Helen M. Gehring

Mechanisms of relaxin action in the reproductive tract : Studies in the relaxin-deficient (Rlx-/-) mouse

Abstract: The major functions of relaxin (RLX) are associated with female reproductive tract physiology, namely, the regulation of biochemical processes involved in remodeling of extracellular matrix components in the cervix and vagina at term. Studies in RLX‐deficient mice (Rlx−/−) demonstrate that although females give birth to live young without apparent dystocia, the pubic symphysis is not elongated, and they have abnormal cervical and vaginal morphology. The current study examined phenotypic differences in collagen, matrix metalloproteinases (MMP), and estrogen receptors (ERs) in the cervix and vagina of pregnant Rlx+/+ and Rlx−/− mice. Neither collagen nor TGFβ1 mRNA levels in the cervix and vagina differed significantly between Rlx+/+ and Rlx−/− at any stage of gestation, except on gestation day 18.5, with an increase in α1‐I collagen and TGFβ1 expression in Rlx−/− mice. MMP gene expression was also increased in Rlx−/− mice, especially at term. Administration of recombinant H2 RLX (0.05 μg/μL/h) to Rlx−/− mice for 6 d from gestation day 12.5 caused a significant decrease in α1‐I collagen and MMP‐13 gene expression in the cervix and vagina, but it had no effect on TGFβ1. There was also a significant reduction in ERβ expression in RLX‐treated Rlx−/− mice. Interestingly, RLX treatment caused a significant decrease in LGR7 expression in these reproductive tissues. In summary, these data show increases in MMP gene expression in Rlx−/− mice that are not correlated with changes in collagen expression. Furthermore, we report a novel ER phenotype in the cervix and vagina of Rlx−/− mice.

Evolution of the CDKN1C-KCNQ1 imprinted domain

BackgroundGenomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.ResultsDespite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed.ConclusionCDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.

Up-Regulation of Mesotocin Receptors in the Tammar Wallaby Myometrium Is Pregnancy-Specific and Independent of Estrogen

Abstract The oxytocin-like peptide of most Australian marsupials is mesotocin, which stimulates uterine contractions and is important for normal birth in the tammar wallaby. Female marsupials have two uteri and, in monovular species such as the tammar, one uterus is gravid with a single fetus, whereas the contralateral uterus is nongravid. A significant increase in myometrial mesotocin receptor concentrations occurs only in the gravid uterus on Day 23 of the 26-day gestation. This study examined whether or not mesotocin receptors are present in the myometrium and are up-regulated at the equivalent stage of the luteal phase in unmated tammars. In contrast to the marked increase in mesotocin receptor mRNA and protein concentrations in the myometrium of the gravid uterus during pregnancy, receptors did not increase in the unmated animals. There were also no significant differences between the two uteri, except on Day 27. Plasma profiles of peripheral estradiol-17β and progesterone did not differ significantly between pregnant and nonpregnant cycles. However, progesterone concentrations were significantly lower on Day 1 postpartum compared with Day 27 of the nonpregnant cycle. In pregnant tammars, the molar ratio of circulating estradiol-17β to progesterone increased significantly between Day 25 of gestation and 1 day postpartum, but was not correlated with an increase in mesotocin receptor concentrations in either uterus. The data confirm that a local fetal influence is more important than systemic factors, such as estrogen, in the regulation of uterine mesotocin receptors in the tammar wallaby.

Steroid-Independent Regulation of Uterine Oxytocin Receptors

The oxytocin receptor is an important contractile‐associated protein, up‐regulated at term in the myometrium in many mammalian species. We conducted studies in a novel animal model to challenge the general view that gonadal steroids are a major regulatory factor of uterine oxytocin receptors. Female marsupials have separate uteri and, in monovular species such as the tammar wallaby, the conceptus is present in one uterus whereas the contralateral uterus is empty. A marked increase in myometrial oxytocin receptors occurs only in the gravid uterus. Fetectomy experiments demonstrated that local embryo‐derived factors stimulate this gravid uterus‐specific increase in oxytocin receptors, and that uterine distension is probably not a key component in this regulatory pathway. Unilateral ovariectomy has no significant effect on uterine oxytocin receptors, emphasizing the impact of the conceptus on oxytocin receptor regulation and the minimal influence of gonadal steroids on parturition in this species. Our data highlight that regulation of uterine oxytocin receptor expression is multifactorial, and does not necessarily rely on gonadal steroids.

