Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

Immunocytochemical localization of atrial natriuretic factor (ANF) in rat female reproductive tract: evidence for a potential hormonal regulation

In the present studies atrial natriuretic factor (ANF) was characterized immunocytochemically in the reproductive tract of immature female rats, and changes of ANF levels in response to different hormonal conditions were demonstrated. Administration of pregnant mare serum gonadotropin (PMSG) to immature animals has shown to be a useful method to synchronize growth, differentiation and atresia of ovarian follicles. ANF immunoreactivity was investigated in rat uterus and oviduct during follicular growth and estrogenic dominance (48 h after PMSG treatment) and during follicular atresia and progesterone dominance (96 h after PMSG treatment). Our immunocytochemical results showed that in rat uterus ANF was localized in endometrial mucosal and glandular epithelium and smooth muscle cells of the myometrium. In the oviduct ANF immunoreactivity was observed in mucosal cells and muscle layers. Immunocytochemical staining patterns and Western blot analysis revealed that ANF levels in rat uterus and oviduct are modulated by the hormonal status. ANF immunoreactivity was elevated during estrogenic dominance (48 h after PMSG) in uterus and oviduct. However, during progesterone dominance (96 h after PMSG) elevation of ANF immunoreactivity was observed in the uterus only. These results raise the possibility that ANF expression in rat oviduct is positively controlled by estrogen and negatively by progesterone. ANF staining in uterus during progesterone phase provides evidence that both estrogen and progesterone regulate ANF levels in uterus. The observed staining patterns indicate that ANF may have intracellular functions as well as a role in priming the extracellular environment. Accordingly, the possibility that ANF might be an important regulatory molecule for autocrine/paracrine communication within the female reproductive tract should be considered.     

Fibroblast growth factor-10: a stromal mediator of epithelial function in the ovine uterus

Fibroblast growth factor-10 (FGF-10) is a stromal-derived paracrine growth factor considered to be important during embryogenesis; however, its expression by cells in the female reproductive tract has not been investigated. Therefore, an ovine FGF-10 cDNA was cloned from an ovine endometrial cDNA library to investigate expression and potential paracrine characteristics of FGF-10 in the ovine uterus. The ovine FGF-10 cDNA encodes a protein of 213 amino acids and possesses an unusually long 5' untranslated region (UTR). In situ hybridization demonstrated that ovine FGF-10 mRNA was expressed by endometrial stromal cells and by mesenchymal cells of the chorioallantoic placenta. The mRNA for FGF-7, a homologue of FGF-10, was localized in the tunica muscularis of blood vessels in endometrium and myometrium. In contrast, FGF receptor 2IIIb, the high-affinity receptor for both FGF-10 and FGF-7, was expressed exclusively in luminal epithelium, glandular epithelium, and placental trophectoderm. The in vivo spatial expression pattern suggests that FGF-10 is a novel endometrial stromal cell-derived mediator of uterine epithelial and conceptus trophectodermal functions. The nonoverlapping spatial patterns of expression for FGF-10 and FGF-7 in ovine uterus and conceptus suggest independent roles in uterine function and conceptus development.     

Characterization of an immortalized oviduct cell line from the cynomolgus monkey (Macaca fascicularis)

To establish reproductive biological techniques in mammals, it is important to understand the growth environment of the embryo. Oviduct epithelial cells are in close proximity to the embryo during pre-implantation development. We, therefore, established an immortalized oviduct epithelial cell line from the cynomolgus monkey, evaluated the usefulness of these cells as feeder cells for embryo culture, and investigated the gene expression of several growth factors and cytokines in the cells. The immortalized cells were positive for the anti-cytokeratin antibody, as determined by immunocytochemistry, indicating that they are epithelial. They also expressed oviductin, which is specific to oviduct epithelial cells, glyceraldehyde-3-phosphate dehydrogenase (control), leukemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor 1, transforming growth factor beta-2, and interleukin 4. Mouse embryo development was improved when the immortalized cells were used as feeder cells. This cell line is also useful for studying the factors secreted by oviduct epithelial cells.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.     

Regulation of polypeptide growth factor synthesis and growth factor-related gene expression in the rat and mouse uterus before and after implantation

It is apparent that the uterus is a rich source of growth factors, the synthesis of which may be induced by female sex steroids. These growth factors appear to be involved in complex autocrine/paracrine regulatory circuits which in turn interact with steroid hormones. With the exception of CSF-1, however, detailed studies of the appearance of these growth factors and their receptors during pregnancy and under different hormonal regimens have yet to be performed. Furthermore, causative roles have not been established for any of these growth factors in uterine biology and pregnancy. In the near future we can expect that, by using in-situ techniques, the producing and responding cells will be identified. Cell culture models will also have to be established to investigate specific growth factor-induced functions. In the longer term, once more is known about the regulation of uterine growth factors, imaginative new experiments need to be designed, perhaps involving transgenic animals, to establish causative roles for growth factors in uterine biology.     

