Perinatal Bisphenol A Exposure in C57B6/129svj Male Mice: Potential Altered Cytokine/Chemokine Production in Adulthood

Pregnant mice (n = 3) were exposed to BPA by intraperitoneal injection, from gestation day 9.5 until end of lactation. Male offspring were evaluated for cytokine production at 20 wk-of-age. One pregnant control mouse produced no males, precluding statistical analysis. However, recurring shifts in cytokines were suggested in the adult BPA offspring. Serum showed a numeric increase in 16 of 21 basal cytokine levels. ConA-stimulated splenocytes showed a numeric increase in 17 of 21 cytokines, and LPS-stimulated splenocytes an increase in 18 of 21 cytokines. The cytokine profile was one of TH1 up-regulation more than TH2, and with skewing toward TH17 responses

Progesterone receptor-mediated regulation of N-acetylneuraminate pyruvate lyase (NPL) in mouse uterine luminal epithelium and nonessential role of NPL in uterine function.

N-acetylneuraminate pyruvate lyase (NPL) catalyzes N-acetylneuraminic acid, the predominant sialic acid. Microarray analysis of the periimplantation mouse uterine luminal epithelium (LE) revealed Npl being the most downregulated (35×) gene in the LE upon embryo implantation. In natural pregnant mouse uterus, Npl expression increased 56× from gestation day 0.5 (D0.5) to D2.5. In ovariectomized mouse uterus, Npl was significantly upregulated by progesterone (P4) but downregulated by 17β-estradiol (E2). Progesterone receptor (PR) antagonist RU486 blocked the upregulation of Npl in both preimplantation uterus and P4-treated ovariectomized uterus. Npl was specifically localized in the preimplantation D2.5 and D3.5 uterine LE. Since LE is essential for establishing uterine receptivity, it was hypothesized that NPL might play a critical role in uterine function, especially during embryo implantation. This hypothesis was tested in the Npl ((-/-)) mice. No significant differences were observed in the numbers of implantation sites on D4.5, gestation periods, litter sizes, and postnatal offspring growth between wild type (WT) and Npl ((-/-)) females from mating with WT males. Npl ((-/-))xNpl ((-/-)) crosses produced comparable little sizes as that from WTxWT crosses. Comparable mRNA expression levels of several genes involved in sialic acid metabolism were observed in D3.5 uterus and uterine LE between WT and Npl ((-/-)), indicating no compensatory upregulation in the D3.5 Npl ((-/-)) uterus and LE. This study demonstrates PR-mediated dynamic expression of Npl in the periimplantation uterus and dispensable role of Npl in uterine function and embryo development

Distinct spatiotemporal expression of serine proteases Prss23 and Prss35 in periimplantation mouse uterus and dispensable function of Prss35 in fertility.

PRSS23 and PRSS35 are homologous proteases originally identified in mouse ovaries. In the periimplantation mouse uterus, Prss23 was highly expressed in the preimplantation gestation day 3.5 (D3.5) uterine luminal epithelium (LE). It disappeared from the postimplantation LE and reappeared in the stromal compartment next to the myometrium on D6.5. It was undetectable in the embryo from D4.5 to D6.5 but highly expressed in the embryo on D7.5. Prss35 became detectable in the uterine stromal compartment surrounding the embryo on D4.5 and shifted towards the mesometrial side of the stromal compartment next to the embryo from D5.5 to D7.5. In the ovariectomized uterus, Prss23 was moderately and Prss35 was dramatically downregulated by progesterone and 17β-estradiol. Based on the expression of Prss35 in granulosa cells and corpus luteum of the ovary and the early pregnant uterus, we hypothesized that PRSS35 might play a role in female reproduction, especially in oocyte development, ovulation, implantation, and decidualization. This hypothesis was tested in Prss35((-/-)) mice, which proved otherwise. Between wild type (WT) and Prss35((-/-)) mice, superovulation of immature females produced comparable numbers of cumulus-oocyte complexes; there were comparable numbers of implantation sites detected on D4.5 and D7.5; there were no obvious differences in the expression of implantation and decidualization marker genes in D4.5 or D7.5 uteri. Comparable mRNA expression levels of a few known protease-related genes in the WT and Prss35((-/-)) D4.5 uteri indicated no compensatory upregulation. Comparable litter sizes from WT × WT and Prss35((-/-))× Prss35((-/-)) crosses suggested that Prss35 gene was unessential for fertility and embryo development. Prss35 gene has been linked to cleft lip/palate in humans. However, no obvious such defects were observed in Prss35((-/-)) mice. This study demonstrates the distinct expression of Prss23 and Prss35 in the periimplantation uterus and the dispensable role of Prss35 in fertility and embryo development

