Effects of colostrum, feeding method and oral IGF1 on porcine uterine development.

Nursing ensures lactocrine delivery of maternally derived, milk-borne bioactive factors to offspring, which affects postnatal development of female reproductive tract tissues. Disruption of lactocrine communication for two days from birth (postnatal day (PND) 0) by feeding milk replacer in lieu of nursing or consumption of colostrum alters porcine uterine gene expression globally by PND 2 and inhibits uterine gland genesis by PND 14. Here, objectives were to determine effects of: (1) nursing or milk replacer feeding from birth; (2) a single dose of colostrum or milk replacer and method of feeding and (3) a single feeding of colostrum or milk replacer, with or without oral supplementation of IGF1, administered at birth on aspects of porcine uterine development at 12-h postnatally. Results indicate nursing for 12 h from birth supports rapid establishment of a uterine developmental program, illustrated by patterns of endometrial cell proliferation, expression of genes associated with uterine wall development and entry into mitosis and establishment of a uterine MMP9/TIMP1 system. A single feeding of colostrum at birth increased endometrial cell proliferation at 12 h, regardless of method of feeding. Oral supplementation of IGF1 was sufficient to support endometrial cell proliferation at 12 h in replacer-fed gilts, and supplementation of colostrum with IGF1 further increased endometrial cell proliferation. Results indicate that lactocrine regulation of postnatal uterine development is initiated with the first ingestion of colostrum. Further, results suggest IGF1 may be lactocrine-active and support a 12-h bioassay, which can be used to identify uterotrophic lactocrine activity.

Maternal lactocrine programming of porcine reproductive tract development.

The lactocrine hypothesis for maternal programming of female reproductive tract development is based on the idea that non-nutritive, milk-borne bioactive factors (MbFs), delivered from mother to offspring during nursing, play a role in determining the trajectory of development with long-term consequences in the adult. Porcine female reproductive tract development is completed postnatally, and the period during which maternal support of neonatal growth derives exclusively from colostrum/milk defines a window of opportunity for lactocrine programming of reproductive tissues. Beyond nutrition, milk serves as a delivery system for a variety of bioactive factors. Porcine relaxin is a prototypical MbF. Present in colostrum at highest concentrations at birth, relaxin is transmitted into the circulation of nursing piglets where it can act on Relaxin receptors found in neonatal female reproductive tract tissues. This process is facilitated by the physiology of the maternal-neonatal dyad and the fact that the neonatal gastrointestinal tract is open to absorb macromolecules for a period of time postnatally. Age at first nursing and duration of nursing from birth are also important for porcine female reproductive tract development. These parameters affect both the quality and quantity of colostrum consumed. Disruption of lactocrine signaling by feeding milk replacer from birth altered porcine uterine, cervical, and testicular development by postnatal Day 2. Moreover, insufficient colostrum consumption in nursing piglets can impair uterine capacity to support viable litters of optimal size in adulthood. In the pig, lactocrine signaling supports neonatal organizational events associated with normal reproductive development and may program adult uterine capacity.

Effects of age, nursing, and oral IGF1 supplementation on neonatal porcine cervical development.

Nursing supports neonatal porcine uterine and testicular development, however, lactocrine effects on cervical development are undefined. Studies were conducted to determine the effects of i) age and the imposition of the lactocrine-null state from birth (postnatal day 0 (PND0)) by milk replacer feeding on cervical histology; ii) imposition of the lactocrine-null state for 2 days from birth on cervical cell proliferation, as reflected by proliferating cell nuclear antigen immunostaining; and iii) a single feeding of colostrum or milk replacer, administered at birth, with or without oral IGF1, on cervical cell proliferation and phosphorylated AKT (pAKT) and B-cell lymphoma 2 (BCL2) protein levels at 12 h postnatal. Cervical crypt depth and height of luminal epithelium (LE) increased with age by PND14, when both responses were reduced in replacer-fed gilts. Cell proliferation was reduced in LE at PND2, and in crypt epithelium and stroma by PND14 in replacer-fed gilts. Returning replacer-fed gilts to nursing on PND2 did not rescue the cervical phenotype by PND14. A single feeding of colostrum, but not milk replacer, was sufficient to support cervical cell proliferation at 12 h postnatal. IGF1 supplementation induced cell proliferation in replacer-fed gilts, and increased cervical pAKT and BCL2 levels in colostrum-fed gilts and replacer-fed gilts at 12 h postnatal. Results indicate that age and nursing support porcine cervical development, support is initiated at first ingestion of colostrum, IGF1 may be lactocrine-active, and identification of lactocrine-active factors can be accomplished by 12 h postnatal using this bioassay system.

