We compared the efficacy of the microdrop and minimum volume cooling (MVC) methods for the vitrification of in vitro-produced porcine zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents. Zygotes and blastocysts were equilibrated in 2% (v/v) ethylene glycol and 2% (v/v) propylene glycol for 13–15 min. Then, they were vitrified in a medium comprised of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3 M sucrose, and 50 mg/ml polyvinylpyrrolidone either by either dropping them directly into liquid nitrogen (microdrop method) or placing them on Cryotop sheets in a minimum volume of medium and plunging into liquid nitrogen (MVC method). Both zygotes and blastocysts were successfully vitrified. For the vitrification of zygotes, the MVC and microdrop methods were equally effective; however, for blastocyst vitrification, MVC was superior. For both methods, the vitrification of zygotes produced higher-quality embryos than the vitrification of blastocysts.

Previously, our group demonstrated that recombinant porcine oviductin (pOVGP1) binds to the zona pellucida (ZP) of in vitro-matured (IVM) porcine oocytes with a positive effect on in vitro fertilization (IVF). The fact that pOVGP1 was detected inside IVM oocytes suggested that this protein had a biological role during embryo development. The aim of this study was to evaluate the effects of pOVGP1 on bovine in vitro embryo development. We applied 10 or 50 µg/ml of pOVGP1 during IVF, embryonic in vitro culture (IVC), or both, to evaluate cleavage and embryo development. Blastocyst quality was assessed by analyzing the expression of important developmental genes and the survival rates after vitrification/warming. pOVGP1 was detected in the ZP, perivitelline space, and plasma membrane of blastocysts. No significant differences (P > 0.05) were found in cleavage or blastocyst yield when 10 or 50 µg/ml of pOVGP1 was used during IVF or IVC. However, when 50 µg/ml pOVGP1 was used during IVF + IVC, the number of blastocysts obtained was half that obtained with the control and 10 µg/ml pOVGP1 groups. The survival rates after vitrification/warming of expanded blastocysts cultured with pOVGP1 showed no significant differences between groups (P > 0.05). The use of pOVGP1 during IVF, IVC, or both, increased the relative abundance of mRNA of DSC2, ATF4, AQP3, and DNMT3A, the marker-genes of embryo quality. In conclusion, the use of pOVGP1 during bovine embryo in vitro culture does not affect embryo developmental rates but produces embryos of better quality in terms of the relative abundance of specific genes.

Studies on mouse and rat pituitaries reported that Sox2-expressing cells play roles as stem/progenitor cells in the adult pituitary gland. The presence of cells with stem cell-like properties in the pituitary adenoma and SOX2-positive cells has been demonstrated in the human pituitary. However, considering the difficulty in fully examining the stem/progenitor cell properties in the human pituitary, in the present study, we analyzed the SOX2-positive cells in the pituitary of the adult common marmoset (Callithrix jacchus), which is used as a non-human primate model. Immunohistochemistry demonstrated that localization pattern of SOX2-positive cells in the common marmoset pituitary was similar to that observed in the rodent pituitary, i.e., in the two types of niches (marginal cell layer and parenchymal-niche) and as scattered single cells in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells express S100 and were located in the center or interior of LAMININ-positive micro-lobular structures. Collectively, the present study reveals properties of SOX2-positive cells in the common marmoset pituitary and suggests that the common marmoset proves to be a useful tool for analyzing pituitary stem/progenitor cells in a non-human primate model.

The role of oocytes in follicular antrum formation is not well understood. We examined the effect of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the formation of antrum-like structures by cultured bovine oocyte-granulosa cell complexes (OGCs). OGCs containing growing oocytes (105–115 µm in diameter) were collected from early antral follicles (1.2–1.8 mm) and used to prepare oocytectomized complexes (OXCs) and granulosa cell complexes (GCs). The mRNAs of GDF9 and BMP15 were expressed in the oocytes, but not in the granulosa cells. The complexes were cultured for five days with or without GDF9 and BMP15 either alone or in combination. The OGCs maintained their complex integrity and developed antrum-like structure, whereas OXCs and GCs neither maintained their integrity nor developed any antrum-like structure without growth factors. GDF9 or BMP15 alone increased the integrity of these complexes and induced antrum-like structures in OXCs and GCs. Moreover, the combination of GDF9 and BMP15 was more potent for both phenomena in all types of complexes. In OXCs and GCs cultured without GDF9 and BMP15 or with BMP15 alone, outgrowing granulosa cells differentiated into fibroblast-like cells. The combination of GDF9 and BMP15 suppressed the appearance of fibroblast-like cells in OXCs and GCs during incubation. Instead, the granulosa cells appeared rhomboid and pebble-like in shape, similar to those in OGCs cultured without supplementation of GDF9 and BMP15. These results suggest that oocytes maintain complex integrity by preventing granulosa cell differentiation and participate in follicular antrum formation via GDF9 and BMP15.