Altered Spatiotemporal Expression of Collagen Types I, III, IV, and VI in Lpar3-Deficient Peri-Implantation Mouse Uterus1

Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3−/− uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3−/− uterus. An observed delay of COL III and COL VI clearance in the Lpar3−/− uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3−/− uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3−/− uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3−/− uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus

High Serum FSH is Associated with Brown Oocyte Formation and a Lower Pregnacy Rate in Human IVF Parctice

Background/Aims: To investigate whether brown zona pellucida (ZP) of oocytes affects the outcome of fertilization, embryo quality and pregnancy rate in in vitro fertilization-embryo transfer (IVF-ET). Methods: Based on the ZP color of their oocytes, a total number of 703 patients dated from 2012 to 2014 were divided into a normal egg group (group A) and a brown oocyte group (group B), with 629 and 74 cases, respectively. Clinical characteristics, gonadotropin (Gn) days, Gn dosage, serum hormone levels on the day of human chorionic gonadotropin (HCG) injection, ZP thickness (ZPT) of the eggs, fertilization rate, rescue intracytoplasmic sperm injection (rICSI) rate, good-quality embryo rate and pregnancy rate were compared between the two groups. Results: No significant differences were found in the duration and the causes of infertility, and their basal level of endocrine hormone before IVF-ET between normal egg group and brown egg group. The level of serum hormone including estradiol, progesterone and luteinizing hormone on the day of HCG injection were again similar. Moreover, there were no differences in number of mature oocytes, oocyte fertilization rates and rICSI rates after IVF between the two groups. However, we observed that the ZPT of brown oocytes (group B) was higher than that of normal oocytes (group A). Moreover, the Gn dosage and FSH levels on the day of HCG injection were significantly higher in group B than in group A and the good-quality embryo rate and pregnancy rate in group B were lower than those in group A. Conclusion: Compared with normal eggs, oocytes with a brown ZP were found to have a higher ZPT, lower embryo quality and lower pregnancy rate, which might be due to a high Gn dosage injection and high serum FSH levels during IVT-ET cycles

Deletion of <i>Prss35</i> on the expression of a few protease-related genes in D4.5 uterus.

<p>+/+, wild-type; −/−, <i>Prss35</i>^(−/−). X-axis indicated the names of different genes. Y-axis showed the normalized mRNA expression levels by <i>GAPDH</i> x the number in the parentheses following each gene on X-axis in order to present the relative expression levels of these genes in the same figure. <i>Prss35</i> was included to confirm that <i>Prss35</i> was deleted in the <i>Prss35</i>^(−/−) uterus. N = 6 (+/+) and N = 4 (−/−). Error bars, standard deviation.</p

Expression of <i>Prss23</i> in the periimplantation uterus by <i>in situ</i> hybridization.

<p>A. Gestation day 3.5 (D3.5), wild-type (WT, +/+) uterus. B. D3.5 <i>Lpar3</i>^(−/−) uterus. C. D4.5 WT uterus. D. D4.5 <i>Lpar3</i>^(−/−) uterus. E. D5.5 WT uterus. F. D6.5 WT uterus. G. D7.5 WT uterus. H. D7.5 WT embryo enlarged from the black rectangle in G. I. D7.5 WT uterus enlarged from the red rectangle in G. J. D4.5 WT ovary as a positive control, red arrow indicating granulosa cells. <i>Prss23</i> sense probe was used as a negative control, no specific signal was detected (data not shown). Red star, embryo; LE, luminal epithelium; Str, stroma; Dec, decidual zone; Myo, myometrium; scale bar, 100 µm.</p

Deletion of <i>Npl</i> on embryo implantation and the expression of implantation and decidualization markers in gestation day 4.5 (D4.5), D5.5 and D7.5 uteri.

