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INTRODUCTION

Lactation is the defi ning characteristic of mam-mals (Pond, 1984; Blackburn et al., 1989; Peaker, 2002). Neonatal consumption of milk, including “fi rst milk” or colostrum, confers essential nutritive and protective support to newborn mammals as they make the transi-tion from prenatal to postnatal life (Burrin et al., 1997; Wheeler et al., 2007; McClellan et al., 2008; Buddington and Sangild, 2011; Hinde and Milligan, 2011; Hurley and Theil, 2011). The nature of maternal investments in support of development from conception to weaning varies by species. Nevertheless, all mammals display

LACTATION BIOLOGY SYMPOSIUM: Lactocrine signaling and developmental programming1,2

F. F. Bartol,*3 A. A. Wiley,* D. J. Miller,* A. J. Silva,* K. E. Roberts,* M. L. P. Davolt,* J. C. Chen,† A.-L. Frankshun,† M. E. Camp,† K. M. Rahman,† J. L. Vallet,‡ and C. A. Bagnell†

*Department of Anatomy, Physiology and Pharmacology, Cellular and Molecular Biosciences Program, Auburn University, Auburn, AL 36849-5517; †Department of Animal Sciences, Endocrinology and Animal Biosciences Program, Rutgers, The State University of New Jersey, New Brunswick 08901;

and ‡USDA ARS, US Meat Animal Research Center, Clay Center, NE 68933-0166.

ABSTRACT: Lactocrine signaling is defi ned as trans-mission of bioactive factors from mother to offspring as a consequence of nursing. Lactocrine transmission of signaling molecules may be an evolutionarily conserved process through which bioactive factors necessary for support of neonatal development are delivered postna-tally. Dependence on maternal resources for develop-ment in eutherian mammals extends into neonatal life for at least that period of time when nutrition is obtained solely from fi rst milk (i.e., colostrum). Data for the pig (Sus scrofa domesticus) provide evidence of lactocrine mediated effects on development of the female repro-ductive tract and other somatic tissues. Porcine uterine gland development, an estrogen receptor-alpha (ESR1)-dependent process, begins within 2 d of birth [postnatal day (PND) 0]. A lactocrine-driven, ESR1-mediated pro-cess was proposed as a regulatory mechanism govern-ing onset of uterine gland development and endometrial

maturation in the neonatal pig. Gilts maintained in a lactocrine-null state for 2 d from birth by milk-replacer feeding displayed altered patterns of endometrial gene expression and retarded uterine gland development by PND 14. In lactocrine-null gilts, inhibition of endome-trial and cervical ESR1 and vascular endothelial growth factor (VEGFA) expression observed on PND 2 persist-ed to PND 14, even after gilts were returned to nursing on PND 2. Collectively, data support a role for lacto-crine signaling in regulation of critical neonatal devel-opmental events. Maternal lactocrine programming of postnatal development may help to insure healthy devel-opmental outcomes. A systems biology approach will be required to defi ne and understand mechanistic dynamics of lactocrine signaling events that may ultimately con-nect genotype to phenotype and establish the parameters of reproductive potential.

Key words: colostrum, development, female reproductive tract, lactocrine, neonate

© 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2013.91:696–705 doi:10.2527/jas2012-5764

1Based on a presentation at the Lactation Biology Symposium titled “The long-term impact of epigenetics and maternal infl uence on the neonate through milk-borne factors and nutrient status” at the Joint Annual Meeting, July 15–19, 2012, Phoenix, AZ, with publication sponsored by the Journal of Animal Science and the American Society of Animal Science.

2Work reviewed here was supported by National Research Initiative Competitive Grants 2003-35203-13572 and 2007-35203-18098 (to FFB and CAB) from the USDA National Institute of Food and Agriculture and by NSF-EPS-0814103 and 1158862 (to FFB). The authors thank L. Comerford, K. Mezey, B. Anderson, the staffs of the Auburn University and Rutgers University animal care programs, and a multitude of un-dergraduate students for their contributions to this work. FFB and CAB contributed equally to this work.

3Corresponding author: bartoff@auburn.eduReceived August 16, 2012.Accepted September 7, 2012.

Published December 2, 2014

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a postgestational period during which maternal support of offspring comes uniquely from milk (Langer, 2008). Moreover, many milk proteins and other components of the lactation system in monotreme, marsupial, and euthe-rian mammals are highly conserved (Lemay et al., 2009; Lefevre et al., 2010). Collectively, these observations em-phasize the central role of lactation in the grand scheme of mammalian reproduction and development.

