Androgen synthesis cell-specific CREBZF deficiency alters adrenal cortex steroid secretion and develops behavioral abnormalities in adult male mice.

The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells in vitro; however, further validation through in vivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17ß-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.

LncRNA STAT3-AS regulates endometrial receptivity via the STAT3 signaling pathway.

Endometrial receptivity is critical for the successful establishment of pregnancy in ruminants. Interferon tau (IFNT) plays a key role in promoting embryo attachment by activating the Janus kinase/signal transducer and activator of transcription pathway, which induces the expression of a series of interferon-stimulated genes (ISGs). In our previous study, sequencing analysis of goat endometrial epithelial cells (gEECs) treated with 20 ng/mL IFNT revealed a differentially expressed long non-coding RNA located on the STAT3 antisense chain, which we designated STAT3-AS. The aim of this study was to investigate the role and mechanism of STAT3-AS in establishing endometrial receptivity in goats. The results showed that STAT3-AS was expressed in both the nucleus and cytoplasm of gEECs, and its expression increased significantly in the uterus on day 15 of pregnancy. STAT3-AS expression was upregulated in gEECs treated with IFNT alone or in combination with progesterone and estradiol. Knockdown of STAT3-AS using specific short interfering RNA significantly inhibited the expression of classical ISGs such as interferon-stimulated gene 15 and 2',5'-oligodenylate synthetase 2, as well as uterine endometrial receptivity-related genes including homeobox gene A11, integrin beta 3, and vascular endothelial growth factor. Moreover, gEEC proliferation and the STAT3 pathway were suppressed in the absence of STAT3-AS. However, pretreatment with the STAT3 activator RO8191 restored the effect of silencing STAT3-AS on endometrial receptivity. Overall, these results suggest that STAT3-AS is an important regulator of endometrial receptivity in goats and that it regulates endometrial receptivity through the STAT3 pathway.

Resveratrol and lycium barbarum polysaccharide improve Qinling giant panda (Ailuropoda melanoleuca Qinlingensis) sperm quality during cryopreservation.

BACKGROUND: Semen cryopreservation has become an essential tool for conservation efforts of the giant panda (Ailuropoda melanoleuca); however, it is severely detrimental to sperm quality. Evidence has shown that antioxidants have the potential to reverse cryopreservation-induced damage in sperm. The purpose of this study was to screen effective antioxidants that could retain sperm quality during cryopreservation and to determine the optimal dose. Seven antioxidant groups, including resveratrol (RSV = 50 µM, RSV = 100 µM, RSV = 150 µM), lycium barbarum polysaccharide (LBP = 2 mg/mL, LBP = 4 mg/mL), laminaria japonica polysaccharides (LJP = 1 mg/mL) or combination (LBP = 2 mg/mL, LJP = 1 mg/mL and RSV = 100 µM) were assessed. RESULTS: RSV, LBP, LJP, or a combination of RSV, LBP, and LJP added to the freezing medium significantly improved sperm progressive motility, plasma membrane integrity, acrosome integrity, and mitochondrial activity during the cryopreservation process. Furthermore, the activities of glutathione peroxidase and superoxide dismutase were also improved. The levels of reactive oxygen species and malondialdehyde in semen were notably reduced. Hyaluronidase activity and acrosin activity were significantly increased in LBP-treated sperm. However, sperm total motility and DNA integrity were not significantly different between the groups. CONCLUSIONS: RSV (50 µM) or LBP (2 mg/mL) are the best candidate antioxidants for inclusion in the freezing medium to improve the quality of giant panda spermatozoa during semen cryopreservation.

Transcriptomic Analysis of STAT1/3 in the Goat Endometrium During Embryo Implantation.

Interferon tau (IFNT), a pregnancy recognition signal in ruminants, promotes the establishment of embryo implantation by inducing the expression of interferon-stimulated genes (ISGs) via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. However, the precise regulatory mechanism of IFNT in goat embryo implantation remains largely unknown. In this study, we performed RNA sequencing of goat endometrial epithelial cells (gEECs) with or without 20 ng/mL IFNT treatment. Differential comparison showed that there were 442 upregulated differentially expressed genes (DEGs) and 510 downregulated DEGs. Bioinformatic analyses revealed that DEGs were significantly enriched in immune-related functions or pathways. The qRT-PCR validation results showed that the expression levels of STAT family members (STAT1, STAT2, and STAT3) were significantly upregulated in gEECs after IFNT treatment, which is in agreement with the RNA-seq data. Meanwhile, the protein levels of p-STAT1 and p-STAT3 increased significantly in gEECs after 6 and 24 h of IFNT treatment, respectively. Further in vivo experiments also confirmed that both mRNA and protein phosphorylation levels of STAT1 and STAT3 in the uterus on day 18 of pregnancy (P18) were significantly increased compared to those on day 5 (P5) and day 15 of pregnancy (P15). On P5, STAT1 and STAT3 proteins were primarily located in the uterine luminal epithelium (LE) and glandular epithelium (GE), and were also detected in the stromal cells. The intense immunostaining of STAT1 and STAT3 proteins were decreased on P15 and then increased on P18, especially in the superficial GE and subepithelial stromal cells. Moreover, p-STAT1 and p-STAT3 were highly expressed in the deep GE on P18. Collectively, these results highlight the role of IFNT in regulating endometrial receptivity in gEECs and uncover the temporal and spatial changes in the expression of STAT1/3 during embryo implantation in the goat endometrium.

