Creation, effects on embryo quality, and clinical outcomes of a new embryo culture medium with 31 optimized components derived from human oviduct fluid: A prospective multicenter randomized trial.

Purpose: Our aim is to make an ideal embryo culture medium close to human oviduct fluid (HOF) components, and to evaluate the quality of this medium with embryo quality and clinical outcomes in assisted reproductive technology (ART) by a prospective randomized controlled trial (RCT). Methods: Study I: HOF was collected laparoscopically from patients (n = 28) with normal pelvic findings. According to HOF analysis results, the new medium "HiGROW OVIT®" (OVIT) was designed. Study II: Embryos (2 pronuclei (2PN) = 9633) were assigned from 1435 patients. The blastulation rate (BR), good BR (gBR), utilized (transferred/cryo-preserved) BR (uBR), pregnancy rate (PR), and miscarriage rate (MR) were compared between the OVIT and control groups by RCT. Results: The novel medium 'OVIT' was produced according to 31 HOF components. The concentrations of essential amino acids (e-AAs) were lower in OVIT than in current media, yet the opposite was true for ne-AA concentrations. gBR and uBR were higher in the OVIT group than in the control group. In the older female group, gBT and uBR were significantly higher in the OVIT group. Conclusions: The novel medium 'OVIT' was produced according to HOF data. The OVIT had significantly better embryo quality and clinical outcomes than the current media.

Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes.

The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Size of the perivitelline space and incidence of polyspermy in rabbit and hamster oocytes.

Purpose: The size of the perivitelline space and the incidence of polyspermy were observed in ovulated and cultured oocytes from rabbits and hamsters with or without treatment by 4-methylumbelliferone (MU), an inhibitor of hyaluronic acid (HA) synthase, in order to examine the relationship between the incidence of polyspermy and the size of the perivitelline space. The amount of HA in the medium with MU-treated hamster oocytes was measured and compared with that in the medium with untreated oocytes. Methods: The perivitelline space of oocytes with 1st polar bodies was measured by use of a micrometer under a microscope, and the incidence of polyspermy was observed in the oocytes after insemination. The amount of HA in the medium was examined using an enzyme-linked immunosorbent assay. The distribution of cortical granules was observed by staining with FITC-conjugated LCA. Results: In both rabbit and hamster, the mean size of the perivitelline space was significantly smaller and the incidence of polyspermy was significantly higher in the cultured and MU-treated oocytes than in the ovulated and untreated oocytes. The mean amount of HA in the medium with MU-treated oocytes (8.96 pg) was significantly smaller than that in the medium with untreated oocytes (21.77 pg). The distribution of cortical granules did not differ among the ovulated, cultured, and MU-treated oocytes. Conclusions: These findings suggest that the size of the perivitelline space is closely related to the incidence of polyspermy, and that the oocyte itself synthesizes and secretes the HA involved in the enlargement of the perivitelline space.

Time-lapse videomicrographic observations of blastocyst hatching in cattle.

Morphological changes of cultured bovine blastocysts during hatching were observed using time-lapse videomicrography in order to investigate the patterns of the hatching process that occurred in the blastocysts and to determine whether the hatching patterns differed between blastocysts developed from fresh and cryopreserved embryos. Compacted morulae (CMs) were collected from superovulation-treated Japanese Black and Holstein dairy cattle and cultured in a medium in a CO(2) culture chamber equipped with an inverted microscope at 38.5 C. Images of resultant blastocysts during the period from blastocoel formation to completion of hatching were taken at 4-sec intervals by a CCD color camera connected to an inverted microscope and recorded by a time-lapse video cassette recorder. In blastocysts developed from fresh CMs, hatching was found to begin with protrusion of trophectoderm cells from zonae pellucidae at the expanded stage. Protrusion of the cells occurred in any site of the trophectoderm. After protrusion, a large or small slit was formed in the zona pellucida in all blastocysts as a result of blastocyst expansion or enlargement of the protrusion. Then, blastocysts completely escaped from the zona pellucida through the slit in the state of expansion. From these findings, the hatching patterns of cattle blastocysts could be classified into 5 types. In blastocysts developed from frozen-thawed CMs, the hatching pattern and length of time needed for hatching are similar to those in blastocysts developed from fresh CMs. In addition, the pregnancy rate of recipients following transfer of frozen-thawed CMs (52.4%) did not differ from that of recipients following transfer with fresh CMs (58.3%). These results suggested that the quality of frozen-thawed cattle embryos is comparable to that of fresh embryos and that there could be a relationship between the hatching pattern of blastocysts and the viability of embryos after transfer.

