Oocyte quality is affected by many factors, among which the environment of growth and maturation seems to be crucial. Studies show that well balanced oocyte energy metabolism has a significant impact on several elements of cytoplasmic and nuclear maturation as well as further embryo developmental competence. Therefore homeostasis between metabolism of glucose and fatty acids in the oocyte is being widely described nowadays. This review aims to discuss the follicular (in vivo) or maturation media (in vitro) environments with regard to glucose and fatty acid metabolism, as the main sources of the energy for the oocyte. A great emphasis is given on the balance between those two metabolic pathways and its further impact on female fertility.

Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.

One major side effect of chemotherapy that young women with cancer suffer from is ovarian damage. Therefore, it is necessary to study the pathogenesis of chemotherapeutic drugs in order to develop pharmaceutical agents to preserve fertility. Epirubicin is one of the commonly used chemotherapy drugs for breast cancer patients. This research explored the side effects of epirubicin in mice. We found that epirubicin significantly reduced the body weight, the weight of the ovaries and uteri, and the pups’ number, while melatonin, which is extremely resistant to oxidation, significantly reduced these damages. Moreover, co-treatment with melatonin prevented epirubicin-induced decrease in E₂ and progesterone, and the loss of follicles. Mechanism study showed that melatonin significantly reduced the levels of proapoptotic genes p53, Caspase3, and Caspase9 while it upregulated antiapoptotic factors Bcl-2 and Bcl2l1, and antioxidant genes superoxide dismutase 1 and catalase compared with the epirubicin group. In addition, melatonin markedly reduced reactive oxygen species (ROS) and the transcription of Caspase12 and Chop, which is vital in endoplasmic reticulum stress (ERS)-mediated apoptosis. These results indicate melatonin protects against epirubicin-induced ovarian damage by reducing ROS-induced ERS. Therefore, melatonin has a therapeutic potential for the protection of ovarian function and preservation of fertility during chemotherapy.

In female mammals, luteal cells rapidly proliferate and form corpora lutea (CLs) after ovulation. The corpus luteum (CL) plays crucial roles in establishing and maintaining pregnancy. To gain further insights into the role of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptosis factor that is structurally similar to procaspase-8 but lacks proteolytic enzyme activity, we examined the expression in CLs of Thai swamp buffalos (Bubalus bubalis) during the early, mid, and late stage of the estrous cycle and pregnancy. cFLIP short form and long form (cFLIP_S and cFLIP_L, respectively) mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction and western blotting, respectively. cFLIP_S mRNA levels were low in the mid and late stages of the estrous cycle and increased during pregnancy (P < 0.05). cFLIP_L mRNA was highly expressed in CLs during pregnancy and was lower in the mid and late stages of the estrous cycle. The level of cFLIP_S protein was high in CLs during pregnancy and low levels were noted in the mid stage of the estrous cycle (P < 0.05). Higher levels of cFLIP_L protein were demonstrated in CLs during pregnancy and lower levels were found in CLs during the early stage of the estrous cycle. Strong positive immunohistochemical staining for cFLIP_S/L proteins was observed in luteal cells during pregnancy. The present findings revealed that cFLIP was at the highest level in CLs during pregnancy, and this may act as a dominant survival anti-apoptotic factor by inhibiting intracellular apoptosis signal transduction in luteal cells of CLs during pregnancy.

Embryo transfer uses embryos developed in vivo or in vitro for cattle production, however there is a difference in the quality of the embryos obtained by the two methods. This study addresses the differences in gene expression between blastocysts developed in vitro and in vivo. In vivo blastocysts were flushed from the uteri of super-ovulated cows and blastocysts developed in vitro were derived from in vitro matured and fertilized embryos. The same batch of frozen bull sperm was used for insemination and in vitro fertilization. Blastocysts were then subjected to RNA sequencing. Differentially expressed genes upregulated in in vitro blastocysts were annotated to focal adhesion, extracellular matrix (ECM)-receptor interaction, and PI3K-Akt signaling and the genes that were upregulated in in vivo blastocysts were annotated to oxidation-reduction processes, mitochondrion organization, and mitochondrial translation. Although the total cell number of the two types of blastocysts was similar, the mitochondrial quantity (determined by mitochondrial DNA copy numbers and expression levels of TOMM20), and ATP content in the blastocysts were lower in in vivo blastocysts compared with those developed in vitro. In conclusion, RNAseq revealed differential molecular backgrounds between in vitro and in vivo developed blastocysts and mitochondrial number and function are responsible for these differences.

Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

In bovine placentomes, the inflammatory response is considered important for the detachment of the fetal membrane from the caruncle after parturition. Glucocorticoids, a trigger of the onset of parturition, facilitate functional maturation of placentomes via prostaglandin (PG) and estrogen production in cattle. This study investigated how exogeneous glucocorticoids, which exert immunosuppressive effects, affect placental inflammation at parturition. Placentomes were collected immediately after spontaneous or induced parturition. Parturition was conventionally induced using PGF2α or dexamethasone or with a combination of triamcinolone acetonide and high-dose betamethasone (TABET treatment). Polymerase chain reaction (PCR) array analysis indicated that 9/13 C–C motif chemokine ligands (CCLs) were upregulated > two-fold in spontaneous parturition, with CCL2 and CCL8 being highly expressed. The expressions of CCL2, CCL8, C–C motif chemokine receptor 1 (CCR1), and CCR5 in caruncles were significantly higher in spontaneous parturition than in induced parturition. Although the clinical dose of dexamethasone did not influence the expression of these CCLs and CCRs, TABET treatment increased CCR1 expression. CCL8, CCR1, CCR2, and CCR5 were localized in the caruncular epithelial cells. CCR2 was also localized in the epithelial cells of the cotyledonary villi. This study is the first report to reveal the disruption in CCL and CCR expression in bovine placentomes at induced parturition. Enhanced glucocorticoid exposure for the induction of parturition may upregulate CCR1 expression in placentomes, but the treatment does not adequately promote CCL expression. Additionally, immunohistochemistry suggested that the CCL–CCR system is involved in the functional regulation of maternal and fetal epithelial cells in placentomes at parturition.

Testis-specific genes are prone to affect spermatogenesis or sperm fertility, and thus may play pivotal roles in male reproduction. However, whether a gene really affects male reproduction in vivo needs to be confirmed using a gene knock-out (KO) model, a ‘gold standard’ method. Increasing studies have found that some of the evolutionarily conserved testis-enriched genes are not essential for male fertility. In this study, we report that 1700121C10Rik, a previously uncharacterized gene, is specifically expressed in the testis and produces two long noncoding RNAs (lncRNAs) in mouse: Transcript 1 and Transcript 2. qRT-PCR, northern blotting, and in situ hybridization revealed that expression of both the lncRNAs commenced at the onset of sexual maturity and was predominant in round and elongating spermatids during spermiogenesis. Moreover, we found different subcellular localization of Transcript 1 and Transcript 2 that was predominant in the cytoplasm and nucleus, respectively. 1700121C10Rik-KO mouse model disrupting Transcript 1 and Transcript 2 expression was generated by CRISPR/Cas9 to determine their role in male reproduction. Results showed that 1700121C10Rik-KO male mice were fully fertile with approximately standard testis size, testicular histology, sperm production, sperm morphology, sperm motility, and induction of acrosome reaction. Thus, we conclude that both the testis-specific 1700121C10Rik-produced lncRNAs are dispensable for male fertility in mice under standard laboratory conditions.

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52–53% or 48–51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

Unlike sex steroids, mineralocorticoids have attracted limited attention in ovarian physiology. Recent studies on primates have indicated possible local synthesis and action of mineralocorticoids in the ovary. Here, we examined developmental changes in the levels of mineralocorticoids and expression of genes encoding their biosynthetic enzymes and receptor in the bovine ovary. The follicles and corpora lutea (CL) were collected from F1 heifers. Expression levels of 21α-hydroxylase (CYP21A2), 11β-hydroxylase-1 (CYP11B1), and the mineralocorticoid receptor (NR3C2) in granulosa cells (GC), thecal layers (TL), and CL tissues were quantified by real-time PCR, whereas mineralocorticoids in the follicular fluid were measured by enzyme immunoassay (EIA). TL and GC expressed CYP21A2 and NR3C2, whereas CYP11B1 was expressed at very low or undetectable levels. The expression levels of these genes were not significantly different among small/large and healthy/atretic follicles but were higher in TL than in GC. CYP21A2 and NR3C2 were expressed in all CL stages with higher expression observed in the mid-stage. CYP11B1 expression was only apparent in the mid-stage CL. Aldosterone was detected in all follicles, and its concentration was not significantly different among the follicular groups. In paired large-healthy/atretic follicles, the concentration of deoxycorticosterone, a precursor of aldosterone, was approximately ten-fold higher than that of aldosterone and not significantly different between healthy and atretic follicles. In conclusion, the presence of mineralocorticoids and expression of NR3C2 in the bovine follicle together with the developmental change in the expression of CYP21A2, CYP11B1, and NR3C2 in the CL suggest possible endocrine/paracrine/autocrine roles of mineralocorticoids in the bovine ovary.

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.

To prevent twin pregnancies in cattle, a simple transvaginal device can be used for follicular drainage. This study examines embryo survival following follicular drainage of the smaller pre-ovulatory follicle at timed artificial insemination (AI) in cows with a pre-ovulatory follicle in each ovary. The study groups established were a control group of 289 monovular cows, a non-drainage group of 114 bi-ovular cows and a follicular drainage group of 113 bi-ovular cows. All cows undergoing drainage developed a corpus luteum (CL) in the drained ovary. Pregnancy loss was recorded 56 days post-AI in 19.5% of the 149 cows that became pregnant. Pregnancy loss in the drainage group cows not suffering heat stress (3.8%) was significantly lower (P < 0.05) than in control non-heat stressed cows (20.9%) and heat-stressed non-drainage group cows (25%). Results indicate that CL induction by follicular drainage for twin pregnancy prevention may reduce the incidence of pregnancy loss.