Nursing for 48 hours from birth supports porcine uterine gland development and endometrial cell compartment-specific gene expression.

The first 2 wk of neonatal life constitute a critical period for estrogen receptor alpha (ESR1)-dependent uterine adenogenesis in the pig. A relaxin receptor (RXFP1)-mediated, lactocrine-driven mechanism was proposed to explain how nursing could regulate endometrial ESR1 and related gene expression events associated with adenogenesis in the porcine neonate during this period. To determine effects of nursing on endometrial morphogenesis and cell compartment-specific gene expression, gilts (n = 6-8/group) were assigned at birth to be either 1) nursed ad libitum for 48 h, 2) gavage fed milk replacer for 48 h, 3) nursed ad libitum to Postnatal Day (PND) 14, or 4) gavage fed milk replacer for 48 h followed by ad libitum nursing to PND 14. Uteri were collected on PND 2 or PND 14. Endometrial histoarchitecture and both ESR1 and proliferating cell nuclear antigen (PCNA) labeling indexes (LIs) were evaluated. Laser microdissection was used to capture epithelium and stroma to evaluate treatment effects on cell compartment-specific ESR1, VEGFA, and RXFP1 expression. Imposition of a lactocrine-null state by milk replacer feeding for 48 h from birth retarded endometrial development and adenogenesis. Effects of replacer feeding, evident by PND 2, were marked by PND 14 when endometrial thickness, glandularity, and gland depth were reduced. Consistently, in lactocrine-null gilts, PCNA LI was reduced in glandular epithelium (GE) and stroma on PND 14, when epithelial ESR1 expression and ESR1 LI in GE were reduced and stromal VEGFA and RXFP1 expression increased. Results establish that lactocrine signaling effects morphogenetic changes in developing uterine tissues that may determine reproductive capacity later in life.

Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus.

Lactocrine communication of milk-borne bioactive factors (MbFs) from mother to offspring through nursing can affect neonatal development with lasting consequences. Relaxin (RLX), a lactocrine-active peptide found in porcine colostrum, stimulates estrogen receptor-α (ESR1) expression required for uterine development shortly after birth (postnatal day=PND 0). Whether other MbFs or cooperative lactocrine mechanisms affect the neonatal uterine developmental program is unknown. To determine the effects of age, nursing, and exogenous RLX on gene expression associated with uterine development, gilts (n=4-5/group) were assigned to nurse ad libitum or to receive milk replacer, with or without exogenous RLX (20 µg/kg BW i.m./6 h for 48 h), from birth to PND 2 when uteri were collected. Body weight and uterine weight increased (P<0.05) similarly from birth to PND 2 in all gilts. However, colostrum consumption was required for normal uterine ESR1, vascular endothelial growth factor (VEGFA), matrix metalloproteinase 9 (MMP9), and RLX receptor (RXFP1) protein and/or transcript expression on PND 2. Uterine ESR1, VEGFA, and MMP9 protein levels were below (P<0.01) the assay sensitivity in replacer-fed gilts. Supplemental RLX increased (P<0.05) uterine ESR1 protein and mRNA in nursed gilts, as well as VEGFA protein in nursed and VEGFA mRNA in both nursed and replacer-fed gilts. RLX treatment did not affect uterine MMP9 mRNA levels. When compared with replacer-fed gilts on PND 2, uterine RXFP1 mRNA was reduced (P<0.05) in nursed gilts and in RLX-supplemented replacer-fed gilts. These results constitute the first evidence that establishment of the neonatal porcine uterine developmental program requires maternal lactocrine support.

Transient estrogen exposure from birth affects uterine expression of developmental markers in neonatal gilts with lasting consequences in pregnant adults.

