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OONO–/MMP2/MMP9 pathway-mediated apoptosis of porcine granulosa cells is associated with DNA damage

Kun Lei, Quanwei Wei, Ying Cheng, Zhe Wang, Haoze Wu, Fang Zhao, Wei Ding, and Fangxiong Shi

In brief

The apoptosis of granulosa cells (GCs) is the main reason for porcine follicular atresia. This study provides a novel mechanism for peroxynitrite anion-mediated GC apoptosis and follicular atresia in porcine ovary.

Abstract

Granulosa cells play a crucial role in the development of follicles, and their cell apoptosis in the porcine ovary is a major contributor to follicular atresia. Here, we provide a new mechanism for follicular atresia by describing a crucial mechanism by which peroxynitrite anion (OONO–) may cause GC death. We discovered that nitric oxide, oxidative stress level, and OONO– were positively correlated with porcine follicular atresia, which was accompanied by high expression of matrix metalloproteinase 2 (MMP2) and MMP9. We created a model of OONO–-induced apoptosis in GCs and discovered that OONO– could boost the expression of MMP2 and MMP9 and increase the expression of pro-apoptotic proteins and DNA damage. Furthermore, by inhibiting the activities of MMP2 and MMP9, we found that SB-3CT (a specific inhibitor for MMP2 and MMP9) alleviated the decrease in cell survival rates and DNA damage caused by OONO–, which may have been impacted by reducing the cleavage of PARP1 by MMP2 and MMP9. Therefore, our findings imply that OONO– can cause DNA damage to GCs, participating in mediating the expression of pro-apoptotic proteins and inhibiting DNA repair by preventing the activity of PARP1 through MMP2 and MMP9. These results help explain how OONO–/MMP2/MMP9 affects porcine follicular atresia and GC apoptosis.

DOI:
https://doi.org/10.1530/REP-22-0295
Volume 165: Issue 4,
Pages:
431–443 (13 total)
Restricted access

PCSK9 involves in the high-fat diet-induced abnormal testicular function of male mice

Zhi-hui Cui, Yong-dan Ma, Yi-cheng Wang, Huan Liu, Jia-wei Song, Li-xue Zhang, Wen-jing Guo, Xue-qin Zhang, Sha-sha Tu, Dong-zhi Yuan, Jin-hu Zhang, Li Nie, and Li-min Yue

In brief

Impaired spermatogenesis resulting from disturbed cholesterol metabolism due to intake of high-fat diet (HFD) has been widely recognized, however, the role of preprotein invertase subtilin 9 (PCSK9), which is a negative regulator of cholesterol metabolism, has never been reported. This study aims to reveal the role of PCSK9 on spermatogenesis induced by HFD in mice.

Abstract

Long-term consumption of a high-fat diet (HFD) is an important factor that leads to impaired spermatogenesis exhibiting poor sperm quantity and quality. However, the mechanism of this is yet to be elucidated. Disrupted cholesterol homeostasis is one of many crucial pathological factors which could contribute to impaired spermatogenesis. As a negative regulator of cholesterol metabolism, preprotein invertase subtilin 9 (PCSK9) mediates low density lipoprotein receptor (LDLR) degradation to the lysosome, thereby reducing the expression of LDLR on the cell membrane and increasing serum low-density lipoprotein cholesterol level, resulting in lipid metabolism disorders. Here, we aim to study whether PCSK9 is a pathological factor for impaired spermatogenesis induced by HFD and the underlying mechanism. To meet the purpose of our study, we utilized wild-type C57BL/6 male mice and PCSK9 knockout mice with same background as experimental subjects and alirocumab, a PCSK9 inhibitor, was used for treatment. Results indicated that HFD induced higher PCSK9 expression in serum, liver, and testes, and serum PCSK9 is negatively correlated with spermatogenesis, while both PCSK9 inhibitor treatment and PCSK9 knockout methodologies ameliorated impaired lipid metabolism and spermatogenesis in mice fed a HFD. This could be due to the overexpression of PCSK9 induced by HFD leading to dyslipidemia, resulting in testicular lipotoxicity, thus activating the Bcl-2–Bax–Caspase3 apoptosis signaling pathway in testes, particularly in Leydig cells. Our study demonstrates that PCSK9 is an important pathological factor in the dysfunction of spermatogenesis in mice induced by HFD. This finding could provide innovative ideas for the diagnosis and treatment of male infertility.

