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ABSTRACT. This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor. (LIFR), and glycoprotein ...

BIOLOGY OF REPRODUCTION 63, 508–512 (2000)

Leukemia Inhibitory Factor, Leukemia Inhibitory Factor Receptor, and Glycoprotein 130 in Rhesus Monkey Uterus During Menstrual Cycle and Early Pregnancy1 Zhan-Peng Yue,3 Zeng-Ming Yang,2,3 Peng Wei,3 Shi-Jie Li,3 Hong-Bin Wang,4 Jing-He Tan,3 and Michael J.K. Harper5 Department of Biotechnology3 and Department of Animal Medicine,4 Northeast Agricultural University, Harbin 150030, China CONRAD Program,5 Arlington, Virginia 22209-2111 ABSTRACT

cy, just preceding blastocyst implantation [4–6]. Experiments with homozygous LIF2/LIF2 mice have shown that maternal LIF expression is essential for mouse blastocyst implantation [7]. During the human menstrual cycle, a higher level of LIF mRNA and protein were found during the luteal phase than during the proliferative phase [8, 9]. The expression of LIF, LIFR, and gp130 was also investigated in human endometrium during the menstrual cycle and the three trimesters of pregnancy [10]. Recent studies suggest that reduced endometrial LIF level contributes to human infertility [11, 12]. The menstrual cycles are similar between rhesus monkeys and humans, and the study was undertaken to investigate the changes of LIF, LIFR, and gp130 in the monkey uterus during the menstrual cycle and the peri-implantation period.

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.

MATERIALS AND METHODS

Animals

Rhesus monkeys (Macaca mulatta) at Fujian Province Non-Human Primate Research Center in China were caged individually in a controlled environment with a cycle of 12L:12D. Animals were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Male rhesus monkeys of proven fertility from previous matings were used for mating. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey. Vaginal smears were examined the next morning for the presence of sperm. The day that a positive smear for sperm was found was designated Day 1 of pregnancy. All of the monkeys were killed by an overdose injection of ketamine (Harbin Chemical Company, Harbin, China). The uteri were removed and treated as described.

female reproductive tract, implantation/early development, menstrual cycle, pregnancy, uterus

INTRODUCTION

Leukemia inhibitory factor (LIF) is a glycoprotein (gp) with pleiotropic activity on a wide variety of cell types in vitro. LIF exerts its effects by binding to cell surface receptors. Equilibrium analysis of the binding of radio-iodinated LIF led to the classification of two receptor types, low affinity and high affinity [1]. The cloned LIF receptor (LIFR) only binds LIF with low affinity; however, subsequent binding of gp130 transforms low affinity LIFRb into a high affinity form and activates signal transduction. Gp130 is a signal-transducing receptor component of LIF, interleukin-6 (IL-6), IL-11, oncostatin M, and ciliary neurotrophic factor receptors, but by itself, does not bind LIF [2, 3]. LIF mRNA and protein in the mouse uterus show two distinct peaks of expression: one during proestrus/estrus, when ovulation occurs; and a second on Day 4 of pregnan-

LIF Immunoblot

An immunoblot was done to verify the specificity of the goat anti-recombinant human LIF (anti-rhLIF) antibody (R&D Systems, Minneapolis, MN) with the scraped monkey uterine endometrium on Day 20 of the menstrual cycle. Monkey uterus was homogenized and the supernatant from ultracentrifugation was run on a 10% SDS-PAGE gel under reduced conditions. After transfer to a polyvinylidene difluoride (PVDF) membrane, individual lanes were cut and blocked with 5% milk/TBS (20 mM Tris-HCl, pH 7.5; 150 mM NaCl) for 1 h, followed by incubation at 208C for 1 h with either a) normal goat immunoglobulin G (IgG) or b) goat anti-rhLIF antibody. The membranes were washed three times for 5 min each in 5% milk/TBS and incubated with rabbit anti-goat IgG (Sigma, St. Louis, MO; 1:1000)

Supported by U.S. CICCR/CONRAD grant CIG-96-07, Eastern Virginia Medical School, and Chinese National Natural Science Foundation grants 39770090 and 39825120. 2 Correspondence. FAX: 86 451 5303336; e-mail: [email protected] 1