<p><i>+/+</i>, wild type (WT); <i>−/−</i>, <i>Npl</i>^(−/−). A. A representative uterus from D4.5 WT mice. B. A representative uterus from D4.5 <i>Npl</i>^(−/−) mice. Red arrow, implantation site. C. the number of implantation sites on D4.5. N, the number of female mice in each group; error bars, standard deviation. D–G. Expression of implantation markers in D4.5 uterus by <i>in situ</i> hybridization using <i>Prap1</i> and <i>Abp1</i> antisense probes, respectively. D. <i>Prap1</i> in WT uterus. E. <i>Prap1</i> in <i>Npl</i>^(−/−) uterus. F. <i>Abp1</i> in WT uterus. G. <i>Abp1</i> in <i>Npl</i>^(−/−) uterus. H–K. Expression of decidualization marker, <i>Dtprp</i>, in D5.5 and D7.5 uterus by <i>in situ</i> hybridization using <i>Dtprp</i> antisense probes. H. <i>Dtprp</i> in D5.5 WT uterus. I. <i>Dtprp</i> in D5.5 <i>Npl</i>^(−/−) uterus. J. <i>Dtprp</i> in D7.5 WT uterus. K. <i>Dtprp</i> in D7.5 <i>Npl</i>^(−/−) uterus. Red star, embryo; LE, luminal epithelium; D, decidual zone; scale bar, 100 µm (D–G) and 500 µm (H–K). N = 2–3. No signals were detected using <i>Prap1</i>, <i>Abp1</i>, or <i>Dtprp</i> sense probes (data not shown).</p

Deletion of <i>Prss35</i> on embryo implantation and the expression of implantation markers in D4.5 uterus.

<p>A. A representative uterus from D4.5 WT mice. B. A representative uterus from D4.5 <i>Prss35</i>^(−/−) mice. A & B. Red arrows, implantation sites. C. The average number of implantation sites per mouse in the WT (+/+, N = 8) or <i>Prss35</i>^(−/−) (−/−, N = 8) females. All the mating males were <i>Prss35</i>^(−/−) for both groups except 2 WT males mated with 2 females in the WT group. Error bars, standard deviation. D∼K. Gene expression in D4.5 uterus by <i>in situ</i> hybridization. D. <i>Prss35</i> in WT uterus. E. <i>Prss35</i> in <i>Prss35</i>^(−/−) uterus. F. <i>Gjb2</i> in WT uterus. G. <i>Gjb2</i> in <i>Prss35</i>^(−/−) uterus. H. <i>Gja1</i> in WT uterus. I. <i>Gja1</i> in <i>Prss35</i>^(−/−) uterus. J. <i>Cox-2</i> in WT uterus. K. <i>Cox-2</i> in <i>Prss35</i>^(−/−) uterus. Red star, embryo; LE, luminal epithelium; Dec, decidual zone; scale bar<b>s</b>, 100 µm.</p

Deletion of <i>Prss35</i> on embryo implantation and the expression of decidualization markers in D7.5 uterus.

<p>A. A representative uterus from D7.5 WT mice. B. A representative uterus from D7.5 <i>Prss35</i>^(−/−) mice. A & B. Red arrows, implantation sites. C. The average number of implantation sites per mouse in the WT (+/+, N = 7) or <i>Prss35</i>^(−/−) (−/−, N = 7) females. All the mating males were <i>Prss35</i>^(−/−) for both groups except one WT male mated with one female in the WT group. Error bars, standard deviation. D∼I. Expression of decidualization markers in D7.5 uterus by <i>in situ</i> hybridization. D. <i>Dtprp</i> in WT uterus. E. <i>Dtprp</i> in <i>Prss35</i>^(−/−) uterus. F. <i>Prlpi</i> in WT uterus. G. <i>Prlpi</i> in <i>Prss35</i>^(−/−) uterus. H. <i>Abp1</i> in WT uterus. I. <i>Abp1</i> in <i>Prss35</i>^(−/−) uterus. Red star, embryo; M, mesometrial side; AM, antimesometrial side; scale bars, 500 µm.</p