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In developing ovarian oocytes of 2-day-old, 12-day-old and adult hamsters, oxidative enzyme activity as demonstrated by the cerium technique is strongest at 12 days and occurs principally in those oocytes which have begun rapid growth. The reaction is associated with the outer mitochondrial membrane, nearby cytoplasm, endoplasmic reticulum and nuclear envelope. Once antrum formation has begun and the mitochondria have reached the dormant (storage) stage, cerium reactivity ceases. In adults, reaction occurs at the same stages of oocyte development but is weaker and limited to the mitochondria and their immediate vicinity.

Osmium-pyroantimonate solutions for the precipitation of cations are unsuitable for use with delicate mammalian oocytes. A variant of the pyroantimonate technique employing a mixture of pyroantimonate and glutaraldehyde has been found to give successful and repeatable results if a fixation time of 4 hr is used. Calcium-containing antimonate precipitates were localized principally in nuclei, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and cytoplasmic processes of both oocytes and follicle cells, and along the plasma membrane in small oocytes. Deposits were also concentrated around the periphery of lipid droplets in the follicle cells. The presence of calcium in the precipitates was confirmed by x-ray microprobe analysis.

The differentiation of the endoplasmic reticulum (ER) in the developing oocyte of the golden hamster is accompanied by changes in susceptibility to impregnation with a zinc iodide-osmium tetroxide (ZnOs) mixture. The staining of two of the three categories of oocyte ER is first seen at or about the time when rapid oocyte growth is initiated. Staining reaches a peak before antrum formation, then declines. A third category of ER remains unstained at all stages. Aberrant reactivity to ZnOs is seen on oocytes which become atretic, and differs with the stage of oocyte development at which atresia occurs. Relationships between the three categories are described, and an attempt made to relate changes in form and distribution to developmental processes. The frequent contact/continuity between ER and mitochondria is discussed with regard to its possible role in lipid metabolism.

A coating of electron dense material is present on the cytoplasmic surface of outer mitochondrial membranes in medium-sized hamster oocytes. The coating is not present at earlier or later stages of oocyte development. Its possible relationship to the synthesis and transport of mitochondrial protein is discussed. Associations between endoplasmic reticulum, ribosomes, glycogen and the outer mitochondrial membrane are also described and discussed.

Endoplasmic reticulum complexes consisting of up to twenty layers of rough cisternae interleaved with rounded vesicles of semismooth membrane have been observed in large oocytes of the hamster in animals older than 3 weeks of age. The appearance of the complexes varies with the mode of tissue preparation. The possible function of the complexes is discussed.

Circumstantial evidence suggests that sodium cacodylate may be a far greater health hazard than has been generally assumed. Until further information is available, electron microscopists should see that extreme care is exercised during the preparation of cacodylate buffers by their staff.

Phosphorus-containing standards for use during quantitative electron probe X-rays microanalysis of ultrathin tissue sections can be quickly and simply prepared using organo-phosphorus compounds which are soluble in Spurr's epoxy resin mixture. Trials of five such compounds (one solid, the rest liquid) are described.

A cytochemical study of the Golgi apparatus in the developing oocyte of the golden hamster was carried out using the TPPase, AcPase and zinc iodide-osmium tetroxide (ZnOs) techniques. Tissue from both immature and sexually mature animals was investigated. Peak TPPase activity was found in pre-growth oocytes in ovaries from sexually mature adults. Some activity was also present in SER in the peripheral cytoplasm of growing oocytes. AcPase activity was found only after the onset of oocyte growth. It was present in Golgi cisternae and associated vesicles and in some profiles of peripheral SER. No structures corresponding to GERL were identified. Strong staining with ZnOs was seen, at all stages studied, in certain Golgi vesicles and short tubules but not in the cisternae unless the oocyte was atretic. Weaker ZnOs staining was characteristic of ER throughout the oocyte. With all techniques there was a falling off of reactivity as oocyte size increased. Within a single oocyte some Golgi bodies were negative while others were positive, with both TPPase and AcPase techniques. This suggests that two or more functional types of this organelle are present within the developing oocytes.

When thiamine pyrophosphate is used as substrate, heavily stained complexly branching cytoplasmic structures are seen in developing oocytes of the hamster between 8 and 12 days after birth. This coincides with the onset of the dictyate stage of first meiotic prophase. The structures appear to arise from the oocyte plasma membrane in two ways: (1) by proliferation and infolding of the membrane, and (2) by membrane being pushed into the cytoplasm by invaginating follicle cell processes. This appears to be followed by proliferation of the invaginated plasma membrane within the oocyte cytoplasm. Incubations with ATP or beta-glycerophosphate, with and without the inhibitor L-p-bromotetramisole, indicate that the staining is not due to the action of either ATPase or alkaline phosphatases. The origin and possible developmental significance of the reactive structures are discussed.

Size variations and ultrastructural changes in mitochondria of developing germ cells of the female hamster were analyzed. Mitochondria in oogonia of foetus and newborn were elongate with transverse cristae. During pre-dictyate meiotic prophase they became small, rounded, and electron-dense with pleomorphic cristae. These changes were largely reversed when dictyate was reached. Maximum mitochondrial size and complexity of cristae were reached just at the beginning of the phase of rapid oocyte growth, and thereafter declined. As mitochondrial size and number of cristae decreased in the rapidly enlarging oocyte, the ratio of length to width increased, as did electron density of the matrix, until the formation of an antrum within the follicle. After antrum formation, the mitochondria again became more rounded and cristae were seldom seen. An attempt is made to correlate changes of mitochondrial morphology with other events occurring during oogenesis.

Ovaries of immature and adult hamsters were incubated in medium containing thiamine pyrophosphate (TPP) to determine the age at which TPPase-reactive cytoplasmic structures first appear in the germ cells, and at what age the structures cease to be present. The structures were found only in oocytes from animals 8-15 days of age. They occur in predictyate germ cells in polyovular follicles and in very early dictyate oocytes in unilaminar follicles. The TPPase-reactive structures were never observed in atretic oocytes, in unilaminar follicles of adult animals, nor in multilaminar follicles of animals at any age. Ovaries of 8-12-day-old animals and adults were then incubated in media in which one of the following substrates was substituted for TPP: uridine diphosphate (UDP), inosine diphosphate (IDP), and adenosine monophosphate (AMP). Half of the samples in each experiment were incubated in medium containing the inhibitor L-p-bromotetramisole. beta-Glycerophosphate was used in control incubations, or the substrate was omitted entirely. The cytoplasmic structures were found to be reactive after incubation in UDP-containing media, but not after incubation in media containing AMP. With IDP as substrate, reactions were atypical and confined to peripheral regions of the cytoplasm. Other sites of enzyme activity after incubation with the various substrates (cell membranes, zona pellucida, endoplasmic reticulum and Golgi apparatus) are also described and discussed.

Paired cisternae of rough endoplasmic reticulum linked together by two parallel structures exhibiting periodic striations have been observed in the cytoplasm of small pre-antrum oocytes in the golden hamster. They are present only in oocytes from animals older than 3 weeks of age. Two or more such pairs may be associated with one another, and similar structures have been observed in contact with the nuclear envelope. The peak incidence of the paired membranes coincides with a sharp increase in rough endoplasmic reticulum as the oocyte commences rapid growth. A suggested role for the paired membranes in production of new endoplasmic reticulum is discussed.