Fine structure of the male reproductive system in two species ofHaplognathia sterrer (Gnathostomulida,Filospermoidea)

Summary The structure of the male reproductive systems of two species ofHaplognathia cf.lyra andH. cf.rosacea was described. The structure of the testes and the anterior portions of the sperm ducts in both species was found to be similar. However, considerable species differences were found between the structures of the glands and muscles associated with the reproductive systems. These were more elaborate inH. cf.lyra than inH. cf.rosacea. The former species possessed an H-shaped sperm duct gland, three distinct groups of penis muscles and a penis with two cell types and with a lumen. The latter species had paired sperm duct glands, no specialized penis muscles and a penis with only one cell type and without a detectable lumen. No open gonopore was observed in either species. The sperm presumably exit through a ventral tissue connection observed connecting the penis and the ventral epidermis. These findings were discussed in the light of Mainitz's (1977) theory concerning the primitive penis type within the Gnathostomulida.Abbreviations ap anterior-posterior penis muscles - bm basement membrane - csd common sperm duct - dl dorsal lumen of the penis - dp dorsal gland cells of the penis - dv dorsoventral muscles anterior to the penis - dw sperm duct wall cell - e epidermis - ex exit cell - g intestine - gl gut lumen - n nerve - p penis - sd sperm duct - sdg sperm duct gland - tw testes wall cell - vl ventral lumen of the penis - vp ventral gland cells of the penis This project was supported by NSF grant #GB 42211 (R.M. Rieger P.I.). The line drawings have been executed after our design by Ms. Linda McVay     

On the mechanism of sperm release in three gobiid fishes (Teleostei: Gobiidae)

Synopsis In fish, gamete release is commonly assumed to be synchronous in externally inseminating fishes. By collecting and counting the number of sperm and eggs released during separate matings in three demersal spawners, the mediterranean gobies, Zosterisessor ophiocephalus, Gobius niger, and Knipowitschia panizzae, we observed that gametes are released asynchronously. Males release sperm before females start laying their eggs. Sperm is released in the form of sperm trails laid on the nest surface; subsequently active spermatozoa leave the trails and move in the water for several minutes. Sperm trails consist of bands of viscous material in which sperm is embedded. In most cases eggs are not laid directly over the sperm trail, suggesting that sperm may contact the eggs after the latter are released in the water. Male sperm duct glands, seminal vesicles, known to secrete mucosubstances, are likely involved in the production of sperm trails. The possible influence of this mode of insemination on the mating style of marine gobies is discussed.     

A new species of Alboglossiphonia (Hirudinea: Glossiphoniidae) from Egypt

A new Alboglossiphonia species from Egypt is described and figured. This leech is placed in the genus Alboglossiphonia on the basis of having three pairs of eyes, diffuse salivary glands, seven pairs of crop caeca, and the attachment of its eggs to the venter. It differs from other Alboglossiphonia species with seven pairs of crop caeca in its combination of very prominent dorsal papillae, two annuli between the gonopores, lobed first six pairs of crop caeca, and paired ducts of male system at peak of activity with conspicuously differentiated sperm duct, seminal vesicle and ejaculatory duct regions.     

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca²⁺ and exocytosis of cortical alveoli (CABD). The sperm‐penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at ≤40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6‐dimethylaminopurine (6‐DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short‐lived protein kinase(s), sensitive to 6‐DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Dev. Genet. 25:137–145, 1999. © 1999 Wiley‐Liss, Inc.     

The Sperm‐surface glycoprotein,SGP, is necessary for fertilization in the frog,Xenopus laevis

To identify a molecule involved in sperm‐egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm‐surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti‐SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti‐SGP antibody recognized large molecules, with molecular masses of 65–150 kDa and minor smaller molecules with masses of 20–28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle‐binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm‐egg membrane binding and is responsible for the establishment of fertilization in Xenopus.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin. Mol. Reprod. Dev. 50:493–498, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

Ultrastructure of the Male Accessory Glands and Sperm Ducts of Cylindrotaenia hickmani(Cestoda,Cyclophyllidea)

Abstract The ultrastructure of unicellular accessory glands (= prostate glands) and external male ducts of the cestode Cylindrotaenia hickmaniare described. Accessory glands open into the lumen of the external common sperm duct (= external vas deferens). The gland cells contain abundant endoplasmic reticulum, Golgi bodies and secretory bodies, and have elongate necks that pierce the apical cytoplasm of the duct. Cell contact with the apical cytoplasm of the sperm duct is mediated by septate desmosomes. Accessory glands secrete spherical particles, with a diameter of approximately 70 nm, that adhere to spermatozoa. The roles of these accessory glands may relate to activity of the sperm or development of the female system after insemination. Paired sperm ducts arise from testes, and unite to form a common sperm duct. Each duct consists of a tubular anucleate cytoplasmic region which is supported by nucleated cytons that lie sunken in the parenchyma. The apical cytoplasm of the paired sperm ducts (= vasa efferentia) possesses apical microvilli and abundant mitochondria, but few other cytoplasmic features. The apical cytoplasm of the common sperm duct possesses sparse apical microvilli and numerous electronlucent vesicles. The male gonoducts form an elongate syncytium which is markedly polarized along the length of the ducts. The ducts also display apical–basal polarity in that sunken nucleated cytons support the apical cytoplasm which in turn has distinct basal and apical domains.     

