Review: Maternal programming of development in the pig and the lactocrine hypothesis.

Maternal effects on development are profound. Together, genetic and epigenetic maternal effects define the developmental trajectory of progeny and, ultimately, offspring phenotype. Maternally provisioned environmental conditions and signals affect conceptus, fetoplacental and postnatal development from the time of conception until weaning. In the pig, reproductive tract development is completed postnatally. Porcine uterine growth and uterine endometrial development occur in an ovary-independent manner between birth (postnatal day = PND 0) and PND 60. Milk-borne bioactive factors (MbFs), exemplified by relaxin, communicated from lactating dam to nursing offspring via a lactocrine mechanism, represent an important source of extraovarian uterotrophic support in the neonatal pig. Lactocrine deficiency from birth affects both the neonatal porcine uterine developmental program and trajectory of uterine development, with lasting consequences for endometrial function and uterine capacity in adult female pigs. The potential lactocrine signaling window extends from birth until the time of weaning. However, it is likely that the maternal lactocrine programming window - that period when MbFs communicated to nursing offspring have the greatest potential to affect critical organizational events in the neonate - encompasses a comparatively short period of time within 48 h of birth. Lactocrine deficiency from birth was associated with altered patterns of endometrial gene expression in neonatally lactocrine-deficient adult gilts during a critical period for conceptus-endometrial interaction on pregnancy day 13, and with reduced litter size, estimated at 1.4 pigs per litter, with no effect of parity. Data were interpreted to indicate that reproductive performance of female pigs that do not receive sufficient colostrum from birth is permanently impaired. Observations to date suggest that lactocrine-dependent maternal effects program postnatal development of the porcine uterus, endometrial functionality and uterine capacity. In this context, reproductive management strategies and husbandry guidelines should be refined to ensure that such practices promote environmental conditions that will optimize uterine capacity and fecundity. This will entail careful consideration of factors affecting lactation, the quality and abundance of colostrum/milk, and practices that will afford neonatal pigs with the opportunity to nurse and consume adequate amounts of colostrum.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Postnatal reproductive development and the lactocrine hypothesis.

Maternal effects on development can program cell fate and dictate offspring phenotype. Such effects do not end at birth, but extend into postnatal life through signals communicated from mother to offspring in first milk (colostrum). Transmission of bioactive factors from mother to offspring as a specific consequence of nursing defines a lactocrine mechanism. The female reproductive tract is not fully formed at birth (postnatal day = PND 0). Data for ungulates and mice indicate that disruption of development during neonatal life can have lasting effects on the form and function of uterine tissues. Uterine growth and histogenesis proceed in an ovary-independent manner shortly after birth, suggesting that extra-ovarian inputs are important in this process. Data for the pig indicate that lactocrine signals communicated within 12 to 48 h from birth constitute one source of such uterotrophic support. Disruption of lactocrine signaling, either naturally, by limited colostrum consumption, or experimentally, by milk replacer feeding, alters neonatal porcine uterine development and can have negative consequences for reproductive performance in adults. Substantial differences in endometrial and uterine gene expression between colostrum- and replacer-fed gilts were evident by PND 2, when RNA sequencing revealed over 800 differentially expressed, lactocrine-sensitive genes. Lactocrine-sensitive biological processes identified through transcriptomic studies and integrated microRNA-mRNA pathway analyses included those associated with both cell-cell and ESR1 signaling, and tissue development. Evidence for the pig indicates that colostrum consumption and lactocrine signaling are required to establish a normal uterine developmental program and optimal uterine developmental trajectory.

Timing and duration of nursing from birth affect neonatal porcine uterine matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1.

