Human Amniotic Epithelial Stem Cells Alleviate Autoimmune Premature Ovarian Insufficiency in Mice by Targeting Granulosa Cells via AKT/ERK Pathways.

Autoimmune factors play an important role in premature ovarian insufficiency (POI). Human amniotic epithelial stem cells (hAESCs) have recently shown promising treatment effects on chemotherapy-induced POI. However, the therapeutic efficacy and underlying mechanisms of hAESCs in autoimmune POI remain to be investigated. In this study, we showed for the first time that intravenous transplantation of hAESCs could reside in the ovary of zona pellucida 3 peptide (pZP3) induced autoimmune POI mice model for at least 4 weeks. hAESCs could improve ovarian function and fertility, alleviate inflammation and reduce apoptosis of granulosa cells (GCs) in autoimmune POI mice. The transcriptome analysis of mice ovaries and in vitro co-cultivation experiments suggest that activation of the AKT and ERK pathways may be the key mechanism in the therapeutic effect of hAESCs. Our work provides the theoretical and experimental foundation for optimizing the administration of hAESCs, as well as the clinical application of hAESCs in autoimmune POI patients.

Female fertility and the mammalian egg's zona pellucida.

All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked fibrils that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along fibrils. Fibrils are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP fibrils fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 result in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.

A ZP1 gene mutation in a patient with empty follicle syndrome: A case report and literature review.

Genuine empty follicle syndrome (gEFS) is a rare cause of female infertility; it is defined as the presence of cumulus-oocyte complexes (COCs) in follicular fluid but the absence of oocytes after denudation in an in vitro fertilization (IVF) programme. Mutations in one of the four genes encoding zona pellucida (ZP) proteins have been implicated in gEFS. The objectives of the present study were to explore the molecular basis of idiopathic infertility in a 35-year-old woman with gEFS (observed after four ovarian retrievals), compare her phenotype and genotype with those of other patients described in the literature, and discuss therapeutic approaches that could be adopted by reproductive health centres in this situation. Sequencing of the ZP genes revealed a new homozygous missense variant in ZP1: c.1097G > A;p.(Arg366Gln). The variant is located in the ZP-N domain, which is essential for ZP protein polymerization. An immunohistochemical assessment of an ovarian biopsy confirmed the absence of ZP1 protein. The novel variant appears to prevent ZP assembly, which would explain the absence of normal oocytes after denudation in our patient (and despite the retrieval of COCs). ZP gene sequencing should be considered for patients with a phenotype suggestive of gEFS. An etiological genetic diagnosis enables appropriate genetic counselling and a switch to an IVF programme (with a suitable denudation technique) or an oocyte donation programme.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

Transglutaminase 2 crosslinks zona pellucida glycoprotein 3 to prevent polyspermy.

Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, penetration, and fusion. However, the molecular basis and underlying mechanism for the post-fertilization block to sperm penetration through ZP has not yet been determined. Here, we find that transglutaminase 2 (Tgm2), an enzyme that catalyzes proteins by the formation of an isopeptide bond within or between polypeptide chains, crosslinks zona pellucida glycoprotein 3 (ZP3) to result in the ZP hardening after fertilization and thus prevents polyspermy. Tgm2 abundantly accumulates in the subcortical region of the oocytes and vanishes upon fertilization. Both inhibition of Tgm2 activity in oocytes by the specific inhibitor in vitro and genetic ablation of Tgm2 in vivo cause the presence of additional sperm in the perivitelline space of fertilized eggs, consequently leading to the polyploid embryos. Biochemically, recombinant Tgm2 binds to and crosslinks ZP3 proteins in vitro, and incubation of oocytes with recombinant Tgm2 protein inhibits the polyspermy. Altogether, our data identify Tgm2 as a participant of zona block to the post-fertilization sperm penetration via hardening ZP surrounding fertilized eggs, extending our current understanding about the molecular basis of block to polyspermy.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte-called the inner perivitelline layer-is involved in sperm-zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1-4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte-granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

Adoptive transfers of CD4+ CD25+ Tregs partially alleviate mouse premature ovarian insufficiency.

