Endocrine DOI 10.1007/s12020-013-0140-7

ORIGINAL ARTICLE

Progesterone is critical for the development of mouse embryos Cong Zhang • Bruce D. Murphy

Received: 23 September 2013 / Accepted: 25 November 2013 Ó Springer Science+Business Media New York 2013

Abstract Infertility affects approximately 10–15 % of reproductive-aged couples, and embryo loss due to preimplantation death is common to many mammals. Previous studies showed that a complex series of interactive molecular events are associated with this process, especially hormones (progesterone and estrogens) and growth factors, and are important for the cleavage and differentiation of the blastocysts. Yet, the mechanism of preimplantation embryo development is unclear. Using conditional knockout mice (CKO), we showed the development of blastocyst is tightly controlled by the level of progesterone (P4); furthermore, we found that the time when P4 should increase is also crucial for the formation of blastocysts. In CKO mice whose Lrh1 (liver receptor homolog 1) is deleted under the expression of Cre recombinase driven by progesterone receptor promoter, which reduced P4 synthesis, few of their embryos can reach blastocyst stage. When these CKO mice were supplied with P4 in the afternoon of dpc 1 (day post copulation), most of the embryos can form blastocysts; when CKO mice were supplied with P4 from the morning of dpc1, one-third of the embryos can reach blastocyst stage; however, the supplement of P4 in the morning of dpc 2 made very few of the embryos become blastocysts. We

C. Zhang (&) Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200135, China e-mail: [email protected] B. D. Murphy Centre de recherche en reproduction animale, Universite´ de Montre´al, Saint-Hyacinthe, Que´bec, Canada e-mail: [email protected]

conclude that early exposure to P4 is essential for timely progression of early embryogenesis in the mouse. Keywords Embryo
Blastocyst
Progesterone
Estradiol
Day post copulation

Introduction A substantial embryo loss due to preimplantation death is common to many mammals, and it is considered a selection process that leads to the survival of superior embryos. According to the information from Center for Disease Control and Prevention (CDC), about 11.8 % of women (7.3 million) in the United States aged 15–44 years have difficulty getting pregnant or staying pregnant. Approximately 15 % of pregnancies end in miscarriage [1]. Assisted reproductive technology (ART) is an efficient way to treat infertility; today, over 1 % of all infants born in the United States every year are conceived using ART. In CDC’s 2008 ART Success Rate Report, the rate is about 31 %. The reasons for this high failure rate are mainly because of the poor quality of the embryos, about 33 % of the embryos having an abnormal chromosomal complement [2]. Disclosure of the mechanism of preimplantation embryonic development has been a challenge to reproductive and developmental biologists with the goal of alleviating the problems of human infertility and insuring the birth of quality offspring, or developing novel contraception to restrict world population. A complex series of interactive molecular events are associated with this process. However, the mechanism of how proliferation and differentiation are controlled in the preimplantation embryo has not yet been defined.

