Hiroshi Kusunoki

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whittens medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whittens medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whittens medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whittens medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whittens medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whittens medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.

Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells

OBJECTIVE To examine whether human primary spermatocytes will develop into round spermatids after completing meiosis in an in vitro coculture with Vero cells. DESIGN Prospective, controlled in vitro study. SETTING A private infertility clinic and a university laboratory. PATIENT(S) Five azoospermic men whose spermatogenesis was proved to be arrested at the level of the primary spermatocyte in open biopsies. INTERVENTION(S) In vitro coculture of isolated primary spermatocytes with Vero cells and chromosomal analysis for assessment of the completion of meiosis. MAIN OUTCOME MEASURE(S) Isolated primary spermatocytes were cocultured with Vero cells under various conditions. The number of chromosomes and chromatids in newly generated cells was determined by Giemsa staining after the cells were injected into mouse oocytes. RESULT(S) The generation rates of round spermatids in six types of in vitro culture with Vero cells were 0%-10% (highest rates of division were in minimum essential medium (MEM) + 50% boar rete testicular fluid or in human synthetic oviduct fluid + 10% human serum). The number of chromosomes and chromatids in the newly developed cells was 23. CONCLUSION(S) A single primary spermatocyte was observed to divide into four cells during in vitro coculture with Vero cells. These newly developed cells were proved to be round spermatids by chromosomal analysis. It was verified that a primary spermatocyte developed into round spermatids after completing two cycles of meiosis through in vitro culture.

Human sperm head vacuoles are physiological structures formed during the sperm development and maturation process

OBJECTIVE To clarify whether human sperm vacuoles affected intracytoplasmic sperm injection (ICSI) success rates. DESIGN Retrospective study. SETTING A private infertility clinic. PATIENT(S) Spermatozoa and spermatids were obtained from 11 normozoospermic, 10 oligozoospermic or asthenozoospermic, 4 obstructive azoospermic, and 3 nonobstructive azoospermic men. INTERVENTION(S) Differential interference contrast observation and intracytoplasmic injection of morphologically selected sperm. MAIN OUTCOME MEASURE(S) Incidence, size, and position of vacuoles of sperm cells were recorded. Ability of fertilization and blastocyst development were compared between cells with and without vacuoles. RESULT(S) More than 97.4% of ejaculated, 87.5% of epididymal, 87.5% of testicular spermatozoa, and more than 90.0% of Sc-Sd2 spermatids had vacuoles of various sizes. The incidence of vacuoles on ejaculated cells was significantly higher than that on the other types of cells, but there was no difference between sperm from normozoospermic men and those from the other donors. Removal of plasma membrane and/or acrosome did not affect the incidence of vacuoles. Although more than 60% of spermatozoa had small vacuoles in the acrosomal regions, 52.6% of Sb1-2 spermatids had large vacuoles. After injection of a motile spermatozoon with large and small vacuoles, 60.9% and 85.7% of metaphase II oocytes could be normally fertilized, respectively, and almost half of the zygotes developed to the blastocyst stage. When using sperm without vacuoles, the fertilization rate was 80.0%, but only 25% of them developed to the blastocyst stage. CONCLUSION(S) Human sperm head vacuoles did not affect ICSI outcomes.

Generation of reactive oxygen species in sperms of rats as an earlier marker for evaluating the toxicity of endocrine-disrupting chemicals.

Abstract Bisphenol A (BPA) and diethylstilbestrol (DES) have been reported to cause sperm toxicity. To identify an earlier marker of toxicity of environmental substances or food additives, this study determined whether the levels of reactive oxygen species (ROS) in sperms could serve as indices for the prediction of sperm toxicity and quality. Male Wistar rats were given drinking water containing various doses of BPA or DES for 8 weeks. Some rats were treated with 0.45% N-acetyl cysteine (NAC) for 2 days prior to the administration of DES or BPA. Administration of BPA or DES to rats for 1 week dose-dependently increased the production of ROS, even at doses and time points which had no effect on sperm motility. 4-Hydroxy-2-nonenal modified proteins increased in sperms 8 weeks after BPA or DES treatment. NAC reversed oxidative stress and prevented the loss of sperm function in the DES or BPA-treated group. During observation, changes in the sperm motility, sperm count and morphology were not correlated to the increase in ROS levels. These results suggest that ROS levels may be used as an early indicator of sperm count and quality decreases which result from chronic toxicity.