Nursing for 2 d from birth supports neonatal porcine uterine and cervical development. However, it is not clear how timing or duration of lactocrine signaling from birth (postnatal day = PND 0) affects development of neonatal female reproductive tract tissues. Therefore, studies were conducted to determine effects of age at first nursing and duration of nursing from birth on specific elements of the matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system in uterine and cervical tissues at PND 2. When nursing was initiated at 0 h or 30 min of age, targeted proteins, including proMMP9 and MMP9, were detected in uterine and cervical tissues on PND 2, as was uterine TIMP1. However, these proteins were undetectable when nursing was delayed for 12 h and when gilts were fed milk replacer for 48 h from birth. Increasing the duration of nursing from 30 min to 12 h from birth increased uterine (P < 0.05) and cervical (P < 0.001) MMP9 levels to those observed in gilts nursed for 48 h. Similarly, uterine TIMP1 levels increased with duration of nursing. Uterine MMP2 levels were detectable but unaffected by age at first nursing or duration of nursing from birth. Uterine MMP2 and MMP9 activities, monitored by zymography, reflected immunoblotting data. Results provide evidence for the utility of MMP9 and TIMP1 as markers of age- and lactocrine-sensitive porcine female reproductive tract development.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways.

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and ß (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Nursing supports neonatal porcine testicular development.

The lactocrine hypothesis suggests a mechanism whereby milk-borne bioactive factors delivered to nursing offspring affect development of neonatal tissues. The objective of this study was to assess whether nursing affects testicular development in neonatal boars as reflected by: (1) Sertoli cell number and proliferation measured by GATA-4 expression and proliferating cell nuclear antigen immunostaining patterns; (2) Leydig cell development and steroidogenic activity as reflected by insulin-like factor 3 (INSL3), and P450 side chain cleavage (scc) enzyme expression; and (3) expression of estrogen receptor-alpha (ESR1), vascular endothelial growth factor (VEGF) A, and relaxin family peptide receptor (RXFP) 1. At birth, boars were randomly assigned (n = 6-7/group) to nurse ad libitum or to be pan fed porcine milk replacer for 48 h. Testes were collected from boars at birth, before nursing and from nursed and replacer-fed boars at 50 h on postnatal day (PND) 2. Sertoli cell proliferating cell nuclear antigen labeling index increased (P < 0.01) from birth to PND 2 in nursed, but not in replacer-fed boars. Sertoli cell number and testicular GATA-4 protein levels increased (P < 0.01) from PND 0 to PND 2 only in nursed boars. Neither age nor nursing affected testicular INSL3, P450scc, ESR1, or VEGFA levels. However, testicular relaxin family peptide receptor 1 (RXFP1) levels increased (P < 0.01) with age and were greater in replacer-fed boars on PND 2. Results suggest that nursing supports neonatal porcine testicular development and provide additional evidence for the importance of lactocrine signaling in pigs.

Lactation Biology Symposium: lactocrine signaling and developmental programming.

Lactocrine signaling is defined as transmission 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 postnatally. Dependence on maternal resources for development in eutherian mammals extends into neonatal life for at least that period of time when nutrition is obtained solely from first milk (i.e., colostrum). Data for the pig (Sus scrofa domesticus) provide evidence of lactocrine mediated effects on development of the female reproductive 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 process was proposed as a regulatory mechanism governing 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 endometrial and cervical ESR1 and vascular endothelial growth factor (VEGFA) expression observed on PND 2 persisted to PND 14, even after gilts were returned to nursing on PND 2. Collectively, data support a role for lactocrine signaling in regulation of critical neonatal developmental events. Maternal lactocrine programming of postnatal development may help to insure healthy developmental outcomes. A systems biology approach will be required to define and understand mechanistic dynamics of lactocrine signaling events that may ultimately connect genotype to phenotype and establish the parameters of reproductive potential.

Uterine gland development begins postnatally and is accompanied by estrogen and progesterone receptor expression in the dog.

