Kisspeptin, encoded by KISS1/Kiss1 gene, is now considered a master regulator of reproductive functions in mammals owing to its involvement in the direct activation of gonadotropin-releasing hormone (GnRH) neurons after binding to its cognate receptor, GPR54. Ever since the discovery of kisspeptin, intensive studies on hypothalamic expression of KISS1/Kiss1 and on physiological roles of hypothalamic kisspeptin neurons have provided clues as to how the brain controls sexual maturation at the onset of puberty and subsequent reproductive performance in mammals. Additionally, emerging evidence indicates the potential involvement of extra-hypothalamic kisspeptin in reproductive functions. Here, we summarize data regarding kisspeptin inside and outside the hypothalamus and revisit the physiological roles of central and peripheral kisspeptins in the reproductive functions of mammals.
Progranulin (PGRN) is a multifunctional growth factor with functions in neuroprotection, anti-inflammation, and neural progenitor cell proliferation. These functions largely overlap with the actions of estrogen in the brain. Indeed, we have previously shown that PGRN mediates the functions of estrogen, such as masculinizing the rodent brain and promoting adult neurogenesis. To evaluate the underlying mechanism of PGRN in mediating the actions of estrogen, the localization of estrogen receptor α (ERα) in the brains of wild-type (WT) and PGRN-deficient (KO) mice was investigated. First, double-labeling immunofluorescence was performed for ERα with neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP), as markers for neurons, microglia, and astrocytes, respectively, in female mice in diestrous and estrous stages. ERα-immunoreactive (IR) cells were widespread and co-localized with NeuN in brain sections analyzed (bregma –1.06 to –3.16 mm) of both WT and KO mice. In contrast, expression of ERα was not observed in Iba1-IR cells from both genotypes. Interestingly, although ERα was co-localized with GFAP in WT mice, virtually no ERα expression was discernible in GFAP-IR cells in KO mice. Next, the brains of ovariectomized adult female, adult male, and immature female mice were subjected to immunostaining for ERα and GFAP. Again, co-localization of ERα with GFAP was observed in WT mice, whereas this co-localization was not detected in KO mice. These results suggest that PGRN plays a crucial role in the expression of ERα in astrocytes regardless of the estrous cycle stage, sex, and maturity.
Because latent form of matrix metallopeptidase-9 (proMMP9) levels are positively related to blastocyst development, it was hypothesized that addition during maturation may improve development of heat-stressed oocytes. To test hypothesis, 0, 30 or 300 ng/ml human proMMP9 (hMMP9) was added at 18 h of in vitro maturation (hIVM) to cumulus-oocyte complexes matured at 38.5 or 41.0ºC (first 12 h only). Heat stress decreased 24 hIVM proMMP9 levels only in 0 and 30 ng/ml groups and increased progesterone in 0 and 300 ng/ml hMMP9 groups. Heat stress decreased cleavage and blastocyst development. Independent of maturation temperature, hMMP9 at 18 hIVM decreased blastocyst development. In a second study, cumulus-oocyte complexes were matured for 24 h at 38.5 or 41.0ºC (HS first 12 h only) with 0 or 300 ng/ml hMMP9 added at 12 hIVM. Without hMMP9, heat stress decreased 24 hIVM proMMP9 levels and increased progesterone production. Addition of 300 ng/ml of hMMP9 produced equivalent levels of proMMP9 at 24 hIVM (271 vs. 279 ± 77 for 38.5ºC and 41.0ºC treated oocytes, respectively). Heat stress did not affect ability of oocytes to cleave but reduced blastocyst development. Independent of temperature, hMMP9 decreased cleavage and blastocyst development. In summary, hMMP9 supplementation during IVM did not improve development of heat-stressed oocytes even when it was added for the entire maturation period. At doses tested, hMMP9 appeared detrimental to development when supplemented during the last 12 or 6 h of oocyte maturation.
Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation.
Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2–2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication.
We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work.
The aim of this study was to investigate possible relationships between morphometric characteristics of the bovine reproductive tract and measures of fertility, such as antral follicle counts (AFCs) and the number and quality of recovered cumulus oophorus complexes (COCs). First, the genital tracts of 360 abattoir Zebu/Holstein crossbred cows were studied. Rima vulvae length (RL), vulvar width (VW), ovarian size, pelvic fat, number of aspirated COCs, and AFCs were recorded. An index of COC quality (CQI) was established, which weighed overall gamete quality based on the IETS classification. Second, the same external measurements and AFCs (by ultrasound) were analyzed in live Tabapuã (Zebu) cows (n = 48). Relative RL and vulvar width were defined as the original measurements divided by the body weight of each cow for statistical analyses. In abattoir tracts, the AFC was smaller (P < 0.05) in animals with large VW (34.88 ± 3.50) than in animals with small (45.71 ± 2.57) and intermediate (42.25 ± 2.45) VW. The CQI was higher (P < 0.05) when the pelvic fat score was intermediate (3.22 ± 0.06) than that in lean (2.99 ± 0.08) and obese (2.90 ± 0.12) animals. The CQI was higher (P < 0.05) in the intermediate ovary group (3.19 ± 0.08) than in the small (2.96 ± 0.08) and large (2.95 ± 0.09) ovary groups. In live cows, the AFC was higher (P = 0.035) in females with large relative rima length than in those with small relative rima length (18.96 ± 1.97 and 14.76 ± 1.51, respectively). Similarly, AFC was greater (P = 0.0001) in females with large relative VW than in those with small relative VW (20.08 ± 1.36 and 11.16 ± 1.60, respectively). In conclusion, larger external genitalia relative to body size were good predictors of the ovarian follicular reserve in live animals.
Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.
Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection.
The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme’s adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity.
In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation.
Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids.
A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples. The serum concentrations of estradiol measured by PATHFAST also correlated well with FIA. Circulating estradiol increased during mid-gestation and high levels of serum estradiol were maintained in late gestation, followed by an abrupt decline to term. These results demonstrate the utility of PATHFAST in equine clinics as an accurate diagnostic tool for the rapid assay of estradiol within 26 min using unextracted whole blood.
Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.
In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.