TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm-egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

Mouse strain-dependent egg factors regulate calcium signals at fertilization.

Calcium (Ca2+ ) signals triggered at fertilization initiate resumption of the cell cycle and initial steps of embryonic development. In mammals, the sperm factor phospholipase Cζ triggers the release of Ca2+ from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca2+ transients that is modulated by egg factors including Ca2+ influx channels, Ca2+ transporters, and phosphoinositide-regulating enzymes. Here we compared characteristics of Ca2+ oscillations following in vitro fertilization (IVF) and ER Ca2+ stores among nine common laboratory mouse strains: CF1, C57BL6, SJL, CD1, DBA, FVB, 129X1, BALBc, 129S1, and the F1 hybrid B6129SF1. Sperm from B6SJLF1/J males was used for all IVF experiments. There were significant differences among the strains with respect to duration and maximum amplitude of the first Ca2+ transient, frequency of oscillations, and ER Ca2+ stores. With male strain held constant, the differences in Ca2+ oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg-intrinsic properties in determining Ca2+ oscillation patterns and have important implications for the interpretation and comparison of studies on Ca2+ dynamics at fertilization.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca(2+) release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼ 20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca(2+) release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca(2+) increases that were sufficient to cause premature zona pellucida conversion. Rgs2(-/-) females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2(-/-) eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca(2+) release in eggs that are poised on the brink of development.

Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development in the mouse.

Understanding factors that regulate zygotic genome activation (ZGA) is critical for determining how cells are reprogrammed to become totipotent or pluripotent. There is limited information regarding how this process occurs physiologically in early mammalian embryos. Here, we identify a mediator complex subunit, MED13, as translated during mouse oocyte maturation and transcribed early from the zygotic genome. Knockdown and conditional knockout approaches demonstrate that MED13 is essential for ZGA in the mouse, in part by regulating expression of the embryo-specific chromatin remodeling complex, esBAF. The role of MED13 in ZGA is mediated in part by interactions with E2F transcription factors. In addition to MED13, its paralog, MED13L, is required for successful preimplantation embryo development. MED13L partially compensates for loss of MED13 function in preimplantation knockout embryos, but postimplantation development is not rescued by MED13L. Our data demonstrate an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during ZGA.

CaV3.2 T-type channels mediate Ca²âº entry during oocyte maturation and following fertilization.

Initiation of mouse embryonic development depends upon a series of fertilization-induced rises in intracellular Ca(2+). Complete egg activation requires influx of extracellular Ca(2+); however, the channels that mediate this influx remain unknown. Here, we tested whether the α1 subunit of the T-type channel CaV3.2, encoded by Cacna1h, mediates Ca(2+) entry into oocytes. We show that mouse eggs express a robust voltage-activated Ca(2+) current that is completely absent in Cacna1h(-/-) eggs. Cacna1h(-/-) females have reduced litter sizes, and careful analysis of Ca(2+) oscillation patterns in Cacna1h(-/-) eggs following in vitro fertilization (IVF) revealed reductions in first transient length and oscillation persistence. Total and endoplasmic reticulum (ER) Ca(2+) stores were also reduced in Cacna1h(-/-) eggs. Pharmacological inhibition of CaV3.2 in wild-type CF-1 strain eggs using mibefradil or pimozide reduced Ca(2+) store accumulation during oocyte maturation and reduced Ca(2+) oscillation persistence, frequency and number following IVF. Overall, these data show that CaV3.2 T-type channels have prev8iously unrecognized roles in supporting the meiotic-maturation-associated increase in ER Ca(2+) stores and mediating Ca(2+) influx required for the activation of development.

Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Deletion of Kallikrein-related peptidases ( Klks ) has no effect on fertility in mice.

Kallikreins (KLKs) are serine peptidases. It was established that Klks are estrogen-target genes in mouse uteri. However, the functional requirement of KLK family in the uterine function during reproduction is unknown. Here we generated a compound deletion of Klk1b3, Klk1b4, Klk1b5, and Klk1 in a mouse model using CRISPR/Cas9 strategy with four single guide RNAs (sgRNAs) to target the second exon of these four genes that are aligned back-to-back in a single locus spanning 32.95 kb on chromosome 7. We found that both male and female knockout mice are fertile with no apparent health defect compared to wild-type controls. Our data suggest that Klk1b3, Klk1b4, Klk1b5, and Klk1 are not necessary for male and female reproductive function in mice.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

ARRDC5 expression is conserved in mammalian testes and required for normal sperm morphogenesis.

