Chemotherapy can potentially impair fertility in premenopausal cancer patients. Female fertility preservation has been mainly focused on the ovarian aspects and benefited greatly from assisted reproductive technologies, such as in vitro fertilization (IVF). The rate-limiting step for the success of IVF is embryo implantation in the uterus. Doxorubicin (DOX) is a widely used chemotherapeutic agent with ovarian toxicity. It remains unknown if the uterus is a direct target of DOX. To circumvent the indirect uterine effect from ovarian toxicity of DOX and to investigate potential long-term impact of DOX on the uterus, young adult ovariectomized CD-1 mice were given an intraperitoneal injection once with PBS or DOX (10 mg/kg, a human relevant chemotherapeutic dose), and 30 days later, each set of mice was randomly assigned into three groups and subcutaneously injected with oil, 17β-estradiol (E2, for 6 h), and progesterone (P4, for 54 h), respectively. Uterine transcriptomic profiles were determined using RNA-seq. Principal component analysis of the uterine transcriptomes revealed four clusters from the six treatment groups: PBS-oil & DOX-oil, PBS-P4 & DOX-P4, PBS-E2, and DOX-E2, indicating that DOX treatment did not affect the overall uterine transcriptomic profiles in the oil and P4-treated mice but altered uterine responses to E2 treatment. DAVID analysis indicated that the top-affected gene cluster was “Glycoprotein”. These data demonstrate that DOX can directly target the uterus and has a long-term impact on uterine responses to E2.

Summary Sentence

Our finding that a single dose of doxorubicin relevant to human treatment level can change uterine gene expression in response to estrogen in ovariectomized mice one month later indicates that the uterus is a direct target of doxorubicin and doxorubicin has a long-term impact on the uterus.

Mechanistic target of rapamycin (mTOR) signaling functions as a central regulator of cellular metabolism, growth, and survival in response to hormones, growth factors, nutrients, energy, and stress signals. Mechanistic TOR is therefore critical for the growth of most fetal organs, and global mTOR deletion is embryonic lethal. This review discusses emerging evidence suggesting that mTOR signaling also has a role as a critical hub in the overall homeostatic control of fetal growth, adjusting the fetal growth trajectory according to the ability of the maternal supply line to support fetal growth. In the fetus, liver mTOR governs the secretion and phosphorylation of insulin-like growth factor binding protein 1 (IGFBP-1) thereby controlling the bioavailability of insulin-like growth factors (IGF-I and IGF-II), which function as important growth hormones during fetal life. In the placenta, mTOR responds to a large number of growth-related signals, including amino acids, glucose, oxygen, folate, and growth factors, to regulate trophoblast mitochondrial respiration, nutrient transport, and protein synthesis, thereby influencing fetal growth. In the maternal compartment, mTOR is an integral part of a decidual nutrient sensor which links oxygen and nutrient availability to the phosphorylation of IGFBP-1 with preferential effects on the bioavailability of IGF-I in the maternal–fetal interface and in the maternal circulation. These new roles of mTOR signaling in the regulation fetal growth will help us better understand the molecular underpinnings of abnormal fetal growth, such as intrauterine growth restriction and fetal overgrowth, and may represent novel avenues for diagnostics and intervention in important pregnancy complications.

Summary Sentence

Emerging evidence suggest that mTOR signaling in the fetal liver, trophoblast, and decidua serves as a critical hub in the overall homeostatic control of fetal growth, adjusting the fetal growth trajectory according to the ability of the maternal supply line to support fetal growth.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. For decades, attempts to efficiently derive ESCs in animal livestock species have been unsuccessful, but this goal has recently been achieved in cattle. Together with the recent reconstitution of the germ cell differentiation processes from ESCs in mice, these achievements open new avenues for the development of promising technologies oriented toward improving health, animal production, and the environment. In this article, we present a strategy that will notably accelerate genetic improvement in livestock populations by reducing the generational interval, namely in vitro breeding (IVB). IVB combines genomic selection, a widely used strategy for genetically improving livestock, with ESC derivation and in vitro differentiation of germ cells from pluripotent stem cells. We also review the most recent findings in the fields on which IVB is based. Evidence suggests this strategy will be soon within reach.

Summary Sentence

In vitro breeding constitutes a fast and intense selection strategy to genetically improve livestock populations.

