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Deciphering early folliculogenesis: Jun Amino Terminal Kinase (JNK) plays a key role in preantral follicle growth
tolerated, and well absorbed, producing notable plasma leuprolide levels that approximate those seen following traditional SQ administration.
Monday, October 13, 2003 2:45 P.M. O-67 Progestogen effects on mood and cognition in early menopause. Robert Krikorian, Jeffrey A. Welge, Marcelle D. Shidler, Ken Muse, James H. Liu. Univ of Cincinnati, Cincinnati, OH; Univ of Kentucky, Lexington, KY; Case Western Univ, Cleveland, OH. Objective: To evaluate progesterone and differing progestogen treatments with respect to cognitive function and mood in healthy women in early menopause. Design: This was a randomized, placebo-controlled, double-blind trial with longitudinal assessments of neuropsychological function and mood at baseline and 6 and 24 months. In addition to placebo, there was a progesterone and three progestogen treatment groups, one with estradiol augmentation. Materials and Methods: Subjects were recruited from regions served by two university medical centers in the midwestern U.S. Prospective subjects who had used hormone replacement therapy previously, who had increased risk for cancer, or had a potentially contributing psychiatric or neurological condition were excluded. One hundred forty-two healthy women who had not experienced a menstrual cycle in at least 12 months following either surgery or natural menopause were randomized to one of five treatment groups, including micronized oral progesterone, 400 mg/day; norethindrone (19-nortestosterone), 1.0 mg/day; medroxyprogesterone acetate (3 methyl analogue progesterone), 10 mg/day; medroxyprogesterone acetate, 10 mg/ day plus micronized estradiol, 1 mg/day; and placebo for 24 months. Eighty-four women completed the study. A comprehensive neuropsychological protocol was administered to all subjects at pretreatment baseline and after 6 and 24 months of treatment. The outcome measures included tests of executive function abilities, attention, episodic memory, motor function, and mood. Repeated measures analyses of variance for each domain of functioning were performed to identify changes from baseline at 6 and 24 months. Results: There was no difference between groups at baseline in age (F ⫽ .48, ns), educational level (F ⫽ .66, ns), IQ estimate (F ⫽ 1.56, ns), or level of depression (F ⫽ .1.19, ns). Mean group scores at baseline on the Hamilton depression scale were well below clinical threshold and ranged from 5.4 to 7.8 (overall M ⫽ 6.3). There was no treatment effect on the neuropsychological measures: executive planning ability (F ⫽ 1.63, ns); working memory (F ⫽ 0.26, ns); verbal attention (F ⫽ 0.17, ns); spatial attention (F ⫽ .41, ns); episodic memory (F ⫽ 0.57, ns); and fine motor speed (F ⫽ 0.23, ns). However, there was a significant group effect for mood (F ⫽ 2.77, p ⬍ .05). This latter effect was attributable to a decline in total mood disturbance in the norethindrone group from baseline (M ⫽ 20.9) to 6 months (M ⫽ 5.2), and this decline was maintained until study termination at 24 months (M ⫽ 6.3). Conclusion: These findings indicate that short- and long-term progesterone and progestogen therapy, alone or in combination with estradiol, do not affect cognition positively or negatively in healthy, postmenopausal women. On the other hand, norethindrone produced improvement in overall mood, independent of cognitive function, and this specific effect persisted for the duration of the trial. This improvement in mood is particularly notable given that women were not clinically depressed at baseline and may reflect the greater androgenic nature of this particular progestogen.
Monday, October 13, 2003 3:00 P.M. O-68 The impact of the ACGME workweek requirements on resident participation in REI clinics and surgical cases. Eve C. Feinberg, Magdy P. Milad. Northwestern University’s Feinberg Sch of Medicine, Chicago, IL. Objective: To assess the impact of the ACGME 80-hour workweek requirements on resident education in REI.