Endocrine and paracrine control of sperm production in stallions.

The specific nature and relative contribution of the major hormones involved in regulation of reproductive function of the stallion are not well defined nor have paracrine or autocrine factors been identified. Over the last 12 years, our laboratory has been engaged in characterizing the hypothalamic-pituitary-testicular axis (HPT) in stallions. A number of endocrine factors and mechanisms important for normal reproductive function have been investigated. Studies investigating poor fertility in stallions suggest that a closer look at the testicular level is warranted. For a complete understanding of intratesticular control mechanisms including cell-to-cell interactions in the stallion, studies on the actions of paracrine/autocrine factors such as growth factors, inhibin, activin, and oxytocin are needed. In other species, paracrine/autocrine systems appear to be important in modulating endocrine control of testicular function and spermatogenesis.     

Sostdc1 Secreted from Cutaneous Lymphatic Vessels Acts as a Paracrine Factor for Hair Follicle Growth

In our previous study, we found that lymphatic vessels stimulate hair follicle growth through paracrine effects on dermal papilla cells. However, the paracrine factors secreted from cutaneous lymphatic vessels that can activate dermal papilla cells are still unknown. In this study, we investigated whether lymphatic endothelial cells might secrete paracrine factors that activate dermal papilla cells in vitro. We found that Sostdc1 was more expressed in lymphatic endothelial cells compared with blood vascular endothelial cells. In addition, Sostdc1 expression levels were significantly increased during the anagen phase in the back skin of C57BL/6J mice, as compared to the telogen phase. We also observed that incubation of dermal papilla cells with 200 ng/mL Sostdc1 for 72 h induced the expression levels of Lef-1, a downstream target of Wnt signaling. Taken together, our results reveal that Sostdc1, a BMP antagonist, secreted from cutaneous lymphatic vessels, may act as a paracrine factor for hair follicle growth.     

Keratinocyte growth factor: expression by endometrial epithelia of the porcine uterus

Keratinocyte growth factor/fibroblast growth factor-7 (KGF/FGF-7) is an established paracrine mediator of hormone-regulated epithelial growth and differentiation. In all organs studied, KGF is uniquely expressed in cells of mesenchymal origin. To determine whether KGF and its receptor, keratinocyte growth factor receptor (KGFR) or fibroblast growth factor receptor-2IIIb, were expressed in the porcine uterus as a potential paracrine system mediating progesterone action, we cloned KGF and KGFR partial cDNAs from the porcine endometrium. KGF and KGFR expression was detected in endometrium by Northern blot hybridization. Interestingly, in situ hybridization results demonstrated that KGF was expressed by endometrial epithelia and was particularly abundant between Days 12 and 15 of the estrous cycle and pregnancy. KGF secretion into the lumen of the porcine uterus was also detected on Day 12 of the estrous cycle and pregnancy. KGFR was expressed in both endometrial epithelia and conceptus trophectoderm. These novel findings suggest that KGF may act on the uterine endometrial epithelium in an autocrine manner and on the conceptus trophectoderm in a paracrine manner in the pig, which is the only species possessing a true epitheliochorial type of placentation.     

Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture

Summary The role of polypeptide growth factors (estromedins) as mediators of estrogen-responsive mammary tumor growth is studied in this report. Three possible new mechanisms were investigated that include endocrine, autocrine, and paracrine related growth factors. The first hypothesis being tested is whether estrogens interact with target tissues and cause the biosynthesis and secretion of polypeptide growth factors, which then act as mitogens for normal and neoplastic mammary tissues. Data presented suggest that this mechanism involves estrogen interaction with uterus, kidney, and pituitary gland causing production of growth factors, which then enter the general circulation and promote growth of distant target tissues. This is an endocrine type mechanism. Another type of estromedin control (autocrine control) may be exerted in an autostimulatory way in which the target tissue produces the polypeptide factors for its own growth in response to estrogen stimulation. A variation of the autocrine mechanism may be a paracrine mechanism in which some cells of an estrogen-responsive normal or neoplastic tissue produce growth factors that act on adjacent or neighboring cells. From the data available, all three possible types of growth factors could be functioning synergistically to yield the final result of continuous estrogen responsive tumor growth in vivo. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by American Cancer Society Grant BC-255; D. A. S. is the recipient of an American Cancer Society Faculty Research Award, FRA-212. D. D. is supported by a Rosalie B. Hite predoctoral fellowship from the Rosalie B. Hite Foundation, Houston, Texas. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Reproductive fitness in roe bucks (Capreolus capreolus): seasonal timing of testis function

The exact seasonal timing of normal testis function is a crucial precondition for the reproductive fitness of roe bucks and for successful breeding during rut in July–August. Production of spermatozoa and testosterone requires both endocrine regulation and local testicular control by autocrine/paracrine factors. These local control mechanisms include the action of several growth factors. Our short review assigns histological organization of roe deer testis to new data on the involvement of several growth factors in its regulation. The expression of growth factors is season-specific and cell-type-specific. This suggests its functional role in the complex interaction between germinative and somatic cells for the regulation of testis growth, spermatogenesis and function of hormone-producing cells. The authors dedicate this review to Prof. Dr. Christian Pitra who celebrates his 65th birthday in April 2006.     

