Naoaki Kuji

Integrins and reproductive physiology: expression and modulation in fertilization, embryogenesis, and implantation

OBJECTIVE To review the available information regarding the role of integrins in reproductive physiology and to discuss their potential clinical implications. DESIGN Studies that specifically relate to the expression and modulation of integrins in fertilization, embryogenesis, and implantation were identified through the literature and Medline searches. RESULT(S) Integrins are a class of adhesion molecules that participate in cell-to-cell and cell-to-substratum interactions and are present on essentially all human cells. All mammalian eggs express integrins at their surface, and the integrin alpha 6 beta 1 serves as a sperm receptor that mediates sperm-egg binding. In addition, certain integrin moieties appear to be regulated within the cycling endometrium. Specifically, the expression of beta 1 integrins in the early proliferative phase is restricted to the glandular epithelium, whereas stromal cells also express beta 1 integrins in the midsecretory phase. The expression of beta 1 integrins increases at the time of implantation and remains elevated in the decidua during early pregnancy. A disruption of integrin expression is associated with certain types of infertility in women. The apical surface of the mural trophectoderm does indeed possess functional integrins, and trophoblast interactions with extracellular matrix proteins largely depend on the integrin family of adhesion receptors. CONCLUSION(S) Integrins play particularly important roles in both fertilization and embryogenesis, including the process of implantation.

Global gene expression profiling of preimplantation embryos

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 μm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

An evaluation of semen processing methods for eliminating HIV-1

ObjectiveWhether artificial insemination can provide adequate protection for discordant couples where the man is HIV-1 positive and the woman is HIV-1 negative is uncertain because of the paucity of HIV-1 elimination data assessing current sperm-washing techniques. We evaluated how effectively these techniques eliminate HIV-1 RNA and proviral DNA from semen. MethodsSpermatozoa were separated from semen samples from HIV-1-positive patients with haemophilia by discontinuous Percoll gradient centrifugation and the ‘swim-up’ method. The HIV-1 RNA and proviral DNA were measured by a highly sensitive PCR. In another test 5 × 106 copies of HIV-1 RNA (LAI strain) were added to semen from healthy donors and then assessed after single and combined procedures. ResultsSwim-up processing after Percoll gradient centrifugation reduced HIV-1 RNA and HIV-1 proviral DNA in semen to undetectable levels in the original specimen. Although discontinuous and continuous Percoll gradient centrifugation respectively reduced HIV-1 RNA added to seminal plasma specimens from healthy donors to less than < 1 copy from 105 and about 1 copy per 103 pre-separation copies, the discontinuous method left detectable HIV-1 RNA and proviral DNA in one out of 12 samples from patients with HIV-1 infection (8%). HIV-1 RNA and proviral DNA were decreased to undetectable levels after adding the swim-up procedure. ConclusionsSwim-up separation following Percoll gradient centrifugation should offer adequate protection for HIV-1-discordant couples.

Complete removal of HIV-1 RNA and proviral DNA from semen by the swim-up method: Assisted reproduction technique using spermatozoa free from HIV-1

Background:Use of antiretroviral drugs has reduced the mortality rate for HIV infection and many HIV-discordant couples wish to have children. It is possible for an HIV-infected man to father children without risk of HIV transmission if HIV-free spermatozoa can be obtained from his semen. Methods:An improved swim-up method was used to collect HIV-free spermatozoa from the semen of HIV-positive males. Diluted semen was layered over a Percoll solution with a continuous density gradient of 30–98%, and then centrifuged. The bottom layer was collected by cutting the end from the tube and the sperm suspension was collected using the swim-up method. Spermatozoa were tested by nested polymerase chain reaction (PCR) for HIV-1 RNA and DNA, with a detection limit of one copy. Spermatozoa were used for assisted reproduction in 43 couples. Results:HIV-1 RNA and proviral DNA were not detected by nested-PCR assay in all 73 of the collected spermatozoa samples from 52 patients. The HIV-1-negative sperm was used for in vitro fertilization in 12 couples and for intracytoplasmic sperm injection in 31 couples. No detection of HIV-1 RNA or proviral DNA in the culture medium of the fertilized eggs was confirmed again before embryo transfer. Of the 43 female partners, 20 conceived and 27 babies were born. HIV antibodies, HIV RNA and proviral DNA were negative in all of the females and babies. Conclusions:HIV-negative spermatozoa could be obtained from semen of HIV-positive men. The method involves no risk of HIV transmission to female partners and their children.