123

Endocrine

Mouse embryos can develop to blastocyst stage in culture media without hormones, and when these kinds of blastocysts are transplanted into pseudopregnant females, viable babies can be born [3]. This experiment evidences that hormones might not be necessary for embryo development up to the blastocyst stage. There are also evidences that the development of mouse embryos in vitro is slower than in vivo, perhaps because of the different physical and nutritive environment or hormonal deficiency. Bowman and Mclaren had demonstrated that mouse embryos developed in vivo from the 2-cell to the blastocyst stage had a constant cell doubling time of about 10 h, while embryos cultured in vitro over the same period showed declined rate of cleavage from initially almost as great as in the reproductive tract to subsequently about 24 h, addition of estrogen to the cultural medium increased the diameter of blastocysts but did not increase cell number [4]. Roblero found the lower cleavage rates of embryos of ovariectomized pregnant mice can be restored to normal values by progesterone treatment [5]. Roblero and Izquierdo [6] showed that the addition of serum and progesterone (P4) results in an increased cleavage rate and a significant increase in the number of blastomeres/blastocyst. Wu et al. showed that, in ovariectomized rats, 72 % of the embryos were in blastocyst stage compared with 99 % in intact controls [7]. Roblero and Garavagno demonstrated that in ovariectomized mice, only 37.9 % of the embryos can develop to blastocysts compared with 89.3 % in control group; treatment with estrogen and P4 reverses these defects; the ratio of blastocysts in ovariectomized group reached 94.4 % [8]. All these experiments indicate that the hormones are necessary for embryos’ development to blastocysts. On the other hand, some in vitro studies have shown that P4 has an inhibitory effect on the early development of mammalian embryos as judged by the arrest of cleavage and much lower ratio of blastocysts, and the embryos at different stages have different sensitivity, the younger the embryos are, the more serious the damage will be, and once the early blastula stage is attained, there is little or no detrimental effect [9–11]. Since there is no evidence that estrogen and/or P4 act directly on the preimplantation embryo, embryonic development is considered to depend on growth promoting factors originating from the reproductive tract under the influence of these hormones. The mouse and human preimplantation embryos themselves also express many growth factors and their receptors and embryos are exposed to growth factors as the developing embryos move down the reproductive tracts. Whether growth factors/cytokines of embryonic origin and reproductive tract origin regulate preimplantation development of the embryos in autocrine/ paracrine manner is of ongoing research interest.

123

Adamson added IGF-2, insulin, platelet-derived growth factor-a (PDGF-a), transforming growth factor-b (TGF-b), and EGF to cultured preimplantation embryos and found that they stimulated DNA and RNA synthesis and increased cell numbers [12]; Bhatnagar et al. found that CSF-1 specifically increased trophoblast cell numbers [13]. Similarly, Paria and Dey showed that culture of murine preimplantation embryos in groups improved their development, maybe because of increase of the concentrations of endogenously produced growth factors which have a stimulatory effect, and they also found EGF and TGF-a had beneficial effects on preimplantation embryo development in an autocrine/paracrine manner [14]; addition of antisense oligonucleotides to the EGFr retarded blastocoele formation, but had no significant effect on cell proliferation [15]. Dadi et al. showed knockdown of EGF, EGFR, and TGF-a caused by siRNA decreased blastocyst formation, increased the number of apoptotic cells, and reduced the total number of differentiated cells in blastocysts [16]. Unlike the above factors, Pampfer found TNF-a reduced inner cell mass proliferation [17]. However, so many attempts have been made to study whether preimplantation development is regulated by either endogenous or exogenous factors. Actually all these observations about preimplantation development were done using in vitro culture medium known to be suboptimal for embryonic growth. Embryos cultured in these media exhibited diverse defects, such as failure to develop beyond the two-cell stage, a reduction in cleavage rate resulting in embryos having a reduced cell number compared to those developing in vivo, and a failure to hatch from the zona [4, 18]. Therefore, about the cleavage and differentiation of the blastocyst, a huge amount of information has accumulated describing the spatial and temporal roles of hormones and patterns of growth factors. However, only with the application of gene manipulation techniques, in particular using mice carrying mutant genes of interest, it has become possible to determine which factors are essential for these events [19]. Here, we have a perfect model to study the embryo development using conditional knockout mice (CKO). In this system (Cre-loxP), Cre recombinase is driven by the P4 receptor promoter (PrCre), while the loxP sites flanked in exons 3 and 4 in gene liver receptor homolog1 (Lrh1fl/fl), an orphan nuclear receptor that regulates various metabolic and hormone synthetic processes. PrCre mice were bred to the Lrh1fl/fl mice. The resultant female offspring (PrCre/?; Lrh1fl/fl) designated conditional knockout mice were used for the experiment. These female CKO mice cannot synthesize P4 after ovulation, but their E2 is normal. Using these kinds of animals, we found that mouse embryo development is tightly regulated by P4; furthermore, we found that embryos are

Endocrine

very sensitive to P4 concentration and the time when the P4 concentration should increase.

