Vasoactive Intestinal Peptide and Its Receptors in Human Ovarian Cortical Follicles

BACKGROUND: Ovarian cryopreservation is one option for fertility preservation in patients with cancer. The danger of reseeding malignancies could be eliminated by in vitro maturation of primordial follicles from the frozen-thawed tissue. However, the development of this system is hindered by uncertainties regarding factors that activate primordial follicles. Neuronal growth factors such as vasoactive intestinal peptide (VIP) play important roles in early mammalian folliculogenesis. There are no data on the expression of VIP and its vasoactive intestinal peptide pituitary adenylate cyclase 1 and 2 receptors (VPAC1-R and VPAC2-R) in human preantral follicles. METHODOLOGY/PRINCIPAL FINDINGS: Tissue samples from 14 human fetal ovaries and 40 ovaries from girls/women were prepared to test for the expression of VIP, VPAC1-R, and VPAC2-R on the protein (immunohistochemisty) and mRNA (reverse transcription polymerase chain reaction) levels. Immunohistochemistry staining was mostly weak, especially in fetal samples. The VIP protein was identified in oocytes and granulosa cells (GCs) in the fetal samples from 22 gestational weeks (GW) onwards. In girls/women, VIP follicular staining (oocytes and GCs) was identified in 45% of samples. VPAC1-R protein was identified in follicles in all fetal samples from 22GW onwards and in 63% of the samples from girls/women (GC staining only in 40%). VPAC2-R protein was identified in follicles in 33% of fetal samples and 47% of the samples from girls/women. The mRNA transcripts for VIP, VPAC1-R, and VPAC2-R were identified in ovarian extracts from fetuses and women. CONCLUSIONS: VIP and its two receptors are expressed in human ovarian preantral follicles. However, their weak staining suggests they have limited roles in early follicular growth. To elucidate if VIP activates human primordial follicles, it should be added to the culture medium

Controlled ovarian hyperstimulation - A state of endothelial activation

Problem: To aim of the study was to investigate whether controlled ovarian hyperstimulation (COH) causes endothelial activation and whether there is a correlation between endothelial activation and serum sex-steroid levels. Method of study: The study population consisted of 14 consecutive patients undergoing our routine IVF long gonadotropin-releasing hormone-analog protocol. Blood was drawn three times during the COH cycle: (1) day when adequate suppression was obtained (Day-S); (2) on the day of or the day prior to hCG administration (Day-hCG); and (3) on the day of ovum pick-up (Day-OPU). The levels of sex steroids and plasma soluble endothelial (E)-selectin were compared among the time points. Soluble E-selectin was measured with a commercial sandwich enzyme-linked immunosorbent assay. Results: Soluble E-selectin levels were significantly higher on Day-OPU than Day-S and Day-hCG, whereas no difference was observed between Day-hCG and Day-S. No significant correlations were found between soluble E-selectin level and patient age, number of gonadotropin ampoules used, number of oocytes retrieved, or serum estradiol, progesterone and human chorionic gonadotropin levels. Conclusions: Human chorionic gonadotropin administration leads to endothelial activation regardless of the degree of ovarian response. Further studies are required to elucidate the relationship between COH and endothelial activation. These findings may lead to new strategies for predicting and preventing complications of COH, such as severe ovarian hyperstimulation syndrome

IMH photographs of VPAC2-R protein expression.

<p>(A) Section of a human ovary from the same fetus as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037015#pone-0037015-g001" target="_blank">Figure 1</a> (A) and (D). Note the red-brown staining indicating VPAC2-R expression in the oocytes (full weak cytoplasmic staining with nuclear staining), in a portion of the GC and stroma cells. Original magnification X400. (B) Section of human ovary from the same woman as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037015#pone-0037015-g002" target="_blank">Figure 2</a> (A) and (B). Note the secondary follicle (full cytoplasmic staining with nuclear staining), with red-brown staining in the GC and stroma cells. Original magnification X400. (C) Negative control for the same ovarian section as in panel A. Note the primordial follicles with overall blue staining and lack of red-brown staining. Original magnification X400. (D) Negative control for the same ovarian section as in panel B. Note the primordial follicles with overall blue staining and lack of red-brown staining. Original magnification X400.</p

Protein expression (IMH) of VIP, VPAC1-R and VPAC2-R in human ovaries.

<p><u>Note</u>: IMH = immunohistochemistry, GW = gestational weeks. Percentages represent the proportion of the samples with follicular staining.</p><p>Staining intensities: + = positive staining; full/partial = full or partial cytoplasmic staining.</p

IMH photographs of VIP protein expression.

<p>(A) Section of human ovary from a 22-GW-old fetus. Note the primordial follicles, weak red-brown staining indicating VIP expression in oocytes (full cytoplasmic and nuclear staining), and weak staining in a portion of the GC and stroma cells. Original magnification X400. (B) Section of human ovary from a 6-year-old girl. Note the primordial follicles with red-brown staining indicating VIP protein expression in the oocyte (mainly cytoplasmic staining and nuclear staining), and in a portion of the GC and a portion of the stroma cells. Original magnification X400. (C) Positive control for VIP protein expression of section of mouse brain. Note the red-brown staining in the sample. Original magnification X400. (D) Negative control for the same ovarian section as in panel A. Note the primordial follicles with the overall blue staining and the lack of red-brown staining. Original magnification X400. (E) Negative control for the same ovarian section as in panel B. Note the primordial follicles with overall blue staining and the lack of red-brown staining. Original magnification X400.</p

Representative RT-PCR gel illustrating expression of the VIP, VPAC1-R and VPAC2-R genes in fetal and adult ovaries.

<p>(A) VIP gene (134 base pairs), (B) VPAC1-R gene (97 base pairs), (C) VPAC2-R gene (155 base pairs), (D) DHFR gene as positive control (231base pairs). Sample 1: Ovary from a 21-year-old woman, Sample 2: Ovary from a 29-year-old woman, Sample 3: Ovary from a 39-year-old woman, Sample 4: Ovary from a 21-GW-old fetus, Sample 5: Ovary from a 23-GW-old fetus, Sample 6: Ovary from a 25-GW-old fetus, Sample 7: Ovary from a 27-GW-old fetus. M: Marker (100 base pair DNA ladder, QIAGEN), +: RT presence, -: RT absence.</p