Received: 2 July 1999. First decision: 11 August 1999. Accepted: 23 March 2000. Q 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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LIF, LIFR, AND gp130 IN RHESUS MONKEY UTERUS TABLE 1. Degree of LIF, LIFR, and gp130 staining in rhesus monkey uterus during menstrual cycle. Menstrual Cycle (day) Luminal epithelium Glandular epithelium (next to luminal epithelium) Glandular epithelium (next to myometrium) Stroma Myometrium Serosa

Antigen

1

6

9

12

16

20

25

LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130

1/2 1/2 1/2 1 1/2 1/2 1 1 1 2 2 2 1/2 1/2 1/2 2 2 2

1/2 1/2 1/2 1 1 1/2 1 1 1 2 2 1/2 1/2 1/2 1/2 2 2 2

1 1 1/2 1 1 1 11 11 1 2 2 1 1 1/2 1/2 2 2 2

1 1 1 1 1 1 11 11 11 2 2 1 1 1/2 1/2 2 2 2

11 11 1 111 111 111 1111 1111 111 1/2 1/2 11 11 11 11 2 2 2

1 1 1 11 11 11 111 111 11 1/2 1/2 1 11 11 1 2 2 2

1/2 1 1 1 1 1 11 11 1 1/2 1/2 1/2 1 1 1/2 2 2 2

LIF, Leukemia inhibitory factor; LIFR, LIF receptor; 2, no staining; 1/2, slight staining. The larger number of plus signs indicates a greater degree of staining.

conjugated with horseradish peroxidase in 5% milk/TBS for 1 h. The membranes were washed in TBS three times for 5 min each followed by 1 min of incubation with ECL Western Blot reagent (Boehringer-Mannheim, Mannheim, Germany), then exposed on x-ray film for 24 h. Immunohistochemistry

The uteri were immediately cut into small pieces and fixed in Bouins solution for 24 h. The fixed tissues were rinsed in distilled water, dehydrated with graded ethanol, and embedded in paraffin. Sections (8 mm) were cut and deparaffinized. Nonspecific binding was blocked in 3% BSA (Sigma) for 1 h. The sections were incubated with goat anti-rhLIF (1 mg/ml, 1:70; R&D Systems) or goat anti-rhLIFR (250 mg/ml, 1:30; R&D Systems) in 3% BSA at 208C for 2 h. The sections were then incubated with biotinylated secondary antibody followed by an avidin-alkaline phosphatase complex and Vector Red according to the manufacturer’s protocol (Vectastain ABC-AP kit; Vector Laboratories, Burlingame, CA). Vector Red was visualized as a red reaction product. Endogenous alkaline phosphatase activity was inhibited with levamisole (Sigma). Moreover, goat anti-rhLIF or goat anti-hLIFR was replaced by normal goat IgG as a negative control. For the immunostaining of gp130, the deparaffinized sections were treated with 3% H2O2 to quench endogenous peroxidase and then incubated in 3% BSA for 1 h to block the nonspecific binding. The sections were then incubated with guinea pig anti-recombinant human gp130 antibody (rhgp130, TOSOH Corp., Osaka, Japan; 1:50; kindly provided by Professor Kiyoshi Yasukawa) in 3% BSA at 208C for 2 h, and with horseradish peroxidase-conjugated secondary antibody. 3Amino-9-ethylcarbazole (AEC) was used as a substrate and visualized as a red reaction product. Guinea pig antirhgp130 was replaced by normal guinea pig IgG as a negative control. All the sections were counterstained with hematoxylin and mounted. The degree of staining on the slides was subjectively assessed by two independent investigators.

Injection of Anti-Recombinant Human LIF Antibody In Vivo

Pregnant monkeys (n 5 36) on Day 8 of pregnancy were randomly assigned to two groups (18 per group). Under general anesthesia, monkey uteri were exposed by abdominal surgery for intraluminal injection through the fundus with a 25-ml glass microsyringe. In the treatment group, 2 mg of goat anti-rhLIF IgG dissolved in 25 ml of 0.85% NaCl was administered into the uterine lumen on Day 8 of pregnancy, while controls received 2 mg of normal goat IgG dissolved in 25 ml of 0.85% NaCl. Pregnancy was assessed with a B-type ultrasonoscope and rectal palpation on Day 25 of pregnancy. RESULTS