Spectral pattern of urinary water as a biomarker of estrus in the giant panda

Near infrared spectroscopy (NIRS) has been successfully used for non-invasive diagnosis of diseases and abnormalities where water spectral patterns are found to play an important role. The present study investigates water absorbance patterns indicative of estrus in the female giant panda. NIR spectra of urine samples were acquired from the same animal on a daily basis over three consecutive putative estrus periods. Characteristic water absorbance patterns based on 12 specific water absorbance bands were discovered, which displayed high urine spectral variation, suggesting that hydrogen-bonded water structures increase with estrus. Regression analysis of urine spectra and spectra of estrone-3-glucuronide standard concentrations at these water bands showed high correlation with estrogen levels. Cluster analysis of urine spectra grouped together estrus samples from different years. These results open a new avenue for using water structure as a molecular mirror for fast estrus detection.

Anti-inflammatory effects of hepatocyte growth factor on the vicious cycle of macrophages and adipocytes

The paracrine loop involving inflammatory cytokines between adipocytes and macrophages establishes a vicious cycle that aggravates pro-inflammatory changes in adipose tissue. The serum level of hepatocyte growth factor (HGF) is increased in metabolic syndrome, but whether HGF is beneficial or detrimental in inflammatory conditions is unclear. We previously reported that HGF has strong anti-inflammatory effects. We therefore hypothesized that HGF may inhibit chronic inflammation in adipose tissue by inhibiting the vicious cycle between adipocytes and macrophages. We stimulated the macrophage cell line RAW264 with HGF and evaluated pro-inflammatory cytokines. Coculturing differentiated 3T3-L1 adipocytes with RAW264 results in marked upregulation of pro-inflammatory cytokines. We examined whether HGF suppresses the upregulation of pro-inflammatory cytokines in this coculture system. Cardiac-specific HGF transgenic mice (HGF-Tg) were crossed with ApoE KO (knockout) mice, to yield ApoE KO/HGF-Tg mice, which were treated with a high-fat diet (HFD) and received angiotensin (Ang) II. The phenotypes of ApoE KO and ApoE KO/HGF-Tg mice were compared. Treatment with HGF reduced the expression of pro-inflammatory cytokine genes (Tumor necrosis factor-α, monocyte chemoattractant protein-1 and interleukin-6) in RAW264 and the coculture system. The anti-inflammatory effects of HGF were also confirmed in in vivo studies. Macrophage infiltration in epididymal adipose and fatty liver was reduced in ApoE KO/HGF-Tg. Adipocyte diameter was reduced in ApoE KO/HGF-Tg, and the serum adiponectin level was upregulated. These beneficial effects in ApoE KO/HGF-Tg mice under HFD and Ang II infusion were abrogated by an anti-HGF neutralizing antibody. These results suggest that HGF inhibits the vicious cycle of adipocytes and macrophages through the inhibition of macrophage-mediated pro-inflammatory cytokines and upregulation of adiponectin in adipocytes. These favorable effects may suppress chronic inflammation in adipose tissue.

Fourteen babies born after round spermatid injection into human oocytes

Significance Men without spermatozoa or elongating spermatids in their testes have been considered sterile and are advised to consider using a sperm donor. However, these men may have round spermatids. We have been able to accurately identify these cells based on their structural and physical characteristics (verified by karyotyping and FISH). Round spermatid injection was effectively used in our clinic and resulted in the birth of 14 healthy babies. Although the current success rate of round spermatid injection is not very high compared with intracytoplasmic sperm injection, this procedure can be the last resort for men who cannot produce spermatozoa but wish to use their own genetic material to produce offspring. During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.