DOI:
https://doi.org/10.1530/REP-22-0216
Volume 165: Issue 4,
Pages:
457–474 (18 total)
Restricted access

Sperm utilize tumor necrosis factor alpha to shut down a 2-methoxyestradiol nongenomic pathway that accelerates oviductal egg transport in the rat

María L Oróstica, Patricia Reuquen, Emanuel Guajardo-Correa, Alexis Parada-Bustamante, Hugo Cardenas, and Pedro A Orihuela

In brief

Mating shuts down the 2-methoxyestradiol (2ME) nongenomic pathway that accelerates oviductal egg transport in the rat. This study shows that sperm cells, but not vaginocervical stimulation, utilize TNF-α to shut down this 2ME nongenomic pathway.

Abstract

The transport of oocytes or embryos throughout the oviduct to the implantation site in the uterus is defined as egg transport. In the rat, 2-methoxyestradiol (2ME) accelerates egg transport through the oviduct via a nongenomic pathway. Mating is known to shut down this 2ME pathway and then trigger an estradiol genomic pathway that accelerates egg transport. Here, we tested whether intrauterine insemination (IUI) or vaginocervical stimulation (VCS) shuts down the 2ME nongenomic pathway that accelerates egg transport, and if these mating components require tumor necrosis factor alpha (TNF-α). Levels of TNF-α and the mRNA for TNF-α receptors were measured in the oviduct of IUI or VCS rats. The tissue distribution of TNF-α receptor proteins and the concentration of the mRNA for catechol-O-methyl transferase (Comt) and 2ME were also analyzed in the oviduct. Finally, we assessed whether 2ME accelerates egg transport in IUI or VCS rats previously treated with the TNF-α antagonist W9P9QY. Results show that IUI, but not VCS, increased TNF-α and their receptors in the oviduct. IUI and VCS did not change the tissue distribution of TNF-α receptors; however, both decreased the oviductal concentration of Comt and 2ME. IUI and VCS each blocked the 2ME-induced egg transport acceleration; however, only the IUI was antagonized by the TNF-α antagonist. We concluded that IUI and VCS inhibit the 2ME nongenomic pathway that accelerates egg transport; however, the vias of action are distinct, with a TNF-α increase on spermatozoa presence being required for the shutdown of the 2ME pathway.

DOI:
https://doi.org/10.1530/REP-22-0289
Volume 165: Issue 4,
Pages:
383–393 (11 total)
Free access

Synthetic embryos: a new venue in ethical research

Adrian Villalba, Jon Rueda, and Íñigo de Miguel Beriain

In brief

Two independent groups have reported the development of ‘artificial embryos’. Those are in vitro models made of mouse embryonic stem cells, without the need for egg or sperm, and grown ex utero without requiring implantation. This system might open new venues in bioethical research if human cells show the ability to replicate this system.

Abstract

The recent publications reported in 2022 reveal the possibility of obtaining mouse embryos without the need for egg or sperm. These ‘artificial embryos’ can recapitulate some stages of development ex utero – from neurulation to organogenesis – without implantation. Synthetic mouse embryos might serve as a valuable model to gain further insights into early developmental stages. Indeed, it is expected for these models to be replicated by employing human cells. This promising research raises ethical issues and expands the horizon of ethics in regard to the development of the human embryo. From this point of view, we state some of the new open venues for bioethical research.

DOI:
https://doi.org/10.1530/REP-22-0416
Volume 165: Issue 4,
Pages:
V1–V3
Restricted access

Endometrial stromal cells from women with repeated implantation failure display impaired invasion towards trophoblastic spheroids

Qi Qiu, Yijing Li, Sze Wan Fong, Kai Chuen Lee, Andy Chun Hang Chen, Hanzhang Ruan, Kai-Fai Lee, Hang Wun Raymond Li, Ernest Hung Yu Ng, William Shu Biu Yeung, and Yin lau Lee

In brief

Implantation failure can occur even after the transfer of good-quality embryos. This study showed that the migration of human endometrial stromal cells towards embryonic trophoblasts is higher in women with live births in the first in vitro fertilization cycle than those with repeated implantation failure, suggesting that the chemotactic response of stroma cells is associated with successful pregnancy.