Insulin receptor substrate 2 (IRS2) is a component of the insulin/insulin-like growth factor 1 (IGF1) signaling cascade, which plays an important role in mouse hypothalamic and ovarian functions. The present study was conducted to investigate the role of IRS2 in steroidogenesis, apoptosis, cell cycle and proliferation in mouse granulosa cells (GCs). Flow cytometry and CCK8 assay showed that IRS2 knockdown inhibited cell proliferation, reduced cell viability, and increased apoptosis in GCs. The study also revealed that the expression of Cyclin A1, Cyclin B1 and Bcl2 was downregulated, while the expression of Bax, Cyclin D1 and Cyclin D2 was upregulated. ELISA analysis showed that IRS2 knockdown decreased the concentrations of estradiol (E₂) and progesterone (P₄), which was further validated by the decreased expression of Star, Cyp11a1, and Cyp19a1. Moreover, IRS2 knockdown altered the expression of Has2 and Ptgs2, which are essential for folliculogenesis. In addition, we found that IRS2-mediated cell viability and hormone secretion are dependent on the PI3K/AKT signaling pathway. Collectively, this study demonstrated that IRS2 plays an important role in the regulation of cell proliferation and steroidogenesis in mouse GCs via the PI3K/AKT signaling pathway.

Intracytoplasmic sperm injection (ICSI) has become critical for the treatment of severe male infertility. The principal feature of ICSI is the direct injection of spermatozoon into an oocyte, which facilitates the production of fertilized embryos regardless of semen characteristics, such as sperm concentration and motility. However, the chromosomal integrity of ICSI zygotes is degraded compared to that of zygotes obtained via in vitro fertilization. This chromosomal damage may occur due to the injection of non-capacitated, acrosome-intact spermatozoa, which never enter the oocytes under natural fertilization. Furthermore, it is possible that the in vitro incubation and pre-treatment of spermatozoa during ICSI results in DNA damage. Chromosomal aberrations in embryos induce early pregnancy losses. However, these issues may be overcome by embryo production using gametes with guaranteed chromosomal integrity. Because conventional chromosome analysis requires fixing cells to obtain the chromosome spreads, embryos cannot be produced using the nucleus that has been analyzed. On the other hand, genome cloning using androgenic or gynogenic embryos provides an additional nucleus for chromosome analysis following embryo production. Thus, this review aims to highlight the hazardous nature of chromosomal aberrations in sperm during ICSI and to introduce a method for the prezygotic examination for chromosomal aberrations.

This study was conducted to clarify the relationships of plasma concentrations of insulin-like peptide 3 (INSL3), testosterone, inhibin, and insulin-like growth factor-I (IGF-I) with scrotal circumference and testicular weight in Japanese Black beef bull calves (n = 20), from birth to pre-puberty. Monthly blood sampling (0 to 7 months) and scrotal circumference measurements (0 to 7 months) were performed. Testicular weight was recorded immediately after castration at 7 months. Plasma INSL3, testosterone, inhibin, and IGF-I concentrations were measured either by enzyme immunoassay or time-resolved fluorescence immunoassay. The correlation coefficients of these hormonal concentrations with scrotal circumference were significant (P < 0.0001) and it was higher for INSL3 (r = 0.647) than for testosterone (r = 0.597), IGF-I (r = 0.400), and inhibin (r = –0.453). Calves with heavier testes (> 60 g) at castration (7 months) had higher (P < 0.05) plasma INSL3 (from 3 to 7 months) and inhibin (from 1 to 4 months) concentrations than those with lighter testes (< 60 g). The calves with heavier testes at castration had larger (P < 0.05) scrotal circumference than those with lighter testes from 3 to 7 months. In conclusion, blood INSL3 concentrations may be the best functional indicator among the hormones analyzed for determining total testicular volume during pre-puberty in bull calves. In addition, inhibin and INSL3 concentrations in early calfhood may be functional predictors for testicular weight at pre-puberty.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.