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Abstracts
Design: Cross-sectional survey administration via e-mail. Materials and Methods: A survey was sent to Ob/Gyn residency program directors addressing the amount of time per year residents spend on the REI rotation, frequency of night call, the existence of a night float system, percent time spent in various areas (clinic, OR, minor procedures) and teaching methodology. Program directors were asked which aspects of REI clinical training would change with the new workweek requirements. Results: Of the 184 programs surveyed, 36 responded (20% response rate). Of these, 22 (61%) were from university-based programs and 4 (11%) were from New York and already in compliance. The average number of residents per year is 5.5. Residents spend 9.76 weeks (range 4-20 weeks) on the REI service during their four years in training with the following breakdown: 3 weeks (1-6 weeks) during PGY-1, 5.7 weeks (2-8 weeks) during PGY-2, 7.5 weeks (1-16 weeks) during PGY-3 and 4.8 weeks (1-8 weeks) during PGY-4. The bulk of REI training occurs during PGY-3. Only one program anticipated a reduction in the number of weeks on the rotation however 20 programs expected a decline in clinical experience to accommodate a night float system. To compensate for decreased clinical time, 20% of programs are increasing didactic time and nonclinical teaching. Conclusion: Currently, REI rotations account for a small percentage of the overall training during an Obstetrics and Gynecology residency. With accommodation of the 80-hour workweek restrictions, many residents are going to be experiencing declines in clinical education in REI. Further studies are needed to elucidate the impact of declining REI experience on the level of comfort of graduates managing patients with endocrine and infertility complaints.
Monday, October 13, 2003 4:00 P.M. O-69 Deciphering early folliculogenesis: Jun Amino Terminal Kinase (JNK) plays a key role in preantral follicle growth. Kutluk H. Oktay, Erkan Buyuk, Ozgur Oktem, Maja Oktay, Zev Rosenwaks, Filippo Giancotti. Weill Medical Coll of Cornell Univ, New York, NY; Montefiore Medical Ctr, Dept of Pathology, New York, NY; Memorial Sloan Kettering Cancer Ctr, New York, NY. Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK. Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles. Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day. Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2⫾6.5m vs. day 9: 114.56⫾5.8m, p ⬍ 0.001). Whereas in controls, follicles continued to grow (144.88⫾17.5m vs. 164.34⫾19.1m, p ⫽ 0.001).
Vol. 80, Suppl. 3, September 2003
microns in diameter that were spherical with a centrally located oocyte, had an intact basement membrane, and included a theca layer were placed individually in culture wells or culture tubes. Culture medium was alphaMEM with ITS⫹, 8-br-cGMP, antibiotics with or without recombinant FSH (100 ng/ml). Culture tubes were placed in an orbital rotator at 15 RPM. Both sets of cultures (plates and tubes) were incubated at 37 degrees and 5% CO2 for 72 hours. Follicle diameter in three dimensions was measured daily. Flattening was mathematically defined as asymmetry in the follicle diameter along the different dimensional axes. A subset of individual follicles from each group underwent DNA quantification using a fluorescent dye and a microplate fluorescence reader in order to verify that an increase in follicle size was accompanied with increased cell number. Results: As expected, FSH induced growth of follicles in both plates and tubes. Follicles cultured in 96-well plates in the absence of FSH did not have an increase in diameter. Follicles cultured with FSH had a 20.5% increase in diameter (p ⬍ 0.05) relative to control. However, over half of the follicles (53.5%) underwent flattening of an average of 14% (range 5.1-26%). Rupturing of follicles also occurred frequently 16.3%, most often between day 2 and day 3 of culture. Of note, the follicles that remained spherical had a slower rate of growth than the flattened or ruptured follicles (12.5% vs 25%, p ⬍ 0.05). Follicles cultured in tubes in the absence of FSH exhibited minimal increase in follicle diameter. However, follicles grown in tubes in the presence of FSH underwent a marked 42% increase in follicle diameter and had twice the DNA content of follicles cultured in the absence of FSH. No follicles cultured in tubes underwent flattening or rupture. Conclusions: Culture of follicles in a suspension culture system results in rapid follicular growth while simultaneously maintaining a more normal follicle anatomy. This represents an advance over culture on a flat surface in which there is significant distortion of follicle anatomy and follicle loss. The ability to culture larger follicles, while maintaining the anatomic relationships between theca, granulosa, and oocyte is a step toward longterm culture of follicles of larger mammals. This is essential if we are to develop reliable in vitro culture of cryopreserved follicles as a treatment for infertility.
Monday, October 13, 2003 4:30 P.M. O-71 Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth. 1. Oktay et al. Biol Reprod 2000(63):457. Supported by: NIH HDO43339-01 (to K.O).
