Meiosis Arrest Research Articles

PKD regulates mitophagy to prevent oxidative stress and mitochondrial dysfunction during mouse oocyte maturation

Mitochondria play dominant roles in various cellular processes such as energy production, apoptosis, calcium homeostasis, and oxidation–reduction balance. Maintaining mitochondrial quality through mitophagy is essential, especially as its impairment leads to the accumulation of dysfunctional mitochondria in aging oocytes. Our previous research revealed that PKD expression decreases in aging oocytes, and its inhibition negatively impacts oocyte quality. Given PKD's role in autophagy mechanisms, this study investigates whether PKD regulates mitophagy to maintain mitochondrial function and support oocyte maturation. When fully grown oocytes were treated with CID755673, a potent PKD inhibitor, we observed meiosis arrest at the metaphase I stage, along with decreased spindle stability. Our results demonstrate an association with mitochondrial dysfunction, including reduced ATP production and fluctuations in Ca2+ homeostasis, which ultimately lead to increased ROS accumulation, stimulating oxidative stress-induced apoptosis and DNA damage. Further research has revealed that these phenomena result from PKD inhibition, which affects the phosphorylation of ULK, thereby reducing autophagy levels. Additionally, PKD inhibition leads to decreased Parkin expression, which directly and negatively affects mitophagy. These defects result in the accumulation of damaged mitochondria in oocytes, which is the primary cause of mitochondrial dysfunction. Taken together, these findings suggest that PKD regulates mitophagy to support mitochondrial function and mouse oocyte maturation, offering insights into potential targets for improving oocyte quality and addressing mitochondrial-related diseases in aging females.

Estrogen influences the transzonal projection assembly of cumulus-oocyte complexes through G protein-coupled estrogen receptor during goat follicle development.

Estrogen is an important hormone that plays a role in regulating follicle development and oocyte maturation. Transzonal projections (TZPs) act as communication bridges between follicle somatic cells and oocytes, and their dynamic changes are critical for oocyte development and maturation. However, the roles and mechanisms of estrogen in regulating TZPs during follicular development are not yet understood. We found that the proportion of oocytes spontaneously resuming meiosis increases as the follicle grows, which is accompanied by rising estrogen levels in follicles and decreasing TZPs in cumulus-oocyte complex. To further explore the effect of elevated estrogen levels on TZP assembly, additional estrogen was added to the culture system. The increased estrogen level significantly decreased the mRNA and protein expression levels of TZP assembly-related genes. Subsequent research revealed that TZP regulation by estrogen was mediated by the membrane receptor GPER and downstream ERK1/2 signaling pathway. In summary, our study suggests that estrogen may regulate goat oocyte meiosis arrest by decreasing TZP numbers via estrogen-mediated GPER activation during follicle development.

Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC–MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

New allele of mouse DNA/RNA helicase senataxin causes meiotic arrest and infertility.

An unbiased screen for discovering novel mouse genes for fertility identified the spcar3, spermatocyte arrest 3, mutant phenotype. The spcar3 mutation identified a new allele of the Setx gene, encoding senataxin, a DNA/RNA helicase that regulates transcription termination by resolving DNA/RNA hybrid R-loop structures. The Setxspcar3 mutant mice exhibit male infertility and female subfertility. Histology of the Setxspcar3 mutant testes revealed absence of spermatids and mature spermatozoa in the seminiferous tubules. Cytological analysis of chromosome preparations of the Setxspcar3 mutant spermatocytes revealed normal synapsis, but aberrant DNA damage in the autosomes, defective formation of the sex body, and arrest of meiosis in mid-prophase. Additionally, Setxspcar3 testicular cells exhibit abnormal accumulation of R-loops. Transient expression assays identified regions of the senataxin protein required for sub-nuclear localization. Together, these results not only confirm that senataxin is required for normal meiosis and spermatogenesis but also provide a new resource for determination of its role in maintaining R-loop formation and genome integrity.

SARS-CoV-2 infection reduces quality of sperm parameters: prospective one year follow-up study in 93 patients.

