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1.
Zoolog Sci ; 41(3): 302-313, 2024 Jun.
Article En | MEDLINE | ID: mdl-38809869

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.


Sperm Motility , Spermatozoa , Male , Animals , Spermatozoa/physiology , Salamandridae/physiology , Fertilization/physiology , Hydrogen-Ion Concentration , Female , Adenylyl Cyclases/metabolism , Adenylyl Cyclases/genetics , Signal Transduction
2.
Reprod Sci ; 31(6): 1757-1762, 2024 Jun.
Article En | MEDLINE | ID: mdl-38653856

Endometriosis, affecting approximately 10% of reproductive-aged women globally, poses significant challenges, including chronic pelvic pain, dysmenorrhea, and infertility. In low- and middle-income countries like India, accessibility to affordable infertility care remains a concern. This multicenter prospective cohort study, conducted across six tertiary care hospitals in India from 2017 to 2022, aims to explore the natural progression of conception and pregnancy outcomes in women with endometriosis. Of the 257 participants, 19.1% conceived during the study, revealing significant geographic and income-based variations (p < 0.001, p = 0.01). Dysmenorrhea (p < 0.001) and dyspareunia (p=0.027) were correlated with conception, while no such associations were found with chronic pelvic pain or menstrual factors. Lesion type, number, and severity showed no conclusive link with conception. Natural conception occurred in 70% of cases, with an average post-surgery conception time of 282.1 days. Live birth rate was 85.7%, while complications included placenta previa (16.4%), preeclampsia (4.1%), and preterm births (4.1%). This study, one of the first in India on endometriosis-related fertility progression, emphasizes the need for comprehensive understanding and management of conception and pregnancy outcomes. Considering India's substantial endometriosis burden, the study recommends prioritizing larger multicenter investigations for a better understanding and effective strategies for infertility management.


Endometriosis , Fertilization , Pregnancy Outcome , Humans , Female , Endometriosis/complications , Endometriosis/epidemiology , Endometriosis/diagnosis , Pregnancy , Adult , Pregnancy Outcome/epidemiology , Longitudinal Studies , India/epidemiology , Fertilization/physiology , Prospective Studies , Infertility, Female/epidemiology , Infertility, Female/therapy , Infertility, Female/etiology , Pregnancy Complications/epidemiology
3.
Hum Reprod ; 39(5): 902-911, 2024 May 02.
Article En | MEDLINE | ID: mdl-38461455

STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.


Flow Cytometry , Semen Analysis , Seminal Plasma Proteins , Spermatozoa , Male , Humans , Spermatozoa/physiology , Flow Cytometry/methods , Semen Analysis/methods , DNA Fragmentation , Sperm Motility , Phosphoinositide Phospholipase C/metabolism , Adult , Microfluidics/methods , Fertilization/physiology , Microfluidic Analytical Techniques/methods , Cell Separation/methods , Carrier Proteins/metabolism
4.
Nat Plants ; 10(2): 268-282, 2024 02.
Article En | MEDLINE | ID: mdl-38287093

During double fertilization in angiosperms, the pollen tube delivers two sperm cells into an embryo sac; one sperm cell fuses with an egg cell, and the other sperm cell fuses with the central cell. It has long been proposed that the preference for fusion with one or another female gamete cell depends on the sperm cells and occurs during gamete recognition. However, up to now, sperm-dependent preferential fertilization has not been demonstrated, and results on preferred fusion with either female gamete have remained conflicting. To investigate this topic, we generated Arabidopsis thaliana mutants that produce single sperm-like cells or whose egg cells are eliminated; we found that although the three different types of sperm-like cell are functionally equivalent in their ability to fertilize the egg and the central cell, each type of sperm-like cell fuses predominantly with the egg cell. This indicates that it is the egg cell that controls its preferential fertilization. We also found that sperm-activating small secreted EGG CELL 1 proteins are involved in the regulation of egg-cell-dependent preferential fertilization, revealing another important role for this protein family during double fertilization.


Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Seeds/metabolism , Fertilization/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Pollen Tube
5.
Reprod Sci ; 31(5): 1345-1352, 2024 May.
Article En | MEDLINE | ID: mdl-38172334

The purpose of this study was to determine whether utilization of assisted reproductive technology following clearance of endometrial intraepithelial neoplasia (EIN) or early endometrial cancer (EC) shortens time to conception (TTC) and reduces recurrence. Patients aged 18 to 45 with EIN or early EC who achieved pathologic response following progesterone treatment were identified via retrospective chart review. Study groups included patients who pursued ovulation induction (OI), in vitro fertilization (IVF), and spontaneous pregnancy. Primary outcomes were TTC and recurrence rate. Three hundred forty-six charts were reviewed, with 86 patients meeting inclusion criteria and 53 attempting pregnancy. Of those 53 patients, 11 became pregnant and seven had a live birth. Median times to pregnancy were 183 days for IVF, 54 days for OI, and 347 days for spontaneous conception (p < 0.05). No differences were seen in recurrence or progression based on attempted pregnancy method, nor with duration of fertility treatment. Forty-two of 86 patients (49%) were lost to follow-up. For patients with a history of treated EIN or EC, OI may decrease TTC. Larger prospective studies are needed to definitively answer this question. Although no differences in recurrence or progression were identified, the significant loss to follow-up rate in this study is concerning and warrants further investigation.


Endometrial Neoplasms , Ovulation Induction , Humans , Female , Adult , Pregnancy , Retrospective Studies , Ovulation Induction/methods , Fertility , Middle Aged , Carcinoma in Situ/therapy , Carcinoma in Situ/pathology , Young Adult , Fertilization in Vitro/methods , Pregnancy Rate , Adolescent , Time-to-Pregnancy , Neoplasm Recurrence, Local , Time Factors , Fertilization/physiology , Infertility, Female/therapy , Infertility, Female/etiology
6.
J Clin Invest ; 134(1)2024 Jan 02.
Article En | MEDLINE | ID: mdl-38165034

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.


Infertility, Male , Semen , Child , Humans , Male , Semen/physiology , Calcium Channels/genetics , Sperm Motility/physiology , Spermatozoa/physiology , Infertility, Male/therapy , Infertility, Male/genetics , Fertilization in Vitro , Fertilization/physiology
7.
Nat Commun ; 15(1): 792, 2024 Jan 26.
Article En | MEDLINE | ID: mdl-38278786

In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.


Caenorhabditis elegans , Ubiquitin , Animals , Male , Caenorhabditis elegans/genetics , Ubiquitin/metabolism , Ligases/metabolism , Semen , Fertilization/physiology , Spermatozoa/metabolism , Oocytes/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Chitin/metabolism , Sperm-Ovum Interactions/physiology
8.
FEBS J ; 291(1): 114-131, 2024 01.
Article En | MEDLINE | ID: mdl-37690456

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin-substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.


Fibroblasts , Semen , Male , Animals , Mice , Zona Pellucida Glycoproteins/metabolism , Fibroblasts/metabolism , Semen/metabolism , Metalloproteases/metabolism , Mammals/metabolism , Endopeptidases , Fertilization/physiology
9.
Zygote ; 32(1): 38-48, 2024 Feb.
Article En | MEDLINE | ID: mdl-38050697

The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.


Actins , Spectrum Analysis, Raman , Animals , Male , Actins/metabolism , Semen , Actin Cytoskeleton/metabolism , Fertilization/physiology , Sea Urchins/metabolism , Ovum/metabolism
10.
Reprod Sci ; 31(3): 697-703, 2024 Mar.
Article En | MEDLINE | ID: mdl-37814199

Our main objective was to identify the male and female parameters associated with total fertilization failure (TFF) in IVF with nonmasculine indications. The present work, IRB equivalent INS 63209, is a case-control study that evaluated all cases with TFF after conventional IVF at the Center for Human Reproduction from January 2010 to December 2019 (n = 154). As a control group, we analyzed all patients who did not experience fertilization failure after conventional IVF in the same period (n = 475). We evaluated various parameters, both male and female, assessed during infertility treatment, and only cases without masculine etiology (normal seminal parameters) were included. Ages (female and male) were not different between the groups. Moreover, AMH (anti-Müllerian hormone), semen volume, preprocessing concentration and preprocessing motility were not significantly different (P > 0.05). However, the number of collected oocytes (study versus control groups, median [25-75 interquartile]: 2 [1-5] and 5 [3-8]); MII (2 [1-4] and 5 [2-7]); and postprocessing motility (85 [70-90] and 90 [80-95]) were significantly different between both groups (P < 0.05). Furthermore, a logistic regression analysis including all significant data demonstrated that the number of collected oocytes was significantly related to IVF failure. Patients with fewer than 5 oocytes had an OR of - 1.37 (- 0.938 to - 1.827) for TFF after conventional IVF. Our results showed that a lower follicular response to controlled ovarian stimulation, evidenced by a decreased number of collected oocytes, was the most important parameter associated with IVF failure in nonmasculine infertility.