LIF Immunostaining

A band of about 22 kDa was detected in monkey uterus using goat anti-rhLIF antibody in immunoblots, but not when the goat anti-rhLIF antibody was replaced by normal goat IgG (data not shown). Moreover, there was no specific immunostaining when goat anti-rhLIF antibody was replaced by normal goat IgG in uterine sections. The degree of LIF protein staining during the menstrual cycle is summarized in Table 1. On Days 1 and 6 of the menstrual cycle, only slight staining for LIF protein was detected on the luminal and glandular epithelia (Fig. 1A). LIF staining began to increase on Day 9 and reached its highest level on Day 16 of the menstrual cycle (Fig. 1B). Expression of LIF began to decrease on Day 20 of the menstrual cycle (Fig. 1, C and D). Slight LIF staining was detected on the stroma of the uteri from Day 16 to Day 25. LIF staining was also detected on the myometrium, where it remained relatively stable during the menstrual cycle. LIF staining was not detectable on the serosa. The degree of LIF immunostaining during early pregnancy is presented in Table 2. LIF staining in the glandular and luminal epithelium was weak on Days 1 and 2 of pregnancy (Fig. 1E), it gradually increased from Day 3 of pregnancy, and reached its highest level on Day 9 (Fig. 1, F and G). LIF staining was maintained at a high level on Day

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on Day 8 of pregnancy, controls received 2 mg of normal goat IgG. Pregnancy rate was decreased from 66.7% in controls (12 of 18) to 22.2% in the treated group (4 of 18) (P , 0.05, calculated by the Fisher exact test) when pregnancy was confirmed by both B-type ultrasonoscope and rectal palpation on Day 25 of pregnancy. Examination with a Btype ultrasonoscope on Day 25 of pregnancy revealed that the fetus was visible in the uterine lumen and that the size of the uterus was sharply increased. The lumen of a nonpregnant uterus was closed (shaped like a line) and the size of the uterus was relatively small. LIFR Immunostaining

FIG. 1. LIF immunostaining in monkey endometrium. On Days A) 6, B) 12, C) 20, and D) 25 of the menstrual cycle, and on E) Days 2, F) 5, G) 9, and H) 11 of early pregnancy. 3325.

11 (Fig. 1H). LIF staining was also detected on the myometrium, where it remained relatively stable during early pregnancy. Effects of Goat Anti-rhLIF Antibody on Monkey Implantation

In treated animals, 2 mg of goat anti-rhLIF IgG was administered into the uterine lumen by abdominal surgery

The degree of LIFR staining during the menstrual cycle is summarized in Table 1. LIFR was localized mainly in the glandular and luminal epithelia. LIFR uterine staining during the luteal phase was stronger than it was during the proliferative phase (Fig. 2, A–C) and was greatest during the mid-luteal phase of the menstrual cycle (Fig. 2C). Slight LIFR staining was also detected on the stroma of the uteri from Day 16 to Day 25. LIFR staining was also detected on the myometrium, where it remained relatively stable during the menstrual cycle. LIFR staining was not detectable on the serosa. There was no specific staining when goat anti-rhLIFR antibody was replaced with normal goat IgG. The degree of LIFR staining during early pregnancy is summarized in Table 2. LIFR staining in the glandular and luminal epithelium was weak on Days 1 and 2, it gradually increased from Day 3 of pregnancy, and reached its highest level on Day 9 (Fig. 2D). LIFR staining was maintained at a high level on Day 11 (Fig. 2E). LIFR staining was also detected on the myometrium, where it remained relatively stable during early pregnancy. Gp130 Immunostaining

The degree of gp130 staining during the menstrual cycle is summarized in Table 1. Gp130 was mainly localized in the glandular epithelium. Gp130 staining during the luteal phase was stronger than it was during the proliferative phase (Fig. 2, F and G). The highest level of gp130 staining

TABLE 2. Degree of LIF, LIFR, and gp130 staining in rhesus monkey uterus during early pregnancy. Early Pregnancy (day) Luminal epithelium Glandular epithelium (next to luminal epithelium) Glandular epithelium (next to myometrium) Stroma Myometrium Serosa

