Number Of Gametes Research Articles

Sexual selection and life history interact to influence the evolution of paternal care.

Parental care is essential to offspring survival in many species. Understanding why males of some species provide care, whereas others do not, has received substantial attention. Previous research has found that sexual selection can favor paternal care, yet we still do not fully understand why sexual selection favors male care in some species but not others. It is also unclear when paternal care versus other preferred male trait(s) will be favored by sexual selection. We hypothesize that sexual selection can interact with basic life history to influence the conditions under which paternal care and/or another preferred male trait will be favored by sexual selection. We used a mathematical approach in which males alone provide parental care and exhibit a non-care trait that is preferred in mate choice. Using this approach, we demonstrate that life-history characteristics (stage-specific mortality, fertilization success, gamete numbers) can interact with sexual selection to influence the evolution of paternal care and/or a preferred non-care trait. In particular, whether (1) adult mortality, egg mortality, and fertilization success are high versus low and (2) a tradeoff exists between paternal care and a non-care preferred trait will influence whether selection most strongly favors additional paternal care or a non-care preferred trait. In general, we would expect strong selection for more male care when it is preferred in mate choice. In some cases, mate preferences for paternal care can inhibit selection for a preferred non-care trait. Mate preferences for paternal care can also broaden the life-history conditions under which we would expect the elaboration of male care to occur.

Sex-specific growth and lifespan effects of germline removal in the dioecious nematode Caenorhabditis remanei.

Germline regulates the expression of life-history traits and mediates the trade-off between reproduction and somatic maintenance. However, germline maintenance in itself can be costly, and the costs can vary between the sexes depending on the number of gametes produced across the lifetime. We tested this directly by germline ablation using glp-1 RNA interference (RNAi) in a dioecious nematode Caenorhabditis remanei. Germline removal strongly increased heat-shock resistance in both sexes, thus confirming the role of the germline in regulating somatic maintenance. However, germline removal resulted in increased lifespan only in males. High costs of mating strongly reduced lifespan in both sexes and obliterated the survival benefit of germline-less males even though neither sex produced any offspring. Furthermore, germline removal reduced male growth before maturation but not in adulthood, while female growth rate was reduced both before and especially after maturation. Thus, germline removal improves male lifespan without major growth costs, while germline-less females grow slower and do not live longer than reproductively functional counterparts in the absence of environmental stress. Overall, these results suggest that germline maintenance is costlier for males than for females in C. remanei.

Allochronic reproductive cycles among colonies of the Caribbean octocoral Antillogorgia americana

AbstractThe reproductive biology of the branching octocoral Antillogorgia americana was studied at a site on the Caribbean coast of Panama in 1990–1991 by examining the reproductive status of 11 colonies across 14 months. Colonies were gonochoric. The presence of large and mature eggs or spermaries was allochronic across colonies and months, with peak gonad volumes occurring in months ranging from October through May. Reproductive effort varied between branches on a colony, with variation between branches and branchlets accounting for 25% of the random variation between polyps. Branchlets at the tip of the colony had fewer mature eggs than those lower on the branch, and polyps at the tips of the branchlets had fewer still. Although the simultaneous release of eggs and sperm is critical to reproductive success, the lack of synchrony among colonies on the scale of months may reflect less need for all colonies to spawn in a single event among abundant species that release large numbers of gametes. Such a strategy also spreads the risk of reproductive failure due to environmental conditions during any single month. The presence of multiple spawning episodes can also drive the reproductive isolation of populations and may reflect the presence of cryptic species within the taxon. Studies of reproductive timing can be an important adjunct in identifying variation in life history strategies as well as assessing the validity of species boundaries.

Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

Two distinct mechanisms lead to either oocyte or spermatocyte decrease in C. elegans after whole developmental exposure to γ-rays.