Short- and long-term implications of SARS-CoV-2 on the quality of the sperm and the results of this on fertility remain largely unknown due to lack of longitudinal studies. In this longitudinal observational cohort study, we aimed to analyse the differential effect and the impact of SARS-CoV-2 infection on different semen quality parameters. Sperm quality was assessed using the World Health Organization criteria, DNA damage to sperm cells by quantifying the DNA fragmentation index (DFI) and the high-density stainability (HDS), IgA- and IgG-anti-sperm antibodies (ASA) were assessed with light microscopy. SARS-CoV-2 infection was associated with sperm parameters that were independent of spermatogenic cycle like progressive motility, morphology, DFI and HDS, as well as spermatogenic cycle dependent parameters such as sperm concentration. Detection of IgA- and IgG-ASA allowed classification of patients in three different groups according to its sequence of appearance in sperm during post-COVID-19 follow-up. The maximum progressive motility was lowest during follow-up in patients without ASA (41.9%), intermediate in patients with only IgA-ASA (46.2%) and highest inpatients who had both IgA- and IgG-ASA (54.9%). SARS-CoV-2 infection was associated with changes of all analysed sperm parameters to a different degree which is also observed in their return to normality and is suggestive of individual variations in the patient's immune system performance. Firstly, sperm production is decreased through temporal immune mediated arrest of active meiosis, and secondly immune induced sperm DNA damage prevents fertilization if transferred to the oocyte. Both mechanisms are temporal, and most sperm parameters return to baseline after infection. AML (R20-014), Femicare.

(088) The Essential Role of O-GlcNAcylation in the Testis Aging

Abstract Introduction With the delay of reproductive age, reproductive disorders related to advanced age have gradually attracted widespread attention. Studies have shown that the decline in male fertility due to aging is manifested as a decrease in semen parameters and spermatogenetic cells histologically. O-GlcNAc (O-acetylglucosamine) glycosylation is a post-translational modification that exists widely in cells. The level of UDP-GlcNAc, which is the substrate of O-GlcNAcylation, changes rapidly with various nutrients and external factors. Therefore, O-GlcNAcylation can respond to glucose, amino acid, fatty acid, and nucleoside metabolism, and is considered to be an important sensor of cellular metabolism. The previous study of our project found that the level of O-GlcNAc modification in aged testis tissue was significantly increased, so this study aimed to explore the specific mechanism of O-GlcNAc modification involved in testicular aging. Objective 1.Explore the modification pattern of O-GlcNAcylation in testis 2.Mimic the high O-GlcNAcylation state of aging mice in young mice, to study the effect of elevated high O-GlcNAcylation level on testis. 3.Reveal the role of key proteins modified by O-GlcNAc in testicular aging Methods Mice were injected intraperitoneally with OGA inhibitor Thiamet-G for 35 days to increase the level of O-GlcNAc to simulate aging. CASA system was used to detect semen parameters, and chromosome spreading technology was used to study mouse testis meiosis. Using protein profiling technology, the differentially modified O-GlcNAc proteins in aged testis and young testis were screened, and point mutations were made at their O-GlcNAc sites to study the specific mechanism of key O-GlcNAc proteins involved in testis aging. Results The O-GlcNAc modification level in the testis of aged mice was significantly increased. After intraperitoneally injecting Thiamet-G into mice for 35 days, the level of O-GlcNAcylation in the testis tissue was significantly increased. High level of O-GlcNAcylation in testis damaged the sperm parameter, decreased the thickness of seminiferous epithelium and increased apoptosis in testis. Chromosome spreading experiments showed that, γH2Ax, which is the marker of DNA double strand breaks, stained unusually at autosomes in pachytene spermatocytes. The elevation of γH2Ax affected the transition of pachytene spermatocytes to diplotene. Importantly, we found that the O-GlcNAcylation of Hyou1 protein was significantly increased in the testis of aged mice. O-GlcNAcylation of Hyou1 promotes the degradation of Hyou1. An O-GlcNAc site of Hyou1 was identified at S634 and mutation of this site decreased the O-GlcNAcylation, increased the protein stability and exerts protective roles in aging testis. Conclusions In the testis of aged mice, the level of O-GlcNAcylation is increased, which leads to the arrest of spermatocyte meiosis by affecting the DNA damage repair pathway. Hyou1 is the key O-GlcNAc protein in testicular aging and mutation of this site exerts protective roles. This study revealed for the first time the specific mechanism of O-GlcNAc involved in testicular aging, and provided a new therapeutic target for clinically delaying testicular aging and treating male infertility. Disclosure No