Abstract

The success rate of in vitro fertilization (IVF) remains limited in some women despite transfers of good-quality embryos in repeated attempts. There is no reliable tool for assessing endometrial receptivity. This study aimed to assess the interaction between decidualized human primary endometrial stromal cells (1°-EnSC) and human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) and to compare the invasion ability of decidualized 1°-EnSC towards BAP-EB between women attaining live birth in the first IVF cycle and those with repeated implantation failure (RIF). The invasion of the decidualized human endometrial cell line (T-HESC) and 1°-EnSC towards BAP-EB was studied. Real-time quantitative PCR and immunocytochemistry were employed to determine the expression of decidualization markers at mRNA and protein levels, respectively. Trophoblast-like BAP-EB-96h, instead of early trophectoderm (TE)-like BAP-EB-48h, facilitated the invasion ability of decidualized T-HESC and decidualized 1°-EnSC. Human chorionic gonadotropin at supra-physiological levels promoted the invasiveness of decidualized 1°-EnSC. The extent of BAP-EB-96h-induced invasion was significantly stronger in decidualized 1°-EnSC from women who had a live birth in the first IVF cycle when compared to those with RIF. While no difference was found in the expression of decidualization markers, PRL and IGFBP1 among two groups of women, significantly lower HLA-B was detected in the non-decidualized and decidualized 1°-EnSC from women with RIF. Collectively, the findings suggested that the invasion of decidualized 1°-EnSC towards trophoblast-like BAP-EB-96h was higher in women who had a live birth in the first IVF cycle than those with RIF.

DOI:
https://doi.org/10.1530/REP-22-0282
Volume 165: Issue 3,
Pages:
335–346 (12 total)
Open access

Functional role of GATA3 and CDX2 in lineage specification during bovine early embryonic development

Yan Shi, Bingjie Hu, Zizengchen Wang, Xiaotong Wu, Lei Luo, Shuang Li, Shaohua Wang, Kun Zhang, and Huanan Wang

In brief

The lineage specification during early embryonic development in cattle remains largely elusive. The present study determines the effects of trophectoderm-associated factors GATA3 and CDX2 on lineage specification during bovine early embryonic development.

Abstract

Current understandings of the initiation of the trophectoderm (TE) program during mammalian embryonic development lack evidence of how TE-associated factors such as GATA3 and CDX2 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on the expression of lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6, and SOX17, by using cytosine base editor system. We successfully knockout GATA3 or CDX2 in bovine embryos with a robust efficiency. However, GATA3 or CDX2 deletion does not affect the developmental potential of embryos to reach the blastocyst stage. Interestingly, GATA3 deletion downregulates the NANOG expression in bovine blastocysts. Further analysis of the mosaic embryos shows that GATA3 is required for NANOG in the TE of bovine blastocysts. Single blastocyst RNA-seq analysis reveals that GATA3 deletion disrupts the transcriptome in bovine blastocysts. Altogether, we propose that GATA3 plays an important role in maintaining TE lineage program in bovine embryos and the functional role of GATA3 is species-specific.

DOI:
https://doi.org/10.1530/REP-22-0269
Volume 165: Issue 3,
Pages:
325–333 (9 total)
Restricted access

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro

Yuki Yamamoto, Maho Kurokawa, Taiji Ogawa, Sayaka Kubota, and Koji Kimura

In brief

Spontaneous contraction of oviductal smooth muscle is essential for gamete transport to the fertilization site in mammals. This study sheds light on the mechanism of elevated contraction amplitude in the bovine oviductal isthmus just before ovulation.

Abstract

Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0–4 days after ovulation), at the mid-late luteal stage (10–17 days), and at the follicular stage (1–3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher in the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

DOI:
https://doi.org/10.1530/REP-22-0174
Volume 165: Issue 3,
Pages:
301–312 (12 total)
Open access

Metformin improves ovarian function and increases egg production in broiler breeder hens

Evelyn A Weaver and Ramesh Ramachandran

In brief

The pathophysiology of the ovarian dysfunction encountered in broiler breeder hens remains poorly understood but is similar to a condition in women known as polycystic ovary syndrome. This study reveals that metformin may provide a cheap and effective method of improving ovarian function in broiler breeder hens.

Abstract

Broiler breeder hens, the parent stock of commercial broiler chickens, have poor reproductive efficiency associated with aberrant and excessive recruitment of ovarian follicles which results in sub-optimal egg production, fertility, and hatchability. The reproductive dysfunction observed in these hens resembles polycystic ovary syndrome in women, a condition wherein metformin is prescribed as a treatment. The main objectives of this study were to determine the effect of metformin on body weight, abdominal fat pad weight, ovarian function, and plasma steroid hormone concentrations. Broiler breeder hens were treated with 0, 25, 50, or 75 mg/kg body weight of metformin mixed in the diet for 40 weeks (n =  45 hens/treatment; 2565 weeks of age). At 65 weeks of age, hens that received the highest dose of metformin had significantly lower body and abdominal fat pad weights (P  < 0.05) than the control. Metformin treatment, at all levels, normalized the preovulatory and prehierarchical ovarian follicular hierarchy. Metformin (50 or 75 mg/kg body weight) significantly increased the total number of eggs laid per hen during the entire production period and these hens had significantly greater fertility and hatchability at 65 weeks of age compared to the control (P  < 0.05). Metformin treatment at all levels altered the plasma profile of reproductive hormones, with significantly lower plasma testosterone concentrations and a decreased testosterone to androstenedione ratio in hens that received metformin (P  < 0.05). Future studies should focus on the mechanisms underlying the beneficial effects of metformin in improving the reproductive efficiency of broiler breeder hens.