Consumption of colostrum and milk confers nutri-tive, protective, and adaptive advantages to newborn mammals (Wheeler et al., 2007; McClellan et al., 2008; Langer, 2009; Hinde and Milligan, 2011). Less well un-derstood are the roles played by milk-borne bioactive factors (MbF), including peptide and steroid hormones, cytokines, and microRNA, in support of neonatal de-velopment (Simmen et al., 1990; Grosvenor et al., 1993; Donovan and Odle, 1994; Koldovsky, 1995; Playford et al., 2000; Blum, 2006; Blum and Baumrucker, 2008; Chen et al., 2010b; Hondares et al., 2010; Frankshun et al., 2011). The term “lactocrine” was coined (Yan et al., 2006b) as a unifying descriptor of mechanisms through which MbF are communicated from mother to offspring as a specifi c consequence of nursing. Subsequently, the lactocrine hypothesis for maternal programming of female reproductive tract (FRT) tissues was proposed (Bartol et al., 2008), supported by data indicating that nursing is re-quired for establishment of the neonatal porcine uterine developmental program (Chen et al., 2011). The objective of this review is to discuss the potential role of lactocrine signaling in regulation of neonatal epigenotype and to present evidence of the importance of lactocrine signaling in support of FRT development.

Programming and Epigenotype

Successful development depends upon establishment and maintenance of an organizational program such that the trajectory of development is optimal, as defi ned by the capacity of an adult organism to adapt to its environ-ment, thrive, and, ultimately, reproduce. Mechanistically, elements of a developmental program are defi ned by the myriad of processes connecting genotype to phenotype. Over 90 yr ago, Stockard (1921) provided an analysis of the “causes and conditions which determine the usual type of structural expression or form.” Later, recogniz-ing that mechanisms connecting genotype to phenotype were neither simple nor linear, Waddington (1939, 1942) proposed the term “epigenotype” to describe the complex network of processes that ultimately defi ne phenotype. Today, such functional relationships might be described as a developmental interactome (Vidal et al., 2011). Both Stockard (1921) and Waddington (1942) recognized that even small changes in the developmental program or epigenotype early on can have profound and lasting con-

sequences later in life, including alterations in tissue and even organismal phenotype. Taken together with the idea that there might be “critical moments” or periods of time during which interruption of development could be ex-pected to have such lasting effects (Stockard, 1921), the idea of developmental programming was born.

In recent years, a great deal of attention has been focused on identifi cation of critical periods during pre-, peri- and postnatal life when macro- (e.g., climate, diet) and/or microenvironmental conditions (e.g., metabolic, endocrine, immunological) have the potential to affect developmental programming of uteroplacental, fetal, and/or neonatal tissues with lasting consequences (Barker, 1998; Gluckman et al., 2005; Wu et al., 2006; Burdge et al., 2007; Foxcroft et al., 2009; Symonds et al., 2009; Funston et al., 2010; Hanley et al., 2010; Burton and Fowden, 2012; Reynolds and Caton, 2012). In this regard, Gluckman et al. (2005) observed that it is important to distinguish between those environmental effects acting during development that are disruptive in nature from those effects that have adaptive value, indicating that ef-fects with adaptive value are most likely to be associated with programming events.

Evolutionarily and functionally, lactation and, by association, lactocrine signaling represent adaptive pro-cesses of the highest order (Peaker, 2002; Langer, 2008; Hinde and Milligan, 2011; Bartol and Bagnell, 2012). Not only does lactocrine signaling extend the period of ma-ternal communication with offspring into postnatal life (Bartol et al., 2008; Bartol and Bagnell, 2012), it also pro-vides an opportunity for information related to the nature of postnatal environmental conditions to be passed on ef-fi ciently, thereby enhancing the capacity of neonates to sense, respond, and adapt to the circumstances into which they are born.