During neonatal and juvenile life, mammalian uteri undergo extensive structural and functional changes, including uterine gland differentiation and development. In sheep and mice, inhibition of neonatal uterine gland development induced by progestin treatment led to a permanent aglandular uterine phenotype and adult infertility, suggesting that this strategy might be useful for sterilizing dogs and other companion animals. The goal of this study was to define temporal patterns of adenogenesis (gland development), cell proliferation, and progesterone and estrogen receptor expression in uteri of neonatal and juvenile dogs as a first step toward determining whether neonatal progestin treatments might be a feasible contraceptive approach in this species. Uteri obtained from puppies at postnatal wk 1, 2, 4, 6, or 8 were evaluated histologically and immunostained for MKI67, a marker of cell proliferation, estrogen receptor-1, and progesterone receptor. Adenogenesis was under way at 1 wk of age, as indicated by the presence of nascent glands beginning to bud from the luminal epithelium, and rapid proliferation of both luminal epithelial and stromal cells. By Week 2, glands were clearly identifiable and proliferation of luminal, glandular, and stromal cells was pronounced. At Week 4, increased numbers of endometrial glands were evident penetrating uterine stroma, even as proliferative activity decreased in all cell compartments as compared with Week 2. Whereas gland development was most advanced at Weeks 6 to 8, luminal, glandular, and stromal proliferation was minimal, indicating that the uterus was nearly mitotically quiescent at this age. Both estrogen receptor-1 and progesterone receptor were expressed consistently in uterine stromal and epithelial cells at all ages examined. In summary, canine uterine adenogenesis was underway by 1 wk of age and prepubertal glandular proliferation was essentially complete by Week 6. These results provided information necessary to facilitate development of canine sterilization strategies based on neonatal progestin treatments designed to permanently inhibit uterine gland development and adult fertility.

Xenomitochondrial mice: investigation into mitochondrial compensatory mechanisms.

Xenomitochondrial mice, harboring evolutionarily divergent Mus terricolor mitochondrial DNA (mtDNA) on a Mus musculus domesticus nuclear background (B6NTac(129S6)-mt(M. terricolor)/Capt; line D7), were subjected to molecular and phenotypic analyses. No overt in vivo phenotype was identified in contrast to in vitro xenomitochondrial cybrid studies. Microarray analyses revealed differentially expressed genes in xenomitochondrial mice, though none were directly involved in mitochondrial function. qRT-PCR revealed upregulation of mt-Co2 in xenomitochondrial mice. These results illustrate that cellular compensatory mechanisms for mild mitochondrial dysfunction alter mtDNA gene expression at a proteomic and/or translational level. Understanding these mechanisms will facilitate the development of therapeutics for mitochondrial disorders.

Epigenetic programming of porcine endometrial function and the lactocrine hypothesis.

Epigenetic programs controlling development of the female reproductive tract (FRT) are influenced by the effects of naturally occurring bioactive agents on patterns of gene expression in FRT tissues during organizationally critical periods of foetal and perinatal life. Aberrations in such important cellular and molecular events, as may occur with exposure to natural or manmade steroid or peptide receptor-modulating agents, disrupt the developmental program and can change the developmental trajectory of FRT tissues, including the endometrium, with lasting consequences. In the pig, as in other mammals, maternal programming of FRT development begins pre-natally and is completed post-natally, when maternal effects on development can be communicated via signals transmitted in milk. Studies involving relaxin (RLX), a prototypic milk-borne morphoregulatory factor (MbF), serve as the basis for ongoing efforts to identify maternal programming events that affect uterine and cervical tissues in the neonatal pig. Data support the lactocrine hypothesis for delivery of MbFs to neonates as a specific consequence of nursing. Components of a maternally driven lactocrine mechanism for RLX-mediated signalling in neonatal FRT tissues, including evidence that milk-borne RLX is delivered into the neonatal circulation where it can act on RLX receptor (RXFP1)-positive neonatal tissues to affect their development, are in place in the pig. The fact that all newborn mammals drink milk extends the timeframe of maternal influence on neonatal development across many species. Thus, lactocrine transmission of milk-borne developmental signals is an element of the maternal epigenetic programming equation that deserves further study.

Uterine development and endometrial programming.