Fertilization in Vitro , Infertility , Humans , Male , Female , Pregnancy , Sperm Injections, Intracytoplasmic , Case-Control Studies , Infertility/therapy , Oocytes , Anti-Mullerian Hormone , Fertilization/physiology , Pregnancy Rate
11.
Biochem J ; 480(24): 2023-2035, 2023 12 20.
Article En | MEDLINE | ID: mdl-38014506

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.


Calcium , Thimerosal , Mice , Male , Animals , Thimerosal/pharmacology , Semen , Spermatozoa/physiology , Adenosine Triphosphate , Fertilization/physiology
12.
Sci Rep ; 13(1): 20342, 2023 11 20.
Article En | MEDLINE | ID: mdl-37990051

JUNO-IZUMO1 binding is the first known physical link created between the sperm and egg membranes in fertilization, however, how this initiates sperm-egg fusion remains elusive. As advanced structural insights will help to combat the infertility crisis, or advance fertility control, we employed all-atom Molecular Dynamics (MD) to derive dynamic structural insights that are difficult to obtain experimentally. We found that the hydrated JUNO-IZUMO1 interface is composed of a large set of short-lived non-covalent interactions. The contact interface is destabilized by strategically located point mutations, as well as by Zn2+ ions, which shift IZUMO1 into the non-binding "boomerang" conformation. We hypothesize that the latter might explain how the transient zinc spark, as released after sperm entry into the oocyte, might contribute to block polyspermy. To address a second mystery, we performed another set of simulations, as it was previously suggested that JUNO in solution is unable to bind to folate despite it belonging to the folate receptor family. MD now suggests that JUNO complexation with IZUMO1 opens up the binding pocket thereby enabling folate insertion. Our MD simulations thus provide crucial new hypotheses how the dynamics of the JUNO-IZUMO1 complex upon solvation might regulate fertility.


Membrane Proteins , Receptors, Cell Surface , Male , Humans , Receptors, Cell Surface/metabolism , Membrane Proteins/metabolism , Sperm-Ovum Interactions/genetics , Molecular Dynamics Simulation , Semen/metabolism , Fertilization/physiology , Spermatozoa/metabolism , Folic Acid/metabolism , Immunoglobulins/metabolism
13.
J Gen Physiol ; 155(10)2023 10 02.
Article En | MEDLINE | ID: mdl-37561060

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCß. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCß. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.


Calcium , Fertilization , Animals , Male , Fertilization/physiology , Xenopus laevis/metabolism , Calcium/metabolism , Semen/metabolism , Spermatozoa/metabolism
14.
Int J Mol Sci ; 24(13)2023 Jun 26.
Article En | MEDLINE | ID: mdl-37445840

The extracellular ubiquitin-proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.


Fertilization , Urochordata , Animals , Female , Male , Humans , Fertilization/physiology , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Proteasome Endopeptidase Complex/metabolism , Urochordata/genetics , Urochordata/metabolism , Semen/metabolism , Spermatozoa/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism
15.
Mol Reprod Dev ; 90(6): 369-377, 2023 06.
Article En | MEDLINE | ID: mdl-37486100

Throughout the reproductive life of women, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment, and act as a gatekeeper for oocyte developmental potential. One such mechanism relies on the hypoxia-tolerance response, which, with age, decreases systematically, including in the ovary. We aimed to evaluate the association between gene expression related to hypoxia and aging in CC and reproductive results in in vitro fertilization cycles. We recruited 94 women undergoing controlled ovarian stimulation. Total RNA was extracted from pooled CCs collected after oocyte pick-up (OPU) and reverse-transcribed to complementary DNA using random hexamers to test 14 genes related to hypoxia response via HIF1α activation, oxidative stress, and angiogenic responses. The expression of CLU, NOS2, and TXNIP had a positive correlation with age (rs = 0.25, rs = 0.24, and rs = 0.35, respectively). Additionally, NOS2 and HMOX1 expression correlated positively with the retrieval of immature oocytes (rs = 0.22 and rs = 0.40, respectively). Moreover, VEGFC levels decreased overall with increasing fertilization rate, independently of age (rs = -0.29). We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia with age, pointing at NOS2, HMOX1, and VEGFC expression as markers for oocyte maturation and fertilization success.


Cumulus Cells , Oogenesis , Female , Humans , Cumulus Cells/metabolism , Fertilization/physiology , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Hypoxia/genetics , Hypoxia/metabolism , In Vitro Oocyte Maturation Techniques , Nitric Oxide Synthase Type II/metabolism , Oocytes/metabolism , Oogenesis/physiology
16.
Theriogenology ; 207: 96-109, 2023 Sep 01.
Article En | MEDLINE | ID: mdl-37271105

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.