Evaluation of MYBL1 as the master regulator for pachytene spermatocyte genes dysregulated in interspecific hybrid dzo

Misregulation of spermatogenesis transcription factors (TF) in hybrids can lead to misexpression, which is a mechanism for hybrid male sterility (HMS). We used dzo (male offspring of Bos taurus ♂ × Bos grunniens ♀) in bovines to investigate the relationship of the key TF with HMS via RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses. RNA sequencing showed that the widespread misexpression in dzo was associated with spermatogenesis-related genes and somatic or progenitor genes. The transition from leptotene or zygotene spermatocytes to pachytene spermatocytes may be the key stage for meiosis arrest in dzo. The analysis of TF-binding motif enrichment revealed that the male meiosis-specific master TF MYB proto-oncogene like 1 (MYBL1, known as A-MYB) motif was enriched on the promoters of downregulated pachytene spermatocyte genes in dzo. Assay for transposase-accessible chromatin with high-throughput sequencing revealed that TF-binding sites for MYBL1, nuclear transcription factor Y, and regulatory factor X were enriched in the low-chromatin accessibility region of dzo. The target genes of the MYBL1-binding motif were associated with meiosis-specific genes and significantly downregulated in dzo testis. The transcription factor MYBL1 may be the candidate master regulator for pachytene spermatocyte genes dysregulated in interspecific HMS dzo. This study reported that a few upstream TF regulation changes might exert a cascading effect downstream in a regulatory network as a mechanism for HMS.

Bi-allelic MEI1 variants cause meiosis arrest and non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is characterized by the failure of sperm production due to testicular disorders and represents the most severe form of male infertility. Growing evidences have indicated that gene defects could be the potential cause of NOA via genome-wide sequencing approaches. Here, bi-allelic deleterious variants in meiosis inhibitor protein 1 (MEI1) were identified by whole-exome sequencing in four Chinese patients with NOA. Testicular pathologic analysis and immunohistochemical staining revealed that spermatogenesis is arrested at spermatocyte stage, with defective programmed DNA double-strand breaks (DSBs) homoeostasis and meiotic chromosome synapsis in patients carrying the variants. In addition, our results showed that one missense variant (c.G186C) reduced the expression of MEI1 and one frameshift variant (c.251delT) led to truncated proteins of MEI1 in in vitro. Furthermore, the missense variant (c.T1585A) was assumed to affect the interaction between MEI1 and its partners via bioinformatic analysis. Collectively, our findings provide direct genetic and functional evidences that bi-allelic variants in MEI1 could cause defective DSBs homoeostasis and meiotic chromosome synapsis, which subsequently lead to meiosis arrest and male infertility. Thus, our study deepens our knowledge of the role of MEI1 in male fertility and provides a novel insight to understand the genetic aetiology of NOA.

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

BackgroundZearalenone (ZEA) widely exists in moldy grains, which seriously destroys the fertility of females. Isorhamnetin, a natural flavonoid, has extensive of pharmacological activities. However, the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.MethodsOocytes were treated with different concentrations of ZEA (3, 5, 8 and 10 μmol/L) and isorhamnetin (5, 10, 20 and 30 μmol/L) for 44 h at 39 ℃. ZEA (5 μmol/L) and isorhamnetin (10 μmol/L) were selected for subsequent studies. Polar body exclusion rate, apoptosis rate and apoptosis related proteins, ROS levels and SOD2 protein, mitochondrial membrane potential and distribution, endoplasmic reticulum distribution and proteins expression, and PI3K, Akt and p-Akt proteins expression of oocytes were detected. In addition, the effect of PI3K antagonist (LY294002) on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.ResultsOur findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes. Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes. Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production. Moreover, isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA. Changing the expression of endoplasmic reticulum stress-related marker proteins (CHOP, GRP78) and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress. Mechanistically, isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.ConclusionsCollectively, these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway, which enhances meiotic maturation and mitochondrial function, and inhibits early apoptosis, oxidative stress and endoplasmic reticulum stress in porcine oocytes. Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.Graphical A possible mechanism by which isorhamnetin protected porcine oocytes from ZEA-induced damage. Isorhamnetin inhibited meiosis arrest and apoptosis of porcine oocytes induced by ZEA through the PI3K/Akt signaling pathway. Moreover, isorhamnetin repaired ZEA-induced oocyte damage by alleviating oxidative stress, mitochondrial dysfunction and ER stress.