Reduced expression of αvβ3 integrin in the endometrium of unexplained infertility patients with recurrent IVF-ET failures: Improvement by danazol treatment

AbstractPurpose: To determine whether there is any association between the expression of endometrial integrin α v β3 and repeated IVF-ET failure and to examine the effect of danazol treatment on α v β3 expression. Methods: This prospective study was performed using a semiquantitative immunohistochemical analysis on the staining intensity of α v β3 in the mid-secretory endometria derived from 10 fertile women and 57 infertile patients with a history of repeated IVF-ET failures. Nine patients randomly selected from these 22 patients with unexplained infertility were then treated with oral danazol administration for 12 weeks and reexamined at the first mid-secretory phase after the danazol treatment. Result(s): The levels of endometrial α v β3 expression were lower in 22 patients with unexplained infertility than in the fertile control and 35 patients with explained infertility. The 9 patients treated with danazol showed a significant increase in the α v β3 staining. Conclusion(s): The significantly decreased expression of endometrial integrin α v β3 suggested that functional, but not morphological, endometrial defect may be one of the causes for the patients with unexplained infertility. Danazol may have a therapeutic potential in improving endometrial function together with up-regulation of α v β3.

Ureteral catheter placement for prevention of ureteral injury during laparoscopic hysterectomy

Aim:  Ureteral injury is among the most devastating complications of gynecologic surgery. Estimated incidence of ureteral injury during laparoscopic hysterectomy is 2.6–35 times (0.2–6.0%) that in abdominal hysterectomy. We investigated preoperative ureteral catheter (UC) placement as a way to prevent ureteral injury in laparoscopic hysterectomy.

Interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system in follicular growth and ovulation.

The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.

A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13C8] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

Expression of human oocyte-specific linker histone protein and its incorporation into sperm chromatin during fertilization

OBJECTIVE To investigate the expression of oocyte-specific linker histone protein (hH1FOO) in human ovaries and its incorporation into sperm chromatin after intracytoplasmic sperm injection (ICSI). DESIGN Laboratory study. SETTING University hospital. PATIENT(S) Human ovarian tissues were obtained from patients at oophorectomy. Human oocytes and embryos were obtained from infertile patients undergoing IVF and ICSI. INTERVENTION(S) A polyclonal rabbit antibody targeting the predicted hH1FOO protein was used for immunohistochemical analysis. Western blot analysis and the reverse transcriptase-nested polymerase chain reaction were done to detect hH1FOO in chromatin of germinal vesicle-stage oocytes through to two-cell embryos. MAIN OUTCOME MEASURE(S) The hH1FOO antibody reactivity of oocytes, ovarian tissues, and sperm chromatin after ICSI. RESULT(S) hH1FOO protein was localized in all oocytes from primordial to Graafian follicles. In unfertilized oocytes after ICSI, the chromatin of injected sperm was condensed without hH1FOO incorporation in 45.5% of oocytes, condensed with hH1FOO incorporation in 9%, and decondensed with hH1FOO incorporation in 45.5%. None of the oocytes contained decondensed sperm chromatin without hHFOO incorporation. CONCLUSION(S) hH1FOO protein was expressed by human oocytes from primordial follicles to early embryogenesis. Sperm nuclei that were still condensed after ICSI could be separated into two categories by hH1FOO incorporation, which might provide valuable information regarding failed fertilization.

Role of β1 Integrins in Human Endometrium and Decidua during Implantation

The present study was undertaken to investigate the expression and function of β₁ integrins in human endometrium and decidua. Fluorescence-activated flow cytometry demonstrated the greater expression of the β₁, α₁, α₂, and α₅ subunits of the β₁ integrin family in cultured stromal cells from the midsecretory phase than in those of the early proliferative phase. The addition of estradiol (E₂) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of β₁ integrins in vitro. The immunohistochemical distribution of β₁ integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and stromal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of β₁, α₁, α₂, α₃, α₅, and α₆ subunits of β₁ integrin family in cultured decidual cells. Immunohistochemistry confirmed the β₁ integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In the subsequent experiment, the effects of antibodies against specific β₁ integrin heterodimers on mouse embryo attachment and spreading were tested to identify the role of β₁ integrins in early implantation. We developed assays for the attachment of mouse embryos and for trophoblastic spreading on cultured human decidual cells. The addition of antibodies directed against β₁ and α integrin subunits to cultured decidual cells did not affect the rates of hatching or attachment of the blastocysts, whereas the outgrowth of embryos on the decidual cells was inhibited by their antibodies in a dose-dependent manner. Thus, β₁ integrin in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation.