits, the DMH was highly responsive to mating: the number of c-FOS-IR cells increased by more than sevenfold after copulation (Fig. 5).
69
Fig. 4 – Upper panels: photographs of c-FOS-IR cells visualized in the INF of biostimulated unmated or mated rabbits. Calibration bar¼ 200 lm. Lower panel: number of c-FOS-IR cells quantified in the INF of female rabbits from the six experimental groups. No significant interactions between mating and reproductive condition were found (F5,29 ¼ 6.358; po0.128) but significant overall effects of mating (F5,29 ¼25.804; po0.0001) and reproductive condition (F5,29 ¼ 10.27; po0.001) occurred. Mating markedly reduced the number of c-FOS-IR cells in lactating and in biostimulated rabbits but it provoked the reverse effect in estrous does. Suppression of a nursing episode on lactation day 10 increased the number of c-FOS-IR cells by more than two-fold in the absence of mating (i.e., biostimulatedunmated vs lactating-unmated does). apo0.0001; bpo0.002.
In the LS no significant interaction between mating and reproductive condition (F5,29 ¼ 8.7; po0.098) nor specific effects of mating (F5,29 ¼ 1.049; po0.315) were evident (Fig. 6). Thus, copulation did not significantly modify the number of c-FOS-IR cells in either estrous or biostimulated does (po0.261 and po0.095, respectively) but provoked a significant decline in the lactating group (po0.002). Moreover, each of the four groups of nursing does (i.e., biostimulated and lactating, mated or not) showed significantly fewer c-FOS-IR cells in the LS than did the estrous groups (po0.001), coinciding with a significant overall effect of reproductive condition (F5,29 ¼ 224.357; po0.0001). The VMH did not respond to either mating or biostimulation; the six experimental groups showed similar and low numbers of c-FOS-IR cells (Fig. 6).
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Fig. 5 – Upper panels: photographs of c-FOS-IR cells visualized in the DMH of biostimulated unmated or mated rabbits. Calibration bar¼200 lm. Lower panel: number of cFOS-IR cells quantified in the DMH of female rabbits from the six experimental groups. A significant interaction between reproductive condition and mating was evident (F5,29 ¼35.01; po0.027), as well as overall effects of each of these factors alone (mating: F5,29 ¼ 46.62; po0.0001; reproductive condition: F5,29 ¼3.739; po0.037). In biostimulated and in lactating does, but not in estrous rabbits, mating increased the number of c-FOS-IR cells by more than seven-fold. a po0.0001.
3.
Discussion
The present results show that copulation in doe rabbits selectively stimulates the expression of the c-FOS protein in specific forebrain nuclei and not in others. Some of these results agree with earlier findings from us (Reyna-Neyra et al., 2000) and others (Caba et al., 2000b), such as the large activation of the POA. This, in turn, coincides with the fact that: a) GnRH-producing neurons, in rabbits, are located precisely in that region (Foster and Younglai, 1991); b) an increased number of double-labeled cFOS/GnRH cells is observed in the preoptic region (specifically, the anteroventral periventricular nucleus) at 90 min post-copula (Caba et al., 2000b); c) the perfusion of certain agents (like norepinephrine and some progestins) through “push-pull” cannulae placed in the POA stimulates the release of GnRH in unmated estrogen-treated does (Ramírez and Beyer, 1988). The present results extend such findings by showing that the responsiveness to mating differs among estrous, biostimulated,
Fig. 6 – Upper panel: number of c-FOS-IR cells quantified in the LS of female rabbits from the six experimental groups. No significant interaction between mating and reproductive condition (F5,29 ¼ 8.7; po0.098) nor specific effects of mating (F5,29 ¼1.049; po0.315) were evident. Copulation provoked a significant decline in the lactating group. bpo0.002. Lower panel: Number of c-FOS-IR cells quantified in the VMH of female rabbits from the six experimental groups. No effects of mating or reproductive condition were observed.
