Meiosis-I in Mesostoma ehrenbergii spermatocytes includes distance segregation and inter-polar movements of univalents, and vigorous oscillations of bivalents.

In this article, we describe meiosis-I in spermatocytes of the free-living freshwater flatworm Mesostoma ehrenbergii. The original observations of Oakley (1983, 1985) and Fuge (Eur J Cell Biol 44:294-298, 1987, Cell Motil Cytoskeleton 13:212-220, 1989, Protoplasma 160:39-48, 1991), the first to describe these cells, challenge our understanding of cell division, and we have expanded on these descriptions with the aim of laying the framework for further experimental work. These cells contain three bivalents and four univalent chromosomes (two pairs). Bivalent kinetochores oscillate vigorously and regularly throughout prometaphase, for up to several hours, until anaphase. Anaphase onset usually begins in the middle of the kinetochore oscillation cycle. Precocious cleavage furrows form at the start of prometaphase, ingress and then remain arrested until the end of anaphase. The four univalents do not pair, yet by anaphase there is one of each kind at each pole, an example of "distance segregation" (Hughes-Schrader in Chromosoma 27:109-129, 1969). Until proper segregation is achieved, univalents move between spindle poles up to seven times in an individual cell; they move with velocities averaging 9 µm/min, which is faster than the oscillatory motions of the bivalent kinetochores (5-6 µm/min), and much faster than the anaphase movements of the segregating half-bivalents (1 µm/min). Bipolar bivalents periodically reorient, most often resulting in the partner kinetochores exchanging poles. We suggest that the large numbers of inter-polar movements of univalents, and the reorientations of bivalents that lead to partners exchanging poles, might be because there is non-random segregation of chromosomes, as in some other cell types.

Methods for rearing Mesostoma ehrenbergii in the laboratory for cell biology experiments, including identification of factors that influence production of different egg types.

Mesostoma ehrenbergii spermatocytes are uniquely useful to study various aspects of cell division. Their chromosomes are large in size and few in number, with only three bivalent and four univalent chromosomes. During prometaphase, bipolar bivalents oscillate regularly to and from the poles for 1-2 hours. The univalents remain at the poles but occasionally move from one pole to the other. In addition, a precocious cleavage furrow forms during prometaphase and remains partially constricted until anaphase. Attempts to rear these animals indefinitely in laboratory conditions, however, have been mostly unsuccessful because of their reproductive strategy. M. ehrenbergii are hermaphroditic flatworms that can produce viviparous offspring (termed S eggs) and/or diapausing eggs (termed D eggs) and they follow either one of two reproductive patterns: (1) they first form S eggs and following the delivery of these eggs produce D eggs, or (2) they only produce D eggs. When only D eggs are formed, which is common under laboratory conditions, the stocks die out until the D eggs hatch, which is irregular and creates unpredictable wait times. Consequently, in order to maintain M. ehrenbergii stocks to study their spermatocytes, we examined various factors that might influence egg-type production. Feeding them daily and keeping them at 25°C favours S egg production. Currently, our cultures have reached the 53rd generation. We herein describe our rearing and dissection methods, and some experiments which led to our present rearing methods.

Mesostoma ehrenbergii spermatocytes--a unique and advantageous cell for studying meiosis.

Mesostoma ehrenbergii have a unique male meiosis: their spermatocytes have three large bivalents that oscillate for 1-2 h before entering into anaphase without having formed a metaphase plate, have a precocious ('pre-anaphase') cleavage furrow, and have four univalents that segregate between spindle poles without physical interaction between them, that is via 'distance segregation'. These unique and unconventional features make Mesostoma spermatocytes an ideal organism for studying the force produced by the spindle to move chromosomes, and to study cleavage furrow control and 'distance segregation'. We review the literature on meiosis in Mesostoma spermatocytes and describe our current research with Mesostoma spermatocytes, rearing the animals in the laboratory using methods that described in our companion article [Hoang et al. (2013); Cell Biol Int].

Measurements of forces produced by the mitotic spindle using optical tweezers.

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and inward movements of spindle poles after laser cuts across Potorous tridactylus (rat kangaroo) kidney (PtK2) cell half-spindles. Mesostoma spermatocyte kinetochores execute oscillatory movements to and away from the spindle pole for 1-2 h, so we could trap kinetochores multiple times in the same spermatocyte. The trap was focused to a single point using a 63× oil immersion objective. Trap powers of 15-23 mW caused kinetochore oscillations to stop or decrease. Kinetochore oscillations resumed when the trap was released. In crane-fly spermatocytes trap powers of 56-85 mW stopped or slowed poleward chromosome movement. In PtK2 cells 8-mW trap power stopped the spindle pole from moving toward the equator. Forces in the traps were calculated using the equation F = Q'P/c, where P is the laser power and c is the speed of light. Use of appropriate Q' coefficients gave the forces for stopping pole movements as 0.3-2.3 pN and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes as 2-3 and 6-10 pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700 pN measured previously in grasshopper spermatocytes.

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations.

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.