Monday, October 13, 2003 4:15 P.M. O-70 Suspension culture of intact ovarian follicles maintains spherical threedimensional structure better than traditional tissue culture. Elizabeth A. McGee, Neshat Rowgani, Matthew Heise, Dan McKeel, Richard Koepsel, Alan Russell. Univ of Pittsburgh, Pittsburgh, PA. Objective: Though it is possible to culture intact mouse follicles to maturity, culture of follicles from larger mammals has not been as successful. We have found that rat follicles grown in traditional flat tissue culture wells often become flattened and lose anatomic integrity just before or just after the acquisition of an antrum. In this study, we have compared the effect of culture on a flat surface with culture in suspension on the anatomic integrity and growth rate of preantral follicles derived from rats. Design: Mechanically dissected follicles from immature rats were cultured in traditional 98-well plates or in culture tubes in an orbital rotator. Growth rate and degree of flattening or rupture were determined for follicles in each system in the presence and absence of FSH. Materials and Methods: Preantral follicles were mechanically dissected from ovaries of juvenile Sprague-Dawley rats. Follicles between 140 –150
FERTILITY & STERILITY威
Acrogranin (Granulin Epithelin Precursor, PCDFG) is crucial for mouse embryo receptivity and implantation. Christian M. Perez, Laura Diaz-cueto, Dustin Van der voort, Juan E. Schwarze, George L. Gerton. Univ of Pennsylvania, Philadelphia, PA; Unidad de Invest en Medicine reproductiva. IMSS, Mexico D.F., Mexico. Introduction: Acrogranin, also called the granulin-epithelin precursor or PC-derived growth factor PCDGF is a 67,000 Mr glycoprotein with growth regulatory activity and potent mitogenic effects. It has been encountered in many tissues and is highly expressed in the kidney, uterus, and trophectoderm of preimplantation mouse embryos, suggesting an important role in uterine biology and reproductive function. Objective: To determine the acrogranin gene and protein statement during the estrous cycle and early implantation period. Design: Prospective experimental laboratory study. Materials and Methods: We approached the problem using the in-vivo mouse model during the estrous cycle, normal pregnancy and delayed implantation and performing SDS-page, Western immunoblotting, immunohistochemistry, in-situ hybridization, real time RT-PCR, and antibody function-blocking experiments. Results: Acrogranin protein and transcripts were found mainly in epithelial endometrial cells but also and weakly in glandular, stromal, and smooth muscle cells of the mouse uterus. Expression levels were found to be cyclical during estrous cycle being weak at proestrous, intermediate at estrous, and robust at diestrous stage, meaning that acrogranin expression is hormonally influenced as the uterus prepares for embryo receptivity or implantation. We found that acrogranin mRNA and protein amounts in female adult mice were low before implantation, increased 6-7 fold during peri-implantation, and decreased again thereafter to previous levels. During normal pregnancy acrogranin protein and mRNA expression patterns were strongly localized in the implantation chamber around the embryo and signals were very low or absent in the interimplantation zones (tissue between two
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Monday, October 13, 2003 2:45 P.M. O-67 Progestogen effects on mood and cognition in early menopause. Robert Krikorian, Jeffrey A. Welge, Marcelle D. Shidler, Ken Muse, James H. Liu. Univ of Cincinnati, Cincinnati, OH; Univ of Kentucky, Lexington, KY; Case Western Univ, Cleveland, OH. Objective: To evaluate progesterone and differing progestogen treatments with respect to cognitive function and mood in healthy women in early menopause. Design: This was a randomized, placebo-controlled, double-blind trial with longitudinal assessments of neuropsychological function and mood at baseline and 6 and 24 months. In addition to placebo, there was a progesterone and three progestogen treatment groups, one with estradiol augmentation. Materials and Methods: Subjects were recruited from regions served by two university medical centers in the midwestern U.S. Prospective subjects who had used hormone replacement therapy previously, who had increased risk for cancer, or had a potentially contributing psychiatric or neurological condition were excluded. One hundred forty-two healthy women who had not experienced a menstrual cycle in at least 12 months following either surgery or natural menopause were randomized to one of five treatment groups, including micronized oral progesterone, 400 mg/day; norethindrone (19-nortestosterone), 1.0 mg/day; medroxyprogesterone acetate (3 methyl analogue progesterone), 10 mg/day; medroxyprogesterone acetate, 10 mg/ day plus micronized estradiol, 1 mg/day; and placebo for 24 months. Eighty-four women completed the study. A comprehensive neuropsychological protocol was administered to all subjects at pretreatment baseline and after 6 and 24 months of treatment. The outcome measures included tests of executive function abilities, attention, episodic memory, motor function, and mood. Repeated measures analyses of variance for each domain of functioning were performed to identify changes from baseline at 6 and 24 months. Results: There was no difference between groups at baseline in age (F ⫽ .48, ns), educational level (F ⫽ .66, ns), IQ estimate (F ⫽ 1.56, ns), or level of depression (F ⫽ .1.19, ns). Mean group scores at baseline on the Hamilton depression scale were well below clinical threshold and ranged from 5.4 to 7.8 (overall M ⫽ 6.3). There was no treatment effect on the neuropsychological measures: executive planning ability (F ⫽ 1.63, ns); working memory (F ⫽ 0.26, ns); verbal attention (F ⫽ 0.17, ns); spatial attention (F ⫽ .41, ns); episodic memory (F ⫽ 0.57, ns); and fine motor speed (F ⫽ 0.23, ns). However, there was a significant group effect for mood (F ⫽ 2.77, p ⬍ .05). This latter effect was attributable to a decline in total mood disturbance in the norethindrone group from baseline (M ⫽ 20.9) to 6 months (M ⫽ 5.2), and this decline was maintained until study termination at 24 months (M ⫽ 6.3). Conclusion: These findings indicate that short- and long-term progesterone and progestogen therapy, alone or in combination with estradiol, do not affect cognition positively or negatively in healthy, postmenopausal women. On the other hand, norethindrone produced improvement in overall mood, independent of cognitive function, and this specific effect persisted for the duration of the trial. This improvement in mood is particularly notable given that women were not clinically depressed at baseline and may reflect the greater androgenic nature of this particular progestogen.