E2/ER signaling mediates the meiotic arrest of goat intrafollicular oocytes induced by follicle-stimulating hormone.

The increased production of high-quality oocytes lies at the heart of the search to accelerate the reproduction of high-quality breeding livestock using assisted reproductive technology. Follicle-stimulating hormone (FSH) maintains the arrest of oocyte meiosis during early follicular development in vivo and promotes the synchronous maturation of nucleus and cytoplasm to improve oocyte quality. However, the mechanism by which FSH maintains meiotic arrest in oocytes is still not fully understood. Oocytes spontaneously resume meiosis once released from the arrested state. In this study, we isolated goat antral follicles with a diameter of 2.0-4.0 mm, cultured them in vitro either with or without added FSH, and finally collected the oocytes to observe their meiotic state. The results showed that FSH effectively inhibited the meiotic recovery of oocytes in follicles [4 h: control (n = 84) vs. with FSH (n = 86), P = .0115; 6 h: control (n = 86) vs. FSH (n = 85), P = 0.0308; and 8 h: control (n = 95) vs. FSH (n = 101), P = 0.0039]. FSH significantly inhibited the downregulation of natriuretic peptide receptor 2 (NPR2) expression and cyclic guanosine monophosphate (cGMP) synthesis during follicular culture in vitro (P < 0.05). Further exploration found that FSH promoted the synthesis of 17β-estradiol (E2) (P = .0249 at 4 h and P = .0039 at 8 h) and maintained the expression of the estrogen nuclear receptor ERβ, but not the estrogen nuclear receptor ERα during follicle culture in vitro (P = .0190 at 2 h, and P = .0100 at 4 h). In addition, E2/ER (estrogen nuclear receptors ERα and ERβ) mediated the inhibitory effect of FSH on the downregulation of NPR2 expression and cGMP synthesis, ultimately preventing the meiotic recovery of oocytes (P < .05). In summary, our study showed that FSH-induced estrogen production in goat follicles, and the E2/ER signaling pathway, both mediated meiotic arrest in FSH-induced goat oocytes.

Decabromodiphenyl ethane induces male reproductive toxicity by glycolipid metabolism imbalance and meiotic failure

Decabromodiphenyl ethane (DBDPE) is a typical flame retardant found in various electrical and textile items. DBDPE is abundantly available in the surrounding environment and wild animals based on its persistence and bioaccumulation. DBDPE has been shown to cause apoptosis in rat spermatogenic cells, resulting in reproductive toxicity. However, the toxicity of DBDPE on the male reproductive system and the potential mechanisms are still unclear. This study evaluated the effect of DBDPE on the reproductive system in male SD rats and demonstrated the potential mechanisms of reproductive toxicity. DBDPE (0, 5, 50, and 500 mg/kg/day) was administered via gavage to male SD rats for 28 days. DBDPE caused histopathological changes in the testis, reduced sperm quantity and motility, and raised the malformation rate in rats, according to the findings. Furthermore, it caused DNA damage to rat testicular cells. It inhibited the expressions of spermatogenesis‐and oogenesis‐specific helix‐loop‐helix transcription factor 1 (Sohlh1), piwi-like RNA-mediated gene silencing 2 (MILI), cyclin-dependent kinase 2 (CDK2), and CyclinA, resulting in meiotic failure, as well as the expressions of synaptonemal complex proteins 1 and 3 (SYCP1 and SYCP3), leading to chromosomal association disorder in meiosis and spermatocyte cycle arrest. Moreover, DBDPE induced glycolipid metabolism disorder and activated mitochondria-mediated apoptosis pathways in the testes of SD rats. The quantity and quality of sperm might be declining due to these factors. Our findings offer further evidence of the harmful impact of DBDPE on the male reproductive system.