Disruption of estrogen-sensitive, estrogen receptor (ER)-dependent events during porcine uterine development between birth (postnatal day=PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Objectives here were to determine effects of EV exposure from birth through PND 13 on neonatal uterine and adult endometrial markers of growth, patterning, and remodeling. Targets included the relaxin receptor (RXFP1), estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), morphoregulatory markers HOXA10 and WNT7A, and the matrix metalloproteinases (MMP)2 and MMP9. Gilts were treated daily with EV (50 microg/kg body weight per day, i.m.) or corn oil vehicle from birth through PND 13. Uteri were obtained from neonates on PND 14 and from adults on pregnancy day 12 (PxD 12). In neonates, EV exposure from birth increased uterine RXFP1 gene expression, and both ESR1 and VEGFA proteins. At PxD 12, endometrial RXFP1 mRNA remained elevated, while ESR1 protein was reduced. Early EV treatment decreased neonatal uterine WNT7A, but increased HOXA10 expression. WNT7A expression was reduced in EV-treated adults. Transient EV exposure increased MMP9 transcripts at PND 14, whereas both latent and active MMP9 activity was increased due to early EV treatment in adults on PxD 12. Results support the hypothesis that transient, estrogen-induced disruption of porcine uterine development from birth alters early programming events that lead to functional consequences in the adult.

Equine endometrial development during late fetal and postnatal periods.

Equine uterine development, including endometrial histogenesis, begins prenatally and is completed postnatally. Little is known about this process in the horse. Uterine tissue was acquired from 38 foals, ranging in developmental age from gestational day (GD) 300 to postnatal day (PND) 180, for assessment of endometrial histogenesis. Patterns of endometrial cell proliferation were evaluated by multispectral imaging of uterine tissue sections stained immunofluorescently for Ki-67. Labeling index (LI, % labeled cells) for Ki-67 was calculated for each endometrial cell compartment (luminal epithelium, glandular epithelium, stroma). Histologically, nascent endometrial glands were present in all pre- and postnatal uterine tissues. Overall, Ki-67 LI increased (P < 0.0001) from the pre-to postnatal periods, and was higher (P < 0.0001) in epithelium as compared to stroma. Postnatally, endometrial Ki-67 LI increased (P < 0.0001) from week 1 to week 24. Our findings confirm that, in contrast to neonatal patterns of uterine development described for domestic ungulates, equine endometrial histogenesis begins prenatally, marked by the appearance of uterine glands as early as GD 300. Epithelial proliferation associated with maturation of the equine endometrium is pronounced by postnatal week 24.

Relaxin (RLX) and estrogen affect estrogen receptor alpha, vascular endothelial growth factor, and RLX receptor expression in the neonatal porcine uterus and cervix.

The porcine female reproductive tract undergoes estrogen receptor (ER) alpha-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERalpha, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17beta (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERalpha increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERalpha and VEGF expression may be important for neonatal reproductive tract development.

Endosymbionts of Siboglinum fiordicum and the phylogeny of bacterial endosymbionts in Siboglinidae (Annelida).

Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known-vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic gamma-proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of gamma-proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.

Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin.

In the pig, temporospatially regulated proliferation of uterine luminal (LE) and glandular (GE) epithelium between birth (postnatal day=PND 0) and PND 15 is essential for success of endometrial development. Exposure of gilts to estrogen (E) or relaxin (RLX) during this period disrupts uterine development, and neonatal E exposure can compromise adult uterine function. Neonatal uterotrophic effects of E and RLX, administered for 2 days beginning on PND 12, can be inhibited with the antiestrogen ICI 182,780 (ICI) indicating crosstalk between RLX and E signaling systems. Here, objectives were to determine effects of: (study 1) neonatal age and (study 2) exposure to E, RLX, and ICI on porcine neonatal uterine histoarchitecture and patterns of epithelial cell proliferation as reflected by proliferating cell nuclear antigen labeling index. In study 1, uteri were obtained on PND 0, 3, 6, 9, 12 and 15. Glandular epithelium, absent at birth, was observed by PND 3. Overall, epithelial labeling index increased from birth to PND 3, declined from PND 6-9 in LE and GE, and increased to PND 15 in GE. In study 2, uteri were collected on PND 14 after administration of vehicle, E, or RLX for 2 days, or following pretreatment with ICI. Alone, E was uterotrophic and adenogenic and increased labeling index in both LE and GE. Both RLX and ICI increased proliferation in GE. Effects of E and RLX were attenuated by ICI, providing further support for crosstalk between these signaling systems in the developing neonatal porcine endometrium.

Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling.