and lactating does. Thus, mating had practically no effect on the number of c-FOS-IR cells in the POA of nursing rabbits agreeing with their state of anestrus (García Dalmán and GonzálezMariscal, 2012). By contrast, copulation provoked, in the POA, a much larger increase in the number of c-FOS-IR cells in estrous than in biostimulated does. This finding coincides with the fact that our non-lactating does were in a state of estrus, as expected of rabbits kept under a long photoperiod, with an adequate food supply (Brecchia et al., 2006; Hudson et al., 1994). Although biostimulation increases fertility above that seen in estrous rabbits (Theau-Clément et al., 1998) the lack of an effect of mating on the number of c-FOS-IR cells may indicate that, although the “general” reactivity of the POA to mating (as determined by the c-FOS protein) is smaller in biostimulated rabbits, the responsiveness of specific subnuclei directly involved in triggering ovulation may be larger. Further studies in particular subnuclei of the POA, using double-label immunocytochemistry to c-FOS and specific peptides promoting ovulation are warranted to explore such possibility. Of particular interest are: a) neuropeptide Y, which is co-released with GnRH in the
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rabbit hypothalamus in vivo (Pau et al., 1989) and promotes the release of GnRH and pituitary gonadotropins in vitro (Khorram et al., 1988) and b) kisspeptin. In addition the finding that the number of c-FOS-IR cells in the POA did not differ between biostimulated-unmated and lactating-unmated does coincides with the recent report that the number of PER1 protein-IR cells is not significantly reduced in the POA of does deprived of nursing for 24 h (Meza et al., 2015). Moreover, the present finding contributes to the understanding of the ways by which the biostimulation method promotes fertility without abolishing nursing behavior, as the POA is crucial for both ovulation (Foster and Younglai, 1991; Caba et al., 2000b) and maternal care (Cruz and Beyer, 1972; González-Mariscal et al., 2009b) in rabbits. The time of nursing used in the present study (10:00 h) is different from the one at which lactating does spontaneously suckle the litter under laboratory conditions, namely: 03:52 h (González-Mariscal et al., 2013). Yet, we have found that nursing the kits at 10:00 h triggers a robust c-FOS response in the LS, PVN, and SON (González-Mariscal et al., 2009b). Moreover, the PER1 protein shows a rhythm that shifts in parallel to the time of nursing in both the POA and the LS (Meza et al., 2015). These findings indicate that the brain's responsiveness to suckling can be effectively measured by such methods at different times of day. In the PVN a clear effect of biostimulation was evident: in unmated does this group showed significantly more c-FOS-IR cells than (unmated) lactating or estrous animals. To our knowledge this is the first evidence showing that the restoration of estrus, provoked by suppressing a single nursing bout, is associated with an increase in the activity of a specific brain region. Although we ignore the phenotype of the c-FOS-IR cells counted it is tempting to speculate that many of them are oxytocinergic as the PVN of rabbits is rich in oxytocin (OT)producing neurons, especially during lactation (Caba et al., 1996). The secretion of this peptide is stimulated by copulation (Todd and Lightman, 1986) and by nursing (Fuchs et al., 1984) so it seems puzzling that (regular) lactating mated females, which received both of those stimuli, showed fewer c-FOS-IR cells than estrous does, which were only mated. This issue needs to be further explored by using double-immunostaining to c-FOS and OT to determine if the proportion of double-stained neurons is larger in lactating-mated rabbits than in estrous ones. Both groups of estrous rabbits (i.e., mated and unmated) showed practically the same number of c-FOS-IR cells in the LS, a finding strongly suggesting that such region is probably not related with the perception of sensory stimulation received during copulation nor with the release of GnRH. Mated lactating and mated biostimulated does showed similar counts of cFOS-IR cells which, in turn, were about three-fold lower than those seen in estrous does. In agreement with our earlier study (González-Mariscal et al., 2009b) c-FOS immunoreactivity in the LS was stimulated by suckling as unmated lactating rabbits showed significantly more c-FOS-IR cells than did unmated biostimulated does, which missed a nursing bout. These results agree with a recent publication showing that canceling a single nursing episode reduces (by approximately 50%) the number of PER1 protein-IR cells in the dorsal and ventral septum (Meza et al., 2015). In mated animals the difference between lactating and biostimulated does disappeared suggesting that mating can blunt the responsiveness of the LS to suckling.