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin–proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

Summary Sentence

SCF complexes are involved in normal oocyte maturation, polysperm defense, and correct course of preimplantation development.

Homocysteine (Hcy) is an intermediate in the one-carbon metabolism that donates methyl groups for methylation processes involved in epigenetic gene regulation. Although poor oocyte quality in polycystic ovarian syndrome (PCOS) patients is associated with elevated Hcy concentration in serum and follicular fluid, whether Hcy directly affects oocyte quality and its mechanisms are poorly understood. Here we show that Hcy treatment impaired oocyte quality and developmental competence, indicated by significantly reduced survival rate, polar body extrusion rate, and cleavage rate. Hcy treatment resulted in mitochondrial dysfunction, with increased production of mitochondrial ROS, reduced mtDNA copy number, and the expression of 7 out of 13 mtDNA-encoded genes and 2 ribosome RNA genes, 12S rRNA and 16S rRNA. Upon Hcy treatment, the expression of one-carbon metabolic enzymes and DNMT1 was enhanced. Interestingly, DNA methyltransferase inhibitor 5′AZA rescued Hcy-induced mitochondrial dysfunction, impaired oocyte quality and developmental competence. Concurrently, expression of one-carbon metabolic enzymes and methylation status of mtDNA coding sequences were also normalized, at least partially, by 5′AZA treatment. Our findings not only extend the understanding about how Hcy induces poor oocyte quality, but also contribute to a novel angle of identifying targets for enhancing the quality of oocyte from PCOS patients.

Summary Sentence

Homocysteine disrupts one-carbon metabolism, which leads to hypermethylation of mitochondrial DNA, mitochondrial dysfunction, and impaired oocyte quality.

Endometriosis is a prevalent gynecological disorder that eventually gives rise to painful invasive lesions. Increased levels of transforming growth factor-beta 1 (TGF-B1) have been reported in endometriosis. However, details of the effects of high TGF-B1 on downstream signaling in ectopic endometrial tissue remain obscure. We induced endometriotic lesions in mice by surgical auto-transplantation of endometrial tissues to the peritoneal regions. We then treated endometriotic (ectopic and eutopic endometrial tissues) and nonendometriotic (only eutopic endometrial tissues) animal groups with either active TGF-B1 or PBS. Our results demonstrate that externally supplemented TGF-B1 increases the growth of ectopically implanted endometrial tissues in mice, possibly via SMAD2/3 activation and PTEN suppression. Adhesion molecules integrins (beta3 and beta8) and FAK were upregulated in the ectopic endometrial tissue when TGF-B1 was administered. Phosphorylated E-cadherin, N-cadherin, and vimentin were enhanced in the ectopic endometrial tissue in the presence of TGF-B1 in the mouse model, and correlated with epithelial-mesenchymal transition (EMT) in ovarian endometriotic cells of human origin. Furthermore, in response to TGF-B1, the expression of RHOGTPases (RAC1, RHOC, and RHOG) was increased in the human endometriotic cells (ovarian cyst derived cells from endometriosis patient) and tissues from the mouse model of endometriosis (ectopic endometrial tissue). TGF-B1 enhanced the migratory, invasive, and colonizing potential of human endometriotic cells. Therefore, we conclude that TGF-B1 potentiates the adhesion of ectopic endometrial cells/tissues in the peritoneal region by enhancing the integrin and FAK signaling axis, and also migration via cadherin-mediated EMT and RHOGTPase signaling cascades.

Summary Sentence

TGF-B1 regulates the pathophysiology of endometriosis by specifically enhancing the migration, attachment, proliferation, colonization, and invasiveness of floating endometriotic cells or tissues, via the integrin-FAK, cadherin, and RHOGTPase signaling cascades.