Estrogen receptor-dependent organizational events between birth [postnatal day (PND) 0] and PND 14 affect development and function of porcine uterine tissues. Observations that uterotrophic effects of relaxin (RLX) in neonatal gilts were inhibited by the antiestrogen ICI 182,780 suggested that a RLX signaling system, capable of cross-talk with the estrogen receptor, evolves during a critical period for uterine programming (PND 0-14). Objectives were to determine 1) effects of age and estrogen exposure from birth on porcine uterine RLX/insulin-like 3 receptor (LGR7/LGR8) expression and 2) whether milk serves as a natural source of RLX in neonatal pigs. Uterine LGR7/LGR8 expression, detected by RT-PCR and in situ hybridization on PND 0, 7, and 14, was predominantly stromal for LGR7, myometrial for LGR8, and increased with age and after treatment with estradiol valerate (50 microg/kg body weight x d) from birth. Stromal expression of LGR7 was also detected immunohistochemically. Milk RLX concentrations declined (P < 0.001) from 17.3 +/- 1.4 ng/ml (lactation d 0) to 1.7 +/- 0.3 ng/ml (lactation d 14). RLX, present in the serum of nursing pigs on PND 0 and 1, was undetectable before nursing and in neonates fed RLX-free milk replacer for 12 h. Thus, a developmentally regulated, estrogen-sensitive LGR7 and LGR8 receptor system is present in the porcine uterus at birth and may be activated by milk-borne RLX delivered into the circulation during the first 48 h of postnatal life. Maternal lactocrine contributions to the neonatal hormonal milieu could affect the developmental programming of uterine and other somatic tissues.

Tissue-specific effects of relaxin on the reproductive tract of neonatal gilts.

In the pig, relaxin treatment for 2 d from birth (postnatal day 0, PND 0) was not uterotrophic, but it increased cervical size. To understand the mechanism underlying this tissue-specific response, relaxin receptor (LGR7) expression in the neonatal uterus and cervix was investigated. At PND 2, quantitative RT-PCR analysis showed that LGR7 levels were higher in the cervix than in the uterus. Interestingly, relaxin decreased cervical LGR7 expression when compared to that of the controls. Differential expression of LGR7 between uterine and cervical tissues may contribute to tissue-specific relaxin responsiveness in the neonatal porcine reproductive tract.

Effects of relaxin on neonatal porcine uterine growth and development.

Relaxin (RLX), a key reproductive hormone in pigs, stimulates uterine growth in pregnant and prepubertal gilts and in neonates 2 weeks after birth. The neonatal uterotrophic response to RLX is developmentally regulated and estrogen receptor dependent because RLX fails to increase uterine weight in the absence of estrogen receptor (ER)-alpha or when the ER is chemically inactivated. However, the role of RLX and insulin-like peptide-3 receptors, LGR7 and LGR8, respectively, in the neonatal uterotrophic response is unknown. Current studies focus on direct (LGR7/8-mediated) and indirect (ER-mediated) effects of RLX in the neonatal porcine uterus. Porcine LGR7 and LGR8 cDNAs were cloned and used as probes to identify uterine transcripts for LGR7 and LGR8, which increased from birth (postnatal day [PND] 0) to PND 14, a critical period for porcine uterine development. In situ hybridization showed that endometrial signals for both LGR7 and LGR8 are predominantly stromal during this period. Administration of RLX on PND 0, before onset of uterine ER expression, increased uterine luminal epithelial height (P < .05) but not uterine weight in the LGR7/8-positive uterus on PND 2. However, RLX increased both uterine weight and luminal epithelial height by PND 14 (P < .05), after overt endometrial ER expression. Aberrant ER activation between PND 0 and 14 alters the uterine organizational program and affects the function of adult porcine uterine tissues. Present data suggest that crosstalk between LGR7/8 and ER may be involved in estrogen-sensitive morphoregulatory events that are central to the development of an optimally functional adult uterus in the pig.

Relaxin and maternal lactocrine programming of neonatal uterine development.

In mammals, including the pig (Sus scrofa domesticus), structural patterning and functional programming of uterine tissues involve events that occur shortly after birth. Porcine endometrial development between birth (postnatal day 0 [PND 0]) and PND 15 is estrogen receptor (ER) dependent and estrogen sensitive. The endometrium is relaxin (RLX) receptor (RXFP1) positive and ERalpha negative at birth. Uterine expression of RXFP1 and ERalpha, detectable by PND 2, increases with age from PND 0 to 14. Estrogen exposure during this period sufficient to affect uterine developmental trajectory and adult uterine phenotype also alters uterine RXFP1 gene expression patterns in the neonatal uterus. Data implicate RXFP1 as an element of the uterine developmental program. Uterotrophic effects documented for both estrogen and RLX in the neonatal pig are age-specific and most pronounced after onset of ERalpha expression. Patterns of inhibition of RLX effects on uterine development induced with ICI 182,780, an ER antagonist, indicate that cross talk between RLX and estrogen signaling systems evolve with age in the porcine uterus. Given that RLX administered from birth stimulates uterine ERalpha expression and that estrogen administered from birth stimulates RXFP1 expression by PND 2, a feed-forward relationship between these signaling systems is envisioned. Evidence that RLX is present in porcine milk and in the circulation of nursing offspring supports the lactocrine hypothesis for maternal programming of uterine tissues in which milk-borne RLX, acting via RXFP1, sustains ERalpha expression and porcine endometrial development in the neonate.