This study was designed to investigate the protective effect of CD4+ CD25+ regulatory T cells (Tregs) against zona pellucida glycoprotein 3 peptide (pZP3) immunization-induced premature ovarian insufficiency (POI) in mice. A mouse POI model was induced by two subcutaneous injections of pZP3 (50 nmol/L). Mice in the pZP3-Treg group were intraperitoneally injected with 5 × 105 CD4+ CD25+ Tregs after the POI model was established. Sex hormone levels, follicle numbers, apoptotic events, and the Akt/FOXO3a signaling pathway molecules in the ovaries were assessed. Compared with control group, the weight of ovaries in both pZP3 group and pZP3-Treg group was decreased and no difference was found between them. The number of follicles in the Treg transferred mice, like in pZP3 group, was significantly reduced compared to the control group, but showed a modest improvement when compared the pZP3 group alone. Significantly lower serum concentrations of follicle-stimulating hormone, luteinizing hormone, and anti-zona pellucida antibodies (AZPAbs) were found, while the concentrations of estradiol and anti-Mullerian hormone increased. In mechanism, Treg cell transfer to ZP3 treated mice restored the levels of Caspase3 to control levels, and partially restored Bax, however, had no effect on Bcl-2. Moreover, Treg cell transfer to ZP3 treated mice partially restored the levels of Akt and FOXO3a, and partially restored the ratios of p-Akt/Akt and p-FOXO3a/FOXO3a. In conclusion, Treg cells improved some aspects of ZP3-induced POI which may be mediate by suppressing ovarian cells apoptosis and involving the Akt/FOXO3a signaling pathway. Therefore, Treg cells may be protective against autoimmune POI.

Mammalian egg coat modifications and the block to polyspermy.

Fertilization by more than one sperm causes polyploidy, a condition that is generally lethal to the embryo in the majority of animal species. To prevent this occurrence, eggs have developed a series of mechanisms that block polyspermy at the level of the plasma membrane or their extracellular coat. In this review, we first introduce the mammalian egg coat, the zona pellucida (ZP), and summarize what is currently known about its composition, structure, and biological functions. We then describe how this specialized extracellular matrix is modified by the contents of cortical granules (CG), secretory organelles that are exocytosed by the egg after gamete fusion. This process releases proteases, glycosidases, lectins and zinc onto the ZP, resulting in a series of changes in the properties of the egg coat that are collectively referred to as hardening. By drawing parallels with comparable modifications of the vitelline envelope of nonmammalian eggs, we discuss how CG-dependent modifications of the ZP are thought to contribute to the block to polyspermy. Moreover, we argue for the importance of obtaining more information on the architecture of the ZP, as well as systematically investigating the many facets of ZP hardening.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Immunomodulatory effect of human amniotic epithelial cells on restoration of ovarian function in mice with autoimmune ovarian disease.

Autoimmune ovarian disease (AOD) is considered to be a major cause of premature ovarian failure (POF). The immunomodulatory properties of human amniotic epithelial cells (hAECs) have been studied in many disease models. We previously reported that hAECs restored ovarian function in chemotherapy-induced POF mice, but the immunomodulatory mechanism of hAECs is still unclear. To investigate the effect of hAECs on recipient mice, especially on regulatory Treg cells, hAECs and hAEC-conditioned medium (hAEC-CM) were intravenously injected into AOD mice immunized with zona pellucida protein 3 peptides (pZP3). Ovarian function was evaluated through estrous cycle, hormone secretion, follicle development, and cell apoptosis analysis. Immune cells including CD3, CD4, CD8 and Treg cells in the spleens were tested by flow cytometry. To elucidate the effect of hAEC-CM on macrophage function, inflammation model in vitro was established in RAW264.7 cells induced by lipopolysaccharide (LPS). hAECs and hAEC-CM regulated estrous cycles, promoted follicle development, ameliorated cell apoptosis and fibrosis in ovaries of AOD mice. In addition, hAECs significantly reversed the decrease of pZP3-induced Treg cells in the spleens. In vitro, hAEC-CM significantly inhibited the inflammatory reaction induced by LPS in RAW264.7 cells via up-regulating the expression of M2 macrophage genes. Further study demonstrated that hAEC-secreted transforming growth factor-beta and macrophage inhibitory factor played important roles in the macrophage polarization and migration under inflammatory stimulation. Taken together, hAECs restored ovarian function by up-regulating Treg cells in the spleens and reduced the inflammatory reaction via modulating the activated macrophage function in a paracrine manner in the ovaries of AOD mice.

Human placenta-derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice.

Previous studies have shown that the ovarian failure in autoimmune-induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta-derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol-requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X-box binding protein 1 (XBP1), up-regulated 78 kDa glucose-regulated protein (GRP78) and caspase-12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis-induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation-mediating ovarian function recovery.