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In the INF the highest number of c-FOS-IR cells was observed in unmated biostimulated animals followed by unmated lactating ones. These findings are consistent with the activity of as yet unidentified secretory neurons involved in the regulation of lactation (Tucker, 1994). Moreover, these results indicate that the INF is one of the brain structures whose activity is clearly increased following the suppression of one nursing bout and which may be related to the facilitation of ovulation and fertility through biostimulation (Bonanno et al., 2002; Theau-Clément et al., 1998). The impact of reproductive condition on the number of c-FOS-IR cells following mating was especially clear in the SON while estrous mated rabbits showed an insignificant increase in c-FOS-IR cells relative to their unmated counterparts in both groups of lactating does mating provoked a reduction in such parameter. Moreover, in unmated females, biostimulation markedly increased the number of such cells above the levels seen in lactating or estrous rabbits. Many of such cells could be oxytocinergic or vasopressinergic neurons that are far more abundant in lactating than in estrous rabbits (Caba et al., 1996). Copulation did not impact the number of c-FOS-IR cells in the DMH of estrous rabbits, a finding in agreement with the idea that this structure does not have a known role in ovulation or sexual behavior. However, mated lactating and mated biostimulated does showed two to three times more c-FOS-IR cells than estrous animals. Because: a) the DMH plays an important role in the regulation of feeding (Yang et al., 2001) and b) nursing rabbits increase their food intake across lactation (González-Mariscal et al., 1994, 2009a) we believe that the larger number of c-FOS-IR cells observed in the DMH of lactating mated does is related with such phenomena. Yet, the mechanisms by which the condition of lactation increases the responsiveness to mating (relative to the state of estrus) remain to be determined. In contrast with the above regions, no significant differences were seen among the six experimental groups in the VMH. This is a puzzling finding as this region is rich in estradiol receptor alpha (Caba et al., 2003) and estradiol implants in the VMH stimulate sexual receptivity in ovariectomized rabbits (Melo et al., 2008). This result apparently also contradicts our earlier finding that mating promotes an increased expression of the c-FOS gene in the hypothalamus of estrous does, as determined by RT-PCR (Reyna-Neyra et al., 2000). These differences may be due to the lack of fine neuroanatomical resolution associated with the RT-PCR method and/or the activation of a specific neuronal phenotype, not identified in the present study, by mating. Indeed, a marked increase in the number of double-labelled c-FOS/ GnRH cells was found at 90 min post-coitus in the ventrolateral hypothalamus of estrous does (Caba et al., 2000b). On the other hand, the lack of differences between estrous-mated and biostimulated-mated does may be related with the fact that our mother–litter separation lasted only 24 h. Indeed, an interval of 48 h is necessary to provoke a massive increase in the concentration of estradiol in plasma (Ubilla et al., 2000) and longer mother–litter separations have been reported to provoke larger increases in fertility (Alvariño et al., 1998). However, the 24 h separation is usually preferred in rabbit farms as it both enhances fertility and allows the survival of the 10-day-old kits. Finally, we must consider that lactational anestrus is a complex phenomenon revealed through several
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physiological and behavioral indicators. We have found, for instance, that: a) the magnitude of estrus inhibition is dependent on the number of suckling young and b) three behaviors characteristic of estrous does (i.e., ambulation, scent-marking, and lordosis) have different thresholds of inhibition by suckling (García Dalmán and GonzálezMariscal, 2012). Accordingly, a 24-hr doe–litter separation may not be enough for the “state of estrus” to be reflected in terms of c-FOS-IR cells in the VMH. Clearly, more work is needed to unravel the ways by which nursing inhibits and biostimulation promotes estrus and fertility. In summary, our work has found that the reproductive state of female rabbits determines the responsiveness of specific forebrain regions to mating. This information can be a starting point to begin investigating the neuroendocrine signals and neuronal pathways involved in enhancing (biostimulation) or decreasing (lactation) the reactivity to copulatory stimulation in a reflex ovulator.