Developmental Plasticity and the Neonatal Female Reproductive Tract

The ability of identical genotypes to produce differ-ent phenotypes is defi ned as developmental plasticity (Hochberg et al., 2011). Divergence from a developmen-tal program leading to alternate developmental trajecto-ries and associated, divergent phenotypes and physiology is thought to refl ect environmentally induced changes in epigenotype (Bateson et al., 2004; Hochberg et al., 2011; Guerrero-Bosagna and Skinner, 2012). Epigenetic pro-gramming can occur throughout development. However, the potential for environmentally induced changes to af-fect developmental trajectory with lasting consequences tends to be greatest early in development, especially when cells and tissues are differentiating, during periods associ-ated with what Hochberg et al. (2011) called “life-history phases” (Fig. 1). Developmental plasticity allows organ-

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isms to adapt, acutely and chronically, to environmental change. Still, as Stockard (1921) observed, disruption of development during critical organizational periods can produce extreme phenotypes, often at signifi cant cost to reproductive success (Bateson et al., 2004).

Developmental disruption by aberrant exposure to bioactive compounds, such as steroid hormones and structurally related pharmaceuticals or environmen-tally derived endocrine disruptive chemicals (Zoeller et al., 2012), can change tissue developmental trajectories. Pathways through which such developmentally disrup-tive signals may be transmitted include 1) ligand acti-vation of nuclear receptors and transcription factors, 2) canonical membrane receptor signaling cascades, and 3) metabolic activators and inhibitors of epigenetic machin-ery (Diamanti-Kandarakis et al., 2009; Hochberg et al., 2011; Guerrero-Bosagna and Skinner, 2012). Regardless of mechanism, signaling pathways must be in place for potentially disruptive factors to affect the developmental program, defi ned as the sequence of events that specify cell fate and determine cell and tissue identity and func-tion (Frankshun et al., 2012). Windows of sensitivity to developmental disruption evolve in association with tem-porospatial patterns of expression and ontogeny of the functional elements of signal transduction systems in de-veloping cells and tissues.

The neonatal period constitutes a phase of life his-tory (Fig. 1) during which cells and tissues can be subject to environmentally induced epigenetic programming or misprogramming (Hochberg et al., 2011). In this regard,

developmental plasticity of neonatal FRT tissues is well documented in both murine and domestic ungulate spe-cies (Bartol et al., 1999, 2006; Ma, 2009; Cooke et al., 2012; Spencer et al., 2012). Uterine wall development, marked by differentiation and proliferation of nascent glandular epithelium (GE) or adenogenesis, is complet-ed postnatally in most, if not all, mammals (Gray et al., 2001a; Spencer et al., 2005). Disruption of neonatal endo-metrial development can have profound effects on adult uterine morphology and function.

For example, exposure of ewe lambs to a 19-nor-progestin from birth [postnatal day (PND) 0] inhibited adenogenesis, resulting in adult ewes lacking endome-trial glands and establishing the “uterine gland knockout” (UGKO) phenotype (Bartol et al., 1988, 1999; Gray et al., 2000). Similarly, exposure of beef heifer calves to a com-bination of progesterone and estradiol benzoate (PEB)from birth reduced adult uterocervical wet weights by 35%, myometrial area by 23%, and endometrial area by 27% when compared with unexposed controls (Bartol et al., 1995). Uterine gland density was reduced by 65% in adult heifers exposed to PEB from birth and the glandless UGKO phenotype was observed in some animals. These effects were attenuated systematically when initial PEB exposure was delayed until PND 21 or PND 45 (Bartol et al., 1995). Consistently, when compared with vehicle-treated controls, mice injected with progesterone from PND 3 to 9, 5 to 9, or 3 to 7 lacked endometrial glands at PND 10 whereas progesterone treatment from PND 3 to 5 or 5 to 7 did not affect endometrial glandularity (Cooke et al., 2012). Importantly, progesterone exposure from PND 3 to 9 was the only condition in which the glandless, UGKO phenotype persisted in adult mice (Cooke et al., 2012). Similar fi ndings were reported recently by Filant et al. (2012). Experimental evidence for both sheep (Gray et al., 2001b) and mice (Cooke et al., 2012; Filant et al. 2012) indicates that neonatal induction of the UGKO phe-notype renders the adult uterus incapable of supporting pregnancy. This defect is manifest in recurrent pregnancy loss due to an implantation defect. In the pig, neonatal uterine estrogen sensitivity is age dependent (Spencer et al., 1993; Tarleton et al., 1998, 1999). Disruption of estrogen-sensitive, ESR1-dependent porcine endome-trial development by administration of estradiol valerate (EV) for 2 wk from birth did not affect adult endometrial morphology (Bartol et al., 2006). However, neonatal EV exposure did result in smaller adult uteri (Tarleton et al., 2003), reduced uterine capacity for conceptus support, al-terations in the adult endometrial proteome at pregnancy d 12 (Bartol et al., 2006), and long-term effects on endo-metrial gene expression during the periattachment period in adult gilts (Chen et al., 2010a).