Monday, October 13, 2003 3:00 P.M. O-68 The impact of the ACGME workweek requirements on resident participation in REI clinics and surgical cases. Eve C. Feinberg, Magdy P. Milad. Northwestern University’s Feinberg Sch of Medicine, Chicago, IL. Objective: To assess the impact of the ACGME 80-hour workweek requirements on resident education in REI.
S26
Abstracts
Design: Cross-sectional survey administration via e-mail. Materials and Methods: A survey was sent to Ob/Gyn residency program directors addressing the amount of time per year residents spend on the REI rotation, frequency of night call, the existence of a night float system, percent time spent in various areas (clinic, OR, minor procedures) and teaching methodology. Program directors were asked which aspects of REI clinical training would change with the new workweek requirements. Results: Of the 184 programs surveyed, 36 responded (20% response rate). Of these, 22 (61%) were from university-based programs and 4 (11%) were from New York and already in compliance. The average number of residents per year is 5.5. Residents spend 9.76 weeks (range 4-20 weeks) on the REI service during their four years in training with the following breakdown: 3 weeks (1-6 weeks) during PGY-1, 5.7 weeks (2-8 weeks) during PGY-2, 7.5 weeks (1-16 weeks) during PGY-3 and 4.8 weeks (1-8 weeks) during PGY-4. The bulk of REI training occurs during PGY-3. Only one program anticipated a reduction in the number of weeks on the rotation however 20 programs expected a decline in clinical experience to accommodate a night float system. To compensate for decreased clinical time, 20% of programs are increasing didactic time and nonclinical teaching. Conclusion: Currently, REI rotations account for a small percentage of the overall training during an Obstetrics and Gynecology residency. With accommodation of the 80-hour workweek restrictions, many residents are going to be experiencing declines in clinical education in REI. Further studies are needed to elucidate the impact of declining REI experience on the level of comfort of graduates managing patients with endocrine and infertility complaints.
Monday, October 13, 2003 4:00 P.M. O-69 Deciphering early folliculogenesis: Jun Amino Terminal Kinase (JNK) plays a key role in preantral follicle growth. Kutluk H. Oktay, Erkan Buyuk, Ozgur Oktem, Maja Oktay, Zev Rosenwaks, Filippo Giancotti. Weill Medical Coll of Cornell Univ, New York, NY; Montefiore Medical Ctr, Dept of Pathology, New York, NY; Memorial Sloan Kettering Cancer Ctr, New York, NY. Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK. Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles. Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day. Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2⫾6.5m vs. day 9: 114.56⫾5.8m, p ⬍ 0.001). Whereas in controls, follicles continued to grow (144.88⫾17.5m vs. 164.34⫾19.1m, p ⫽ 0.001).