Wildlife is subject to various sources of pollution, including ionizing radiation. Adverse effects can impact the survival, growth, or reproduction of organisms, later affecting population dynamics. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help to comprehend species-specific differences in radiosensitivity. From our previous studies, we found that decrease in reproduction is life stage dependent in the roundworm Caenorhabditis elegans, possibly resulting from an accumulation of damages during germ cell development and gamete differentiation. To go further, we used the same experimental design to assess more precisely the molecular determinants of reproductive toxicity, primarily decreases in gamete number. As before, worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiation throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). To enable cross species extrapolation, conserved molecular pathways across invertebrates and vertebrates were analysed: apoptosis and MAP kinase Ras/ERK (MPK-1), both involved in reproduction and stress responses. Our results showed that these pathways are life-stage dependent, resulting from an accumulation of damages upon chronic exposure to IR throughout the life development. The Ras/ERK pathway was activated in our conditions in the pachytene region of the gonad where it regulates cell fate including apoptosis, but not in the ovulation zone, where it controls oocyte maturation and ovulation. Additionally, assessment of germ cell proliferation via Ras/ERK pathway showed no effect. Finally, a functional analysis of apoptosis revealed that while the decrease of the ovulation rate is caused by DNA-damaged induced apoptosis, this process does not occur in spermatocytes. Thus, sperm decrease seems to be mediated via another mechanism, probably a decrease in germ cell proliferation speed that needs further investigation to better characterize sex-specific responses to IR exposure. These results are of main importance to describe radio-induced reprotoxic effects and contribute as weight of evidence for the AOP #396 "Deposition of ionizing energy leads to population decline via impaired meiosis".

Sex-specific selection patterns in a dioecious insect-pollinated plant.

Competition for mate acquisition is the hallmark of any sexual organism. In insect-pollinated plants, competition to attract pollinators is expected to result in pollinator-mediated selection on attractive floral traits. This could overlap with sexual selection if the number of mating partners increases with pollinator attraction, resulting in an improved reproductive success. In this study, we measured a set of floral traits and estimated individual fitness in male and female Silene dioica in an experimental population. Results align with the predictions of Bateman's principles, in the absence of pollen limitation. In females, natural selection acted on traits that are typically linked with fertility (number of flowers and number of gametes), and selection strength was similar in open- and hand-pollinated females, suggesting a limited role of pollinator-mediated selection. In males, flowering duration and corolla width were positively associated with both reproductive success and number of mates, suggesting that sexual selection has played a role in the evolution of these traits. The use of Bateman's metrics further confirmed stronger sexual selection in males than in females. Taken together, our results shed light on the occurrence of sex-specific patterns of selection in an insect-pollinated plant population.

Histomorphological comparisons in testicles between Barb horse, donkey and mule

The mule is a sterile hybrid domestic animal that results from the breeding of a male donkey with a female horse, understanding the reproductive biology of these species is very critical. The goal of this paper was to perform a comparative and more accurate histomorphometric of the testicles in Barb horse, donkeys and mules. Microscopic examinations and histological description were carried on genital tract of horses, donkeys and mules healthy and mature; this study was conducted during April-May 2018. The histological and the morphological results shows a similarity between the two equine species and the infertile hybrid for the testicles, the epididymis and the vas deferens. However, the difference was presented on the morphometric data; vas deferens was more voluminous in the horse and donkey than a mule. Moreover, the differences were significantly higher for the surface of the seminiferous tubules and for the epididymis. The lumen of the seminiferous tubules in mule was significantly higher than in the horse and donkey. Absence of gametes in the epididymal cavity and lower number of gametes in the mule. Furthermore, we have noted the presence of spermatozoa in one mule 16.67%. Therefore, the mule could complete development of spermatogenesis.

Irregularities in Meiotic Prophase I as Prerequisites for Reproductive Isolation in Experimental Hybrids Carrying Robertsonian Translocations

The basic causes of postzygotic isolation can be elucidated if gametogenesis is studied, which is a drastically different process in males and females. As a step toward clarifying this problem, we obtained an experimental inbred lineage of the eastern mole vole Ellobius tancrei, whose founder animals were animals with identical diploid numbers 2n = 50 but with different Robertsonian translocations (Rb), namely 2Rb4.12 and 2Rb9.13 in the female and 2Rb.2.18 and 2Rb5.9 in the male. Here, we analyzed strictly inbred hybrids (F1, fertile and F10, sterile) using immunocytochemical methods in order to study spermatocytes during the meiotic prophase I. Previously, the presence of trivalents was assumed to have no significant effect on spermatogenesis and fertility in hybrids, but we demonstrated that spermatogenesis might be disturbed due to the cumulative effects of the retarded synapses of Rb bivalents as well as trivalents and their associations with XX sex bivalents. Alterations in the number of gametes due to the described processes led to a decrease in reproductive capacity up to sterility and can be examined as a mechanism for reproductive isolation, thus starting speciation.

Reproductive genomics of the mouse: implications for human fertility and infertility.