INCIDENCE OF Y CHROMOSOME MICRODELETIONS AND MICRODISSECTION TESTICULAR SPERM EXTRACTION (MICRO TESE) IN JAPANESE AZOOSPERMIC PATIENTS

After Klinefelter syndrome, Y chromosome microdeletions are the second most frequent genetic cause of male infertility, with a prevalence of 2%-10% in non-obstructive azoospermia (NOA) and three spermatogenesis loci in the Y chromosome long arm (Yq11) have been classified as AZFa, AZFb, and AZFc. The classical correlation of histopathology phenotypes with these three microdeletions comprises of complete absence of germ cells (Sertoli cell-only syndrome) in patients with AZFa microdeletions, maturation arrest of meiosis in patients with AZFb microdeletions, and hypospermatogenesis in patients with AZFc microdeletions, however, individual variation in the extent of deletions has led to various spermatogenic phenotypes.

Implications of High-Density Cholesterol Metabolism for Oocyte Biology and Female Fertility.

Cholesterol is an essential component of animal cells. Different regulatory mechanisms converge to maintain adequate levels of this lipid because both its deficiency and excess are unfavorable. Low cell cholesterol content promotes its synthesis and uptake from circulating lipoproteins. In contrast, its excess induces the efflux to high-density lipoproteins (HDL) and their transport to the liver for excretion, a process known as reverse cholesterol transport. Different studies suggest that an abnormal HDL metabolism hinders female fertility. HDL are the only lipoproteins detected in substantial amounts in follicular fluid (FF), and their size and composition correlate with embryo quality. Oocytes obtain cholesterol from cumulus cells via gap junctions because they cannot synthesize cholesterol de novo and lack HDL receptors. Recent evidence has supported the possibility that FF HDL play a major role in taking up excess unesterified cholesterol (UC) from the oocyte. Indeed, genetically modified mouse models with disruptions in reverse cholesterol transport, some of which show excessive circulating UC levels, exhibit female infertility. Cholesterol accumulation can affect the egg´s viability, as reported in other cell types, and activate the plasma membrane structure and activity of membrane proteins. Indeed, in mice deficient for the HDL receptor Scavenger Class B Type I (SR-B1), excess circulating HDL cholesterol and UC accumulation in oocytes impairs meiosis arrest and hinders the developmental capacity of the egg. In other cells, the addition of cholesterol activates calcium channels and dysregulates cell death/survival signaling pathways, suggesting that these mechanisms may link altered HDL cholesterol metabolism and infertility. Although cholesterol, and lipids in general, are usually not evaluated in infertile patients, one study reported high circulating UC levels in women showing longer time to pregnancy as an outcome of fertility. Based on the evidence described above, we propose the existence of a well-regulated and largely unexplored system of cholesterol homeostasis controlling traffic between FF HDL and oocytes, with significant implications for female fertility.

As3MT-mediated SAM consumption, which inhibits the methylation of histones and LINE1, is involved in arsenic-induced male reproductive damage

Studies have demonstrated that arsenic (As) induces male reproductive injury, however, the mechanism remains unknown. The high levels of arsenic (3) methyltransferase (As3MT) promote As-induced male reproductive toxicity. For As-exposed mice, the germ cells in seminiferous tubules and sperm quality were reduced. Exposure to As caused lower S-adenosylmethionine (SAM) and 5-methylcytosine (5 mC) levels, histone and DNA hypomethylation, upregulation of long interspersed element class 1 (LINE1, or L1), defective repair of double-strand breaks (DSBs), and the arrest of meiosis, resulting in apoptosis of germ cells and lower litter size. For GC-2spd (GC-2) cells, As induced apoptosis, which was prevented by adding SAM or by reducing the expression of As3MT. The levels of LINE1, affected by SAM content, were involved in As-induced apoptosis. Furthermore, folic acid (FA) and vitamin B12 (VB12) supplements restored SAM, 5 mC, and LINE1 levels and blocked impairment of spermatogenesis and testes and lower litter size. Exposed to As, mice with As3MT knockdown showed less impairment of spermatogenesis and testes and greater litter size compared to As-exposed wild-type (WT) mice. Thus, the high As3MT levels induced by As consume SAM and block histone and LINE1 DNA methylation, elevating LINE1 expression and evoking impairment of spermatogenesis, which causes male reproductive damage. Overall, we have found a mechanism for As-induced male reproductive damage, which provides biological insights into the alleviation of reproductive injury induced by environmental factors.