Endometriosis (EM) is a mysterious and complicated disease that has been found to be multifactorial. Recent studies demonstrated that long noncoding RNAs (lncRNAs) play an important role in the pathogenesis of EM. However, the functional and biological mechanisms of lncRNAs in EM remain unknown. Here, we performed microarray analyses to compare the lncRNA expression profiles of four paired ectopic endometrial (EC) tissues and eutopic endometrial (EU) tissues from patients with ovarian EM. A novel lncRNA, CCDC144NL-AS1, was identified as being potentially functional. CCDC144NL-AS1 expression was upregulated in EC tissues compared to EU and normal endometrial (NE) tissues. Its expression was higher in EC tissues than in EU tissues in 86.7% (26/30) of patients with EM. Despite the lack of a significant increase according to revised American Fertility Society (rAFS) stages, approximately 60% of stage VI EM cases exhibited higher CCDC144NL-AS1 levels, many more than in the stage II–III cases. Subcellular fractionation demonstrated that CCDC144NL-AS1 was localized in the cytoplasm and nucleus of the human EM-derived immortalized endometrial stromal cell line hEM15A. CCDC144NL-AS1 depletion suppressed the migration and invasion of hEM15A cells, but exerted no effects on cell adhesion, proliferation, apoptosis, or cell cycle. Knockdown of CCDC144NL-AS1 dramatically altered the distribution of cytoskeletal filamentous actin (F-actin) stress fibers compared to the negative control group treatment. Western blot analysis revealed that knockdown of CCDC144NL-AS1 attenuated the protein levels of vimentin filaments and MMP-9, but not N-cadherin or β-catenin. Collectively, our results suggest that CCDC144NL-AS1 might be involved in the pathogenesis of EM and provide a novel target for ovarian EM.

Summary Sentence

Elevated lncRNA CCDC144NL-AS1 levels contribute to cytoskeleton reorganization, migration, and invasion in endometrial stromal cells via regulating vimentin and MMP-9.

Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and telomeric repeat-containing RNA (TERRA). Proposed TERRA functions are wide ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the presence and role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization, we identify TERRA transcripts in female PGCs at 11.5, 12.5, and 13.5 days postcoitum. In male PGCs, the earliest detection TERRA was at 12.5 dpc where we observed cells with either zero or one TERRA focus. Using qRT-PCR, we evaluated chromosome-specific TERRA expression. Female PGCs showed TERRA expression at 11.5 dpc from eight different chromosome subtelomeric regions (chromosomes 1, 2, 7, 9, 11, 13, 17, and 18) while in male PGCs, TERRA expression was confined to the chromosome 17. Most TERRA transcription in 13.5 dpc male PGCs arose from chromosomes 2 and 6. TERRA interacting proteins were evaluated using identification of direct RNA interacting proteins (iDRiP), which identified 48 in female and 26 in male protein interac-tors from PGCs at 13.5 dpc. We validated two different proteins: the splicing factor, proline- and glutamine-rich (SFPQ) in PGCs and non-POU domain-containing octamer-binding protein (NONO) in somatic cells. Taken together, our data indicate that TERRA expression and interactome during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.

Summary Sentence

TERRA transcription and interacting proteins during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.

Valosin-containing protein (VCP) is a member of the highly conserved AAA (ATPase associated with a variety of cellular activities) superfamily. A previous study has shown that targeted deletion of Vcp in mice results in early embryonic lethality. The aim of the present study was to analyze the expression and localization of VCP and its function in meiotic arrest of mouse oocytes. Vcp mRNA and protein were expressed in multiple mouse tissues. In the ovary, VCP protein was mainly expressed in oocytes and granulosa cells. After ovulation and fertilization, Vcp mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, VCP protein was localized in both the cytoplasm and nucleus of germinal vesicle (GV)-stage oocytes and preimplantation embryos. Moreover, knockdown of Vcp in GV-stage oocytes led to a significantly increased rate of germinal vesicle breakdown (GVBD). In addition, inhibition of VCP protein improved the GVBD rate in mouse GV-stage oocytes. When VCP inhibition was reversed, the final GVBD rate returned to normal. These results provide the first evidence for a novel function of VCP in meiotic arrest of mouse oocytes.

Summary Sentence

A novel function of VCP protein is involved in the control of meiotic arrest in the mouse oocyte.

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

Summary Sentence

A decrease in mitochondrial copy number and function affects oocyte quality in aged females, and is related to increased spindle abnormalities and oxidative stress.