A physiological role of contractions in hatched mouse blastocysts.

Purpose: Using mouse blastocysts with implantation delayed for 7 days (dormant blastocysts) and time-lapse videomicrography, we examined the physiological role of contractions in hatched blastocysts. Methods: The degree and number of contractions in dormant blastocysts were analyzed in images recorded for 48 h of culture, and compared with those in dormant blastocysts in which implantation was induced (activated blastocysts), and dormant blastocysts cultured with estrogen or progesterone. Results: Activated blastocysts exhibited a significantly smaller number of weak contractions and a significantly larger number of strong contractions, compared with dormant blastocysts. Furthermore, the numbers of weak and strong contractions in dormant blastocysts cultured with estradiol-17ß were significantly less frequent or more frequent than those in dormant blastocysts cultured with progesterone and those cultured without steroids. Expression of estrogen receptor mRNA was also detected in dormant blastocysts by the method of reverse transcription-polymerase chain reaction. Conclusion: These results suggested that activated blastocysts enhance the contractions as a preparation for the implantation, and that the enhanced contractions in those serve as a physical stimulation for maternal recognition of pregnancy.

Location and expression of Juno in mice oocytes during maturation.

OBJECTIVE: Oocyte-sperm interaction is the essential step in fertilization. Juno, which has been known as Folate receptor 4, is the Izumo1 receptor expressed on the oocyte membrane. This study aims to investigate the location and expression of Juno in mice oocytes during maturation. METHODS: To confirm the stage at which Juno expression begins in the mice oocytes and its location pattern, we performed immunostaining methods. Next, we evaluated Juno mRNA expression by a half quantitative RT-PCR. Juno knockdown oocytes were generated by microinjecting siRNA into the germinal vesicle (GV) stage oocytes, and analyzed the maturation rate. RESULTS: Our results showed that Juno was expressed on the surface of the oocyte cytoplasmic membrane at the GV stage and it continues to be expressed at similar levels in the metaphase II (MII) stages of oocytes maturation. Interestingly, Juno is also expressed on the first polar body membrane at the MII stage. Fluorescence showing Juno expression was decreased in the oolemma of siRNA injected oocytes, but it was not completely disappearing in knock down oocytes. MII stage-rates of siRNA injected oocytes were not significantly different from sham controls. CONCLUSION: Juno was expressed in oocytes at the GV stage and it continues to be expressed at similar levels in later stages of oocytes maturation. Juno accumulation in oolemma during oocyte maturation is essential for fertilization, such as membrane recognition of both gametes.

Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos.

Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.

Newly established low seizure susceptible and seizure-prone inbred strains of Mongolian gerbil.

Two inbred strains of the Mongolian gerbil with different phenotypes in seizure behaviour and coat color were newly established. LSAG/Nu has low seizure susceptibility and albino phenotypes, whereas SPBG/Nu has seizure-prone and black coat color phenotypes. LSAG was compared with SPBG as to seizure incidence and grade. Mean ages at seizure onset of LSAG and SPBG were 6 and 3 months, respectively. Seizure incidences in over 9 months old LSAG and SPBG gerbils were 37.3% (66/177) and 95.2% (118/124), respectively. LSAG has a significantly lower incidence (p < 0.001) and grade (p < 0.001) of seizures than SPBG. Only a few seizing LSAG gerbils exhibited myoclonus to tonic-clonic seizure progression. These results suggest that LSAG has some mechanisms which delay the onset of seizures and prevent them from becoming serious. Both strains of gerbils can be expected to be useful animal models for the study of human idiopathic generalized epilepsy.

Changes in the amount of cytoplasmic inclusions in mouse oocytes during meiotic maturation in vivo and in vitro.

Background and aims: The changes in cytoplasmic inclusions during meiotic maturation have only been examined in porcine oocytes. In the present study, the amount and the number of cytoplasmic inclusions (glycogen granules, lipid droplets and fibrous structures) were examined in mouse oocytes in the process of in vivo and in vitro maturation. For those inclusions that changed in amount during maturation, we also examined their content in oocytes treated with olomoucine, an inhibitor of cyclin-dependent kinase, in order to clarify the relationship between nuclear maturation and changes in the inclusions. Methods: Nuclear maturation in the oocytes cultured for various periods and those collected from antral follicles and oviducts was examined after staining with aceto-orcein. For the demonstration of glycogen granules and lipid droplets, oocytes were stained with periodic acid-Schiff or Sudan IV. Fibrous structures in the oocytes were observed under an electron microscope. Results: The amount of glycogen granules, Sudanophilic lipid droplets and fibrous structures did not change in the oocytes matured in vivo and in vitro, whereas the number of the lipid droplets increased during maturation. In the oocytes treated with olomoucine, the resumption of nuclear maturation was inhibited, whereas the increase in the number of Sudanophilic lipid droplets was not inhibited. Conclusion: Present findings suggest that the increase in the number of Sudanophilic lipid droplets occurs in the cytoplasm of mouse oocytes during maturation, regardless of in vivo or in vitro maturation, and that such the change in the inclusion is not related to nuclear maturation. (Reprod Med Biol 2004; 3: 231-236).