Buffaloes , Zona Pellucida , Female , Male , Animals , Zona Pellucida/metabolism , Buffaloes/metabolism , Semen/metabolism , Spermatozoa/metabolism , Fertilization/physiology , Polysaccharides , Zona Pellucida Glycoproteins , Lectins/metabolism , Oviducts/metabolism , Trisaccharides/metabolism , Trisaccharides/pharmacology , Epithelium/metabolism , Sperm-Ovum Interactions
17.
Theriogenology ; 206: 189-196, 2023 Aug.
Article En | MEDLINE | ID: mdl-37229958

Ovarian fluid is essential for successful fertilization by maintaining the viability, motility, and velocity of sperm. The organic compounds and inorganic ions in ovarian fluid significantly influence spermatozoa's motility, velocity, and longevity. However, the effect of ovarian fluid on sperm performance is limited in teleost fish. In this study, the effect of ovarian fluid on sperm performance and its components in external fertilization species (Scophthalmus maximus, turbot) and internal fertilization species (Sebastes schlegelii, black rockfish) was investigated using computer-assisted sperm analysis, high-performance liquid chromatography, and metabolome analysis. The ovarian fluid had a distinct and species-specific effect on both species. In the black rockfish, the ovarian fluid from turbot significantly increased sperm motility (74.07% ± 4.09%), as well as VCL (45 ± 1.67 µm/s), VAP (40.17 ± 1.6 µm/s), and VSL (36.67 ± 1.86 µm/s), and longevity (352 ± 11.31 min) (P < 0.05). In the turbot, only the longevity (71.33 ± 5.69 min) and fertilization rate (65.27% ± 11.59%) showed significantly improvement (P < 0.05). The ovarian fluid was rich in organic compounds, suggesting enrichment in the glycolysis/gluconeogenesis pathways. The results suggest that glycometabolism plays a crucial role in improving sperm performance in teleost with internal fertilization. Thus, incorporating ovarian fluid into the sperm activation medium can enhance artificial fertilization in fish breeding.


Flatfishes , Perciformes , Male , Animals , Fertilization/physiology , Semen , Sperm Motility/physiology , Spermatozoa/physiology
18.
Int J Mol Sci ; 24(3)2023 Jan 31.
Article En | MEDLINE | ID: mdl-36768985

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5-6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.


Ciona intestinalis , Fertilization , Animals , Male , Fertilization/physiology , Semen , Spermatozoa/physiology , Hydrogen-Ion Concentration
19.
Int J Mol Sci ; 24(4)2023 Feb 04.
Article En | MEDLINE | ID: mdl-36834494

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.


Epididymis , Extracellular Vesicles , Male , Cats , Animals , Epididymis/metabolism , Galectin 3/metabolism , Semen , Spermatozoa/metabolism , Fertilization/physiology , Proteins/metabolism
20.
Fertil Steril ; 119(2): 313-321, 2023 02.
Article En | MEDLINE | ID: mdl-36402618

OBJECTIVE: To investigate the association between preconception thyroid stimulating hormone (TSH) level and time to pregnancy within a community-based population. DESIGN: A community-based cohort study. SETTING: Two free preconception check-up centers. PATIENT(S): Women who enrolled in the National Free Preconception Check-up Projects from January 1, 2018 to December 31, 2018 in Tianhe and Zengcheng districts of Guangzhou city. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Time to pregnancy. RESULT(S): A total of 1,478 women were eligible for the analysis; of these, 1,401 had a preconception TSH level within the range of 0.50 and 5.59 mIU/L (2.5th-97.5th percentiles) were taken as target study population. Among them, 968 (69.1%) couples achieved pregnancy within the first 6 months and 1,082 (77.2%) within 12 months. Dichotomized by the recommended cut-off value of 2.5 mIU/L, the percentage of women conceived in the high TSH level category (2.50-5.59 mIU/L) was comparable to that of the low category (0.50-2.49 mIU/L) (79.0% vs. 78.1%), with a crude fecundity odd ratio of 0.99 (95% confidence interval at 0.87-1.13). No statistically significant difference was observed after the adjustment in all models. Continuous TSH level was further examined, and the nonlinear association between TSH level and fecundity odds ratios was of no statistical significance. CONCLUSION(S): Preconception TSH level was not associated with fecundity in a healthy community-based population. Women attempting pregnancy with a TSH level ≥ 2.5 mIU/L can be reassured that they are unlikely to have an increased time to pregnancy.


Fertility , Preconception Care , Thyrotropin , Time-to-Pregnancy , Female , Humans , Pregnancy/blood , Cohort Studies , Fertilization/physiology , Health Status , Thyrotropin/blood , Time-to-Pregnancy/physiology , Fertility/physiology
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