Human-specific gene CT47 blocks PRMT5 degradation to lead to meiosis arrest

Exploring the functions of human-specific genes (HSGs) is challenging due to the lack of a tractable genetic model system. Testosterone is essential for maintaining human spermatogenesis and fertility, but the underlying mechanism is unclear. Here, we identified Cancer/Testis Antigen gene family 47 (CT47) as an essential regulator of human-specific spermatogenesis by stabilizing arginine methyltransferase 5 (PRMT5). A humanized mouse model revealed that CT47 functions to arrest spermatogenesis by interacting with and regulating CT47/PRMT5 accumulation in the nucleus during the leptotene/zygotene-to-pachytene transition of meiosis. We demonstrate that testosterone induces nuclear depletion of CT47/PRMT5 and rescues leptotene-arrested spermatocyte progression in humanized testes. Loss of CT47 in human embryonic stem cells (hESCs) by CRISPR/Cas9 led to an increase in haploid cells but blocked the testosterone-induced increase in haploid cells when hESCs were differentiated into haploid spermatogenic cells. Moreover, CT47 levels were decreased in nonobstructive azoospermia. Together, these results established CT47 as a crucial regulator of human spermatogenesis by preventing meiosis initiation before the testosterone surge.

O-207 Incidence of Y chromosome microdeletions and microdissection testicular sperm extraction (micro TESE) in patients with Japanese azoospermic patients

Abstract Study question What is the frequency of azoospermia factor (AZF) microdeletions and sperm retrieval rate (SRR) by micro TESE in patients with these deletions? Summary answer AZFc is most frequent of Y chromosome microdeletions and a predictor of micro TESE outcome in Japanese azoospermic men. What is known already After Klinefelter syndrome, Y chromosome microdeletions are the second most frequent genetic cause of male infertility, with a prevalence of 2%-10% in non-obstructive azoospermia (NOA) and three spermatogenesis loci in the Y chromosome long arm (Yq11) have been classified as AZFa, AZFb, and AZFc. The classical correlation of histopathology phenotypes with these three microdeletions comprises of complete absence of germ cells (Sertoli cell-only syndrome) in patients with AZFa microdeletions, maturation arrest of meiosis in patients with AZFb microdeletions, and hypospermatogenesis in patients with AZFc microdeletions, however, individual variation in the extent of deletions has led to various spermatogenic phenotypes. Study design, size, duration We performed a retrospective study based on two reproduction centers in Japan and evaluated 1373 azoospermic patients in our clinics between September 2013 and December 2021. We investigated the frequency of AZF microdeletions and SRR by micro TESE in patients with these microdeletions and therefore aimed to evaluate the correlation between AZF microdeletions and micro TESE results. Participants/materials, setting, methods A total of 1373 azoospermic were enrolled. After the diagnosis of azoospermia, karyotype analysis and detection of Y chromosome microdeletions were performed on peripheral blood lymphocytes of these patients. Y chromosome microdeletions in AZFa, AZFb, and AZFc regions were detected using Promega Y Chromosome AZF Analysis System version 2.0 (Promega Co.). Twenty sequence-tagged sites within the AZF region of Yq11 and the sex-determining region Y gene were targeted for polymerase chain reaction (PCR) amplification. Main results and the role of chance One hundred and fifty-two AZF microdeletions (11.1%) were detected in the azoospermic patients. The most common deleted region was AZFc (60 cases, 4.4%). Among the patients, 17 (1.2%), 1 (0.1%), 42 (3.1%), 13 (1.0%), and 6 (0.5%) had AZFa, AZFa+b, AZFb+c, AZFb, and AZFa+b+c microdeletions, respectively. When the cases were grouped according to causes of infertility that could be detected, no Y chromosome microdeletions were detected in some groups (cases with Klinefelter Syndrome, hypogonadotropic hypogonadism, congenital absence of vas deferens, and 47, XYY karyotype). Fifty-three azoospermic men with AZFc microdeletions underwent micro TESE, and spermatozoa were detected in 88.7% (47/53) of these men. In contrast, we detected spermatozoa in only 20.4% (109/534) of the azoospermic men without AZF microdeletions. The SRR was much higher in patients with AZFc microdeletions than that of patients without AZF deletions. Although three azoospermic men with AZFb+c microdeletions had also undergone micro TESE following patient request, we did not retrieve spermatozoa. Limitations, reasons for caution We excluded post chemotherapy NOA showing 46, XX and AZFa+b+c deletions post bone marrow transplantation from female donor. Additionally, we did not detect AZFc partial deletion including gr/gr deletion. The cohort size of this study is not small, however, our screened population of infertile men may be biased. Wider implications of the findings NOA patients with AZFc microdeletions had a high percentage of successful sperm retrieval by micro TESE. Our study emphasizes that diagnosis of Y chromosome microdeletions is critical for preconception genetic counseling and provides clinically valuable prognostic information to couples considering surgical sperm retrieval. Trial registration number None