In sexual reproduction, sperm contribute half the genomic material required for creation of offspring yet core molecular mechanisms essential for their formation are undefined. Here, the α-arrestin molecule arrestin-domain containing 5 (ARRDC5) is identified as an essential regulator of mammalian spermatogenesis. Multispecies testicular tissue transcriptome profiling indicates that expression of Arrdc5 is testis enriched, if not specific, in mice, pigs, cattle, and humans. Knockout of Arrdc5 in mice leads to male specific sterility due to production of low numbers of sperm that are immotile and malformed. Spermiogenesis, the final phase of spermatogenesis when round spermatids transform to spermatozoa, is defective in testes of Arrdc5 deficient mice. Also, epididymal sperm in Arrdc5 knockouts are unable to capacitate and fertilize oocytes. These findings establish ARRDC5 as an essential regulator of mammalian spermatogenesis. Considering the role of arrestin molecules as modulators of cellular signaling and ubiquitination, ARRDC5 is a potential male contraceptive target.

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes.

Precise coordination of meiotic progression is a critical determinant of an egg's capacity to be fertilized successfully, and zinc has emerged as a key regulatory element in this process. An early manifestation of a regulatory role for this transition metal is the significant increase in total intracellular zinc. This accumulation is essential for meiotic progression beyond telophase I and the establishment of meiotic arrest at metaphase II. The subsequent developmental event, fertilization, induces a rapid expulsion of labile zinc that is a hallmark event in meiotic resumption. In the present study, we show that the zinc fluxes work, in part, by altering the activity of the cytostatic factor (CSF), the cellular activity required for the establishment and maintenance of metaphase II arrest in the mature, unfertilized egg. We propose a model in which zinc exerts concentration-dependent regulation of meiosis through the CSF component EMI2, a zinc-binding protein. Together, the data support the conclusion that zinc itself, through its interaction with EMI2, is a central component of the CSF.

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway.

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.

Zinc requirement during meiosis I-meiosis II transition in mouse oocytes is independent of the MOS-MAPK pathway.

Zinc is essential for many biological processes, including proper functioning of gametes. We recently reported that zinc levels rise by over 50% during oocyte maturation and that attenuation of zinc availability during this period could be achieved using the membrane-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). This zinc insufficiency resulted in formation of large polar bodies, failure to establish metaphase II arrest, and impaired establishment of cortical polarity. As these phenotypes resemble those of MOS null oocytes, we examined the impact of zinc insufficiency on the MOS-MAPK pathway. Reduced levels of both MOS protein and phosphorylation of MAP2K1/2 are observed in zinc-insufficient oocytes; however, these differences appear only after completion of the first meiotic division. In addition, activation of the downstream effector of the MOS pathway, MAPK3/1, is not affected by zinc insufficiency, and reduced MOS levels are observed only with the presence of TPEN after the first polar body extrusion. These data are inconsistent with the hypothesis that reduced MOS mediates the observed phenotype. Finally, MOS overexpression does not rescue the phenotype of zinc-insufficient oocytes, confirming that the observed disruption of asymmetric division and spindle abnormalities cannot be attributed to impaired MOS signaling. Zinc-insufficient oocytes do not increase maturation promoting factor (MPF) activity following the first meiotic division, and increasing MPF activity through expression of nondegradable cyclin B1 partially rescues the ability of zinc-insufficient oocytes to enter metaphase II. Although we have shown that zinc has a novel role in the meiotic cell cycle, it is not mediated through the MOS-MAPK pathway.

Estrogen actions in the male reproductive system involve estrogen response element-independent pathways.

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs.

Repetitive oscillations in cytoplasmic Ca2+ due to periodic Ca2+ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca2+ to support the refilling of ER stores is required for sustained Ca2+ oscillations, but the mechanisms underlying this Ca2+ influx are controversial. Although store-operated Ca2+ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca2+ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca2+ sensor STIM proteins, and the plasma membrane Ca2+ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca2+ oscillations following fertilization. In addition, no impairment was observed in ER Ca2+ stores or Ca2+ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca2+ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca2+ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca2+ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca2+ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca2+ influx that was previously attributed to SOCE.

Oviductal estrogen receptor α signaling prevents protease-mediated embryo death.

Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception.

Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs.

In last few hours of maturation, the mouse oocyte takes up over twenty billion zinc atoms and arrests after the first meiotic division, until fertilization or pharmacological intervention stimulates cell cycle progression toward a new embryo. Using chemical and physical probes, we show that fertilization of the mature, zinc-enriched egg triggers the ejection of zinc into the extracellular milieu in a series of coordinated events termed zinc sparks. These events immediately follow the well-established series of calcium oscillations within the activated egg and are evolutionarily conserved in several mammalian species, including rodents and nonhuman primates. Functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued cell cycle progression, as increasing zinc levels within the activated egg results in the reestablishment of cell cycle arrest at metaphase. The mammalian egg thus uses a zinc-dependent switch mechanism to toggle between metaphase arrest and resumption of the meiotic cell cycle at the initiation of embryonic development.

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm.

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.