Spernathecal morphology,sperm transfer and a novel mechanism of sperm displacement in the rove beetle,Aleochara curtula (Coleoptera,Staphylinidae)

Summary The mechanism by which sperm are transferred from the male's spermatophore to the female's storing cage is described for the rove beetle Aleochara curtula, emphasizing a novel mechanism of sperm displacement by competing males. The cuticular, U-shaped spermatheca is equipped with a valve structure and two sclerotized teeth. The tube of the spermatophore extends into the spermathecal duct through the guidance of the flagellum of the male endophallus. Further elongation of the spermatophore tube, however, occurs only after separation of the pair. A primary tube bursts at its tip after passing through the valve. Within the lumen of the primary tube, a second tube passes through the valve and continues to extend up to the apical bulb of the spermatheca, doubles back on itself and swells to form a balloon filling most of the spermatheca. The balloon of the spermatophore is pierced within the spermatheca by tooth-like structures pressed against the spermatophore through contraction of the spermathecal muscle. The same process of spermatophore growing and swelling is also observed in mated females. Sperm from previous copulations are backflushed through the valve and the spermathecal duct, indicative of last-male sperm predominance.Abbreviations ad adhesive secretion covering the sperm - sac am amorphous secretion of the spermatophore - as ascending portion of the spermatophore - ds descending portion of the spermatophore - end parts of the male endophallus - ext extended tube - f flagellum - gs genital segment - lt large tooth - m muscle of the spermatheca - nsc non sclerotized cuticle - op opening of the spermathecal gland - pt primary tube - sc sclerotized cuticle - sd spermathecal duct - se secretion of the spermathecal gland - sf secretion flowing out of the primary tube - sg spermathecal gland - sm sperm - smt small tooth - sp spermatheca - ss sperm sac - st secondary tube - vm vaginal muscle     

The anatomy and functional morphology of the large hermaphroditic duct of three species of Aplysia, with special reference to the atrial gland

The anatomy and functional morphology of the large hermaphroditic duct of three species of gastropod mollusc (Aplysia californica, A. dactylomela, and A. brasiliana) were examined. Each duct is composed of two parallel compartments, the red hemiduct (RHD) and the white hemiduct (WHD), which are distinguishable from the outside of the duct. Four secretory regions, all exocrine in morphology, are recognizable: the RHD secretory epithelium, the atrial gland (or atrial gland-like epithelium), the WHD secretory epithelium, and the accessory gland of the copulatory duct (AGCD). Of these regions, only the atrial gland (or atrial gland-like epithelium) contains egglaying activity and only the atrial gland (or atrial gland-like epithelium) is immunocytochemically labeled by serum antibodies generated against low molecular weight A. californica atrial gland peptides. The RHD is the functional oviduct: the egg cordon passes through a channel lined by the RHD secretory epithelium and bordered by the atrial gland (or atrial gland-like epithelium); the eggs are separated from both the WHD secretory epithelium and the AGCD by internal folds of the duct. The WHD is the functional copulatory duct: the penis, exogenous sperm, and endogenous sperm pass directly by the AGCD and in close proximity to the WHD secretory epithelium; they are separated from both the RHD secretory epithelium and the atrial gland (or atrial gland-like epithelium) by internal folds. The atrial gland (or atrial gland-like epithelium) is thus not likely to have a prostatic function or to be directly stimulated by the penis during copulation; it may play a role in oviductal function.     

Asymmetric conspecific sperm precedence in relation to spawning times in the Montastraea annularis species complex (Cnidaria: Scleractinia)

In broadcast spawners, prezygotic reproductive isolation depends on differences in the spatial and temporal patterns of gamete release and gametic incompatibility. Typically, gametic incompatibility is measured in no‐choice crosses, but conspecific sperm precedence (CSP) can prevent hybridization in gametes that are compatible in the absence of sperm competition. Broadcast spawning corals in the Montastraea annularis species complex spawn annually on the same few evenings. Montastraea franksi spawns an average of 110 min before M. annularis, with a minimum gap of approximately 40 min. Gametes are compatible in no‐choice heterospecific assays, but it is unknown whether eggs exhibit choice when in competition. Hybridization depends on either M. franksi eggs remaining unfertilized and in proximity to M. annularis when the latter species spawns or M. franksi sperm remaining in sufficient viable concentrations when M. annularis spawns. We found that the eggs of the early spawning M. franksi demonstrate strong CSP, whereas CSP appears to be lacking for M. annularis eggs. This study provides evidence of diverging gamete affinities between these recently separated species and suggests for the first time that selection may favour CSP in earlier spawning species when conspecific sperm is diluted and aged and is otherwise at a numeric and viability disadvantage with heterospecific sperm.     