TCFL5 deficiency impairs the pachytene to diplotene transition during spermatogenesis in the mouse

Spermatogenesis is a complex, multistep process during which spermatogonia give rise to spermatozoa. Transcription Factor Like 5 (TCFL5) is a transcription factor that has been described expressed during spermatogenesis. In order to decipher the role of TCFL5 during in vivo spermatogenesis, we generated two mouse models. Ubiquitous removal of TCFL5 generated by breeding TCFL5fl/fl with SOX2-Cre mice resulted in sterile males being unable to produce spermatozoa due to a dramatic alteration of the testis architecture presenting meiosis arrest and lack of spermatids. SYCP3, SYCP1 and H1T expression analysis showed that TCFL5 deficiency causes alterations during pachytene/diplotene transition resulting in a meiotic arrest in a diplotene-like stage. Even more, TCFL5 deficient pachytene showed alterations in the number of MLH1 foci and the condensation of the sexual body. In addition, tamoxifen-inducible TCFL5 knockout mice showed, besides meiosis phenotype, alterations in the spermatids elongation process resulting in aberrant spermatids. Furthermore, TCFL5 deficiency increased spermatogonia maintenance genes (Dalz, Sox2, and Dmrt1) but also increased meiosis genes (Syce1, Stag3, and Morc2a) suggesting that the synaptonemal complex forms well, but cannot separate and meiosis does not proceed. TCFL5 is able to bind to the promoter of Syce1, Stag3, Dmrt1, and Syce1 suggesting a direct control of their expression. In conclusion, TCFL5 plays an essential role in spermatogenesis progression being indispensable for meiosis resolution and spermatids maturation.

Novel bi-allelic variants in KASH5 are associated with meiotic arrest and non-obstructive azoospermia.

KASH5 is an essential component of the LINC (linker of the nucleoskeleton and cytoskeleton) complex that regulates chromosome movements and nuclear envelope (NE) remodeling in mouse spermatocytes during meiosis prophase I, but its expression and function in human cells, as well as its association with male infertility are largely unknown. In this study, a novel heterozygous copy number variation (CNV) (seq [GRCh37] del(19) (19q13.33) chr19: g.49894043-49903011del) and a heterozygous loss of function variant (NM_144688: c.979_980del: p.R327Sfs*21) in human KASH5 were identified in a non-obstructive azoospermia (NOA)-affected patient and in his infertile sister by whole-exome sequencing and CNV array. Spermatogenesis in the proband was arrested at zygotene-like stage with a deficiency in homolog pairing and synapsis. KASH5 protein expression in human spermatocytes was evaluated and reported first in this study. Single-cell RNA sequencing demonstrated that the LINC complex and associated genes in human and mouse shared a similar expression pattern, indicating a conserved mechanism in the regulation of chromosome movements and NE remodeling. Kash5 knockout mouse displayed similar phenotypes, including a meiotic arrest at a zygotene-like stage and impaired pairing and synapsis. Collectively, we have identified novel rare variants within human KASH5 in patients with NOA and meiosis arrest. Our study expands the knowledge of KASH5 and associated proteins in regulating human meiosis prophase I progress and provides new insight into the genetic etiology of NOA.