Structural patterning and functional programming of uterine tissues are mechanistically coupled. These processes ensure anteroposterior differentiation of uterine tissues from adjacent segments of the developing female reproductive tract (FRT) and radial patterning that establishes uterine-specific histoarchitecture and functionality. Uterine organogenesis begins prenatally and is completed postnatally. Genes required for FRT development include Pax2, Lim1 and Emx2, genes in the abdominal-B Hoxa cluster, and members of both Wnt and Hedgehog (Hh) gene families. Disruption of morphoregulatory gene expression patterns can prevent FRT development entirely or compromise uterine organogenesis specifically. Oestrogen receptor-alpha (ER) -dependent events associated with development of the neonatal porcine uterus can be altered by administration of oestrogen (E) or relaxin (RLX). Expression of the RLX receptor is detectable in porcine endometrium at birth, before onset of ER expression and uterine gland genesis. Uterotrophic effects of both E and RLX can be inhibited with the ER antagonist ICl 182,780, indicating that RLX may act via crosstalk with the ER system in neonatal tissues. Exposure of neonatal gilts to E alters temporospatial patterns of Hh, Wnt and Hoxa expression in the uterine wall. Oestrogen given for two weeks from birth produced hypoplastic adult porcine uteri that were less responsive to periattachment conceptus signals as reflected by reduced growth response and luminal fluid protein accumulation, altered endometrial gene expression, and reduced capacity for conceptus support. Data reinforce the concept that factors affecting signalling events in uterine tissues that produce changes in morphoregulatory gene expression patterns during critical organisational periods can alter the developmental trajectory of the uterus with lasting consequences. Thus, uterine tissues can be programmed epigenetically for success or failure during perinatal life.

Abnormal morphology of the penis in male rats exposed neonatally to diethylstilbestrol is associated with altered profile of estrogen receptor-alpha protein, but not of androgen receptor protein: a developmental and immunocytochemical study.

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.

Neonatal estradiol exposure alters uterine morphology and endometrial transcriptional activity in prepubertal gilts.

Porcine endometrial development between birth (postnatal day = PND 0) and PND 56 involves differentiation of glandular epithelium (GE) from luminal epithelium (LE) and estrogen receptor-alpha (ER) expression. Juvenile ER architecture evolves after birth, as stroma and nascent GE first express ER. Mature ER architecture is evident after PND 30, when stroma, GE and LE are ER-positive. When administered during discrete periods between PND 0 and 56, effects of estradiol-17beta valerate (EV) on the neonatal porcine uterus relate to endometrial ER architecture. Transient EV exposure from birth reduces embryo survival in pregnant adult gilts. Effects of EV, administered as juvenile endometrial ER architecture develops (P1, PND 0-13), or after mature ER architecture is established (P2, PND 42-55), were evaluated in uteri from gilts treated with corn oil or EV in P1 or P2 and hysterectomized on PND 100 without additional steroids (NSt), on PND 102 after EV on PND100-101 (EV2), or on PND 117 after EV2 followed by progesterone on PND 102-116 (EP). Neonatal EV reduced uterine weight (P < 0.02), size (P < 0.01), luminal protein content (P < 0.07), and percent incorporation of 3H-leucine into nondialyzable endometrial products in vitro (P < 0.01). Group (NSt, EV2, EP) -specific treatment effects detected for endometrial ER, progesterone receptor, uteroferrin, and/or retinol binding protein mRNA levels were frequently related to period (P1,P2). Results support the idea that estrogen-sensitive postnatal organizational events, including those defined, in part, by endometrial ER architecture, are likely components of genetic and epigenetic programs governing uterine morphogenesis and ontogeny of endometrial function in the pig.

Developmental biology of uterine glands.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.

Endometrial glands are required for preimplantation conceptus elongation and survival.