Increasing studies have shown that specific mRNAs and miRNAs expressed in mature sperm may be related to sperm motility. However, the expression profiles and roles of lncRNAs in sperm remain unknown. In the present study, numerous lncRNAs were identified in human sperm, and some lncRNAs were expressed exclusively in sperm and testis. Compared with those in normal sperm, the lncRNA expression profiles in asthenozoospermia (AZS) sperm showed significant differences. Gene ontology and pathway analyses showed that function of differentially expressed lncRNA targets and mRNAs between AZS and normal sperm were closely linked with many processes involved in spermatogenesis and sperm function. Furthermore, among the upregulated lncRNAs in AZS sperm, lnc32058, lnc09522, and lnc98487, which exhibited specific/enriched sperm and testicular expression, increased simultaneously in the same AZS sperm samples, and their expression levels were correlated with sperm progressive motility. This is the first systematic study of lncRNA expression profiles in human mature sperm indicating an association between lncRNA expression and sperm motility. The study provides a preliminary database for identifying lncRNAs crucial for human spermatogenesis and sperm function, and new insights into our understanding of the regulation of sperm motility and causes of male infertility.

Summary Sentence

Numerous lncRNAs were identified and compared between normal and asthenozoospermic patients' sperm, which showed significant differences suggesting that some key sperm/testis-specific/enriched lncRNAs play a role in sperm motility.

Some X-linked genes necessary for spermiogenesis are specifically activated in the postmeiotic germ cells. However, the regulatory mechanism about this activation is not clearly understood. Here, we examined the potential mechanism controlling the transcriptional activation of the mouse testis specific gene A8 (Tsga8) gene in round spermatids. We observed that the Tsga8 expression was negatively correlated with the methylation level of the CpG sites in its core promoter. During spermatogenesis, the Tsga8 promoter was methylated in spermatogonia, and then demethylated in spermatocytes. The demethylation status of Tsga8 promoter was maintained through the postmeiotic germ cells, providing a potentially active chromatin for Tsga8 transcription. In vitro investigation showed that the E12 and Spz1 transcription factors can enhance the Tsga8 promoter activity by binding to the unmethylated E-box motif within the Tsga8 promoter. Additionally, the core Tsga8 promoter drove green fluorescent protein (GFP) expression in the germ cells of Tsga8-GFP transgenic mice, and the GFP expression pattern was similar to that of endogenous Tsga8. Moreover, the DNA methylation profile of the Tsga8-promoter-driven transgene was consistent with that of the endogenous Tsga8 promoter, indicating the existence of a similar epigenetic modification for the Tsga8 promoter to ensure its spatiotemporal expression in vivo. Taken together, this study reports the details of a regulatory mechanism that includes DNA methylation and transcription factors to mediate the postmeiotic expression of an X-linked gene.

Summary Sentence

DNA demethylation is the primary epigenetic modification for the Tsga8 gene expression. Spz1 may enhance the Tsga8 transcription in round spermatids.

Recurrent spontaneous abortion is associated with abnormal maternal tolerance to the semi-allogenic fetus, wherein the Th17/Treg axis plays a crucial role. Adiponectin (APN) is an adipocytokine that is shown to be a novel negative T-cell regulator and induce immune tolerance. The CBA/J × DBA/2 mating was used as an abortion-prone model to investigate whether the addition of recombinant adiponectin (rAPN) improves the pregnancy outcome. Recombinant adiponectin therapy reduced the abortion rate in abortion-prone model. It skewed the ability of serum cytokine production toward a Treg bias and induced APN production. Flow cytometry revealed that rAPN administration expanded the splenic CD4⁺CD25⁺ regulatory T-cell (Treg) population and reduced the Th17 cell populations in CBA/J × DBA/2 matings. RT-PCR revealed that rAPN administration induced the expression of AdipoR1 and AdipoR2 mRNA at the maternofetal interface. Recombinant adiponectin administration induced FoxP3 and reduced RORγt expressions at the maternofetal interface. In vitro experiment also showed that rAPN treatment enhanced the FoxP3 mRNA and protein expression and decreased the RORγt expression in splenic lymphocytes of abortion-prone mice. Blocking the different signal transduction pathways downstream of APN, p38MAPK inhibitor (SB203580) and STAT5 inhibitor (Pimozide) could abrogate the regulatory effect of rAPN on FoxP3 and RORγt expression, while STAT3 inhibitor (Stattic) and AMPK inhibitor (p5499) did not exert any influence. Thus, the current results demonstrated that rAPN therapy improves pregnancy outcome in a murine model of abortion by expanding the Treg cell population and function and decreasing the Th17 cell population and function via a p38MAPK-STAT5 pathway.