DOI:
https://doi.org/10.1530/REP-22-0256
Volume 165: Issue 3,
Pages:
289–300 (12 total)
Restricted access

Modulatory role of mTOR in trophoblast adaptive response in the early stage of placentation in sheep

I Viola, P Toschi, I Manenti, P Accornero, and M Baratta

In brief

Fibroblast growth factor-2 (FGF2) is essential for early placenta development in sheep. This study shows that the mechanistic target of rapamycin is the key modulator of trophoblast adaptive response under FGF2 modulation.

Abstract

During the early stage of placentation in sheep, normal conceptus development is affected by trophoblast cell functionality, whose dysregulation results in early pregnancy loss. Trophoblast metabolism is supported mainly by histotrophic factors, including fibroblast growth factor-2 (FGF2), which are involved in cell differentiation and function through the modulation of specific cellular mechanisms. The mechanistic target of rapamycin (mTOR) is known as a cellular ‘nutrient sensor’, but its downstream regulation remains poorly understood. The hypothesis was that during trophoblast development, the FGF2 effect is mediated by mTOR signalling pathway modulation. Primary trophoblast cells from 21-day-old sheep placenta were characterised and subjected to FGF2 and rapamycin treatment to study the effects on cell functionality and gene and protein expression profiles. The model showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, expressing typical trophoblast markers. FGF2 promoted cell proliferation and migration under normal culture conditions, whereas mTOR inhibition reversed this effect. When the mTOR signalling pathway was activated, FGF2 failed to influence invasion activity. mTOR inhibition significantly reduced cell motility, but FGF2 supplementation restored motility even when mTOR was inhibited. Interestingly, mTOR inhibition influenced endocrine trophoblast marker regulation. Although FGF2 supplementation did not affect ovine placenta lactogen expression, as observed in the control, interferon-tau was drastically reduced. This study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality. In addition, as mTOR is involved in the expression of hormonal trophoblast markers, it may play a crucial role in early placenta growth and fetal–maternal crosstalk.

DOI:
https://doi.org/10.1530/REP-22-0356
Volume 165: Issue 3,
Pages:
313–324 (12 total)
Restricted access

Mouse uterine stem cells are affected by endocrine disruption and initiate uteropathies

Pushpa Singh and Deepa Bhartiya

In brief

Incidence of uteropathies has increased in recent times, possibly due to exposure to endocrine-disrupting chemicals during early development. The present study shows that various uteropathies like endometrial cancer, adenomyosis, and endometriosis are interlinked and occur due to the dysfunction of tissue-resident, very small embryonic-like stem cells (VSELs).

Abstract

Underlying pathomechanisms leading to the initiation of uteropathies including non-receptive endometrium, hyperplasia, adenomyosis, endometriosis, fibroids, and cancer remain elusive. Two populations of stem cells exist in mouse uterus including pluripotent VSELs and ‘progenitors’ termed endometrial stem cells (EnSCs) which express ERα, ERβ, PR, and FSHR, participate in the regular remodelling, and maintain life-long homeostasis. The present study aimed to delineate possible stem cell origins for various uteropathies. For this, mouse pups were treated with oestradiol or diethylstilbestrol and were studied for adult onset of various uteropathies. Treatment resulted in disrupted oestrous cycles, reduced uterine weights, and marked hyperplasia in both epithelial and myometrial compartments, and the stromal compartment was also affected. VSELs were increased in numbers as judged by flow cytometry and increased expression of transcripts specific for Oct-4A, Sox-2, and Nanog, but their further differentiation into a receptive endometrium was affected. Reduced 5-methyl cytosine expression suggested global hypomethylation and was associated with several oncogenic events including loss of tumour-suppressor genes (Pten, p53), dysregulated DNA mismatch repair axis, and repair enzymes. Stem cells were epigenetically altered and showed increased expression of DNMTs, loss of imprinting loci (Igf2-H19, Dlk1-Meg3), and Ezh2. Increased co-expression of CD166 and ALDHA1 with OCT-4 in stem cells was associated with increased Esr-2 and reduced Pr in the endometrium, while both were several folds upregulated in the myometrium. Study results suggest that various uteropathies ensue due to the dysfunction of tissue-resident stem cells and provide huge scope for further research.

DOI:
https://doi.org/10.1530/REP-22-0337
Volume 165: Issue 3,
Pages:
249–268 (20 total)