The Simulated Physiological Oocyte Maturation (SPOM) system in domestic animals: A systematic review

Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation. The system uses cAMP modulators in two steps (pre IVM and IVM) and has presented promising results that are arousing the curiosity of IVF programs in different animal species, generating several papers, adaptations, and controversies worldwide. This study systematically analyses the data in the literature on the use of SPOM and compares the outcomes to the original paper (Albuz et al. Hum. Rep., 25: 2999–3011 2010), classifying them into success or failure. The PubMed, Scopus, and Google Scholar databases were searched and 22 studies were included, from which data on 26 experiments were extracted and evaluated via descriptive statistical analysis. Only experiments that assessed the blastocyst rate (BR) were considered for the success parameter, i.e. success (increase in BR) or failure (either no difference or a reduction in BR). The experiments applied the SPOM system in the following species: cattle, sheep, goats, mice, mares and cats. Three experiments (3/26) could not be evaluated for success or failure, and of the remaining, 34.7% (8/23) succeeded in improving blastocyst production. More than two-thirds (69.2%, 18/26) of experiments were conducted in cattle; of those, 86.8% (13/15) used TCM-199 as the IVM media, and 22.2% did not use forskolin or IBMX modulators as indicated in the original study. Also, 27.7% (5/18) of the experiments in cattle used the same type and dose of FSH, and 22% (4/18) used the same protein source and concentration as indicated in the original study. All experiments conducted in mice (3) kept the parameters of the original study in terms of forskolin and IBMX doses and BSA and FSH concentrations, however, they removed cilostamide from IVM. Cilostamide was used during IVM in more than half (53.8%) of all experiments, but only in cattle and sheep. Considering oocyte and embryo assessments, six experiments assessed cAMP levels and most (5/6) of these observed an increase: in cattle (2), sheep (2), and mice (1). Ten experiments evaluated the effect of SPOM on nuclear maturation, and in 90% (9/10), the SPOM system was able to arrest meiosis (cattle, sheep and mice). Thirteen experiments evaluated the total cell number (cattle, mice and sheep), and six (6/13) showed an increase. Our findings clearly indicate difficulties in reproducing the SPOM system worldwide, demonstrating that the meiosis arrest is not sufficient to ensure successful SPOM application. They also suggest that the different supplements used in the IVM medium and/or their interaction with modulators for different durations may produce a significant bias that affects experimental success.

Triploid-induced complete sterility in the scallop Nodipecten subnodosus might be triggered by an early and sustained DNA damage response

Triploid Nodipecten subnodosus scallops, unlike other mollusks, are completely sterile. In this study we focused on understanding the underlying molecular changes of triploid sterility using a transcriptomic approach. Total RNA from the gonad of diploid scallops in inactive and initial gametogenic stages and triploid scallops of the same cohort and ages were sequenced employing Illumina RNA-Seq. From 68,244 assembled and annotated transcripts, 1120 had terms associated with meiosis checkpoint or arrest, DNA damage response, or recombination. Differential gene expression analyses were conducted by contrasting initial vs. inactive stages of gametogenesis in each ploidy. In diploids, genes participating in homologous recombination during meiosis (msh5 and kdm8), spindle organization (nup62), centrosome formation (cenp-T), and sex differentiation (Ns-dmta2 and pum3), were up-regulated during initial gametogenesis. In triploids, a different set of genes were up-regulated during initial gametogenesis, and included genes involved in the DNA damage response and double strand break repair (rad51-C, xpc, myoVI), in the transition of metaphase/anaphase of mitosis (slp1 and nuf2), as well as genes that trigger both the intrinsic and extrinsic (caspase-3, icad, bmcc1) and extrinsic apoptosis pathways only (tnfr1, dab2ip). The results suggest significant DNA damage in triploids initial gametogenesis, possibly as a consequence of failing to repair double-strand breaks during DNA replication. This coincides with previous observations in which few triploid scallops showed gametic stages more advanced than oogonia or spermatogonia, and when present they were few.