Collectively, these observations provide compelling evidence of the developmentally plastic nature of neo-

Figure 1. Tissue developmental plasticity related to major life-history phases and associated conditions with potential to affect developmental epig-enotype. Generally, developmental plasticity tends to be greatest during early life-history phases but can persist into adulthood. Prenatally, maternal effects on development are profound. However, dependence on maternal resources for development does not end at birth but extends into the early postnatal period via lactocrine effects. Such effects can be modifi ed, complicated, and even inhib-ited by factors presented to the neonate, directly or indirectly (via milk), from the postnatal (extrauterine) environment and associated, integrative (or disinte-grative) actions of endocrine mediated events. Adapted from Langer (2008) and Hochberg et al. (2011).

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natal uterine tissues. Data are consistent with the ideas that 1) there are critical periods of neonatal life during which disruption of steroid hormone-sensitive organiza-tional events can alter the developmental epigenotype and change developmental trajectories of FRT tissues and 2) effects of developmental disruption on FRT tissues tend to be most profound and lasting when disruption begins at or shortly after birth.

Porcine Uterine Development and the Lactocrine Hypothesis

Histogenesis of the porcine uterus (Spencer et al., 1993; Tarleton et al., 1998; Bartol et al., 2006; Masters et al., 2007) and comparative aspects of this process (Gray et al., 2001a; Spencer et al., 2005, 2012) have been de-scribed. Porcine uterine development begins prenatally and is completed postnatally. At birth, the porcine endo-metrium is characterized by a simple luminal epithelium (LE) overlying a densely packed subepithelial zone of stromal cells, supported by more loosely organized stro-ma that extends to the border of the myometrium. Absent at birth, endometrial GE differentiates from LE shortly thereafter. Onset of adenogenesis is marked by ESR1 ex-pression in nascent GE (Fig. 2) as well as rapid prolif-eration of GE between birth and PND 3 (Masters et al., 2007), a period of morphogenetic transition for the neo-natal porcine endometrium. Daily administration of the anti-estrogen ICI 182,780 to gilts from birth inhibited en-dometrial gland genesis in the fi rst 2 wk of neonatal life (Tarleton et al., 1999), indicating that uterine adenogen-esis is not only marked but mediated by ESR1-dependent signaling events. Taken together with data indicating that transient exposure of gilts to EV from birth can affect adult uterine function (Tarleton et al., 2003; Bartol et al., 2006; Chen et al., 2010a), results indicate that conditions affecting ESR1 expression and related signaling events in the neonatal porcine uterus can also affect the uterine developmental epigenotype and the developmental tra-jectory of the endometrium.

A series of experiments involving neonatal gilts es-tablished that 1) uterine relaxin (RLX) receptor (RXFP1) expression is detectable at birth, primarily in endometrial stroma, before onset of endometrial ESR1 expression (Yan et al., 2006b), 2) endometrial RXFP1 expression in-creases with age from PND 0 to PND 14 and in response to estradiol-17β (E) administration during this period (Yan et al., 2006b), 3) treatment with RLX but not E from birth increased the relative abundance of ESR1 protein in uterine and cervical tissues at PND 2 (Yan et al., 2008), and 4) treatment with E but not RLX from birth increased uterine and cervical RXFP1expression at PND 2 (Yan et al., 2008). These results indicated that onset of ESR1 ex-pression in neonatal porcine uterine and cervical tissues

could be supported, in part, by RXFP1-mediated signal-ing events shortly after birth. Furthermore, the fact that uterotrophic effects of RLX in the neonatal porcine uterus were attenuated by co-administration of ICI 182,780 (Yan et al., 2006a, 2008) indicated that RLX support of uter-ine development could involve ESR1-mediated signaling mechanisms.