Materials and methods Mice All the mice used in the experiments are of C57BL/6 background. Animals were maintained under a 14:10 h light: dark cycle provided food and water ad libitum [20]. All animal experiments were approved by the Animal Committee of University of Montreal and done in accordance with the Guide of University of Montreal for the Care and Use of Laboratory Animals. Lrh1 floxed (Lrh1fl/fl, CON) and P4 receptor recombinase (PrCre) [21, 22] mice have been described previously. Lrh1 conditional knockout mice (PrCre/?; Lrh1fl/fl, CKO) were obtained by crossing PrCre mice with Lrh1fl/fl mice. For analysis of embryo development, adult ([60 days of age) female (CON and CKO) mice were mated with males of proven fertility. Animals were checked every morning (09:00) for copulatory plugs, and the day that a vaginal plug was found was designated day post copulation 1 (dpc 1). Animals were terminated at various stages of gestation according to experimental purposes.

Fig. 1 Estradiol in the serum of pregnant animals. Filled square CON, control mice (Lrh1f1/f1); open square CKO, conditional knockout mice (PrCre/?; Lrh1f1/f1, CKO); dpc day post copulation. n = 4–10

Embryo recovery and examination To investigate the development of embryos, mouse oviducts or uteri were flushed with PBS with 1 % BSA, and the recovered embryos were observed under a dissecting microscope and photographed. After implantation, uteri of pregnant females were dissected at dpc 6 (15:00), dpc 7 (15:00), or dpc 8 (15:00) according to experimental design.

Hormone analysis

Embryo culture

Mice were anesthetized using isoflurane (Baxter Corporation, Ontario, Canada), and blood was collected by cardiac puncture. P4 and estradiol levels were analyzed by radioimmunoassay as previously described [23]. The inter- and intra-assay coefficients of variation were 7.76 and 4.66 %, respectively.

Fertilized embryos were collected on dpc1 (09:00), were pooled from several mice in M2 medium (Sigma), and then were washed three times in the same medium. The embryos were cultured in groups of 10 in microdrops (50 ul) of M16 medium (Sigma) under mineral oil in an atmosphere of 5 % CO2 at 37 °C for 96 h. The embryos were observed, and photographs were taken under a Leica microsope.

Exogenous P4 supplementation CKO females were mated with fertile males, and a 2 cm length silastic implant (ID, 3.35 mm; OT, 4.65 mm; Dow Corning, Midland, MI, USA) containing P4 (Sigma Chemical Co., St Louis, MO, USA) was implanted in the dorsal region on dpc1 or dpc2 in the morning (09:00) or the afternoon (17:00) according to procedures previously described [23].

Statistical analyses All data collected were analyzed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). All experiments were repeated at least three times, and all numerical data were represented as mean ± SEM; values were considered significantly different if P B 0.05.

Implantation sites Results Implantation sites on dpc 5 were studied by an intravenous injection of 1 % (W/V, in 19 PBS) Evans blue dye (100 ll) according to procedures described [23]. The injected mice were then terminated 3 min later, and the uteri were dissected for examination.

Estradiol-17b level of the pregnant mice Estradiol-17b in pregnant mice from the CON and CKO was determined from dpc1 to dpc 4 when

123

Endocrine b Fig. 2 Embryo development after mating and before implantation.

The day a copulatory plug was found was designated day post copulation 1 (dpc 1). a1–a5 from CON mice; b1–b5 from CKO mice. a1, b1 2-cell embryos on dpc 1 in the afternoon; a2, b2 embryos on dpc 2 in the morning; a3, b3 morula on dpc 2 in the afternoon; a4, b4 embryos on dpc 3 in the afternoon, embryos from CON mice formed compacted embryos (a4), while half of the CKO embryos could not (b4); a5, b5 blastocysts on dpc 4 in the afternoon. By dpc 4, most of the embryos from CON reached blastocyst stage (a5), while most of embryos from CKO stayed at morula stage (b5). CON control mice (Lrh1f1/f1); CKO conditional knockout mice (PrCre/?; Lrh1f1/f1, CKO). n = 4–10 mice. Bar is 20 lm

Embryo development before implantation At dpc 1 in the afternoon, embryos from both groups were at 2 cell stage (Fig. 2a1, b1). On dpc 2 in the morning, embryos from both groups divided continually; at this time, the embryos contained several blastomeres (Fig. 2a2, b2), and in the same day later, they formed morulae (Fig. 2a3, b3). On dpc 3 in the afternoon, most of the embryos from CON formed compacted embryos, while about half of the CKO embryos could not (Fig. 2a4, b4). By dpc 4, most of the embryos from the CON reached blastocyst stage, while most of embryos from CKO still stayed at morula stage (Fig. 2a5, b5), and only very few had attained blastocyst stage. Embryo development after implantation Since CKO mice have low P4 concentration [23], we inferred this might make the development of the embryos retarded; so, we tracked the development of embryos beyond the time of implantation [24, 25]. As we can see, on dpc 5, CON embryos have implanted denoted by the black bands in Fig. 3a1, while in CKO mouse, there was no black band in the uterus (Fig. 3a2) and the embryos still did not compact well (Fig. 3a3, a4). Given more time, from dpc 6 to dpc 8, in CON uteri, the embryos were further developed (Fig. 3b1, c1, d1); in contrast, in CKO uteri, still no implantation has been observed (Fig. 3b2, c2, d2), and their embryos remained in the similar state (Fig. 3b3, b4, c3), or died (Fig. 3d3). In addition, in some mice, the embryos coexisted with the newly ovulated eggs from the new cycle which can be inferred from the existence of cumulus cells (Fig. 3c4, d4). Embryo development with the P4 supplemented on dpc 1 in the morning

embryos become the blastocysts; during this period of time, there was no difference between the two groups (Fig. 1).

123

Considering the reason of the abnormal embryos in CKO mice, we know that estradiol was normal and P4 was lower compared with CON mice since dpc 2. We explored whether the embryos would be normal with the supplement of P4. So, we implanted P4 pellet on dpc 1 in the morning

Endocrine

Fig. 3 Embryos after implantation. a1, b1, c1, d1 CON; a2–a4, b2–b4, c2–c4, d2–d4 CKO. a1–a4 representative uteri and embryos on dpc 5, black bands were implantation sites, n = 8–12 mice; b1–b4 representative uteri and embryos on dpc 6, n = 5–10 mice; c1–c4 representative uteri and embryos on dpc 7, n = 5–10 mice; d1–d4

representative uteri and embryos on dpc 8, n = 7–10 mice. In CON mice, on dpc 5 and thereafter, the embryos have already implanted, while in CKO mice, rarely could the embryos reach blastocyst stage and there was no implantation. Bar is 20 lm

(09:00) and found, unlike the CON embryo on dpc 4 (Fig. 4a1–a4), about one-third of the embryos from the CKO mice can form blastocysts (Fig. 4b1–b3), while the others could not (Fig. 4b4–b8).

(Fig. 4a1–a4), most of the CKO embryos could develop to blastocysts (Fig. 4c1–c8).

Embryo development with the P4 added on dpc 1 in the afternoon Since only part of the embryos can develop to blastocyst stage with P4 added in the morning (09:00), we thought maybe the adding time of P4 was not appropriate, then we tried to give the P4 in the afternoon (17:00), and tracked the development of the embryos. We found, like CON

Embryo development with the P4 supplied on dpc 2 in the morning To test how long the best adding time of P4 could last, we tried to add P4 on dpc 2 in the morning (09:00) and tracked the development of the embryos. We found unlike CON embryos (Fig. 4a1–a4), very few of the CKO embryos could reach blastocyst stages, while most of the others could not (Fig. 4d1–d8).

