Anne A. Wiley

Developmental biology of uterine glands

Abstract 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-17β, 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.

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 β-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 th...

Ovine Uterine Gland Knock-Out Model: Effects of Gland Ablation on the Estrous Cycle

Abstract 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 F2∝ (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.

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.

IMMUNOLOCALIZATION OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTOR IN THE DEVELOPING TESTIS AND EXCURRENT DUCTS OF GOATS

Because of the significance of androgens and estrogens in prenatal and postnatal differentiation of the testis and excurrent ducts, it is important to understand the developmental pattern of androgen receptor (AR) and estrogen receptor (ER) in these organs.

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.

Regulation of androgen and estrogen receptors in male excurrent ducts of the goat: An immunohistochemical study

Since androgens and/or estrogens must bind with specific receptors in order to elicit a response at the target organ(s), it is important to understand factors that regulate expression of androgen receptors (AR) and estrogen receptors (ER). Hence, the objective of the study is to determine the relative significance between circulating androgen (CA) and luminal androgen (LA) in maintaining normal expression of AR and ER in male excurrent ducts.

Relaxin (RLX) and estrogen affect estrogen receptor α, 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.

Abnormal Morphology of the Penis in Male Rats Exposed Neonatally to Diethylstilbestrol Is Associated with Altered Profile of Estrogen Receptor-α Protein, but Not of Androgen Receptor Protein: A Developmental and Immunocytochemical Study

Abstract Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)α in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 μg 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, ERα-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 ERα.

Neonatal exposure to progesterone and estradiol alters uterine morphology and luminal protein content in adult beef heifers.

Exposure of the developing urogenital tract to steroids can affect structure and function of adult tissues and compromise reproductive performance. This study was conducted to determine 1) if exposure of neonatal heifer calves to progesterone (P) and estradiol benzoate (E), delivered from a commercial growth-promoting implant, would affect adult uterine morphology or uterine luminal protein content; and 2) whether such effects would be related to neonatal age at the first exposure. At birth (Day 0), 20 crossbred beef heifers were assigned to 1 of 4 treatment groups (n = 5 per group), defined by age at implant placement. Heifers either received an implant on Days 0, 21 or 45, or served as untreated controls. The heifers were maintained together and slaughtered at 15 mo of age, during the luteal phase of an induced estrous cycle, when reproductive tracts and blood samples were obtained. Peripheral plasma P concentrations were determined by RIA. Uterocervical wet weights were recorded, and uterine luminal flushings (ULF) were assayed for total protein. Cross-sections of uterine tissues were evaluated histomorphometrically to determine myometrial and endometrial areas and relative endometrial gland density. Treatment did not affect plasma P concentrations (3.2 +/- 0.5 ng/ml). Regardless of age at treatment, neonatal PE exposure reduced uterocervical wet weight by 35% (112.8 < 173.9 +/- 13.9 g; P < 0.01), myometrial area by 23% (125.3 < 162.8 +/- 8.5 mm2; P < 0.02), and endometrial area by 27% (33.3 < 45.4 +/- 2.7 mm2; P < 0.09) compared with the untreated controls. Endometrial gland density was reduced (P < 0.01) by 40% in treated heifers. This effect was related to age at implant placement. Uterine gland density was reduced (P < 0.01) by 65% in heifers treated at birth, while reductions of 22 and 33% were observed for heifers treated on neonatal Day 21 or 45, respectively. Consistently, ULF protein content was lower (P < 0.01) in the treated heifers (2.67 < 4.98 +/-. 72 mg/ULF). Thus, exposure of newborn calves to PE can have profound effects on adult uterine morphology and environment, the extent of which may depend upon the developmental period when exposure occurs. The potential of such alterations to affect reproductive performance in adult beef heifers remains to be investigated.