Michiharu Horikawa

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N‐omega‐nitro‐L‐arginine methyl ester, L‐NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10−7 M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10−3 and 10−5 M L‐NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four‐cell stage was observed by the addition of high concentration of SNP (10−3 M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10−5 and 10−7 M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose‐dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L‐NAME reversed the inhibitory effect by each SNP or L‐NAME treatment. Furthermore, low concentration of SNP (10−7 M) but not high concentration of SNP (10−3 M) significantly stimulated trophoblast outgrowth, whereas the addition of L‐NAME suppressed the spreading of blastocysts in a dose‐dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner. Mol. Reprod. Dev. 58:262–268, 2001.

Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation.

Clinicopathologic risk factors for recurrence of ovarian endometrioma following laparoscopic cystectomy

To identify epidemiologic risk factors and investigate whether the characteristics of removed ovarian tissue during surgery influence the recurrence of endometriomas.

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT-PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT-PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.

Single nucleotide polymorphism in the UBR2 gene may be a genetic risk factor for Japanese patients with azoospermia by meiotic arrest

PurposeTo investigate the association between the UBR2 gene and the risk of azoospermia caused by meiotic arrest.MethodsMutational analysis of the UBR2 gene was performed using DNA from 30 patients with azoospermia by meiotic arrest to 80 normal controls.ResultsThe genotypic and allelic frequencies of c.1,066A>T variant were significantly higher in patient than control groups (p < 0.001).ConclusionThe c.1,066A>T variant in the UBR2 gene is associated with increased susceptibility to azoospermia caused by meiotic arrest.

Single-nucleotide polymorphisms in HORMAD1 may be a risk factor for azoospermia caused by meiotic arrest in Japanese patients

Genetic mechanisms are implicated as a cause of some male infertility, yet are poorly understood. Meiosis is unique to germ cells and essential for reproduction. The synaptonemal complex is a critical component for chromosome pairing, segregation and recombination. Hormad1 is essential for mammalian gametogenesis as knockout male mice are infertile. Hormad1-deficient testes exhibit meiotic arrest in the early pachytene stage and synaptonemal complexes cannot be visualized. To analyze the hypothesis that the human HORMAD1 gene defects are associated with human azoospermia caused by meiotic arrest, mutational analysis was performed in all coding regions by direct sequence analysis of 30 Japanese men diagnosed with azoospermia resulting from meiotic arrest. By the sequence analysis, three polymorphism sites, Single Nucleotide Polymorphism 1 (c. 163A>G), SNP2 (c. 501T>G) and SNP3 (c. 918C>T), were found in exons 3, 8 and 10. The 30 patients with azoospermia and 80 normal pregnancy-proven, fertile men were analyzed for HORMAD1 polymorphisms. Both SNP1 and SNP2 were associated with human azoospermia caused by complete early meiotic arrest (P<0.05). We suggest that the HORMAD1 has an essential meiotic function in human spermatogenesis.

Isolation of the human ePAB and ePABP2 cDNAs and analysis of the expression patterns

PurposeIdentification of the unique genes playing critical roles in human embryo cleavage.MethodsIsolation of human ePAB cDNA using human ovary cDNA libraries and mouse ePAB amino acid sequences, followed by analysis of its expression pattern in various adult tissues and stages during early oocyte development excluding ePABP2.ResultsHuman ePAB encodes a 330-aa protein and is located on chromosome 20q12-q13.1. The amino acid sequence is 72% homologous with that of mouse ePab. Human ePAB has only three RRMs and lacks a PABP domain; the expression pattern is nonspecific in adult tissues and detected in all stages, from oocyte to blastocyst. Human ePABP2 encodes a 282-aa protein and is located on chromosome 16q24.3. The amino acid sequence is 68% homologous with mouse ePabp2.ConclusionsWe identified human ePAB and ePABP2 cDNA. Human ePAB cDNA is not expressed specific to the ovary. Biological discrepancies exist between the human and the mouse.

Single nucleotide polymorphisms in the SEPTIN12 gene may be associated with azoospermia by meiotic arrest in Japanese men

PurposeTo investigate the association between SEPTIN12 gene variants and the risk of azoospermia caused by meiotic arrest.MethodsMutational analysis of the SEPTIN12 gene was performed using DNA from 30 Japanese patients with azoospermia by meiotic arrest and 140 fertile male controls.ResultsThe frequencies of the c.204G>C (Gln38His) allele and the CC genotype were significantly higher in patients than in fertile controls (p < 0.05).ConclusionThe c.204G>C (Gln38His) variant in the SEPTIN12 gene was associated with increased susceptibility to azoospermia caused by meiotic arrest.

Palmoplantar pustular lesions during ovulation inducement therapy: new insight into the pathomechanism of palmoplantar pustulosis?

these lesions improved and worsened cyclically along with buserelin acetate administration, the symptoms were relatively easily controlled with a topical steroid and no arthritis associated with SAPHO syndrome was apparent during all courses. Case 2 is that of a 34-year-old Japanese woman who presented with pustules on her palms and soles ( fig. 1 E). She noticed persistent palmar pustules and vesicles about a month after the administration of buserelin acetate. Although she had also been smoking for several years, she had never experienced palmoplantar lesions until this time. The administration protocol of buserelin acetate and an estrogen patch was the same as that in case 1. Her lesions were recalcitrant to topical steroid, topical maxacalcitol and topical PUVA treatments. The condition improved slightly after the cessation of buserelin acetate ( fig. 1 F). The parts of the skin on which estrogen patches or buserelin acetate were applied did not show any contact dermatitis or pustules and the patient did not have any personal or familial history of PPP or psoriasis just as in case 1. Signs of arthritis suggesting SAPHO syndrome did not appear during all courses.