123

Endocrine

Fig. 4 Embryo development on dpc 4 with P4 supplement at different times. a1–a4 embryos from CON, n = 10 mice; b1–b8 embryos from CKO with P4 supplemented on dpc 1 in the morning (09:00), n = 6 mice; c1–c8 CKO embryos with P4 supplement on dpc

1 in the afternoon (17:00), n = 15 mice; d1–d8 embryos with P4 supplement on dpc 2 in the morning (09:00), n = 9 mice. dpc day post copulation. Bar is 20 lm

Embryo culture in vitro

conditions can never be as the same as the in vivo. The ovariectomy and the embryo transplant are huge stresses to the animals, and might affect the development of the embryos more or less, then the injection of P4 cannot mimic the in vivo situations, not only the injection time, but also the injection amount might be improper to the development of the embryos compared with the in vivo situation, and under such situation, the embryos might still be exposed to the P4 more or less, because in ovariectomized mice, the effect the P4 synthesized during estrous cycle, at any rate, must persist for several days. Using the conditional knockout mice, we can surpass these problems easily, and without affecting the level of estradiol, which has been shown to be important for the development of the embryos and transport of the embryos in the oviducts [8]. Relative to the importance of P4 to the development of embryos, there are three kinds of opinions; the first of all, P4 is not necessary for the development of the embryos, because when the in vitro cultured embryos were transplanted to the foster mothers, viable babies can be born [3]; second of all, P4 is necessary and has a stimulatory effect in vivo upon the development of embryos, and without P4, the blastocyst ratio is lower because the cleavage is lower [4, 5]; the third of all, P4 is inhibitive to the development of embryos [9–11].

Embryos from CON mice were collected on dpc1, cultured, and were observed 96 h after in vitro culture. Compared with the embryos developed in vivo (Fig. 5a–d), embryos maintained in vitro developed slower, and the shapes were inferior (Fig. 5e–h).

Discussion In the present study, we demonstrated P4 is crucial for the development of mouse embryos using CKO mice. Without P4, CKO mouse embryos can rarely reach blastocyst stage, and they even have difficulties to attain the morula stage. With supplemental P4, blastocyst ratio improved, but it depends on the time when the P4 is added, the best time is in the afternoon of dpc 1; on dpc 1 in the morning, it is too early for the embryo development, while in the morning of dpc 2, it is too late. Many researchers tried different ways to explore the mechanism of the embryo development, such as in vitro culture [6, 9], ovariectomy [7, 8], embryo transplant [26], and RNAi [16]. All these methods have their advantages and shortcomings. Like in vitro culture, the cultural

123

Endocrine

Fig. 5 Representative blastocysts of CON mice on dpc 4. a–d blastocysts developed in vivo; e–h blastocysts developed in vitro. n = 6–10 mice. Bars are 20 lm

Our results demonstrated that the development of the embryos requires P4 for optimum development, consistent to the second view. Since our in vitro cultural results showed although the in vitro cultured embryos (both CON and CKO) can reach blastocyst stage, their morphology appeared compromised relative to the in vivo developed counterparts and they developed slower. Given the CKO embryos, when transplanted at the zygote stage to wild type oviducts, the mechanism appears to lie in the reproductive tract. It is most likely related to the paucity of progesterone, which caused the inferior environment of the reproductive tracts. Furthermore, the retention of embryos in the oviducts of ovariectomized females [26], the mouse blastocysts transferred to the oviducts of immature females entered a period of diapauses, which could be activated by culture in vitro or by transfer to pseudopregnant recipients [27], and the delay of transformation of morulae to blastocysts caused by ovariectomy [7] are in agreement with this view. Our results do not agree with the third supposition that P4 has an inhibitory effect upon the cleavage of mouse embryos. The discrepancy with our results can be explained by the fact we supplemented P4 at concentrations of (29.29 ± 2.44 ng/ml), which are close to those of CON mice (16.57 ± 2.36 ng/ml) on dpc 4, while in Kirkpatrick’s report, the concentration was 8 mg/ml [11], that means the concentration was more than 480,000 times higher than the physical concentration. Whitten used a concentration of 4 lg/ml [9], and Daniel used 10 lg/ml [10], both of them were well above physiological levels. Except the too high concentration, their timing of P4 supplementation also had some problems. In ovariectomized mice, embryos can develop to blastocyst stage by