Summary Sentence

Adiponectin, which is essential for lipid metabolism and homeostasis, is beneficial for pregnancy outcome through improving Th17 and Treg populations; the process is associated with the activation of p38MAPK-STAT5 pathway.

The estrous cycle is an iterative change in the anatomy, endocrinology, physiology, and behavior to provide maximum fecundity. Ovarian steroid production involves gonadotropin-induced [Ca²⁺]_i raises due in part to voltage-gated Ca²⁺ channels (VGCCs) whose identity and tissue distribution in situ is largely unknown. Using fluorescence Ca²⁺ imaging and confocal microscopy, we recorded both spontaneous and depolarization-induced Ca²⁺ signals in living mouse ovarian slices. They were most prominent in theca cells (TCs) and oocytes. The presence of Ca²⁺ channel subunits Ca_V 1.2, Ca_V 1.3, Ca_V 2.1, Ca_V 2.2, and Ca_V 3 was examined with immunofluorescence of ovarian sections. Ca_V 1.2 and Ca_V 1.3 (L-type Ca²⁺ channels) are present in the stroma, granulosa cells (GCs), and corpora lutea (CL). Intriguingly subunits that are characteristic of nerve cells are also expressed: P/Q-type (Ca_V 2.1; α1A) in the stroma and CL cells and N-type (Ca_V 2.2; α1B) in perifollicular smooth muscle cells. The expression of α1 subunits fluctuates along the estrous cycle: in metestrus-diestrus (the quiescent stage of the cycle), CL and GCs are similarly stained, while in proestrus (stage of maximal ovarian stimulation) CL staining increases relatively to GCs. Also in proestrus, Ca_V 3 Ca²+ channel subunits are expressed more in CL compared to GC suggesting a more significant role of Ca²⁺ channels. In estrus, Ca_V 3 subunits from mesenchymal and interfollicular stromal cells become intensely stained. Our study represents an important step in understanding the role of VGCCs in ovarian physiology and possibly in ovarian cancer and other reproductive pathologies.

Summary Sentence

Calcium signaling and the expression of voltage-gated Ca²⁺ channels in the ovary is heterogeneous and varies considerably along the estrous cycle.

Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (−/−) mice and examined the detailed function of PATE4 on male fecundity. The morphology and weight of Pate4−/− seminal vesicles were comparable to the control. Although Pate4−/− cauda epididymal spermatozoa have no overt defects during in vitro fertilization, Pate4−/− males were subfertile. We found that the copulatory plugs were smaller in the vagina of females mated with Pate4−/− males, leading to semen leakage and a decreased sperm count in the uterus. When the females mated with Pate4−/− males were immediately re-caged with Pate4+/+ males, the females had subsequent productive matings. When the cauda epididymal spermatozoa were injected into the uterus and plugged artificially [artificial insemination (AI)], Pate4−/− spermatozoa could efficiently fertilize eggs as compared to wild-type spermatozoa. We finally examined the effect of SVSs on AI, and observed no difference in fertilization rates between Pate4+/+ and Pate4−/− SVSs. In conclusion, PATE4 is a novel factor in forming the copulatory plug that inhibits sequential matings and maintains spermatozoa in the uterus to ensure male fecundity.

Summary Sentence

The copulatory plug has dual functions not only to prevent subsequent matings but also to maintain proper sperm count for fertilization in the female reproductive tract, as a winner-take-all strategy to advance male reproduction.

Studies were designed to (a) evaluate the mRNA expression of the C-C motif chemokine receptor 2 (CCR2) and its chemokine ligands, as well as genes related to periovulatory events, within the cumulus oocyte complex (COC) and follicle wall after a luteinizing hormone (LH) stimulus in cultured feline antral follicles; (b) assess the immunolocalization of CCR2 and its main ligand (monocyte chemoattractant protein 1, MCP1) within the feline COC; and (c) examine the direct effects of exogenous recombinant MCP1 on mRNA expression of the CCR2 receptor and MCP1 as well as key periovulatory genes in the COC, using a feline COC culture system. Both culture systems were developed by our laboratory and exhibit physiological response to gonadotropin stimuli. In summary, this study demonstrated mRNA expression of CCR2 receptor and its assessed ligands (MCP1, MCP2, MCP3, and MCP4) within the feline COC and follicle antral wall, and a significant increase in CCR2 mRNA by LH within the COC. Also, CCR2 and MCP1 immunoreactivity was observed in the oocyte and cumulus cells of the feline COC. Remarkably, this is the first report, in any species, describing a direct effect of the recombinant MCP1 in the CCR2/MCP1 system within the COC, by increasing the mRNA levels of key genes involved in the ovulatory cascade, as well as its own receptor CCR2. Together, these data suggest that CCR2 receptor signaling in the COC may regulate events critical for promoting cumulus oocyte expansion and/or oocyte maturation.