Long-Term Monitoring of Fecal Steroid Hormones in Female Snow Leopards (Panthera uncia) during Pregnancy or Pseudopregnancy

Knowledge of the basic reproductive physiology of snow leopards is required urgently in order to develop a suitable management conditions under captivity. In this study, the long-term monitoring of concentrations of three steroid hormones in fecal matter of three female snow leopards was performed using enzyme immunoassays: (1) estradiol-17β, (2) progesterone and (3) cortisol metabolite. Two of the female animals were housed with a male during the winter breeding season, and copulated around the day the estradiol-17β metabolite peaked subsequently becoming pregnant. The other female was treated in two different ways: (1) first housed with a male in all year round and then (2) in the winter season only. She did not mate with him on the first occasion, but did so latter around when estradiol-17β metabolite peaked, and became pseudopregnant. During pregnancy, progesterone metabolite concentrations increased for 92 or 94 days, with this period being approximately twice as long as in the pseudopregnant case (31, 42, 49 and 53 days). The levels of cortisol metabolite in the pseudopregnant female (1.35 µg/g) were significantly higher than in the pregnant females (0.33 and 0.24 µg/g) (P<0.05). Similarly, during the breeding season, the levels of estradiol-17β metabolite in the pseudopregnant female (2.18 µg/g) were significantly higher than those in the pregnant females (0.81 and 0.85 µg/g) (P<0.05). Unlike cortisol the average levels of estradiol-17β during the breeding season were independent of reproductive success. The hormone levels may also be related to housing conditions and the resulting reproductive success in female leopards. The female housed with a male during the non-breeding season had high levels of cortisol metabolites and low levels of estradiol-17β in the breeding season, and failed to become pregnant. This indicates that housing conditions in snow leopards may be an important factor for normal endocrine secretion and resulting breeding success.

Near infrared spectroscopy of urine proves useful for estimating ovulation in giant panda (Ailuropoda melanoleuca)

The usefulness of near infrared spectroscopy (NIRS) to monitor urine estrogen concentrations was studied in order to determine optimal timing for breeding captive female giant pandas. NIR spectra of daily urine samples from a female giant panda (Ailuropoda melanoleuca) were acquired in the period between March 1st and 25th, 2007 (n = 53). Estrone-3-glucuronide (E1G) concentrations in the samples were also measured by enzyme immunoassay (EIA). Transmittance spectra of all urine samples were obtained in the wavelength range from 1100 to 2432 nm (excluding the range from 1884 to 2012 nm) with sample thickness of 1 mm. Partial least square regression was applied to the spectra and good correlation was obtained between E1G concentration measured by EIA and predicted values by NIR (R2 = 0.94, SECV = 10.04 ng ml−1). The results of both soft-independent modeling of class analogy (SIMCA) and moving principal component analysis (MPCA) could detect the time changes in E1G concentration as measured by EIA (the Pearsons correlation coefficients between E1G concentration and the interclass distances of SIMCA or the index of MPCA were r = 0.64 and r = 0.81 respectively, P < 0.01). As for MPCA index, the index sharply dropped on March 24th corresponding to the decrease of the E1G concentration indicating ovulation. Finally, artificial insemination was performed for 3 consecutive days including the peak day, March 24th, and the female became pregnant. These results indicated that NIRS and the following MPCA analysis of the respective urine spectral data could detect the changes of urinary hormones during estrous cycle at a nanogram level. The NIRS can find the optimal timing for breeding quicker and easier than EIA, so this technique can be useful for captive breeding of this threatened species.

A comparative study on testicular microstructure and relative sperm production in gorillas, chimpanzees, and orangutans

We performed histological analyses for comparing testicular microstructure between the gorilla, chimpanzee, and orangutan. Testicular samples were obtained by autopsy or biopsy from 10 gorillas, 11 chimpanzees, and 7 orangutans from several zoos and institutes. The seminiferous epithelia were thick in the chimpanzee and orangutan but thin in the gorilla. Leydig cells in the interstitial tissue were abundant in the gorilla. The acrosomic system was extremely well developed in the orangutans. Our study reveals that the cycle of seminiferous epithelium in orangutan testis can be divided into ten stages, whereas that in human, chimpanzee, and gorilla testes can be divided into only six stages. Phylogenetic analyses of the number of divisions may indicate that the seminiferous epithelium of our common ancestor has changed since the orangutan diverged from it. Furthermore, we performed comparative analyses of testicular microstructure to estimate relative sperm production among these three animals, and proposed a new indicator (namely the spermatogenic index, SI) closely related to sperm production. The SI indicated that a chimpanzee usually produces about 223 times more sperm than a gorilla and about 14 times more than an orangutan. Our data demonstrate the significance of the SI for estimating sperm production, thus aiding our understanding of the reproductive strategy as well as testis weight and relative testis size in investigated primates. Am. J. Primatol. 73: 570‐577, 2011.