Oxytocin and Estrogen Receptor Expression in the Myometrium of Pregnant Relaxin‐Deficient (Rlx−/−) Mice

Abstract: Relaxin decreases oxytocin‐stimulated rat myometrial contractions in vitro. This study used pregnant relaxin‐deficient (Rlx−/−) mice to investigate the interaction between relaxin, oxytocin receptor (OTR), and estrogen receptor (ER) expression in the myometrium. Myometrial OTRs were significantly decreased on gestation day 18.5 in Rlx−/− mice than in Rlx+/+ mice. An increase in ERα in Rlx+/+ mice at term was correlated with a decrease in ERβ, which was not observed in Rlx−/− mice. Treatment of Rlx−/− mice with relaxin had no effect on OTR, LGR7, or ERα expression, but it caused a significant decrease in ERβs.

Effects of Fetectomy on Oxytocin Receptors in the Myometrium of the Tammar Wallaby

Abstract Mesotocin, an oxytocin-like peptide, stimulates uterine contractions during marsupial parturition. Female marsupials have two separate uteri, and in monovular species, the uterus with the conceptus is gravid, whereas the contralateral uterus is nongravid. Marsupials are unique because systemic and feto-placental factors in the regulation of uterine function can be differentiated. In pregnant tammar wallabies, a marked increase in myometrial mesotocin receptors (MTRs) occurs on Day 23 of the 26-day gestation, but only in the gravid uterus. The objective of this study was to investigate the effects of removing the conceptus on this MTR up-regulation. Complete fetectomy on Day 20 of gestation resulted in significantly lower MTR mRNA and receptor concentrations on Day 23 compared with sham-operated controls. In contrast, there was no significant difference in MTR expression between controls and partially fetectomized animals in which uterine distension was maintained in the absence of a conceptus. In a related study, we examined MTRs in the myometrium of animals that appeared to be pregnant with a large, distended uterus. However, these uteri contained an abnormally developed fetus and avascular placenta. In these animals, MTR levels were significantly higher in the distended uterus compared with the nondistended uterus, and did not differ from controls. These data demonstrate that uterine occupancy is essential for the marked increase in uterine MTRs observed on Day 23 gestation. It also appears that distension may be one of the key factors involved.

Effects of the Sucking Stimulus on Relaxin Receptor (LGR7) Expression in the Mammary Apparatus of the Tammar Wallaby (Macropus eugenii)

Abstract: Macropodid marsupials suckle young of different ages simultaneously, a phenomenon known as asynchronous lactation. The growth of the mammary glands supporting young of different ages varies considerably. This study investigates relaxin receptor (LGR7) expression in different mammary glands and nipples during early lactation in the wallaby. LGR7s were upregulated in the nipple and mammary gland supporting the neonate 5‐11 d postpartum. LGR7 mRNA concentrations were also significantly higher in the gland supporting the newborn young than the older young. These data suggest that a local stimulus, that is, continuous sucking by the neonate, may influence LGR7 expression in the mammary apparatus.

Abnormal extracellular matrix remodelling in the cervix of pregnant relaxin-deficient mice is not associated with reduced matrix metalloproteinase expression or activity

Relaxin regulates cervical extracellular matrix (ECM) remodelling during pregnancy by modifying collagen and other ECM molecules by unknown mechanisms. We hypothesised that abnormal collagen remodelling in the cervix of pregnant relaxin-deficient (Rln1-/-) mice is due to excessive collagen (Col1a1 and Col3a1) and decreased matrix metalloproteinases (Mmp2, Mmp9, Mmp13 and Mmp7) and oestrogen receptors (Esr1 and Esr2). Quantitative polymerase chain reaction, gelatinase zymography, MMP activity assays and histological staining evaluated changes in ECM in pregnant wildtype (Rln1+/+) and Rln1-/- mice. Cervical Col1a1, Col3a1 and total collagen increased in Rln1-/- mice and were higher at term compared with Rln1+/+ mice. This was not correlated with a decrease in gelatinase (Mmp2, Mmp9) expression or activity, Mmp7 or Mmp13 expression, which were all significantly higher in Rln1-/- mice. In late pregnancy, circulating MMP2 and MMP9 were unchanged. Esr1 expression was highest in Rln1+/+ and Rln1-/- mice in late pregnancy, coinciding with a decrease in Esr2 in Rln1+/+ but not Rln1-/- mice. The relaxin receptor (Rxfp1) decreased slightly in late-pregnant Rln1+/+ mice, but was significantly higher in Rln1-/- mice. In summary, relaxin deficiency results in increased cervical collagen in late pregnancy, which is not explained by a reduction in Mmp expression or activity or decreased Rxfp1. However, an imbalance between Esr1 and Esr2 may be involved.