Use of in ovo chorioallantoic membrane engraftment to culture testes from neonatal mice.

Many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. The objective of this study was to establish an alternative method of xenotransplantation by developing a new approach for the rapid induction of spermatogenesis by using the chorioallantoic membrane of developing chicken embryos. Fertilized chicken eggs were incubated for 7 d, after which a small window was cut into the shell of the egg. We then transplanted testes from 7- to 8-d-old B6D2F1 mice onto the vessels of the chorioallantoic membrane and incubated them at 35.0 °C for 14 d or 37.5 °C for 12 d. After this in ovo CAM (iCAM) culture, the survival rates of the eggs and testes were assessed histologically and immunohistologically. The transplanted testes in the chicken embryos that survived were supported by the CAM, with an associated chronic vascularization response. The testes cultured at 35.0 °C had lower rates of generation and higher rates of death than did those cultured at 37.5 °C. Histologic examination of the testes cultured at 37.5 °C revealed the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules. The number of cells positive for synaptonemal complex protein 3 in the seminiferous tubules was significantly higher than that in the noniCAM-cultured testes from control mice. These results suggest that iCAM culturing of neonatal donor testis induces androcyte development. This method could be the foundation for a method that would enable in vitro spermatogenesis.

Hatching and distribution of actin filaments in mouse blastocysts whose activities of protein kinase A were suppressed by H-89.

The role of actin filaments and contractions in hatching was determined in mouse blastocysts whose actin filament bundling abilities had been suppressed by H-89, an inhibitor of protein kinase A. The hatching rate of blastocysts developed from morulae in a medium containing H-89 at a concentration of 4.0 microM was 17.2%, which was significantly lower than the 76.7% of the control blastocysts developed from morulae in a medium without H-89. The rates of blastocysts starting hatching and forming a slit in the zona pellucida were significantly lower in H-89-treated blastocysts (84.4 and 21.9%) than in control blastocysts (100.0 and 90.6%). The lengths of time needed for slit formation in the zona pellucida and for completion of hatching were significantly longer in the H-89-treated blastocysts (27.4 and 43.3 h) than in the control blastocysts (6.5 and 18.8 h). Over the course of 32 h after blastocoel formation, the number of strong contractions was similar in the H-89-treated and control blastocysts, but the number of weak contractions was significantly fewer in the H-89-treated blastocysts (2.41 times) than in the control blastocysts (4.19 times). Although the distribution of actin filaments was similar in the H-89-treated and control blastocysts in the pre-hatching, hatching and post-hatching periods, the rate of H-89-treated blastocysts in which most trophectoderm cells possessed the fluorescence of actin filaments (12.7%) was significantly lower than the 95.0% of the control blastocysts in the pre-hatching period. These results suggest that actin filament-mediated movements of trophectoderm cells contribute to hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and by enlarging the protrusion. We also suggest that the low hatching ability of the treated blastocysts is related to weak contractions with a low frequency and to strong contractions requiring a longer time for re-expansion.

Amount of hyaluronan produced by mouse oocytes and role of hyaluronan in enlargement of the perivitelline space.

Production of hyaluronan (hyaluronic acid: HA) was demonstrated in denuded mouse oocytes (DOs) by the enzyme-linked immunosorbent assay, and the role of HA in enlargement of the perivitelline space in the oocytes was examined. The incidence of polyspermy following insemination was also observed in DOs in which HA synthesis was inhibited. HA was not detected in culture medium containing DOs immediately after collection. After culture for 7 h, 4.75 pg of HA per DO was detected in the medium, and the mean amount of HA significantly increased to 20.78 pg 14 h after culture. When DOs were cultured in medium containing 0.25 mM 4-methylumbelliferone (MU), an inhibitor of HA synthase, the mean amount of HA in the culture medium with DOs was 8.61 pg, which was significantly smaller than the amount in the control medium with non-treated DOs (21.59 pg). The mean size of the perivitelline space in oocytes cultured with cumulus cells (5.40 mum) did not differ from that (5.08 mum) of DOs. The mean size of the perivitelline space was significantly smaller in the MU-treated DOs (3.58 mum) than in the control DOs (4.65 mum). The fertilization rate did not differ between the MU-treated DOs (84.9%) and control DOs (81.0%), whereas the incidence of polyspermy was significantly higher in the MU-treated DOs (13.3%) compared with the control DOs (2.1%). These findings clarified that the HA involved in enlargement of the perivitelline space in oocytes is synthesized and secreted by the oocytes themselves. They also suggest that there is a close relationship between the size of perivitelline space and the incidence of polyspermy in mouse oocytes.