Relaxin, a 6 kDa peptide hormone, is as a member of a family of bioactive neohormones that are thought to have evolved in support of viviparity and lactation (Ivell et al., 2007). For species in which RLX is expressed (Sherwood, 2004), luteal tissue is a major source of this peptide (Sherwood and O’Byrne, 1974; Sherwood, 1979). Whereas endometrial RLX expression was reported for the adult pig (Knox et al., 1994) and human (Palejwala et al., 2002), RLX expression by neonatal uterine tissues has yet to be described. In the neonatal pig, adipose tissue may serve as one source of endogenous RLX (Hausman et al., 2006). However, colostrum is the principle source of RLX in neonatal pigs (Yan et al., 2006b; Frankshun et al., 2011), as also described for dogs (Goldsmith et al., 1994; Steinetz et al., 2008) and humans (Eddie et al.,

Figure 2. Ontogeny of estrogen receptor-alpha (ESR1) expression in the neonatal porcine endometrium. Top (panels A and B): Spectrally unmixed composite images, generated using a Nuance FX multispectral imaging camera, depict ESR1 (green) and cytokeratin 8 (yellow) immunofl uorescent staining in uterine endometrial tissues obtained at approximately 24 h (Panel A) and 48 h (Panel B) postnatal from nursed gilts. Differentiation of glandular epithelium (GE) from luminal epithelium (LE) is marked by ESR1 expression in nascent GE, apparent at 24 h (Panel A). Bottom (panels C and D): Wavelength-specifi c images illustrating ESR1-specifi c signal are shown for the same tissues. In the stroma (St), ESR1 signal is evident at 24 h and is more pronounced by 48 h. Immunolocalization of ESR1 was accomplished using a mouse IgG1 antibody (clone [1D5], 1:50; Dako, Carpinteria, CA) and an AlexaFluor 488 goat anti-mouse IgG1(1:400; Invitrogen, Grand Island, NY). Cell nuclei (blue) were identifi ed using POPO-1 (A and B).

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1989). Data indicating that RLX concentrations in milk are greater than in the maternal circulation during lacta-tion (Sherwood et al., 1981; Eddie et al., 1989; Goldsmith et al., 1994) indicate that RLX is either produced by the mammary gland, as documented for both the pig (Frankshun et al., 2011) and rat (Steinetz et al., 2008), or synthesized elsewhere and sequestered in colostrum and milk (Eddie et al., 1989; Bartol et al., 2009).

Prorelaxin is the primary form of bioactive RLX found in porcine milk (Frankshun et al., 2011). Both RLX bioactivity and immunoreactive RLX concentrations are greatest in porcine colostrum within 24 h of birth (Yan et al., 2006b; Frankshun et al., 2011). Consistently, serum RLX concentrations in nursed pigs are greatest during the fi rst 2 d of neonatal life (Yan et al., 2006b), before the es-timated time of gut closure in this species (Lecce, 1973). Critically, RLX was not detectable in the serum of neona-tal pigs at birth, before nursing (Yan et al., 2006b). Taken together with information reviewed above, these data in-dicated that 1) potential endogenous sources of RLX, such as adipose tissue (Hausman et al., 2006), are insuffi cient to affect serum RLX concentrations in the neonatal pig and 2) colostrum is the primary source of RLX found in the porcine neonatal circulation within 2 d of birth. Thus, in the pig, RLX is transferred from mother to offspring as a specifi c consequence of nursing via a prototypical lac-tocrine mechanism (Yan et al., 2006b; Bartol et al., 2008; Bagnell et al., 2009).

Testing the Lactocrine Hypothesis

A feed-forward, lactocrine-driven mechanism was proposed to explain how MbF, exemplifi ed by RLX, could support events central to endometrial adenogenesis in the neonatal pig (Bartol et al., 2009). Elements of this mecha-nism are illustrated in Fig. 3. In this scheme, lactocrine transmission of RLX from mother to offspring allows this MbF to act via its cognate receptor (i.e., RXFP1) in the endometrium to induce and support endometrial ESR1 expression in the neonate. Continued lactocrine signaling provides agonistic input to both RXFP1 and ESR1 sig-nal transduction systems, thereby amplifying lactocrine induction of endometrial ESR1 expression (Fig. 2) as well as the expression of other lactocrine-sensitive fac-tors required to support endometrial adenogenesis (Bartol et al., 2009). These events coincide temporally and spa-tially with onset of uterine gland genesis, as marked by differentiation and increased proliferation of nascent GE within 3 d of birth (Masters et al., 2007). Given this scheme, imposition of the lactocrine-null condition from birth, by substituting a commercial milk replacer for nurs-ing and colostrum consumption, should be antagonistic to uterine and, potentially, FRT development as refl ected by

downregulation of lactocrine-sensitive events in neonatal tissues.

