Centrifugation of 2-cell mouse ova: cytoplasm stratification and recovery

Summary Two-cell mouse ova, which were centrifuged for l h at 70 000–90 000xg, showed a precise stratification of the cytoplasm and an elongation of the nucleus. The ova were fixed at different times and observed by light and electron microscopy using cytochemical methods and detergent extractions. Within 40 min after centrifugation the normal-looking morphology was recovered except for the persisting lipid caps at the centripetal poles of the blastomeres. Cleavage, compaction and blastulation were not prevented by centrifugation. Treatments with colcemid or cytochalasin D delayed but did not impair recovery. These results suggest that a resilient cytoskeletal structure may be involved in this kind of embryonic regulation.     

Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa

In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

Entry of immotile spermatozoa into zona-free hamster ova

We studied six men whose spermatozoa were immotile and possessed a variety of sperm tail structural abnormalities by electron microscopy. The semen of all six subjects had a normal percentage of oval forms and sperm undergoing capacitation and acrosome reaction. Despite the absence of motility, when incubated sperm from these subjects was added to a microdrop of medium containing zona pellucida-free hamster ova, sperm penetration or entry into the cytoplasm of from 1–9% of the eggs was evident with phase contrast microscopy. This latter finding suggests that, at least in this system, oocytes actively facilitate sperm incorporation. Penetration was absent when sperm of fertile men were rendered immotile, though still viable, by heat treatment.     

Fertilizability of cryopreserved zona-free hamster ova

Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at ?50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to ?50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at ?50°C for up to four months. Thawing was performed at 2–4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (?50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm–egg interaction or in the clinical assessment of human sperm quality.     

中国林蛙卵人工孵化试验

中国林蛙 (Rana chinensis)是我国重要的经济蛙类之一。近年来 ,由于人们的生态经济意识不强 ,森林环境破坏严重 ,以及滥捕乱捉 ,使得中国林蛙种群数量急剧下降。但在一些地区 ,由于加强对中国林蛙的管护 ,大力发展林蛙人工养殖业 ,使之成为当地经济发展的一项重要支柱产业。由于林蛙人工饲养刚刚起步 ,饲养经验不完善 ,一些问题有待深入研究。如在自然条件下母蛙产卵时期 (4月上旬 )大致相同 ,但据以往观察 ,幼蛙出池时间却有很大差别 ,而出池时间的早晚表现在秋季回归时的体重差别较大 (约 1倍体重 )。因此缩短卵孵化时间 ,促使幼蛙提早…     

Short-term storage of ova of common carp and tench in extenders

In a study of the effect of short-term storage on the hatching rate of common carp Cyprinus carpio and tench Tinca tinca ova in vitro in various extenders at 21° C under aerobic conditions, the best extender for 30 min storage for common carp appeared to be Dettlaff 1. This gave the same hatching rate as controls without extender (55% v. 56%). For 60 min storage of ova, the best extenders were Dettlaff 2 (24% hatching rate) and Dettlaff 3 (30%), but hatching was significantly lower than in the control (58%). In carp ovarian artificial fluid (CAF) extender, the hatching rate of common carp ova was also high after 10 min, but decreased to 12% after 30 min. In tench, the hatching rate of ova increased after 10 min storage in Dettlaff 5 extender (44%) compared to the control (41%) without extender. However, it was significantly lower after storage in Dettlaff 1, 2, 3, 4, 5 and CAF extenders for 20, 30 and 60 min, compared to controls. Malformations (10–50%) were observed in the tench second control groups without extender after 10, 20 and 30 min storage of ova.     

Ultrastructure of pig tubal ova

Summary The unfertilized ova of the pig are characterized by the first polar body situated in the perivitelline space. The metaphase chromosomes of the ova are found free in a cortical area, predominantly inhabited by the spindle fibers. Mitochondria show morphological changes in the form of swelling of their matrices. Frequently, the membranes of the individual cristae mitochondriales meet each other, forming meeting points, at regular intervals. The endoplasmic reticulum increases in quantity when compared with that of the pig follicular oocytes (Norberg, 1972b). The Golgi complexes are sparse and scattered. Occasionally, remnants of the end bulbs of the corona radiata cell processes occur below the surface membrane of the ova.Usually, the sperm-penetrated ova contain the first and the second polar body within the perivitelline space. Intranuclear annulate lamellae are observed within the male and female pronucleoplasm, and of particular interest are extended linear structures in one of the pronuclei. These structures may be considered as precursor stage in the formation of the intranuclear annulate lamellae. The parapronuclear cytoplasm is rich in organelles, especially the cytoplasmic annulate lamellae. In contrast to the scarcity of Golgi complexes in the unfertilized ova, many newly formed Golgi vesicles and lamellae reappear in the pronuclear stage. The zona pellucida displays ultrastructural changes following sperm penetration of the ova.This work was supported by the Agricultural Research Council of Norway.     

Transgenic medaka enables easy oocytes detection in live fish

Easy oocyte detection in living specimens benefits various developmental biology and environmental toxicology studies. One of the earliest markers of sex differentiation in medaka (Oryzias latipes) is oocyte development. Within the field of toxicology, a simple detection method for induced oocyte in the testis (testis-ova) as a result of endocrine disruption is necessary. In this study we produced transgenic medaka whose oocytes were labeled with fluorescent proteins using the regulatory region of the 42Sp50 gene, an isoform of polypeptide elongation 1-alpha. Short (201 nt) 5'- and 3'-flanking regions were sufficient for reporter gene expression. GFP expression was first observed in female germ cells approximately 5 days post-hatching. In the mature ovaries, germ cells showed such intense fluorescence that the fluorescence was observed from outside the body wall. In contrast, very faint fluorescence was observed in the mature testes. Testis-ova, oocytes artificially induced in the testes, were also labeled with GFP. These findings indicate through the use of transgenic medaka, that detection of female germ cells was straightforward and this transgenic medaka model proves useful for tracking female germ cells in developmental and toxicological studies.     

Eimeria taterae sp. n. and other Intestinal Parasites from the Antelope Rat, Tatera indica in Baghdad District

SYNOPSIS. Of 8 fecal specimens from the antelope rat, Tatera indica Hardwicke examined, 4 were found positive for intestinal parasites. Among these was Eimeria taterae sp. n., sporulated oocysts of which are described, their characteristics serving for the diagnosis of this species. Entamoeba coli cysts, as well as ova of Hymenolepis sp. and Trichuris sp., were also found.