Time-lapse videomicrographic analyses of contractions in mouse blastocysts.

Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts.

Changes in the activities of hydroxysteroid dehydrogenases in mouse oocytes during meiotic maturation.

The activities of hydroxysteroid dehydrogenases (HSDs) were histochemically demonstrated in mouse oocytes in the process of maturation in vivo and in vitro, and the changes in steroid metabolism during meiotic maturation and also the relationship between nuclear maturation and changes in steroid metabolism in the cytoplasm were examined. In mouse oocytes 0 h after human chorionic gonadotrophin (hCG) injection, the activities of Delta(5)-3beta-HSD (with DHA, pregnenolone and 17alpha-hydroxypregnenolone as the substrates), 17beta-HSD (estradiol-17beta and testosterone) and 20beta-HSD (17alpha-hydroxyprogesterone and 20beta-hydroxyprogesterone) were observed in 87 to 97% of those, but that of 20alpha-HSD (20alpha-hydroxyprogesterone) was not. The percentages of oocytes showing the activities of Delta(5)-3beta-HSD, 17beta-HSD and 20beta-HSD did not change during maturation in vivo or in vitro. Oocytes with 20alpha-HSD activity appeared 4 h after the hCG injection or after culture for 4 h and the rates of those reached 92 and 100%, respectively, 14 h after the hCG injection or after culture for 14 h. In oocytes cultured for 8 h with olomoucine or 3-isobutyl-1-methylxanthine, nuclei were almost all in the germinal vesicle stage, and activity of 20alpha-HSD was observed in 84 and 89% of the treated oocytes, respectively. On the other hand, 81% of control oocytes showed 20alpha-HSD activity, with no significant difference from the rate for the olomoucine- or 3-isobutyl-1-methylxanthine-treated oocytes. The present findings suggested that the metabolic abilities of progesterone, 17alpha-hydroxyprogesterone, 17alpha,20beta-dihydroxyprogesterone, 20beta-hydroxyprogesterone, androgen and estradiol-17beta in the cytoplasm are constantly present in mouse oocytes in the process of maturation in vivo and in vitro. The results also suggested that the metabolic ability of 20alpha-hydroxyprogesterone in mouse oocytes increases during maturation, but the change in the metabolic ability of such a steroid is not related to nuclear maturation.

Developmental hormonal profiles in rdw rats with congenital hypothyroidism accompanying increased testicular size and infertility in adulthood.

Congenital hypothyroid mutant male rdw rats have enlarged testes in adulthood with dwarfism accompanied by infertility. To explain how rdw rats acquire enlarged testes in adulthood, we compared age-matched normal (N) rats at various developmental stages for blood levels of hormones, thyroxine (T4), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), and investigated whether T4 therapy (rdw+T4) from 3 weeks of age (w) until adulthood could induce recovery of fertility in rdw rats, as well as how rdw+T4 affected hormonal patterns. Testes weights of rdw rats were higher than those of N rats at 19 w in adulthood though it was low during development. Serum T4 values in rdw rats were markedly lower than those in N rats but steadily increased up to 19 w. The serum FSH values in rdw rats were lower than those in N rats at all ages, and neither serum LH nor T value was significantly different at any age. The testes weight of rdw+T4 rats was significantly higher than that of N rats at 13 w with recovered growth, and was higher than that of rdw rats at 19 w. When they were mated with proestrous females after 16 w, all females became pregnant and gave birth to a normal number of pups. The T4 and FSH values of rdw+T4 rats were significantly higher than those in rdw rats, but similar to those in N rats in adulthood. The results suggest that even low levels of circulating thyroid hormone (TH) in rdw rats stimulate the development of their testes, probably through Sertoli cells, resulting in the enlarged adult testes without fertility, and that a sufficient circulating TH level from the immature stage plays a pivotal role in restoring mating activity, probably through FSH-mediated action towards adulthood.