4.
Experimental procedure
4.1.
Animals
New Zealand white adult (3.5–4.5 kg body weight) virgin rabbits bred in our colony were used. They were housed inside a colony under controlled light (14 h light; 10 h dark; lights on at 07:00 h) and natural temperature (13–25 1C) conditions. They were provided with water and rabbit pellets (Conejina, Purina™) ad libitum. Throughout this work animal care adhered to the Law for the Protection of Animals (Mexico).
4.2.
Experimental groups
4.2.1.
Estrous rabbits
Each female was placed within a round wire mesh arena (1 m in diameter 80 cm height) and a sexually experienced male was then introduced. They were allowed to copulate three times while an observer determined whether lordosis occurred (or not) following each mount from the male. After this test the female was returned to her home cage (estrous mated group; n ¼5). Estrous females in the unmated group
(n ¼5) were placed in the arena alone for about 5 min after which they were returned to their individual wire mesh cages (52 cm long 42 cm wide 41 cm high).
4.2.2.
Lactating rabbits
They were mated as above but were housed in individual maternal cages (90 cm long 60 cm wide 40 cm high) within which a wooden nest box (50 cm long 30 cm wide 32 cm high) was placed to allow the construction of the maternal nest, as previously described (González-Mariscal et al., 1994). From pregnancy day 20 until parturition 300 g of straw were provided daily as material for nest-building. Starting on pregnancy day 30, females were spot-checked across the day to determine the approximate time of delivery. At parturition mothers were left undisturbed for 5–8 h after which kits were removed, litter size was adjusted to eight, and kept (away from the mother) inside a box containing paper shavings, under a mild heat source. On each day when nursing was recorded (see below) litters were weighed and introduced into the nest box at 10:00 h. The mother was allowed to nurse the kits after which they were removed and weighed again; the difference in their body weight (with respect to the one shown before nursing) was taken as a measure of milk output. This method for determining milk yield, reliably used by us in earlier studies (González-Mariscal et al., 1994, 2009b), was repeated for nine consecutive days. On lactation day 10 only half of the does were provided with their litters and allowed to nurse (lactating groups; n¼ 10) while the other half was not given the kits (biostimulated groups; n¼ 10). On the following day all mothers were allowed to nurse at the same time as before (i.e., 10:00 h) and half of them were mated immediately after nursing as described above; the other half was not mated. This strategy produced four experimental groups: lactatingmated (n¼ 5), lactating unmated (n¼5), biostimulated-mated (n¼5), and biostimulated-unmated (n¼ 5). The following diagram summarizes the experimental design:
4.3.
Sacrifice, perfusion, and immunocytochemistry
All females were killed on postpartum day 11 one hour after mating or nursing (or equivalent time of day in the unmated
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group). They were anesthetized with an overdose of sodium pentobartibal and perfused transcardially with saline and 4% para-formaldehyde. Brains were cryoprotected in sucrose (10%, 20%, 30%) and sectioned (40 mm) coronally from the rostral border of the preoptic area to the mammillary bodies. Every single section was collected but only one of every six was reacted with the monoclonal antibody against the c-FOS protein or was stained with cresyl violet for visualization of neuroanatomical structures. Free-floating sections were washed three times in phosphate buffer (PB) 0.1 M, pH 7.4 to remove excess aldehydes and then exposed for 10 min to 0.05% hydrogen peroxide to eliminate endogenous peroxidase activity. After preincubation for 1 h in 1% normal donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA) sections were incubated for 48 h at 4 1C in the monoclonal antibody sc-52 (Santa Cruz Biotechnology), diluted at 1:5000 with 0.3% Triton X-100 (Sigma, St. Louis, MO) in 0.1 M PB. This antibody has been reliably used by us (González-Mariscal et al., 2009b) and others (Caba et al., 2000b; Toledo et al., 2005) to visualize the c-FOS protein in the adult female rabbit brain. Sections were then incubated sequentially in biotinylated donkey antigoat serum IgG (1:200; Vector laboratories, Burlingame, CA) plus avidin–biotin horseradish peroxidase, HRP, complex (1:250; Vector Laboratories). HRP label was visualized using 0.05% diaminobenzidine (Polysciences, Warrington, PA) in 0.1 M PB as the chromogen with 0.01% hydrogen peroxide as substrate. Sections were mounted onto gelatin-subbed slides, dehydrated, and cleared in Hemo-De and then cover-slipped with Permount. Control sections were processed omitting the primary antibody.
