Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion rate was obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.

Gonadotropins are the key regulators of ovarian follicles development. They are applied in therapeutic practice in assisted reproductive technology clinics. In the present review we discuss the basic gonadotropic hormones – recombinant human follicle-stimulating hormone, its derivatives, luteinizing hormone and gonadotropin serum of pregnant mares, their origin, and application in ovarian follicle systems in in vitro culture systems.

Changes in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated egg cells and fusion products of pairs of one egg and one sperm cell of Zea mays L. have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'dihexyloxa-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjuction with computer-controlled video image analysis. In addition, confocal laser scannig microscopy has been used in conjuction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetra cycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+ in cytoplasm is also discrnible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of Cam in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Following in vitro fertilisation of single isolated eggs substantial changes in the Ca2+ levels occur which include an increase in the membrane Ca2+ of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products. Compared with isolated egg cell protolasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescene around the fusion product is visible after 16 h in in culture. The sperm cell chromatin in the fusion product is highly condensed, unlike that of the egg cell, and confocally imaged serial optical sections of the in vitro fusion product reveal the occurrence of karyogamy 35 min following gamete fusion. First visual evidence for intermingling of sperm nuclear chromatin in the zygotic nuclei is also provided.

Potassium (K+) channels are believed to regulate mammalian sperm acquisition of fertilising capacity. However, the molecular identity of these proteins in sperm has not been elucidated. In this report, using immunoconfocal and electron microscopy we show that a minimum of four different classes of K+ channels (Kv1.1, Kv1.2, Kv3.1 and GIRK1) are present and regionally distributed over the surface of mouse epididymal sperm. In addition, the use of reverse transcription polymerase chain reaction on RNA from mouse spermatogenic cells allowed the amplification of multiple transcripts corresponding to the channels identified by immunocytochemistry. Consistent with this, whole-cell patch-clamp recordings showed the expression of at least two different outwardly rectifying K+ currents in spermatogenic cells.

Although the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 °C for 1–2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocysts in vitro (C57BL/6J and B6D2F1/J) and liveborn pups in vivo (B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.

This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2–20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase ‘e’ and ‘p‘, respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen–thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen–thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient’s uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.

Heavy metals are omnipresent in the environment, and industrial use has greatly increased their presence in soil, water and air. Their inevitable transfer to the human food chain remains an important environmental issue as many heavy metals cause a range of toxic effects, including developmental toxicity. Administration of chromium VI (1 and 2 mg/kg as potassium dichromate) through intraperitoneal (i.p.) injection during organogenesis (days 6 to 15 of gestation) in rats revealed embryo- and fetotoxic effects. Reduced fetal weight, retarded fetal development, number of fetuses per mother and high incidences of dead fetuses and resorptions in treated mothers were also observed. Gross morphological abnormalities, such as displayed form of edema, facial defect, lack of tail, hypotrophy, severs subdermal haemorrhage patches and hypotrophy of placenta were observed in fetuses after chromium VI-treated mothers. A skeletal development of fetuses presented an incomplete ossification in nasal, cranium, abdominal or caudal bones in rats treated with 1 mg/kg of chromium, whereas rats treated with 2 mg/kg showed ossification and absence of the sacral vertebrae compared with the control. At a higher dose of chromium, histological changes were found in fetuses with atrophy of theirs vital organs. Placental histological observations revealed a pronounced morphological alteration, with atrophy of decidual cells, a degenerated of chorionic villi and hypertrophy of blood lacuna. The present study suggests a risk to the developing embryo when the mother is exposed to a high concentration of chromium VI during organogenesis.

Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellcida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors – TNFα and actinomycin D – at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFα. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFα and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.

The African catfish Clarias gariepinus Burchell 1822 is a favourite aquaculture fish in many parts of Africa and Asia because of its hardiness and fast growth rate. In this study, early, post-embryonic and larval developmental stages of C. gariepinus were examined chronologically and described. Photomicrographs of unfertilized matured oocytes from 0 min of fertilization through all cell stages to alevin, to complete yolk absorption, to free swimming larval stages are shown and documented live from lateral and top views, with the aid of a light microscope. Extruded oocytes had a mean diameter of 1 ± 0.1 mm, and possessed a thin perivitelline membrane whose space was filled with a protoplasmic layer. Heartbeat was in the range of 115–160/min prior to hatching. Hatchability rate was 85% and hatching occurred at 17 h at a controlled temperature of 28.5 ± 0.5°C, while ontogeny of the eyes and other organs were discernible. At day 4, larvae mean length was 9.3 ± 0.5 mm, exogenous feeding had commenced fully and melanophores spread cephalocaudally but were concentrated significantly on the head parts. This paper, for the first time, presents the significant chronological developmental stages of C. gariepinus embryology that will have significant implications for genetic manipulation and catfish seed production for aquaculture.

Intraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast–cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.

The purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.

The yolk syncytial layer (YSL) has been regarded as one of the main obstacles for a successful cryopreservation of fish embryos. The purpose of this study was to identify and characterize the YSL in Prochilodus lineatus, a fish species found in southeastern Brazil and considered a very important fishery resource. Embryos were obtained through artificial breeding by hormonal induction. After fertilization, the eggs were incubated in vertical incubators with a controlled temperature (28 °C). Embryos were collected in several periods of development up to hatching and then fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Morphological analyses were carried out under either light, transmission or scanning electron microscopy. The formation of the YSL in P. lineatus embryos starts at the end of the cleavage stage (morula), mainly at the margin of the blastoderm, and develops along the embryo finally covering the entire yolk mass (late gastrula) and producing a distinct intermediate zone between the yolk and the endodermal cells. The YSL was characterized by the presence of microvilli on the contact region with the yolk endoderm. A cytoplasmic mass, full of mitochondria, vacuoles, ribosomes, endomembrane nets and euchromatic nuclei, indicated a high metabolic activity. This layer is shown as an interface between the yolk and the embryo cells that, besides sustaining and separating the yolk, acts as a structure that makes it available for the embryo. The structural analyses identified no possible barriers to cryoprotectant penetration.

Embryonic stem cell technology is now well established in the mouse (reviewed by Robertson, 1987). This technology implies the isolation from the preimplantation embrao of a cell line (ES) that is cultured in vitro in an undifferentiated state. Embryonal carcinoma cells (EC) lines obtained from malignant tumours (Martin, 1975), together with all the information available on their culture requirements (reviewed by Heath, 1987), represented a very important starting point for the establishment of ES cells (Martin, 1981). ES cells share many characteristics with EC cells such as the ability to contribute to somatic tissues of animals obtained following injection of cells into a host blastocyst, to differentiate in vitro under appropriate stimuli (Rudnicki & McBurney, 1987) and to form retransplantable tumours. ES cells, however, have substantial advantages over EC cells in that they can be derived directly from a normal embryo, they maintain a normal karyotype and when reintroduced into a host blastocyst they can colonise the germ line (Bradley, 1987). ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent to the cells from differentiating. It has been shown that glycoproteins are responsible for this effect and these have been named according to their different activities: DIA, differentiation inhibitory activity (Smith & Hooper, 1987); LIF, leukaemia inhibiting factor (Smith et al, 1988; Williams et al, 1988); HILDA, human interleukin for DA cells (Moreau et al., 1988). It is now possible to establish and maintain ES cells in culture in the absence of feeders cells but in the presence of such factors (Nichols et al., 1990).

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethyl-aminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy-ethane-N,N,N',N' -tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probaly dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30–40s before the cortical contraction is triggered by fertilisation or ionomycin- induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium the triggered by te sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation eggs appears to involve two independent pathways involving calcium.

This report presents data from ultrastructural and morphometric studies on the germinal-body-like structures, nuage, nuage–mitochondrial clusters and chromatoid bodies in 4.5-day embryo cells and spermatogenic cells of the laboratory mouse Mus musculus. In the 4.5-day embryo cells the germinal-body-like structures that, according to previous data, arise by condensation of mitochondria in Graafian oocytes, were found not to undergo any ultrastructural alterations. In spermatogonia the germinal-body-like structures presumably were transformed into nuage that functioned as ‘intermitochondrial cement’ binding the mitochondrial clusters. In primary spermatocytes mitochondria aggregated by nuage were found with large vacuoles containing membraneous conglomerates that were obviously excreted by organelles into the cytoplasm. The chromatoid bodies that arose in spermatocytes and finally disintegrated in the posterior part of late spermatids seemed not to be implicated in the pathway of the germinal-body-like structure. The dispersion of chromatoid bodies was noted to be accompanied by excretion of membraneous conglomerates by late spermatid mitochondria. The spermatozoa were not found to contain either the germinal-body-like structures or any other germ-plasm-related structures.