Structural and molecular analysis of the cancer prostate cell line PC3: Oocyte zona pellucida glycoproteins.

The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention.

Expression of zona pellucida 3 gene is regulated by 17α-ethinylestradiol in adult topmouth culter Culter alburnus.

Estrogen could lead to abnormal modulation or disruption of physical development, reproduction and sexual behavior in aquatic wildlife, especially in fish. Information on the toxicity of estrogens to native species in that can be used in site-specific risk assessments is scarce. In the present study, one zona pellucida 3 (ZP3) homologue termed CaZP3 was firstly identified from topmouth culter Culter alburnus, following its structural characteristics, tissue distribution and transcriptional modulation to 17α-ethinylestradiol (EE2) exposure were investigated. Meanwhile, vitellogenin (VTG) gene was employed to provide a comparison of the reactive ability to EE2 induction. The CaZP3 characterized with analogical functional domains such as ZP domain, SP, IHP, EHP, 12 cysteine residues, one N-linked glycosylation site and two conserved O-linked glycosylation sites and equal number of eight exons and seven introns with ZP3 counterparts of higher species. CaZP3 mRNA predominantly expressed in ovary, besides, highly expressed in female heart and male muscle and relatively high expressed in testis. CaZP3 has the lower reactive ability to EE2 induction in comparison with VTG, however, CaZP3 transcripts were significantly induced in gonads of both male and female culter by EE2 and could be used as an alternative biomarker to monitor EE2 activity. The present results supplement the database for toxicity of EE2, especially for fish species endemic to China and provide some useful information for the monitoring of EE2 activity in aquatic environment.

The Mouse Egg's Zona Pellucida.

All mammalian eggs are surrounded by a highly specialized extracellular matrix (ECM), called the zona pellucida (ZP), that functions before, during, and after fertilization. Unlike somatic cell ECM the mouse ZP is composed of three different proteins, ZP1-3, that are synthesized and secreted by growing oocytes and assembled into long interconnected fibrils. ECM or vitelline envelope (VE) that surrounds fish, reptilian, amphibian, and avian eggs also consists of a limited number of proteins all closely related to ZP1-3. Messenger RNAs encoding ZP1-3 are expressed only by growing oocytes at very high levels from single-copy genes present on different chromosomes. Processing at the amino- and carboxy-termini of nascent ZP1-3 permits secretion of mature proteins into the extracellular space and assembly into fibrils and matrix. Structural features of nascent ZP proteins prevent assembly within secretory vesicles of growing oocytes. Homozygous knockout female mice that fail to synthesize either ZP2 or ZP3 are unable to construct a ZP, ovulate few if any eggs, and are infertile. ZP1-3 have a common structural feature, the ZP domain (ZPD), that has been conserved through 600 million years of evolution and is essential for ZP protein assembly into fibrils. The ZPD consists of two subdomains, each with four conserved cysteine residues present as two intramolecular disulfides, and resembles an immunoglobulin (Ig) domain found in a wide variety of proteins that have diverse functions, from receptors to mechanical transducers. ZP2 and ZP3 function as receptors for acrosome-reacted and acrosome-intact sperm, respectively, during fertilization of ovulated eggs, but are inactivated as sperm receptors as a result of fertilization.

ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis.

ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD.

The bioactivities of the central segment of Zp2 polypeptide.

In order to understand the role of the protein zona pellucida 2 in fertilization, an antibody against a central segment of the zona pellucida 2 peptide, segment 190-505 (Z2eH), was prepared. The influence of the antibody on sperm-zona interaction was tested using the sperm-egg binding assay. The effect of the antibody on fertility was evaluated by passive immunization with anti-Z2eH antibody. Immunohistochemical assay showed that an antibody from rabbit reacted specifically with the natural zona pellucida on mouse ovarian sections. Immunofluorescence assay showed that the antibody bound specifically to the zonae pellucidae of the ovulated oocytes and 2-cell embryos after passive immunization. The antibody-treated oocytes bound capacitated sperm as control oocytes, passive immunization did not impede the action of sperm to fertilize the oocyte in vivo. These findings suggest that the central peptide of ZP2 (190-505) is immunogenic and contains zona pellucida-specific epitopes, however the central polypeptide might not be the crucial part from which to construct a functional domain to bind sperm.