Genetic analyses of mammalian gametogenesis and fertility have the potential to inform about two important and interrelated clinical areas: infertility and contraception. Here, we address the genetics and genomics underlying gamete formation, productivity and function in the context of reproductive success in mammalian systems, primarily mouse and human. Although much is known about the specific genes and proteins required for meiotic processes and sperm function, we know relatively little about other gametic determinants of overall fertility, such as regulation of gamete numbers, duration of gamete production, and gamete selection and function in fertilization. As fertility is not a binary trait, attention is now appropriately focused on the oligogenic, quantitative aspects of reproduction. Multiparent mouse populations, created by complex crossing strategies, exhibit genetic diversity similar to human populations and will be valuable resources for genetic discovery, helping to overcome current limitations to our knowledge of mammalian reproductive genetics. Finally, we discuss how what we know about the genomics of reproduction can ultimately be brought to the clinic, informing our concepts of human fertility and infertility, and improving assisted reproductive technologies.

Improving Fertility Treatment Documentation - A Survey.

Many couples do not have adequate documentation following fertility treatments. To conduct a survey to understand the information needs of assisted reproductive technology physicians when assessing the reasons affecting treatment outcomes after intrauterine insemination (IUI) and in vitro fertilisation (IVF). Online survey. Anonymous online survey of 14 parameters related to IUI treatment and 21 parameters related to IVF treatment, followed by recommendation of selected parameters for routine documentation. Frequency distribution calculation of responses. For IUI, total motile sperm count and post-wash sperm count and motility and for IVF, the quality and number of gametes, embryo number and morphology were the most important parameters. The study creates recommendations for the minimum information desirable in the fertility treatment documentation given to the couple undergoing treatment.

Female choice of mates in the aphidophagous ladybird beetle, Hippodamia variegata (Coleoptera: Coccinellidae): the effect of male age, sexual status and familiarity

Mating involves the transfer of sperm to females. Female reproductive investment is based on gamete numbers as well as gamete size. Females are more selective than males and this selection is based on numerous factors, like a male’s age, sexual status and familiarity. Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae) is an important natural enemy of many insect pests such as aphids, psyllids, whiteflies and mealybugs in many countries. We investigated female mate choice in Hippodamia variegate caging females with: (i) a young and a middle-aged male, (ii) a virgin and mated male and (iii) a novel and a familiar male. Time to and duration of copulation were recorded. Females preferred to mate with middle-aged and familiar males and did not differentiate between previously mated and virgin males. Females copulated quicker and for longer with young or familiar males.

On the function of flower number: disentangling fertility from pollinator-mediated selection.

In animal-pollinated angiosperms, the 'male-function' hypothesis claims that male reproductive success (RS) should benefit from large floral displays, through pollinator attraction, while female RS is expected to be mainly limited by resource availability. As appealing as this theory might be, studies comparing selection strength on flower number in both sexes rarely document the expected asymmetry. This discrepancy could arise because flower number impacts both pollinator attraction and overall gamete number. In this study, we artificially manipulate floral displays to disentangle the fertility versus pollinator attraction components of selection, both in terms of mating and RS. In females, flower number was under strong fertility selection, as predicted in the absence of pollen limitation. By contrast, in males, flower number was mainly under sexual selection, which in turn increased male RS. However, these selection patterns were not different in males with artificially increased floral displays. This suggests that sexual selection acting on flower number in males does not occur because flower number increases pollinator attraction, but rather because more pollen is available to disperse on more mates. Our study illustrates the power of disentangling various components of selection with potentially sex-specific effects for understanding the evolution of sexual dimorphism.

Sgcocaller and comapr: personalised haplotype assembly and comparative crossover map analysis using single-gamete sequencing data.

Profiling gametes of an individual enables the construction of personalised haplotypes and meiotic crossover landscapes, now achievable at larger scale than ever through the availability of high-throughput single-cell sequencing technologies. However, high-throughput single-gamete data commonly have low depth of coverage per gamete, which challenges existing gamete-based haplotype phasing methods. In addition, haplotyping a large number of single gametes from high-throughput single-cell DNA sequencing data and constructing meiotic crossover profiles using existing methods requires intensive processing. Here, we introduce efficient software tools for the essential tasks of generating personalised haplotypes and calling crossovers in gametes from single-gamete DNA sequencing data (sgcocaller), and constructing, visualising, and comparing individualised crossover landscapes from single gametes (comapr). With additional data pre-possessing, the tools can also be applied to bulk-sequenced samples. We demonstrate that sgcocaller is able to generate impeccable phasing results for high-coverage datasets, on which it is more accurate and stable than existing methods, and also performs well on low-coverage single-gamete sequencing datasets for which current methods fail. Our tools achieve highly accurate results with user-friendly installation, comprehensive documentation, efficient computation times and minimal memory usage.