Summary Sentence

MCP1 directly stimulates the mRNA levels of key ovulatory genes and its own receptor CCR2 within the COC, suggesting that CCR2/MCP1 signaling may regulate events critical for promoting cumulus oocyte expansion and/or oocyte maturation.

Antimicrobial peptides (AMPs) are regarded as host defense peptides that possess bactericidal activity as well as immunomodulatory function. However, the role of AMP in the mammalian ovary is unknown. In the present study, porcine granulosa cells were utilized in a cell model to study the role of porcine beta defensin 2 (pBD2; pDEFB4B) and 3 (pBD3; pDEFB103A) during ovarian follicular development. Granulosa cells were cultured in the absence and presence of 1, 10, and 50 µg/ml of pDEFB4B or pDEFB103A. After 24 h of treatment, pDEFB103A but not pDEFB4B stimulated granulosa cell proliferation in a concentration-dependent manner (P < 0.05). This effect was dependent on the stage of follicular development. In addition, transwell cell migration assay showed that in the presence of pDEFB103A (10 µg/ml), a 2.5-fold increase in cell migration was achieved. Furthermore, further study revealed that pDEFB103A increased the mRNA levels of cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA), both associated with cell proliferation. To study the potential pathway involved in pDEFB103A-induced cell proliferation and migration, western blots were performed. It was found that pDEFB103A significantly increased the phosphorylated-ERK1/2 to nonphosphorylated ratio. Moreover, pretreatment with the U0126, a specific ERK1/2 phosphorylation inhibitor, suppressed PDEFB103A inducing GCs ERK1/2 phosphorylation, as well as proliferation and migration, suggesting that PDEFB103A may act via activating the ERK1/2 pathway. Furthermore, using a signal transduction pathway Elk-1 trans-reporting system, the activation of ERK1/2 pathway by PDEFB103A was further confirmed. Our data suggest that AMP may play a physiological role in the mammalian ovary.

Summary Sentence

Beta defensin 3, regarded as host defense peptide which possess bactericidal activity and immunomodulatory function, enhanced granulosa cell migration and proliferation via its activation of the ERK1/2 pathway.

The mechanism underlying premature ovarian insufficiency remains incompletely understood. Here we report that mice with Per1^m/m; Per2^m/m double mutations display a decrease in female fertility starting approximately at 20 weeks old, with significantly less pups born from 32 weeks old onwards. Histological analysis revealed that a significant reduction of ovarian follicles was observed in the Per1/Per2 mutants compared with the littermate controls examined at 26 and 52 weeks old, while the difference was not statistically significant between the two groups at 3 and 8 weeks old. We further showed that vascular development including the ovarian follicle associated vascular growth appeared normal in the Per1/Per2 mutant mice, although clock genes were reported to regulate angiogenesis in zebrafish. The findings imply that loss-of-function mutations with Per1/Per2 result in a premature depletion of ovarian follicle reserve leading to the decline of reproductive capacity.

Summary Sentence

Disruption of circadian rhythm or its underlying regulatory network contributes to the premature depletion of ovarian follicle reserve.