272. Decreased expression of oestrogen receptor β in the reproductive tract of pregnant relaxin-deficient (Rlx–/–) mice

The peptide hormone relaxin (RLX) is reported to directly affect uterine oestrogen receptors (ERs) in the rat (1). Treatment of immature ovariectomised rats with porcine RLX causes a decrease in uterine ERβ mRNA levels within 6 h. However, RLX has no effect on ERα expression. As both ERβ1 and ERβ2 inhibit ER-mediated transcriptional activity, this RLX-induced downregulation in ERβ could be a prerequisite for oestrogen to exert its effects on target tissues. The aim of the current study was to use relaxin-deficient (Rlx–/–) pregnant mice to investigate if relaxin deficiency results in alterations in either ERβ or ERα mRNA expression in reproductive tissues. Cervix and vagina tissues were obtained from adult C57/Blk6J wild-type mice at five stages of gestation (Days 7.5, 10.5, 14.5, 17.5, 18.5 pc) and Rlx–/– littermates on Days 7.5, 14.5 and 18.5 pc. Q-PCR with TaqMan probes in the Opticon 2 thermal cycler (MJ Research, GeneWorks) was used to quantify ERα and ERα gene expression. ERα mRNA levels were significantly (P < 0.05; ANOVA) increased in the cervix/vagina on Days 17.5 and 18.5 pc in Rlx+/+ mice. The increase in ERα in Rlx+/+ mice was negatively correlated with a significant decrease in ERβ expression from Day 14.5 pc. In contrast, there was no decrease in ERβ expression in the cervix/vagina of Rlx–/– mice; ERβ mRNA levels were significantly (P < 0.05) higher compared to Rlx+/+ mice on Days 14.5 or 18.5 pc. However, there was no corresponding reduction in ERα expression in the cervix/vagina of the Rlx–/– mice, so that ERα mRNA levels were still elevated at term despite the maintenance of high ERα expression. In summary, these data show changes in ERα expression in the cervix/vagina of relaxin-deficient mice, which may subsequently affect ERα-mediated transcriptional activity. (1) Pillai et al. (2002) Biol. Reprod. 67, 1919–1926.

282. Differential expression of the relaxin receptor (LGR7) in the mammary apparatus of the lactating tammar wallaby (Macropus eugenii)

Growth and development of the mammary apparatus (nipple and mammary gland) are important aspects of lactation. Macropodid marsupials can suckle young of two different ages simultaneously, a phenomenon known as asynchronous lactation. As a result, the type of milk produced and the structure of the two mammary glands supporting young of different ages vary considerably. A role for the peptide hormone relaxin in lactation has been demonstrated in relaxin receptor (LGR7)-deficient mice, which fail to deliver milk to their offspring due to impaired nipple development (1). This study investigated the distribution of LGR7 in the different mammary glands and nipples during asynchronous lactation in the tammar wallaby. The specific aim was to determine if the age of the pouch young influences LGR7 gene expression. Tissues were collected from the mammary apparatus sustaining the neonate and an older pouch young in the same mother, between Days 0 and 21 of lactation (n = 5/stage). A partial sequence (250-bp) of the tammar LGR7 was first obtained from a region close to the N-terminus of the soluble ectodomain, with 82% amino acid homology compared to the human LGR7 sequence. LGR7 gene expression was then measured by quantitative-PCR, using a TaqMan probe in the Opticon 2 thermal cycler (MJ Research, GeneWorks). Expression of LGR7 was upregulated in both the nipple and mammary gland supporting the neonate between 5 and 11 days after birth. There was no difference in LGR7 expression between these two tissues. However, LGR7 mRNA concentrations were significantly (P < 0.05: paired t-test) higher in the mammary apparatus supporting the neonate compared with that of the older young. These data suggest that a local stimulus, such as the continuous sucking by the neonate, may influence LGR7 expression in the mammary apparatus. (1) Krajnc-Franken et al. (2004) Mol. Cell. Biol. 24, 687–696.