Two studies were conducted to test the hypotheses that lactocrine signaling is required to 1) establish the neonatal developmental program in FRT tissues, including the uter-us and cervix, and 2) determine tissue developmental tra-jectory (Chen et al., 2011; Frankshun et al., 2012). To test the fi rst of these hypotheses, gilts were assigned at birth to either nurse normally or be maintained in a lactocrine-null state by feeding porcine milk replacer from birth to PND 2, when tissues were collected. Expression patterns of molecular markers and mediators of uterine and cer-vical development, including ESR1, vascular endothelial growth factor (VEGFA), and matrix metalloproteinases (MMP) 2 and 9, were assessed. Results showed that lactocrine signaling is required to support normal uter-ine (Chen et al., 2011) and cervical expression of ESR1, VEGFA, and pro-matrix metalloproteinase-9 (proM-MP9) at PND 2 (Frankshun et al., 2012). Comparisons of ESR1 expression levels representative of uterine and cervical tissues obtained from nursed versus replacer-fed gilts on PND 2 are illustrated in Fig. 4. Analogous effects were observed for other molecular markers of lactocrine signaling (e.g., VEGFA, proMMP9), including cervical antiapoptotic B-cell lymphoma-2 expression (Frankshun et al., 2012). In contrast, uterine and cervical MMP2 ex-pression, observed on PND 2, was unaffected by impo-

Figure 3. Elements of a feed-forward, lactocrine-driven mechanism proposed to explain how milk-borne bioactive factors (MbF), exemplifi ed by relaxin (RLX), support events central to endometrial adenogenesis in the neo-natal pig. Maternal and neonatal compartments are identifi ed (left). Lactocrine transmission of RLX from mother to offspring allows this MbF to act via its cognate receptor [relaxin receptor (RXFP1)] in the endometrium to induce and/or support endometrial estrogen receptor-alpha (ESR1) expression. Continued lactocrine signaling provides agonistic input to both RXFP1 and ESR1 signal transduction systems, thereby amplifying lactocrine induction of endometrial ESR1 expression as well as the expression of other lactocrine-sensitive factors required to support endometrial adenogenesis. These events coincide temporal-ly and spatially with onset of uterine gland genesis within the fi rst few days of life. Factors acting as agonists or antagonists in this scheme have the potential to alter the developmental epigenotype with lasting consequences.

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sition of the lactocrine-null state from birth (Chen et al., 2011; Frankshun et al., 2012), indicating that not all neo-natal uterine and cervical protein expression is lactocrine sensitive.

With respect to the second hypothesis enumerated above, a recent report on the porcine cervix (Frankshun et al., 2012) and data for the uterus (Miller et al., 2013) indicate that lactocrine signaling can affect organizational processes that determine developmental trajectory of FRT tissues. Data for the cervix showed that disruption of lac-tocrine signaling for 2 d from birth induced changes in cervical protein and gene expression patterns at PND 2 that persisted through PND 14, even when replacer-fed gilts were returned to nursing immediately after PND 2 (Frankshun et al., 2012). Similarly, imposition of a lac-tocrine-null state for 48 h from birth by milk-replacer feeding reduced uterine stromal ESR1 labeling index on PND 2 and both epithelial ESR1 mRNA abundance and ESR1 labeling index in GE on PND 14, when uterine gland development was markedly reduced in replacer-fed gilts. Consistently, proliferating cell nuclear antigen labeling index was reduced in nascent GE on PND 2 and in both GE and stroma on PND 14 in replacer-fed gilts, in which endometrial thickness and gland penetration depth were also reduced (Miller et al., 2013). Imposition of the lactocrine-null state for 2 d from birth retarded en-dometrial adenogenesis by PND 14 to a degree similar to that observed in gilts of the same age treated with ICI 182,780 from birth (Tarleton et al., 1999). It is now clear that disruption of neonatal estrogen receptor-dependent, estrogen-sensitive developmental events suffi cient to al-ter patterns of uterine gene expression and development by PND 14 can have lasting effects on adult endometrial

phenotype and functional uterine capacity (Tarleton et al., 1999; Bartol et al., 2006; Chen et al., 2010a). Additionally, disruption of lactocrine signaling for 2 d from birth in-duced biochemical and morphological changes in porcine FRT tissues by PND 14 that are associated with downreg-ulation of ESR1 expression and related signaling events. Given these observations, it is reasonable to suggest that lactocrine signaling should be considered as an element of the organizational pallet of factors required to optimize uterocervical epigenotype and tissue developmental tra-jectory.