Investigation of the effect of COVID-19 infection on sperm DNA fragmentation

Aim. To compare sperm parameters and the DNA fragmentation in patients after the COVID-19 infection and in men without a history of infection.Materials and methods. Sperm parameters and DNA fragmentation index were evaluated in 172 patients examined in 2020–2022. Based on the data on COVID-19 infection in the anamnesis, the patient’s cohort was divided into three groups: group 1 (n = 16) and group 2 (n = 65) – patients who had an infectious disease less than 75 days and more than 75 days before the delivery of the ejaculate, respectively, and group 3 (control, n = 91) – men have no coronavirus infection. A standard semen examination was performed according by the WHO guidelines (2010) recommendation. Sperm DNA fragmentation was evaluated by fluorescent labeling of single- and double-strand DNA breaks (TUNEL method).Results. The percentage of spermatozoa with fragmented DNA in group 1 varies from 2 to 48 %, in group 2 – from 0.9 to 39 %, in group 3 (control group) – from 1.3 to 52.9 %. The average number of gametes with DNA breaks in group 1 (11.7 ± 3.3 %) is greater than in group 2 (10.6 ± 0.8 %) and in group 3 (10.7 ± 0.8%), but without a statistically significant differences. The proportions of patients whose DNA fragmentation index exceeds the reference value (≤15 %) in groups 1, 2 and 3 are 18.8; 13.8 and 18.7 %, respectively. The count of progressively motile (PR) and morphologically normal spermatozoa in group 2 is statistically significantly greater than in group 3 (22.5 ± 1.6 % vs 18.0 ± 1.2 % and 5.52 ± 0.47 % vs 4.03 ± 0.31 %, respectively).Conclusion. The content of sperm with fragmented DNA is higher in men examined less than 75 days after COVID-19 disease, than in patients, examined more than 75 days after COVID-19 and in men who have no history of this infection. In group 1 an increased DNA fragmentation index was noted mainly in patients who had suffered from the disease in a moderate form.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa

BackgroundThe wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry.ResultsPercoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis.ConclusionsWhile creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

Investigation of the role of endogenous miRNAs in determining sterility in triploid Pacific oysters (Crassostrea gigas)

Triploid oysters generate fewer gametes than diploid oysters and exhibit varying degrees of gonad development. The expression of related genes, such as those involved in gametogenesis and energy metabolism, may influence reproductive potential. In addition, microRNAs (miRNAs) are important regulators of gene expression. However, the contribution of miRNAs has not yet been assessed in sterile triploid Pacific oysters (Crassostrea gigas). We therefore obtained extensive miRNA and gene expression data to identify potentially critical miRNA-mRNA interactions that are linked to sterility in triploid C. gigas. Here, we found miR-263-x to be significantly downregulated in the gonads of sterile females. The upregulated target genes were associated with lipid droplet formation, which revealed the likely involvement of nutrient accumulation during gonadal development as regulated by miR-263-x in female sterile triploids. In males, the upregulated miRNAs (miR-1992-y and miR-2001-x) were shown to regulate spermatogenesis-related gene expression, including flagellar formation, and cell proliferation and migration. Our results suggest that energy redistribution regulated by miRNAs may be critical for gonad development in females, indicating that sterile triploids undergo a significant reduction in gamete number and show an increased accumulation of nutrients in the gonads. Our findings further indicate that decreased expression of genes related to spermatogenesis regulated by miRNAs is important in gonadal dysgenesis in sterile triploids. These results shed light on the epigenetic regulatory mechanisms governing sterility in triploid bivalves.

Bateman gradients from first principles

In 1948, Angus Bateman presented experiments and concepts that remain influential and debated in sexual selection. The Bateman gradient relates reproductive success to mate number, and Bateman presented this as the cause of intra-masculine selection. A deeper causal level was subsequently asserted: that the ultimate cause of sex differences in Bateman gradients is the sex difference in gamete numbers, an argument that remains controversial and without mathematical backup. Here I develop models showing how asymmetry in gamete numbers alone can generate steeper Bateman gradients in males. This conclusion remains when the further asymmetry of internal fertilisation is added to the model and fertilisation is efficient. Strong gamete limitation can push Bateman gradients towards equality under external fertilisation and reverse them under internal fertilisation. Thus, this study provides a mathematical formalisation of Bateman’s brief verbal claim, while demonstrating that the link between gamete number and Bateman gradients is not inevitable nor trivial.