Endometrial glands secrete molecules hypothesized to support conceptus growth and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by neonatal progestin exposure. The resulting stable adult uterine gland knockout (UGKO) phenotype was used here to test the hypothesis that endometrial glands are required for successful pregnancy. Mature UGKO ewes were bred repeatedly to fertile rams, but no pregnancies were detected by ultrasound on Day 25. Day 7 blastocysts from normal superovulated ewes were then transferred synchronously into Day 7 control or UGKO ewes. Ultrasonography on Days 25-65 postmating indicated that pregnancy was established in control, but not in UGKO ewes. To examine early uterine-embryo interactions, four control and eight UGKO ewes were bred to fertile rams. On Day 14, their uteri were flushed. The uterus of each control ewe contained two filamentous conceptuses of normal length. Uteri from four UGKO ewes contained no conceptus. Uteri of three UGKO ewes contained a single severely growth-retarded tubular conceptus, whereas the remaining ewe contained a single filamentous conceptus. Histological analyses of these uteri revealed that endometrial gland density was directly related to conceptus survival and developmental state. Day 14 UGKO uteri that were devoid of endometrial glands did not support normal conceptus development and contained either no conceptuses or growth-retarded tubular conceptuses. The Day 14 UGKO uterus with moderate gland development contained a filamentous conceptus. Collectively, these results demonstrate that endometrial glands and, by inference, their secretions are required for periimplantation conceptus survival and development.

Expression and molecular characterization of estrogen receptor alpha messenger RNA in male reproductive organs of adult goats.

The fact that male estrogen receptor alpha (ERalpha) knockout mice are infertile indicates a role for this receptor in male reproduction. Here, objectives were to evaluate ERalpha expression in male goat reproductive tissues at the transcriptional level using RNase protection assay (RPA) and in situ hybridization (ISH), and to clone a partial cDNA for caprine ERalpha using reverse transcription-polymerase chain reaction (RT-PCR). For RPA and ISH procedures, a radiolabeled antisense cRNA probe, generated in vitro from the ovine oER8 cDNA template, was employed. Evaluations were made on individual samples obtained from adult goats. Labeled cRNA sense probe was used as a negative control in ISH. A 530-base pair amplicon was generated by RT-PCR from efferent ductules (EDs), epididymis (EP), and testis, cloned from the ED and EP, and sequenced. The caprine ERalpha (cERalpha) cDNA displayed 81%-96% sequence identity with that of other species. A signal indicative of ERalpha mRNA was identified by both RT-PCR and RPA in all tissues, but was strongest in the ED. Compared with ED, ERalpha signal was sixfold lower in the EP, and 66-fold lower in the testis. Similarly, strong ERalpha expression was observed in ED epithelium, whereas little or no signal was detected in EP or testis by ISH. Thus, among different segments of the male reproductive tract and testis, the highest level of ERalpha mRNA expression was found in epithelium of the ED.

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle.

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.

Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model.

Prolonged exposure of the developing neonatal ovine uterus to a progestin from birth prevents uterine gland development and creates an adult endometrial phenotype characterized by the absence of glandular epithelium, the uterine gland knockout (UGKO) phenotype. This study used endometrium from normal and UGKO sheep to identify messenger RNAs (mRNAs) expressed differentially in the endometrial epithelium using the molecular techniques of mRNA differential display PCR (DD-PCR) and suppression subtractive complementary DNA (cDNA) hybridization (SSH). Sequence analyses of DD- and SSH-identified and cloned cDNAs indicated similarity of some to known mRNAs, including beta-lactoglobulin, alkaline phosphatase, type B and D endogenous sheep retroviruses, gp330/megalin, matrix Gla protein, and others. Other cDNAs were not similar to any known sequences and are considered novel, although some of these match human expressed sequence tags. In situ hybridization analyses of uteri from cyclic and pregnant ewes indicated that all DD-PCR- and SSH-identified mRNAs were expressed in either the endometrial lumenal and/or glandular epithelium, although some were also expressed in other uterine cell types. Northern and in situ hybridization analyses revealed that patterns of mRNA expression for most clones were affected by the day of the estrous cycle and pregnancy in a manner consistent with regulation by progesterone. Studies demonstrate the utility of the ovine UGKO model as a tool with which to identify known and novel uterine epithelial-specific genes. Cloned cDNAs identified here are expressed sequence tags useful for comparative and physical genetic mapping and may be used to reveal new factors and pathways regulating endometrial function.

Endometrial development and adenogenesis in the neonatal pig: effects of estradiol valerate and the antiestrogen ICI 182,780.