Antigen

1

2

3

5

7

9

11

LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130 LIF LIFR gp130

1/2 1 1/2 1 1 1 1 1 1 2 2 1/2 1 1/2 1 2 2 2

1 1 1/2 1 1 11 11 11 11 2 2 1/2 1 1/2 1 2 2 2

11 11 1/2 11 11 11 111 111 11 2 2 1/2 1 1 1 2 2 2

11 11 1/2 11 11 111 111 111 111 2 2 1/2 1 1 1 2 2 2

11 11 1/2 11 11 111 111 111 111 2 2 1/2 1 1 1 2 2 2

111 111 1/2 111 111 111 111 111 111 2 1/2 1/2 1 1 1 2 2 2

111 111 1/2 111 111 111 111 111 111 2 1/2 1/2 1 1 1 2 2 2

LIF, Leukemia inhibitory factor; LIFR, LIF receptor; 2, no staining; 1/2, slight staining. The larger number of plus signs indicates a greater degree of staining.

LIF, LIFR, AND gp130 IN RHESUS MONKEY UTERUS

FIG. 2. LIFR immunostaining shown in monkey endometriumon on Days A) 6, B) 16, and C) 20 of the menstrual cycle, and on Days (D) 9 and (E) 11 of early pregnancy. Gp130 immunostaining in monkey endometrium was shown on Days (F) 16 and (G) 20 of the menstrual cycle, and on Day (H) 9 of early pregnancy. 3325.

was detected during the mid-luteal phase of the menstrual cycle (Fig. 2G). There was slight gp130 staining of the uterine stroma during the menstrual cycle. Gp130 staining was also detected on the myometrium, where it remained relatively stable during the menstrual cycle. There was no detectable gp130 staining on the serosa. There was no specific staining when guinea pig anti-rhgp130 antibody was replaced with normal guinea pig IgG. The degree of gp130 staining during early pregnancy is summarized in Table 2. In early pregnancy, gp130 was also mainly localized in glandular epithelium. Slight staining for gp130 was also detected on the luminal epithelium, stroma, and myometrium. Gp130 staining in the glandular epithelium was weak on Day 1, began to increase on Day 2 of pregnancy, and maintained a high level from Day 5 to Day 11 of pregnancy (Fig. 2H). DISCUSSION

This study examined the presence of LIF, LIFR, and gp130 in the monkey uterus during the menstrual cycle and early pregnancy. The results indicate that the staining of LIF and its receptor during the luteal phase is stronger than it is during the proliferative phase. In the cycle, the staining of LIF and its receptor is greatest during the mid-luteal phase; in early pregnancy, the staining is greatest on Day 9. Rhesus monkeys have a regular menstrual cycle (approximately 28 days), similar to women. During the human menstrual cycle, LIF mRNA and protein are mainly localized in glandular and luminal epithelia. Expression of LIF