injection of P4 daily. This also indicates P4 is necessary for the formation of blastocysts, but this also depends on the time of P4 injection, ovariectomy on the 1st day of pregnancy resulted in virtually no implantation and the survival of very few blastocysts [7]. From our results, in pregnant mice, P4 concentration increases gradually, from 5.43 ± 1.04 ng/ml on dpc 1 to 24.36 ± 5.42 ng/ml on dpc 6, and the difference is only significant from the second day of pregnancy between the pregnant and non pregnant mice. So, P4 supplement should not be added too early, and the concentration should not be too high, which might be inhibitory to the development of the embryos. That is what happened to us when we gave the P4 supplement in the morning of dpc 1. Besides the crucial roles of P4 to the development of embryos, whether embryo development is dependent on the production of endogenous factors has not been established. However, all the growth factors and their receptors, e.g., IGF-1 and 2 [28]; the IGF-1 and 2 receptors [29, 30]; PDGF-A ligand [31], PDGF-a and b receptors [32]; TGF-a [33]; leukemia inhibitory factor (LIF) [34], and the LIF receptor b [35], which are expressed in mouse preimplantation embryos have been disrupted by targeted gene manipulations, and none of these mutants has exhibited any obvious abnormalities up to the time of implantation. Although, absence of TGF-a results in an increase in cell death within the ICM [36], absence of fibroblast growth factor 4 [37] and the EGFr result in lethality shortly after implantation. However, about the role of EGFr, it depends on the mouse strain background on which the mutation is placed, as the same mutation on other backgrounds can survive to midgestation or even parturition [38–40].

123

Endocrine

We conclude that, to the blastocyst stage, P4 is crucial for appropriate development of the mouse embryo. It is unlikely that preimplantation development of the rodent embryos is dependent on exogenous growth factors, and none of the known endogenously produced factors and their receptors are essential from preimplantation development to the blastocyst stage [19]. However, at or shortly after implantation, the need of uterine factors and their receptors does arise, for both the continued embryo development, for hatching from the zona pellucida and for implantation to take place [19]. Acknowledgments This study was funded by the National Natural Science Foundation of China (31172040), Shandong Natural Science Foundation (ZR2011CM047), SRF for ROCS, SEM and 12DZ2260600 to C. Zhang, and OPG 11018 from the Canadian Institutes of Health Research to B.D. Murphy. We are grateful to M. Dobias for hormone analyses and X. Tang for statistical analyses. Conflict of interest The authors declare that they have no conflicts of interest.