L-Proline (proline) in amniotic fluid was markedly increased during pregnancy in both pigs and sheep. However, in vivo data to support a beneficial effect of proline on fetal survival are not available. In this study, pregnant C57BL/6J mice were fed a purified diet supplemented with or without 0.50% proline from embryonic day 0.5 (E0.5) to E12.5 or term. Results indicated that dietary supplementation with proline to gestating mice enhanced fetal survival, reproductive performance, the concentrations of proline, arginine, aspartic acid, and tryptophan in plasma and amniotic fluid, while decreasing the concentrations of ammonia and urea in plasma and amniotic fluid. Placental mRNA levels for amino acid transporters, including Slc36a4, Slc38a2, Slc38a4, Slc6a14, and Na⁺/K⁺ ATPase subunit-1α (Atp1a1), fatty acid transporter Slc27a4, and glucose transporters Slc2a1 and Slc2a3, were augmented in proline-supplemented mice, compared with the control group. Histological analysis showed that proline supplementation enhanced labyrinth zone in the placenta of mice at E12.5, mRNA levels for Vegf, Vegfr, Nos2, and Nos3, compared with the controls. Western blot analysis showed that proline supplementation increased protein abundances of phosphorylated (p)-mTORC1, p-ribosomal protein S6 kinase (p70S6K), and p-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well as the protein level of GCN2 (a negative regulator of mTORC1 signaling). Collectively, our results indicate a novel functional role of proline in improving placental development and fetal survival by enhancing placental nutrient transport, angiogenesis, and protein synthesis.

Summary Sentence

Proline supplementation enhances placental nutrient transport, angiogenesis, and protein synthesis, which are critical for fetal survival in mice.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14–18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 µM/10⁹ spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

Summary Sentence

Thiols regulate stallion sperm functionality.

Many bitterling species are facing extinction because of habitat destruction. Since cryopreservation of fish eggs is still not available to date due to their large size and high yolk content, long-term and stable storage of bitterling genetic resources is currently not possible. We recently discovered that cryopreservation of early-stage germ cells is possible in several fish species and that functional gametes derived from the frozen materials can be produced through their transplantation to embryonic recipients. However, bitterlings have uniquely shaped eggs and their embryos are extremely fragile, making it difficult to perform germ cell transplantation. Therefore, as a first step, we conducted intra-species spermatogonial transplantation using recessive albino Chinese rosy bitterling as donors and wild-type Chinese rosy bitterling as recipients to develop a system to convert freezable early-stage germ cells into functional gametes, particularly eggs. Approximately 3000 testicular cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less recipient embryos produced by dead end (dnd)-knockdown. At 6 months, ten male recipients and nine female recipients produced gametes. Mating studies with the opposite sex of recessive albino control fish revealed that six males and three females produced only albino offspring, suggesting that these recipients' endogenous germ cells were completely removed by dnd-knockdown and they produced only donor-derived gametes. Thus, we successfully established a germ cell transplantation system in an iconic endangered teleost, bitterling. The technology established in this study can be directly applied to produce functional gametes of endangered bitterlings using cryopreserved donor cells.

Summary Sentence

Allogeneic germ cell transplantation system was established using germ cell-less embryonic recipients produced by dead end-knockdown in a bitterling, which is an iconic endangered fish species.

Ubiquitin-specific protease 26 (USP26) is an X-linked gene exclusively expressed in the testis and codes for the USP26, a peptidase enzyme that belongs to the deubiquitinating enzyme family. Recent studies have indicated that mutations in USP26 affect spermatogenesis and are associated with male infertility in humans and mice. However, the exact role of USP26 in spermatogenesis and how it affects male reproduction remains unknown. In this study, we generated a conventional Usp26 knockout mouse model and found that deletion of Usp26 in male mice (Usp26^−/Y) leads to significantly reduced pup numbers per litter and significantly increased intervals between two consecutive offspring. We also found that the serum follicle stimulating hormone and testosterone levels of adult Usp26^−/Y mice were significantly decreased compared to those of Usp26^+/Y mice. Histological examination results showed that Usp26^−/Y mice had significantly increased percentage of abnormal seminiferous tubules at different ages. Flow cytometry results exhibited that Usp26^−/Y mice had significantly reduced percentage of mature haploid cells in the testes compared to Usp26^+/Y mice. Sperm counts in epididymis were also significantly declined in Usp26^−/Y mice compared to those in Usp26^+/Y mice. Immunohistochemistry and immunofluorescence staining and immunoprecipitation analysis results showed that USP26 and androgen receptor were co-localized in mouse testicular cells at different ages and they both had physiological interactions. All these results demonstrated that the loss of Usp26 affects spermatogenesis and hormone secretion and causes male subfertility. Our study also provides the evidence on the interactions between USP26 and androgen receptor in mouse testis, whereby pointing to a potential mechanism.

Summary Sentence

The phenotype of Usp26 knock out in male mice is subfertility, which may be related to the loss of regulation of androgen-AR signaling by USP26 in the testis.