Progressive movement of spermatozoa has conventionally been regarded as a good indicator of motility. However, bull spermatozoa exhibit two types of progressive movement: progressive/planar movement without rotation and progressive/helical movement with rotation. The aim of this study was to reconsider the evaluation criteria of bull ejaculated sperm motility in the context of rotation. Here, we compared the movement patterns of ejaculated spermatozoa with relatively high and low protein kinase A (PKA)-mediated signaling activities, because sperm motility is positively regulated by PKA-mediated signaling activities. We prepared sperm samples with high and low PKA-mediated signaling activities by suspending spermatozoa in media containing either the stimulator (NaHCO₃) or inhibitor (KH-7) of adenylyl cyclase 10, and we then investigated movement patterns and relative velocities using a microscopic high-speed camera and recording system. In the control medium without NaHCO₃ and KH-7, most spermatozoa exhibited round/planar movement without rotation and asymmetrical bends in the principal pieces. NaHCO₃ significantly promoted changes in movement patterns from round/planar movement to progressive/planar movement (without rotation) as well as symmetrization of flagellar bends and increased relative velocities. KH-7 significantly increased spermatozoa exhibiting progressive/helical movement (with rotation), decreased relative velocities, and symmetrized flagellar bends with a reduction in their size. These indicate that progressive/planar movement (without rotation) and fast movement characterize the movement patterns of bull ejaculated spermatozoa with high PKA-mediated signaling activities. A sign of reduced PKA-mediated signaling activity is not only slow movement but also helical movement (with rotation). Thus, it is beneficial to add a new parameter of “rotation” to the evaluation criteria of bull ejaculated sperm motility.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5’ adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.7 ± 3.5 and 9.0 ± 6.0, respectively). Next, we compared in vitro embryo production by ovum pick-up and in vitro fertilization (OPU-IVF) between control (n = 12) and epidural groups (n = 12). The rate of development to transferable blastocysts was higher in the epidural group than in the control (23.3 vs. 10.5%, P < 0.001). In conclusion, a single epidural administration of FSH can induce follicular development comparable to that of the conventional superovulation protocol and may improve the productivity of OPU-IVF.

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP). Then, the sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

Hyaluronan (hyaluronic acid, HA) apparently improves sperm survival in vitro and in vivo (oviduct), maintaining sperm motility and inducing capacitation, but not acrosome exocytosis, either by direct action as a macromolecule or via CD44 membrane receptors. This study explored ejaculated, liquid-extended pig spermatozoa to ascertain (i) the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor, using a specific monoclonal commercial antibody; (ii) whether the CD44 receptor changed location when exposed to bicarbonate, a capacitating trigger, in vitro; and (iii) whether the addition of HA, of molecular size comparable to that produced in the oviduct sperm reservoir (0.0625 to 2.0 mg/ml; 0 HA: control), to semen extenders would improve sperm liquid storage in vitro or cryosurvival post-freezing. Variables tested were sperm velocity and progressive motility (Qualisperm™), sperm viability and acrosome status, membrane integrity and early destabilization, mitochondrial activation, and superoxide production (flow cytometry). The CD44 receptor presence in ejaculated, liquid-stored AI boar spermatozoa, as confirmed by a porcine-specific monoclonal antibody, maintained its membrane location under in vitro capacitation-inducing conditions. HA exposure to 24-, 48-, or 72-h liquid-stored (17–20ºC) spermatozoa lowered sperm velocity in membrane-intact spermatozoa, but increased mitochondrial superoxide production. Finally, HA addition during cooling did not improve cryosurvival but did increase mitochondrial activation and membrane destabilization in surviving cells. These results confirm the existence of a CD44 receptor in pig spermatozoa, but the usefulness of adding HA for long-term storage or cryopreservation of liquid-stored, extended boar semen remains in question, thereby warranting further non-empirical analyses of HA-sperm membrane interactions.

Treatment of fetal rat and embryonic chicken with exogenous glucocorticoids induces premature differentiation of growth hormone (GH) secreting cells. The effect of corticosterone (CORT) on somatotroph differentiation was mostly studied in pituitary cells in vitro. Currently, there is no evidence for glucocorticoid-mediated induction of somatotroph differentiation during pituitary development in bird species other than chicken. In this study, we sought to find out if in ovo injection of corticosterone into developing goose embryos could induce premature increase of GH in somatotrophs. On embryonic day (e) 15, the albumen of fertile goose eggs was injected with 300 μl of 0.9% saline, 300 μl 5 × 10^–8M CORT, or 300 μl 5 × 10^–6 M CORT. Embryos were allowed to develop until e20 and e28 and isolated pituitaries were subjected to quantitative real-time PCR and immunocytochemistry to detect GH mRNA and protein, respectively. At e20 and e28, blood from chorioallantoic vessels was subjected to radioimmunoassay for analysis of plasma GH protein. In ovo administration of exogenous corticosterone brought about a 2.5-fold increase in the expression of GH mRNA and increased the in situ expression of GH protein in goose pituitary cells, and enhanced plasma GH levels compared to that of the respective controls at e20. These findings prove that administration of glucocorticoid could stimulate the expression of GH in somatotrophs during goose embryonic development. Our results suggest the probable involvement of membrane glucocorticoid receptor in the corticosterone mediated expression of GH. Together with previously published data, our results suggests that corticosterone mediated induction of GH expression during embryonic development is relatively conserved among different vertebrates.