slices counted (in our case one of every six); c) the proportion of the height of the slice that will be counted (in our case the middle third); d) the proportion of grid squares on which c-FOSIR cells will be counted (in our case one of every four). We placed a grid with squares of defined size (89 mm2); factor a/p in the formula below) over the specific structure and counted the number of intersections over the area analyzed (number of points). We obtained the distance between the sections analyzed (T) by multiplying the thickness of each slice (40 mm) by the inverse of the slice sampling factor (we selected one slice out of every six). Thus, T¼ 40 E6¼240 mm. We then estimated the volume of each of the structures specified above with the formula \widehatV¼(T) (a/p) (number of points). Next, we counted the number of c-FOS-IR cells (Q) in the selected slices (i.e., one of every six; slice sampling fraction; ssf¼ 1/6), choosing an area sampling fraction (asf) of one fourth (i.e., we counted cFOS-IR cells in one of every four squares of the grid placed over the corresponding slice) and a height sampling fraction (hsf) of one third (i.e., we counted the middle third of the selected 40 mm thick slices). According to the optical fractionator method [34] these factors were introduced into the equation \widehatNV ¼Q 1/asf 1/ssf 1/hsf to estimate the number of particles (\widehatNV) per unit volume in a given structure. This number was then multiplied by the volume of the corresponding brain structure (determined by the Cavalieri method described above) to obtain the total number of c-FOS-IR cells within that structure. Consequently, the c-FOS counts made in all animals in the brain structures selected was not biased as the probability of a cell being counted was not influenced by the size of the region analyzed.
4.4.
4.5.
Quantification of c-FOS-IR cells
Sections were examined under bright-field illumination in an Olympus CH2 microscope and camera lucida drawings were made of all sections. The distribution of c-FOS-IR cells was determined in reference to the rabbit stereotaxic atlas of Girgis and Shih-Chang (1981). The quantification of c-FOS-IR cells per brain region was done manually, by an observer blind to the condition of the animal, using three-dimensional stereology. We adhered to the criteria of Howard and Reed (1988), as described in an earlier publication from us performed in the rabbit brain (González-Mariscal et al., 2009b). The advantages of this method are: a) every cell (or object) within a given structure has the same chance of being selected and counted; b) brain structures are counted in their entirety, thus minimizing the possibility that large structures are sampled more than smaller ones. The nomenclature used and the delineation of the brain structures analyzed was the one adopted in earlier studies from our group (Caba et al., 2003; González-Mariscal et al., 2009b), specifically: preoptic area (POA), paraventricular nucleus (PVN), supraoptic nucleus (SON), lateral septum (LS), dorsomedial hypothalamus (DMH), ventromedial hypothalamus (VMH), and infundibular nucleus (INF). We first estimated the volume \widehatV of each of the above structures by the method of Cavalieri (Gundersen and Jensen, 1987) using Nissl-stained sections. This method takes into account the following factors, which are determined a priori by the investigator and are kept constant for all animals and all brain regions analyzed: a) the thickness of the slices (in our case 40 mm); b) the proportion of
Statistical analysis
A 2-way ANOVA was performed among the six experimental groups, in every brain region analyzed, to determine: a) the interaction between reproductive condition and mating and b) the impact of each factor alone on the number of c-FOS-IR cells. To compare between two specific groups a Student's Ttest was performed.
Acknowledgments This work was supported by CONACYT (National Council of Science and Technology, Mexico) Grant #128625 to GGM.
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