Ufmylation regulates granulosa cell apoptosis via ER stress but not oxidative stress during goat follicular atresia.

Follicular atresia is primarily caused by granulosa cell (GC) apoptosis, although the mechanisms are largely unknown. Ufmylation is a recently identified ubiquitin-like post-translational modifier that plays an important role in cell proliferation and apoptosis. The purpose of this study was to investigate the effects of Ufmylation on GC apoptosis during goat follicular atresia. Ubiquitin-fold modifier 1 (UFM1) and its target DDRGK domain containing 1 (DDRGK1) proteins were identified in granulosa cells (GCs) isolated from all stages of preantral follicles and from healthy (HF), early atretic (EF) and progressed atretic (PF) antral follicles. The expression levels were higher in GCs derived from antral atretic follicles than healthy follicles. Although the viability of GCs was not affected after overexpression of UFM1, siRNA-mediated UFM1 silencing significantly inhibited GC proliferation and induced apoptosis. Notably, components of the ufmylation pathway were significantly upregulated in GCs induced by the ER stress agent tunicamycin (Tm) and thapsigargin (Tg), but not affected by oxidative stress inducer H2O2. Furthermore, UFM1 silencing markedly increased the apoptosis of GCs upon Tg treatment by stimulating the ER stress-related gene expression. Our results provide evidence that UFM1 and its target DDRGK1 are expressed in the goat GCs during follicular development and atresia, and ufmylation may play an important role in the prevention of ER stress but not oxidative stress-induced GCs apoptosis.

UFMylation is associated with LPS-induced inflammatory response in goat endometrial epithelial cells.

The endometrium plays an important role in the defence against invading pathogens, although the mechanisms are not clear. UFMylation is a recently discovered novel ubiquitination-like modification system that plays a pivotal role in inflammation and the immune response. The purpose of this study was to investigate the effects of UFMylation on lipopolysaccharide (LPS)-induced inflammatory responses in immortalized goat endometrial epithelial cells (gEECs). Ubiquitin-fold modifier conjugating enzyme 1 (UFM1) and DDRGK domain containing 1 (DDRGK1) were mainly localized in the luminal epithelium and glandular epithelium of mouse and goat endometrial tissues. The expression levels of UFM1, ubiquitin-like modifier activating enzyme 5 (UBA5), UFM1 specific ligase 1 (UFL1) and DDRGK1, as key components of the UFMylation system, were significantly activated by 5 µg/mL LPS-induced inflammatory response in gEECs for 6 hr. Meanwhile, the expression levels of interleukin-6 (IL-6) were significantly upregulated, and tumour necrosis factor-α (TNF-α) was significantly down-regulated after overexpression of UFM1 in gEECs. Additionally, we observed UFM1 and DDRGK1 were markedly increased on LPS-stimulated mouse endometritis in vivo. In conclusion, the current study demonstrated that UFMylation was significantly activated by LPS and might be involved in regulating inflammatory response in gEECs.

Progesterone-induced RNA Hand2os1 regulates decidualization in mice uteri.

Decidualization is a critical process for successful embryo implantation and subsequent placenta formation. The characterization and physiological function of lncRNA during decidualization remain largely unknown. In the present study, we conducted RNA-sequencing analysis to compare gene expression between decidua of days 6 and 8, and normal pregnant endometrium (day 4). A total of 2332 high-confidence putative lncRNA transcripts were expressed. Functional clustering analysis of cis and trans lncRNA targets showed that differentially expressed lncRNAs may regulate multiple gene ontology terms and pathways that have important functions in decidualization. Subsequent analyses using qRT-PCR validated that eight of all lncRNAs were differentially regulated in mice uteri during decidualization, both in vivo and in vitro. Furthermore, we showed that differentially expressed lncRNA of Hand2os1 was specifically detected in stromal cells on days 2 to 5 of pregnancy and was strongly upregulated in decidual cells on days 6-8 of pregnancy. Similarly, Hand2os1 expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. In uterine stromal cells, P4 was able to significantly upregulate the expression of Hand2os1, but upregulation was impeded by RU486, whereas E2 appeared to have no regulating effect on Hand2os1 expression. Concurrently, Hand2os1 significantly promoted the decidual process in vitro and dramatically increased decidualization markers Prl8a2 and Prl3c1. Our results provide a valuable catalog for better understanding of the functional roles of lncRNAs in pregnant mouse uteri, as it relates to decidualization.