VEGF, VEGFR-1 and VEGFR-2 immunoreactivity in the porcine arteries of vascular subovarian plexus (VSP) during the estrous cycle

Abstract: Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the female reproductive tract. It binds to cell surface through ligand-stimulatable tyrosine kinase receptors, the most important being VEGFR-1 (flt-1) and VEGFR-2 (flk-1). The broad ligament of the uterus is a dynamic organ consisting of specialized complexes of blood vessels connected functionally to the uterus, oviduct and ovary. Endothelial cells form an inner coating of the vessel walls and thus they stay under the influence of various modulators circulating in blood including ovarian steriods involved in developmental changes in the female reproductive system. The aim of the present study was to immunolocalize VEGF and its two receptors: VEGFR-1 and VEGFR-2 in the broad ligament of the uterus in the area of vascular subovarian plexus during different phases of the estrous cycle in pig and to determine the correlation between immunoreactivity of the investigated factors and phases of the estrous cycle. The study was performed on cryostat sections of vascular subovarian plexus stained immunohistochemically by ABC method. Specific polyclonal antibodies: anti-VEGF, anti-VEGFR-1 and anti-VEGFR-2 were used. Data were subjected to one-way analysis of variance. Our study revealed the presence of VEGF and its receptors in endothelial and smooth muscle cells of VSP arteries. All agents displayed phase-related differences in immunoreactivity suggesting the modulatory effect of VEGF, VEGFR-1 and VEGFR-2 on the arteries of the VSP in the porcine broad ligament of the uterus.     

The role of megalin (LRP-2/Gp330) during development

Megalin (LRP-2/GP330), a member of the LDL receptor family, is an endocytic receptor expressed mainly in polarised epithelial cells. Identified as the pathogenic autoantigen of Heymann nephritis in rats, its functions have been studied in greatest detail in adult mammalian kidney, but there is increasing recognition of its involvement in embryonic development. The megalin homologue LRP-1 is essential for growth and development in Caenorhabditis elegans and megalin plays a role in CNS development in zebrafish. There is now also evidence for a homologue in Drosophila. However, most research concerns mammalian embryogenesis; it is widely accepted to be important during forebrain development and the developing renal proximal tubule. Megalin is also expressed in lung, eye, intestine, uterus, oviduct, and male reproductive tract. It is found in yolk sacs and the outer cells of pre-implantation mouse embryos, where interactions with cubilin result in nutrient endocytosis, and it may be important during implantation. Models for megalin interaction(s) with Sonic Hedgehog (Shh) have been proposed. The importance of Shh signalling during embryogenesis is well established; how and when megalin interacts with Shh is becoming a pertinent question in developmental biology.     

Estrogen induces insulin-like growth factor-I expression in the rat uterus

The inability to convincingly demonstrate a mitogenic effect of estrogen on isolated uterine cells in culture suggests that autocrine or paracrine growth factors may be important in the estrogen-induced uterine proliferative response. Here we report that uterine expression of insulin-like growth factor-I (IGF-I), an important mediator of GH action, is increased after 17 beta-estradiol (5 micrograms/100 g bw, ip) administration to ovariectomized prepubertal rats. An increase in uterine IGF-I mRNA abundance, approximately 14-fold above untreated controls, was apparent 6 h after estrogen administration and the level achieved exceeded that seen in the uterus from intact mature rats during diestrus. In contrast to the increase in IGF-I expression in the uterus, no significant change in serum IGF-I concentration or hepatic or renal IGF-I mRNA abundance was demonstrable after 17 beta-estradiol injection of ovariectomized prepubertal rats. The increase in uterine IGF-I expression, was similar in both pituitary-intact and hypophysectomized, ovariectomized rats. We believe this is the first report of induction of IGF-I expression by estrogen in vivo. As such, the finding expands the role and significance of IGF-I as a mediator of growth beyond that related to GH.     

GM-CSF regulation of embryo development and pregnancy

The reproductive tissues undergo profound structural changes and major immune adaptation to accommodate pregnancy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of an array of cytokines with pivotal roles in embryo implantation and subsequent development. Several cell lineages in the reproductive tract and gestational tissues synthesise GM-CSF under direction by ovarian steroid hormones and signalling agents originating in male seminal fluid and the conceptus. The pre-implantation embryo, invading placental trophoblast cells and the abundant populations of leukocytes controlling maternal immune tolerance are all subject to GM-CSF regulation. GM-CSF deficiency in pregnancy adversely impacts fetal and placental development, as well as progeny viability and growth after birth, highlighting this cytokine as a central maternal determinant of pregnancy outcome with clinical relevance in human fertility.