A rapid method for measurement of the passive trans-fer of immunoglobulins from sow to piglet, defi ned as the immunoglobulin immunocrit, was recently validated (Vallet et al., 2013). The immunocrit refl ects colostrum intake in neonatal pigs. In a retrospective test of the lacto-crine hypothesis for maternal programming of reproduc-tive performance, immunocrit values were measured in serum collected from 381 gilts on PND 0 (i.e., on d 1 of life) and the number of piglets born alive was recorded for the same gilts for up to 4 parities at the R.L. Hruska U.S. Meat Animal Research Center (Clay Center, NE). Data, analyzed using PROC MIXED (SAS Inst. Inc., Cary, NC), were best modeled by regression analysis of the recipro-cal of immunocrit values versus number of piglets born alive, with parity as a main effect. Because the linear ef-fect of the reciprocal of the immunocrit did not differ with parity (e.g., no interaction), this term was dropped from the overall model. Gilt was included in the model as a random effect because the number of piglets born alive at each parity represented repeated measures. The relation-ship between immunocrit values and piglets born alive to those adult gilts for which immunocrit values were obtained neonatally is illustrated in Fig. 5. As predicted by the lactocrine hypothesis, results of the overall analy-sis indicated clearly that low immunocrit, equivalent to minimal colostrum consumption, was associated with re-duced litter size (P = 0.01; results from fi rst parity shown in Fig. 5). These data provide compelling support for the idea that lactocrine signaling affects uterine capacity and reproductive performance in adults.

Lactocrine Signals

The array of potentially lactocrine-active MbF in co-lostrum and milk is staggering (Grosvenor et al., 1993; Donovan and Odle, 1994; Koldovsky, 1995; Playford et al., 2000; Blum, 2006; Blum and Baumrucker, 2008; Chen et al., 2010b; Frankshun et al., 2011). Moreover, it is likely that these factors act collectively or cooperatively, more so than independently, to integrate postnatal devel-opmental events that may also be sensitive to endocrine (Fig. 1) and environmental conditions (Koldovsky, 1996; Bartol and Bagnell, 2012). For example, data for both the

Figure 4. Effects of nursing versus milk replacer feeding from birth [postnatal day (PND) 0] on porcine uterine (Panel A) and cervical (Panel B) estrogen receptor-alpha (ESR1) protein concentrations at PND 2. Representative immunoblots are shown. Immunoreactive bands for ESR1 (51 kDa) and actin (43 kDa), included as a loading reference, are indicated. Each lane depicts results for an individual animal. Nursing was required to support detectable concentrations of ESR1 expression in both tissues.

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porcine uterus (Chen et al., 2011) and cervix (Frankshun et al., 2012) indicate that trophic effects of RLX on neo-natal uterine and cervical growth and induction of related gene expression events differ between gilts maintained in a lactocrine-null state and control gilts nursed normally from birth, indicating a role for cooperating lactocrine factors in support of RLX actions in developing FRT tis-sues.

Understanding lactocrine signals is further complicat-ed by the fact that the biochemical nature of colostrum and milk is not static (Klobasa et al., 1987; Blum, 2006; Silva et al., 2010). The array of proteins and peptides in porcine colostrum and milk changes quantitatively and qualita-tively within the fi rst week of lactation (Fig. 6) and can differ from anterior to posterior mammary glands within a single lactating sow (Wu et al., 2010). The fact that bioactive peptides can be encrypted within milk proteins (Coste et al., 1992; Schlimme and Meisel, 1995; Meisel, 2005; Lopez-Exposito and Recio, 2008; Saito, 2008) adds an additional level of complexity to the matter. Latent bioactivity represented by such encrypted peptides can be released through proteolysis and hydrolysis within the gastrointestinal (GI) tract (Schlimme and Meisel, 1995; Meisel and Bockelmann, 1999). Like other potentially lactocrine-active factors, milk protein-derived MbF may traverse the gut and act directly on somatic target tissues or act directly on cells of the GI tract to induce effects on other tissues indirectly (Blum, 2006). Developmentally related changes in neonatal GI tract physiology are also

likely to affect the effi ciency of lactocrine signal transmis-sion (Xu, 1996; Blum, 2006). Much remains to be done to determine which among all of the potential MbF are important developmentally and what conditions are nec-essary to support maternal lactocrine signaling processes.