References 1. R. Rai, L. Regan, Recurrent miscarriage. Lancet 368(9535), 601–611 (2006). doi:10.1016/S0140-6736(06)69204-0 2. N.J. Winston, Developmental failure in preimplantation human conceptuses. Int. Rev. Cytol. 164, 139–188 (1996) 3. L.A. Mc, J.D. Biggers, Successful development and birth of mice cultivated in vitro as early as early embryos. Nature 182(4639), 877–878 (1958) 4. P. Bowman, A. McLaren, Viability and growth of mouse embryos after in vitro culture and fusion. J. Embryol. Exp. Morphol. 23(3), 693–704 (1970) 5. L. Roblero, Effect of progesterone in vivo upon the rate of cleavage of mouse embryos. J. Reprod. Fertil. 35(1), 153–155 (1973) 6. L. Roblero, L. Izquierdo, Effect of progesterone on the cleavage rate of mouse embryos in vitro. J. Reprod. Fertil. 46(2), 475–476 (1976) 7. J.T. Wu, Z. Dickmann, D.C. Johnson, Effects of ovariectomy or hypophysectomy on day one of pregnancy on development and transport of fertilized rat eggs. J. Endocrinol. 49(3), 507–513 (1971) 8. L.S. Roblero, A.C. Garavagno, Effect of oestradiol-17 beta and progesterone on oviductal transport and early development of mouse embryos. J. Reprod. Fertil. 57(1), 91–95 (1979) 9. W.K. Whitten, The effect of progesterone on the development of mouse eggs in vitro. J. Endocrinol. 16(1), 80–85 (1957) 10. J.C. Daniel Jr, Some effects of steroids on cleavage of rabbit eggs in vitro. Endocrinology 75, 706–710 (1964) 11. J.F. Kirkpatrick, Differential sensitivity of preimplantation mouse embryos in vitro to oestradiol and progesterone. J. Reprod. Fertil. 27(2), 283–285 (1971) 12. E.D. Adamson, Activities of growth factors in preimplantation embryos. J. Cell. Biochem. 53(4), 280–287 (1993). doi:10.1002/ jcb.240530403 13. P. Bhatnagar, V.E. Papaioannou, J.D. Biggers, CSF-1 and mouse preimplantation development in vitro. Development 121(5), 1333–1339 (1995)

123

14. B.C. Paria, S.K. Dey, Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc. Natl. Acad. Sci. USA 87(12), 4756–4760 (1990) 15. E.C. Brice, J.X. Wu, R. Muraro, E.D. Adamson, L.M. Wiley, Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies, antisense RNA, and deoxyoligonucleotides. Dev. Genet. 14(3), 174–184 (1993). doi:10.1002/ dvg.1020140304 16. T.D. Dadi, M.W. Li, K.C. Lloyd, Decreased growth factor expression through RNA interference inhibits development of mouse preimplantation embryos. Comp. Med. 59(4), 331–338 (2009) 17. S. Pampfer, Y.D. Wuu, I. Vanderheyden, R. De Hertogh, Expression of tumor necrosis factor-alpha (TNF alpha) receptors and selective effect of TNF alpha on the inner cell mass in mouse blastocysts. Endocrinology 134(1), 206–212 (1994) 18. H.D. McReynolds, R. Hadek, A comparison of the fine structure of late mouse blastocysts developed in vivo and in vitro. J. Exp. Zool. 182(1), 95–118 (1972). doi:10.1002/jez.1401820109 19. C.L. Stewart, E.B. Cullinan, Preimplantation development of the mammalian embryo and its regulation by growth factors. Dev. Genet. 21(1), 91–101 (1997). doi:10.1002/(SICI)15206408(1997)21:1\91:AID-DVG11[3.0.CO;2-D 20. X. Tang, C. Zhang, Relationship between Sloan-Kettering virus expression and mouse follicular development. Endocrine 40(2), 187–195 (2011). doi:10.1007/s12020-011-9477-y 21. A. Coste, L. Dubuquoy, R. Barnouin, J.S. Annicotte, B. Magnier, M. Notti, N. Corazza, M.C. Antal, D. Metzger, P. Desreumaux, T. Brunner, J. Auwerx, K. Schoonjans, LRH-1-mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease. Proc. Natl. Acad. Sci. USA 104(32), 13098–13103 (2007). doi:10.1073/pnas.0702440104 22. S.M. Soyal, A. Mukherjee, K.Y. Lee, J. Li, H. Li, F.J. DeMayo, J.P. Lydon, Cre-mediated recombination in cell lineages that express the progesterone receptor. Genesis 41(2), 58–66 (2005). doi:10.1002/gene.20098 23. C. Zhang, M.J. Large, R. Duggavathi, F.J. Demayo, J.P. Lydon, K. Schoonjans, E. Kovanci, B.D. Murphy, Liver receptor homolog-1 is essential for pregnancy. Nat. Med. 19(8), 1061–1066 (2013). doi:10.1038/nm.3192 24. S.K. Das, X.N. Wang, B.C. Paria, D. Damm, J.A. Abraham, M. Klagsbrun, G.K. Andrews, S.K. Dey, Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 120(5), 1071–1083 (1994) 25. H. Song, H. Lim, B.C. Paria, H. Matsumoto, L.L. Swift, J. Morrow, J.V. Bonventre, S.K. Dey, Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for ‘on-time’ embryo implantation that directs subsequent development. Development 129(12), 2879–2889 (2002) 26. V.E. Papaioannou, K.M. Ebert, Development of fertilized embryos transferred to oviducts of immature mice. J. Reprod. Fertil. 76(2), 603–608 (1986) 27. V.E. Papaioannou, Diapause of mouse blastocysts transferred to oviducts of immature mice. J. Reprod. Fertil. 76(1), 105–113 (1986) 28. J. Baker, J.P. Liu, E.J. Robertson, A. Efstratiadis, Role of insulinlike growth factors in embryonic and postnatal growth. Cell 75(1), 73–82 (1993) 29. M.M. Lau, C.E. Stewart, Z. Liu, H. Bhatt, P. Rotwein, C.L. Stewart, Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8(24), 2953–2963 (1994)