Perinatal zearalenone exposure affects RXFP1, RXFP2, and morphoregulatory gene expression in the neonatal porcine uterus.

The mycotoxin zearalenone (ZEA) is a selective estrogen receptor modulator that can contaminate cereal feeds and lead to reproductive disorders. To determine effects of perinatal ZEA exposure on uterine expression of genes associated with endometrial development in the neonatal gilt, pregnant sows were fed ZEA (1500 microg ZEA/kg of feed/day) or vehicle from 14 days before farrowing through postnatal day (PND) 20-21, when neonatal uterine tissues were collected. At birth, gilts were cross-fostered to generate four ZEA exposure groups (n= 5-6/group): unexposed controls or exposures limited to prenatal, postnatal, or pre- and postnatal (continuous) periods. Results showed that at PND 20-21, uterine Wnt7a, Hoxa10, estrogen receptor alpha, and RXFP2 mRNA levels were decreased in neonates exposed continuously to ZEA (P < 0.05). Uterine RXFP1 transcripts were decreased in postnatally and continuously exposed groups (P < 0.05). Neonatal uterine Wnt4 mRNA levels were unchanged.

Laser microdissection of neonatal porcine endometrium for tissue-specific evaluation of relaxin receptor (RXFP1) expression in response to perinatal zearalenone exposure.

Porcine neonatal uterine relaxin receptor (RXFP1) expression is tissue compartment specific and estrogen sensitive. Here, procedures were established for laser microdissection, tissue capture, and quantification of the effects of perinatal exposure (14 days pre- to 21 days postnatal) to a selective estrogen receptor modulator of environmental origin, zearalenone (ZEA), on endometrial RXFP1 expression. Total RNA from captured endometrium was used to generate cDNA for quantitative reverse transcription-PCR. Cycle threshold values indicated that ZEA reduced (P < 0.06) endometrial RXFP1 expression on postnatal days 20-21.

Estrogen-induced disruption of neonatal porcine uterine development alters adult uterine function.

In the pig, estradiol-17beta valerate (EV) exposure from birth (Postnatal Day [PND] 0) disrupts estrogen receptor-alpha (ER)-dependent uterine development and increases embryo mortality in adults. To determine effects of neonatal EV exposure on adult uterine morphology and function, 36 gilts received corn oil (CO) or EV from PND 0 to PND 13. Cyclic and pregnant (PX) adults from each treatment group were hysterectomized on Day 12 after estrus/mating. Treatment and pregnancy effects were determined for uterine weight and horn volume, uterine luminal fluid (ULF) protein and estradiol content, endometrial incorporation of 3H-leucine (3H-Leu) into nondialyzable product, and endometrial mRNA levels for ER, progesterone receptor (PR), uteroferrin (UF), retinol-binding protein (RBP), and keratinocyte growth factor (KGF). Adults cycled normally and had similar numbers of corpora lutea. Uteri of PX gilts contained tubular/filamentous conceptuses, and ULF estradiol content was unaffected by treatment. However, pregnancy increased uterine weight and size only in CO gilts (Treatment x Status, P < 0.01). Treatment reduced ULF protein content (P < 0.01), endometrial 3H-Leu incorporation (P < 0.05), and the pregnancy-associated increase in ULF protein (Treatment x Status, P < 0.01). Treatment did not affect endometrial ER or PR mRNA levels but attenuated the pregnancy-associated increase in UF mRNA (Treatment x Status; P < 0.01), increased RBP (P < 0.10), and decreased KGF mRNA levels (P < 0.05). These results establish that transient postnatal estrogen exposure affects porcine uterine responsiveness to potentially embryotrophic signals and that estrogen-sensitive postnatal uterine organizational events are determinants of uterine size and functionality.