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from mid- to late-luteal phase is stronger than it is during the proliferative phase [10, 13, 14]. Our results showed that a higher level of LIF staining was present in monkey uterus on Days 16 to 20 of the menstrual cycle, similar to LIF expression in human uterus during the menstrual cycle. In the human uterus, LIFR was mainly localized in luminal epithelium but not in the stroma or glandular epithelium. Gp130 was mainly localized in luminal and glandular epithelium and was expressed throughout the cycle, with expression peaking during the secretory phase of the cycle. The staining of LIFR and gp130 was not detectable in stroma of human uterus [10]. Our results showed that LIFR and gp130 were mainly localized in glandular epithelium, luminal epithelium, and myometrium. The staining of LIFR and gp130 during the mid-luteal phase was stronger than it was during the proliferative phase. LIFR staining was detectable in stroma during the monkey luteal phase. Gp130 staining was detectable in stroma during the monkey menstrual cycle. The expression of LIFR and gp130 in monkey uterus during the menstrual cycle is not entirely similar to the expression of LIFR and gp130 in human uterus. A high level of LIF mRNA and protein in the mouse uterus was detected during estrus, when ovulation occurs, and on Day 4 of pregnancy, which just precedes blastocyst implantation [4–6]. That the two peaks of LIF expression in mouse uterus are consistent with the high level of maternal estrogen, and that estrogen can induce LIF expression in ovariectomied mouse uterus indicates that the expression of LIF in mouse uterus may be regulated by estrogen [15]. In human uterus, LIF expression during the luteal phase is stronger than it is during the proliferative phase. In the cycle, LIF expression is greatest during the mid- to lateluteal phase [14]. Our results showed that a high level of LIF expression appeared during the mid-luteal phase in monkey uterus, not during the ovulatory period. In early pregnancy, a high level of LIF expression appeared on Day 9 of pregnancy, which just precedes blastocyst implantation. The peak of LIF expression in monkey uterus coincided with the high level of maternal progesterone, and their up-regulation of LIF in monkey uterus may be regulated by progesterone. In humans, LIF endometrial production is significantly down-regulated by progesterone both in vivo and in vitro [16]. However, Ghosh et al. [17] reported that there was no difference in LIF protein in Day 6-gestational endometrium between monkeys treated with RU-486 and controls. Because most cells that respond to LIF express both low and high affinity receptors, a functional LIFR complex may require the co-localization of the high affinity conversion component, gp130, and the low affinity LIFRb receptor [2, 18]. Our results showed that high levels of LIFR and gp130 were observed in monkey uterus during Days 16 to 20 of the menstrual cycle and the peri-implantation period. Hence, a functional high affinity LIFR may exist in monkey uterus during these times. This coincided with a high level of LIF staining in monkey uterus. In addition, monkey pregnancy rates could be significantly reduced in controls from 66.7% to 22.2% with an intraluminal injection of goat anti-rhLIF IgG. These results suggest that LIF may also play an important role in monkey implantation, as it does in mouse; however, the mechanism of LIF action in monkey uterus is still unknown. The coexpression of the mRNAs for LIFR and gp130 was detected in human preimplantation embryos at the blastocyst stage [19]. The human embryonic development was also dramatically improved by exogenous LIF [20]. To date, it is still not clear whether monkey em-

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bryos express LIFR and gp130. It is possible that uterine LIF expression in the monkey may be essential for embryo implantation through an autocrine/paracrine interaction between LIF and its receptor in the luminal epithelium. ACKNOWLEDGMENTS We thank Xun-Li Wang and Zhi-Xong Li of the Fujian Non-Human Primate Center for their expert technical assistance.

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9. Chen DB, Hilsenrath R, Yang ZM, Le SP, Kim SR, Chuong CJ, Poindexter III AN, Harper MJK. Leukemia inhibitory factor in human endometrium during the menstrual cycle: cellular origin and action on production of glandular epithelial cell prostaglandin in vitro. Hum Reprod 1995; 10:911–918. 10. Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart CL. Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci U S A 1996; 93:3115–3120. 11. Hambartsoumian E. Endometrial leukemia inhibitory factor (LIF) as a possible cause of unexplained infertility and multiple failures of implantation. Am J Reprod Immunol 1998; 39:137–143. 12. Vogiagis D, Salamonsen LA. The role of leukemia inhibitory factor in the establishment of pregnancy. J Endocrinol 1999; 160:181–190. 13. Senturk LM, Arici A. Leukemia inhibitory factor in human reproduction. Am J Reprod Immunol 1998; 39:144–151. 14. Vogiagis D, Marsh MM, Fry RC, Salamonsen LA. Leukaemia inhibitory factor in human endometrium throughout the menstrual cycle. J Endocrinol 1996; 148:95–102. 15. Stewart CL. Leukemia inhibitory factor and the regulation of preimplantation development of the mammalian embryo. Mol Reprod Dev 1994; 39:233–238. 16. Hambartsoumian E, Taupin JL, Moreau JF, Frydman R, Chaouat G. In-vivo administration of progesterone inhibits the secretion of endometrial leukaemia inhibitory factor in vitro. Mol Hum Reprod 1998; 4:1039–1044. 17. Ghosh D, Kumar PGLL, Sengupta J. Effect of early luteal phase administration of mifepristone (RU486) on leukaemia inhibitory factor, transforming growth factor b and vascular endothelial growth factor in the implantation stage endometrium of the rhesus monkey. J Endocrinol 1998; 157:115–125. 18. Gearing DP. The leukemia inhibitory factor and its receptor. Adv Immunol 1993; 53:31–58. 19. Charnock-Jones DS, Sharkey AM, Fenwick P, Smith SK. Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time. J Reprod Fertil 1994; 101:421–426. 20. Dunglison QF, Barlow DH, Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rate of human embryos cultured in serum free medium. Hum Reprod 1996; 11:191–196.

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