Endocrine 30. T. Ludwig, J. Eggenschwiler, P. Fisher, A.J. D’Ercole, M.L. Davenport, A. Efstratiadis, Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev. Biol. 177(2), 517–535 (1996). doi:10.1006/dbio1996.0182 31. H. Bostrom, K. Willetts, M. Pekny, P. Leveen, P. Lindahl, H. Hedstrand, M. Pekna, M. Hellstrom, S. Gebre-Medhin, M. Schalling, M. Nilsson, S. Kurland, J. Tornell, J.K. Heath, C. Betsholtz, PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85(6), 863–873 (1996) 32. P. Soriano, Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 8(16), 1888–1896 (1994) 33. G.B. Mann, K.J. Fowler, A. Gabriel, E.C. Nice, R.L. Williams, A.R. Dunn, Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73(2), 249–261 (1993) 34. C.L. Stewart, P. Kaspar, L.J. Brunet, H. Bhatt, I. Gadi, F. Kontgen, S.J. Abbondanzo, Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359(6390), 76–79 (1992). doi:10.1038/359076a0 35. C.B. Ware, M.C. Horowitz, B.R. Renshaw, J.S. Hunt, D. Liggitt, S.A. Koblar, B.C. Gliniak, H.J. McKenna, T. Papayannopoulou,

36.

37.

38.

39.

40.

B. Thoma et al., Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121(5), 1283–1299 (1995) D.R. Brison, R.M. Schultz, Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol. Reprod. 56(5), 1088–1096 (1997) B. Feldman, W. Poueymirou, V.E. Papaioannou, T.M. DeChiara, M. Goldfarb, Requirement of FGF-4 for postimplantation mouse development. Science 267(5195), 246–249 (1995) P.J. Miettinen, J.E. Berger, J. Meneses, Y. Phung, R.A. Pedersen, Z. Werb, R. Derynck, Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376(6538), 337–341 (1995). doi:10.1038/376337a0 M. Sibilia, E.F. Wagner, Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269(5221), 234–238 (1995) D.W. Threadgill, A.A. Dlugosz, L.A. Hansen, T. Tennenbaum, U. Lichti, D. Yee, C. LaMantia, T. Mourton, K. Herrup, R.C. Harris et al., Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269(5221), 230–234 (1995)

123