Genetic variation in released gametes produces genetic diversity in the offspring of the broadcast spawning coral Acroporatenuis

All coral species in the genus Acropora are broadcast-spawning hermaphrodites. Fertilization in the ocean requires sufficient numbers of gametes from conspecifics and the contact time for fertilization is thought to be limited by the rapid diffusion of sperm. Many studies have reported a positive correlation between sperm concentration and fertilization success, but it is not clear how gametes diffuse in seawater to produce mixtures of gametes from many colonies, leading to fertilization that improves genetic diversity. To elucidate this, we analyzed the changes in sperm concentration of A.tenuis in situ after spawning and genotyped sperm and fertilized eggs from seawater using seven microsatellite (MS) markers. Results showed that most of the eggs were fertilized at below 106 sperm/mL in situ. MS genotyping showed that the alleles of released sperm were diverse and those alleles also appeared in the fertilized eggs. The MS fragment peak height in released sperm, which presumably reflects the allele frequency of the sperm, was positively correlated with the allele frequencies of the fertilized eggs. Collectively, synchronous spawning populations composed of highly fecund and genetically diverse colonies potentially increases genetic diversity and the number of descendants.

Assessing the biological effects of microwave irradiation on human semen in vitro and determining the role of seminal plasma polyamines in this process.

The aim of the present study was to investigate the influence of millimeter-wave electromagnetic (MW) irradiation on normal and pathological human sperm in vitro, and to evaluate a possible role of polyamines (PA) in this process. The stability of sperm membranes, the number of apoptotic gametes, and the content of seminal plasma PA in the ejaculates of fertile and subfertile men were compared before and after short-term MW electromagnetic exposure in vitro. The ejaculate samples were collected from healthy donors [n=25, age 22-38 years old (y.o.), average age 30.6±1.1 y.o. (mean ± SEM)] and from subfertile men (n=78, age 25-48 y.o., average age 34.1±0.8 y.o.) and exposed to MW radiation. The electromagnetic field had a wavelength of 7.1 mm, a frequency of 42.194 GHz and an exposure time of 20 min. The fragility of sperm membranes was evaluated by their resistance to sodium chloride solution (Milovanov test) and to acetic acid (Joel test). Acrosin activity was assayed spectrophotometrically. Apoptosis was determined by the externalization of phosphatidylserine on the outer side of the sperm membrane and propidium iodide staining. The PA levels were determined by agar gel electrophoretic fractionation. An increase in the resistance of sperm membranes, a decrease in acrosin activity, a decrease in the number of apoptotic gametes and a decrease in the seminal plasma PA concentrations were found after exposure of the native human sperm to low-intensity MW irradiation. Two types of reactions were revealed for the subfertile samples. The results revealed positive bio-effects of specific microwaves on the human semen and the participation of PA in the realization of these effects.

Aquaculture performance comparison of reciprocal triploid C. gigas produced by mating tetraploids and diploids in China

Crassostrea gigas is an important species of oyster culture in northern China, which has important economic value. In recent years, triploid cultivation of C. gigas has gradually emerged because of the advantages of poor fertility and fast growth, but the comparative studies on tetraploid, triploid and diploid C. gigas are not comprehensive and systematic. In addition, forward or reverse mating tetraploids and diploids all can produce triploids, it is necessary to compare the performance of the two triploid groups. This study was conducted to understand the growth, survival, ploidy level and fertility of tetraploids in China, and to figure out how to mass-produce triploids. Aritificial hybridization was made between diploid (D) and tetraploid (T) to produce DD, TD, DT and TT groups (males are listed first). The results showed that the fertilization rate, cleavage rate, D larval rate and larvae survival rate of TD group were significantly higher than those of DT and TT groups, and the larval growth of TD group was significantly faster than that of the other three groups. In cultivation period, the growth and survival rate of TD in the 2 sites were significantly higher, and the 100% triploid rate was stable. In addition, some triploids in TD and DT groups were fertile and could produce functional gametes. But the proportion of female and hermaphrodite in fertile triploids was higher than that in diploids and tetraploids, and the fertility of triploids was still poor overall. However, some tetraploids in TT group would lose chromosomes during growth and become triploids, diploids or aneuploids. Tetraploid had the slowest growth and the lowest survival rate at the both sites, but its fertility was normal and could produce a large number of functional gametes, which could provide sufficient sperm for large-scale production of triploids. All these data support tetraploid ♂ × diploid ♀ is a good strain for commercial promotion in China. In conclusion, this study provides comprehensive data for understanding the performance of tetraploid and the aquaculture potential of triploid C. gigas, and also supports the commercial promotion of triploid C. gigas (tetraploid ♂ × diploid ♀) in China.