Vol. 80, Suppl. 3, September 2003
microns in diameter that were spherical with a centrally located oocyte, had an intact basement membrane, and included a theca layer were placed individually in culture wells or culture tubes. Culture medium was alphaMEM with ITS⫹, 8-br-cGMP, antibiotics with or without recombinant FSH (100 ng/ml). Culture tubes were placed in an orbital rotator at 15 RPM. Both sets of cultures (plates and tubes) were incubated at 37 degrees and 5% CO2 for 72 hours. Follicle diameter in three dimensions was measured daily. Flattening was mathematically defined as asymmetry in the follicle diameter along the different dimensional axes. A subset of individual follicles from each group underwent DNA quantification using a fluorescent dye and a microplate fluorescence reader in order to verify that an increase in follicle size was accompanied with increased cell number. Results: As expected, FSH induced growth of follicles in both plates and tubes. Follicles cultured in 96-well plates in the absence of FSH did not have an increase in diameter. Follicles cultured with FSH had a 20.5% increase in diameter (p ⬍ 0.05) relative to control. However, over half of the follicles (53.5%) underwent flattening of an average of 14% (range 5.1-26%). Rupturing of follicles also occurred frequently 16.3%, most often between day 2 and day 3 of culture. Of note, the follicles that remained spherical had a slower rate of growth than the flattened or ruptured follicles (12.5% vs 25%, p ⬍ 0.05). Follicles cultured in tubes in the absence of FSH exhibited minimal increase in follicle diameter. However, follicles grown in tubes in the presence of FSH underwent a marked 42% increase in follicle diameter and had twice the DNA content of follicles cultured in the absence of FSH. No follicles cultured in tubes underwent flattening or rupture. Conclusions: Culture of follicles in a suspension culture system results in rapid follicular growth while simultaneously maintaining a more normal follicle anatomy. This represents an advance over culture on a flat surface in which there is significant distortion of follicle anatomy and follicle loss. The ability to culture larger follicles, while maintaining the anatomic relationships between theca, granulosa, and oocyte is a step toward longterm culture of follicles of larger mammals. This is essential if we are to develop reliable in vitro culture of cryopreserved follicles as a treatment for infertility.
Monday, October 13, 2003 4:30 P.M. O-71 Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth. 1. Oktay et al. Biol Reprod 2000(63):457. Supported by: NIH HDO43339-01 (to K.O).
Monday, October 13, 2003 4:15 P.M. O-70 Suspension culture of intact ovarian follicles maintains spherical threedimensional structure better than traditional tissue culture. Elizabeth A. McGee, Neshat Rowgani, Matthew Heise, Dan McKeel, Richard Koepsel, Alan Russell. Univ of Pittsburgh, Pittsburgh, PA. Objective: Though it is possible to culture intact mouse follicles to maturity, culture of follicles from larger mammals has not been as successful. We have found that rat follicles grown in traditional flat tissue culture wells often become flattened and lose anatomic integrity just before or just after the acquisition of an antrum. In this study, we have compared the effect of culture on a flat surface with culture in suspension on the anatomic integrity and growth rate of preantral follicles derived from rats. Design: Mechanically dissected follicles from immature rats were cultured in traditional 98-well plates or in culture tubes in an orbital rotator. Growth rate and degree of flattening or rupture were determined for follicles in each system in the presence and absence of FSH. Materials and Methods: Preantral follicles were mechanically dissected from ovaries of juvenile Sprague-Dawley rats. Follicles between 140 –150
FERTILITY & STERILITY威
Acrogranin (Granulin Epithelin Precursor, PCDFG) is crucial for mouse embryo receptivity and implantation. Christian M. Perez, Laura Diaz-cueto, Dustin Van der voort, Juan E. Schwarze, George L. Gerton. Univ of Pennsylvania, Philadelphia, PA; Unidad de Invest en Medicine reproductiva. IMSS, Mexico D.F., Mexico. Introduction: Acrogranin, also called the granulin-epithelin precursor or PC-derived growth factor PCDGF is a 67,000 Mr glycoprotein with growth regulatory activity and potent mitogenic effects. It has been encountered in many tissues and is highly expressed in the kidney, uterus, and trophectoderm of preimplantation mouse embryos, suggesting an important role in uterine biology and reproductive function. Objective: To determine the acrogranin gene and protein statement during the estrous cycle and early implantation period. Design: Prospective experimental laboratory study. Materials and Methods: We approached the problem using the in-vivo mouse model during the estrous cycle, normal pregnancy and delayed implantation and performing SDS-page, Western immunoblotting, immunohistochemistry, in-situ hybridization, real time RT-PCR, and antibody function-blocking experiments. Results: Acrogranin protein and transcripts were found mainly in epithelial endometrial cells but also and weakly in glandular, stromal, and smooth muscle cells of the mouse uterus. Expression levels were found to be cyclical during estrous cycle being weak at proestrous, intermediate at estrous, and robust at diestrous stage, meaning that acrogranin expression is hormonally influenced as the uterus prepares for embryo receptivity or implantation. We found that acrogranin mRNA and protein amounts in female adult mice were low before implantation, increased 6-7 fold during peri-implantation, and decreased again thereafter to previous levels. During normal pregnancy acrogranin protein and mRNA expression patterns were strongly localized in the implantation chamber around the embryo and signals were very low or absent in the interimplantation zones (tissue between two
S27