Molecular Reproduction & Development Volume 78, Issue 7 cover image

Ascidians are hermaphrodites, releasing sperm and eggs nearly simultaneously, but several species including the ascidian (prochodate) Ciona intestinalis are self‐sterile (self‐incompatible). In this species, the self‐incompatibility system is mediated by the vitelline coat ligand v‐Themis‐A/B and the sperm‐side receptor s‐Themis‐A/B. In this issue, it is described that a sperm GPIanchored protein CiUrabin in lipid rafts may play a key role in the primary binding of sperm to the vitelline coat. This image of a C. intestinalis egg was taken by Takako Saito.     

Reproductive histology of Tomeurus gracilis Eigenmann, 1909 (Teleostei: Atherinomorpha: Poeciliidae) with comments on evolution of viviparity in atherinomorph fishes

Tomeurus gracilis is a species long considered pivotal in understanding the evolution of livebearing in atherinomorph fishes. Tomeurus gracilis is a zygoparous or embryoparous poeciliid: internal fertilization is followed by females laying fertilized eggs singly or retaining fertilized eggs until or near hatching. Tomeurus was hypothesized as the sister group of the viviparous poeciliids until it was proposed as a close relative of a derived viviparous poeciliid, Cnesterodon, hence nested among viviparous taxa rather than near the root of the tree. Here, we describe and compare reproductive morphological characters of the little‐known Tomeurus with those of representative atherinomorphs. In Tomeurus and Cnesterodon, sperm are packaged in naked sperm bundles, or spermatozeugmata, in a configuration considered here diagnostic of viviparous poeciliids. Testes are single and free sperm are stored in the ovary in both taxa in contrast to oviparous atherinomorphs in which testes are paired and sperm are not packaged and not stored in the ovary. Efferent ducts in Cnesterodon testes and other viviparous poeciliids have a PAS‐positive secretion demonstrating presence of a glycoprotein that inactivates sperm or prevents final sperm maturation. No PAS‐positive staining secretion was observed in Tomeurus or oviparous atherinomorphs. Tomeurus shares apomorphic reproductive characters, such as sperm bundle and testis morphology and a gonopodium, with viviparous poeciliids and plesiomorphic characters, such as a thick zona pellucida with filaments, with oviparous taxa. We do not postulate loss or reversal of viviparity in Tomeurus, and we corroborate its phylogenetic position as sister to the viviparous poeciliids. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non‐naturally occurring AMPs. We have used a backbone‐cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta‐hairpin‐like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram‐negative, Escherichia coli but improved the activity against gram‐positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram‐negative versus gram‐positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram‐positive bacterium but less active against gram‐negative bacterium. Our results show that there is not always a correlation between improved hairpin‐structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Ultrastructure of the Genital Organs in the Interstitial Syllid Petitia amphophthalma (Annelida,Polychaeta)

Abstract The gonochoristic syllid Petitia amphophthalma is one of the truly interstitial polychaetes. P. amphophthalma does not show any epitokous modifications at maturity such as those that usually occur in syllids. The reproductive structures are unique: the male genital organs consist of a seminal vesicle in chaetigers 6–10, subdivided into a dorsal part tightly filled with spermatozoa and a ventral part with contents in different stages of spermatogenesis, one pair of sperm ducts and conspicuous gland cells situated in chaetigers 10 and 11. Their glandular secretions are discharged into the sperm duct together with those of other types of gland cells that form the duct. The oocytes develop freely within the body cavity of the females. Each of the fertile segments possesses a paired oviduct ending in a large ciliated funnel. Sperm ducts and oviducts are probably modifications of excretory organs; nephridia are absent in segments where gonoducts occur. A direct sperm transfer by lytic opening of the integument of the female and internal fertilization are inferred. Copyright © 1996 Published by Elsevier Science Ltd on behalf of the Royal Swedish Academy of Sciences     

Flow cytometry for assessing the efficacy of interspecific gynogenesis induction in sturgeon

The efficacy of ploidy analysis for separating progeny of Siberian sturgeon Acipenser baerii after induced gynogenesis was demonstrated using sperm of a paternal species differing in ploidy level from the maternal species. Gynogenesis was induced in tetraploid A. baerii with UV‐C irradiated sperm from the diploid sterlet Acipenser ruthenus and vice‐versa. The success of sperm UV irradiation and diploidy restoration by heat‐shock was estimated based on the ploidy level of progeny, confirmed by microsatellite parentage assignment. Hatching rates of interspecific gynogenotes were comparable with rates reported for gynogenesis induction using sperm and eggs of the same species. Juvenile mortality was similar to that observed in the control hybrids. The efficiency and reliability of this method may foster its use for production of gynogenotes in aquaculture, potentially allowing interspecific gynogenesis to replace intraspecific.     

Male age and strain affect ejaculate quality in the Mexican fruit fly

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass‐reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass‐reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass‐reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass‐reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.