We attempted to elucidate female reproduction in long-tailed macaques (Macaca fascicularis). These monkeys have a non-seasonal menstruation cycle, which makes them suitable subjects for studies in a variety fields including medical science and regenerative medicine. We analyzed individual breeding data including time of menarche, start of regular menstruation, and first pregnancy. These three events are related to the maturation of female long-tailed macaques. All research subjects were female long-tailed macaques bred at the Tsukuba Primate Research Center. The study comprised 45 females; we included time of menstruation, male-female cohabitation, and first pregnancy in their growth records. We extracted age and weight data relating to menarche, start of regular menstruation, and first pregnancy from these records. In the two years typically required from menarche to first pregnancy, the body weight increased by approximately 500 g (21% of the weight at menarche); it is clear that there is a significant physical change after menarche. Our findings suggest that female monkeys are not necessarily mature enough for pregnancy at menarche. Therefore, the use of the word “maturity” in terms of fecundity may be more accurate after the start of regular menstruation. This is what we term “adolescence” in the developmental process. Therefore, M. fascicularis monkeys are candidates for an animal model of human adolescence.

In microminipigs, estrus induction with abortion treatment, which is typically performed between 25 and 40 days after mating, is not always successful. Thus, the authors hypothesized that it may be more difficult to induce estrus by treating microminipigs approximately 40 days after mating. Accordingly, in this study, estrus induction was performed with abortion treatment in four microminipigs as follows: 0.3 mg of cloprostenol, a prostaglandin F2-alpha analog, was administered (day 0); after 24 h, 0.15 mg of cloprostenol and 250 IU of equine chorionic gonadotrophin were administered intramuscularly and simultaneously (day 1); after 96 h, 120 IU of human chorionic gonadotropin was injected intramuscularly (day 4). These treatments were compared at two different stages of pregnancy: early treatment (26.5 ± 0.7 days) and late treatment (38.3 ± 0.8 days). In the early treatment, all four microminipigs exhibited estrus on day 5, whereas in the late treatment, estrus was observed clearly in only two pigs on day 6 and slightly in 1 pig on day 10, whereas it was unclear in 1 pig. These results suggest that it is difficult to induce estrus with abortion treatment in microminipigs at approximately 40 days after mating.

Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen–thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen–thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 ―pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze–thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm’s tail.

We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

Spermatogenesis is briefly defined as the production of mature spermatozoa from spermatogonial stem cells at the end of a strictly regulated process. It is well known that, to a large extent, transcriptional activity ceases at mid-spermiogenesis. Several mRNAs transcribed during early stages of spermatogenesis are stored as ribonucleoproteins (RNPs). During the later stages, translational control of these mRNAs is mainly carried out in a time dependent-manner by poly(A)-binding proteins (PABPs) in cooperation with other RNA-binding proteins and translation-related factors. Conserved PABPs specifically bind to poly(A) tails at the 3′ ends of mRNAs to regulate their translational activity in spermatogenic cells. Studies in this field have revealed that PABPs, particularly poly(A)-binding protein cytoplasmic 1 (Pabpc1), Pabpc2, and the embryonic poly(A)-binding protein (Epab), play roles in the translational regulation of mRNAs required at later stages of spermatogenesis. In this review article, we evaluated the spatial and temporal expression patterns and potential functions of these PABPs in spermatogenic cells during spermatogenesis. The probable relationship between alterations in PABP expression and the development of male infertility is also reviewed.

Intracytoplasmic sperm injection (ICSI) was expected to enable more efficient use of sperm from sires with preferable genetic traits and result in a generation containing a larger number of offspring with superior genetic characteristics in livestock. However, the efficiency of the early development of embryos produced by ICSI is still far from satisfactory in cattle. The present study aimed to investigate the effects of the treatment of cryopreserved sperm with glutathione (GSH) on the early development of embryos produced by ICSI in Japanese Black cattle. Moreover, the disulfide bond state and mitochondrial function were investigated in the sperm treated with GSH to confirm the effectiveness of the abovementioned treatment. We also investigated the effect of 7% ethanol activation treatment on the developmental ability of ICSI embryos using GSH-treated sperm. There was no effect on the blastocyst rate from the activation treatment. When sperm-injected oocytes were cultured in vitro, the treatment with GSH significantly improved the early development of embryos. Specifically, the rates of embryos reaching the 4–8-cell stage and blastocyst stage were significantly higher in ICSI with GSH-treated sperm (71.4% and 31.0%, respectively) than that with the control sperm (36.6% and 7.0%, respectively). Moreover, the GSH-treated sperm treatment significantly decreased the number of disulfide bonds in the sperm head (as shown by monobromobimane staining) and enhanced the mitochondrial function in the sperm middle piece (as shown by Rhodamine 123 staining and the adenosine triphosphate-dependent bioluminescence assay). Based on these results, we suggest that the treatment of cryopreserved sperm with GSH might contribute to the improvement of ICSI techniques for the production of blastocysts in Japanese Black cattle.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca²⁺ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ–AID–EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ–AID–EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ–AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG)E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.