In the pig, appearance of endometrial glands between birth (postnatal day [PND] 0) and PND 14 involves development of estrogen receptor-alpha-positive (ER+) phenotype by, and increased DNA synthesis in, nascent glandular epithelium (GE). To determine whether ER activation is required for this process, gilts were treated daily with either vehicle, the antiestrogen ICI 182,780 (ICI), estradiol-17beta valerate (EV), or both ICI and EV. Treatments began on PND 0, before onset of adenogenesis, or on PND 7, after onset of gland proliferation. Uteri obtained on PNDs 7 and 14 (study one) or on PND 14 (study two) were weighed; uterine histology was evaluated; DNA synthesis in luminal epithelium and GE was characterized by determining 5-bromo-2'-deoxyuridine (BrdU) labeling index; and patterns of ER mRNA expression were evaluated in situ (study one). Gland genesis was inhibited by ICI, which decreased gland penetration depth by PND 14 in study one, both endometrial thickness and BrdU-labeling index in GE in study two, and increased stromal cell compaction in both studies. Uterotropic effects of EV included increased gland development and epithelial BrdU labeling and decreased stromal compaction. These effects were inhibited by coadministration of ICI. Treatments did not alter ER mRNA expression, which remained limited to stroma and GE. Data indicate that endometrial maturation and adenogenesis in the neonatal pig require expression and activation of a functional ER system.

Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is an endothelial glycoprotein secreted in lymph nodes that serves as a ligand for leukocyte cell surface selectin and mediates lymphocyte extravasation. In the present studies, rabbit anti-rat GlyCAM-1 IgG was used in immunochemical analyses of GlyCAM-1-like protein in the ovine uterus. In cyclic ewes, GlyCAM-1 expression increased in the endometrial luminal epithelium (LE) and shallow glandular epithelium (cGE) between Days 1 and 5 and then decreased between Days 11 and 15. In pregnant ewes, GlyCAM-1 in the LE and cGE was low on Days 11 and 13, increased on Day 15, and was abundant on Days 17 and 19. Immunoreactive GlyCAM-1 was also detected in the conceptus trophectoderm on Days 13-19. Staining for GlyCAM-1 in the smooth muscle of the vasculature and myometrium was constitutive, and no staining was detected in the stroma. An immunoreactive protein of approximately 45 kDa was identified in endometrial extracts and uterine flushings from cyclic and pregnant ewes. In pregnant ewes, the relative amount of immunoreactive GlyCAM-1 in uterine flushings was low on Days 11 and 13 but high on Days 15 and 17. Results suggest that a GlyCAM-1-like protein may be a secretory product of the endometrial epithelium and/or conceptus trophectoderm. Patterns of distribution observed for immunoreactive GlyCAM-1-like protein in the endometrial epithelium, combined with proposed functions for lymphoid GlyCAM-1, suggest that this mucin glycoprotein may be involved in conceptus-maternal interactions during the periimplantation period of pregnancy in sheep.

Uterine differentiation as a foundation for subsequent fertility.

Uterine differentiation in cattle and sheep begins prenatally, but is completed postnatally. Mechanisms regulating this process are not well defined. However, studies of urogenital tract development in murine systems, particularly those involving tissue recombination and targeted gene mutation, indicate that the ideal uterine organizational programme evolves epigenetically through dynamic cell-cell and cell-matrix interactions that define the microenvironmental context within which gene expression occurs and may ensure adult tissue stability. In the cow and ewe, transient postnatal exposure of the developing uterus to steroids can produce immutable changes in adult uterine tissues that may alter the embryotrophic potential of the uterine environment. Thus, success of steroid-sensitive postnatal events supporting uterine growth and development can dictate the functional potential of the adult uterus. Studies to determine effects of specific steroidal agents on patterns of uterine development during defined neonatal periods, as well as the functional consequences of targeted neonatal steroid exposure in the adult uterus, should enable identification of critical developmental mechanisms and determinants of uterine integrity and function. Extreme adult uterine phenotypes (lesion models) created in cattle and sheep by strategic postnatal steroid exposure hold promise as powerful tools for the study of factors affecting uterine function and the rapid identification of novel uterine genes.