SUMMARY AND CONCLUSIONS

The idea that, in most mammals, lactation has ac-tive rather than passive input into guiding the develop-ment of nursing young is not new (McClellan et al. 2008). Likewise, the idea that postnatal nutrition affects long-term outcomes via developmental programming has been proposed (Hinde and Capitanio, 2010; Wiedmeier et al., 2011). Adoption of the term “lactocrine” enables discus-sion of these complex processes. In an effort to defi ne and refi ne critical questions pertaining to lactation, neonatal nutrition, and development, this term was used in discus-sion of the possibility that cortisol-mediated lactocrine signaling might affect behavioral trajectory in human in-fants (Neville et al., 2012). Similarly, recent data for the dairy cow were interpreted to indicate that nutritional or metabolic programming during the fi rst 2 mo of life can have lifelong implications for milk production via epigen-etic effects modifi ed through a lactocrine-type mechanism in neonatal calves (Soberon et al., 2012). Interestingly,

Figure 5. Relationship between serum immunoglobulin immunocrit values at 1 d of age [postnatal day (PND) 0] for 381 gilts and their fi rst parity litter size as adults, expressed as number born alive. Individual piglet immunocrit values are shown in grey. The complete dataset included observations for 381 gilts for which both immunocrit and litter size data were recorded up to the forth parity. The dark line depicts the regression equation (shown) for fi rst parity only. This asymptotic relationship was best modeled by regression of the reciprocal of the immunocrit values on number born alive. The given P-value (P = 0.01) is for the linear effect of the reciprocal of the immunocrit for all 4 parities. The prediction equation indicates that the number of piglets born alive drops off substantially below an immunocrit of about 0.05. Neonatal immunocrit values, indicative of colostrum consumption, may be predictive of functional uterine capacity in adult gilts.

Figure 6. Proteins and peptides in porcine colostrum [lactation day (LD) 0] and milk (LD 6) identifi ed in 2-dimensional (2D) electrophoretograms of whey proteins. Colostrum or milk samples, obtained on LD 0 and LD 6 from 6 lactating sows, were subjected to 2D electrophoresis. For each sample, duplicate 75 μg aliquots of total whey protein were subjected to immobilized pH gradient, isoelectric focusing in the fi rst dimension followed by equilibration [6 M urea, 2% SDS, 0.375 M Tris-HCL pH 8.8, 20% glycerol, and 2% (wt/vol) dithiothreitol] and separation in the second dimension on 10 to 20% total monomer, gradient polyacrylamide gels with SDS. Individual 2D gels were stained with Sypro-Ruby (Bio-Rad, Hercules, CA) and imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare Life Sciences, Pittsburgh, PA). Digital gel images were then analyzed using PDQuest software (version 8.0.1, Bio-Rad). Shown is a composite 2D master image depicting protein and peptide spots representative of all samples (LD 0 and LD 6) separated by isoelectric point (pH 3 to 10, left to right) and molecular weight (250 to 10 × 10–3, top to bottom). Boxes (A, B, and C) show areas where quantitative and/or qualitative changes were observed in the colostral and milk proteome between LD 0 and LD 6.

Lactocrine programming and development 703

maternal nursing behaviors alone, including pup licking, grooming, and arched-back nursing, were shown to alter the epigenome of offspring in rats (Weaver et al., 2004). Effects included differences in DNA methylation and dif-ferential patterns of histone acetylation and transcription factor (i.e., nerve growth factor-induced protein A) bind-ing at a glucocorticoid receptor gene promoter in the hip-pocampus of neonatal mice that persisted into adulthood (Weaver et al., 2004). Observations of this kind empha-size the idea that even subtle changes in environmental conditions during neonatal life can affect the epigeno-type, leading to stable alterations in phenotype (Weaver et al., 2004). Given such information, it is not diffi cult to envision programming effects occurring as a conse-quence of lactocrine signaling or lack thereof. These data also indicate that environmentally induced phenotypes need not be overt. Cryptic phenotypes, defi ned as those that are only manifest when an individual is exposed to a particular physiological, endocrinological, or environ-mental challenge later in life, can also occur (Burdge and Lillycrop, 2010).

The extent to which maternal factors affect neonatal epigenotype and development is just beginning to be un-derstood. A systems biology approach will be required to defi ne the mechanistic dynamics of lactocrine signaling and the molecular, cellular, physiological, and environ-mental dimensions of the epigenetic interactome that ul-timately connect genotype to phenotype and establish the parameters of reproductive potential.

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