Production Line Hypothesis Research Articles

Examining Variation in Recombination Levels in the Human Female

The risk of aneuploidy increases dramatically with maternal age. The likelihood of having a clinically recognized trisomic pregnancy is 2% to 3% for women in their 20s but increases to greater than 30% for women in their 40s. The basis for the increased risk of aneuploidy in older women is unknown. One of the most cited potential mechanisms is the production-line hypothesis, which was initially proposed in 1968. The hypothesis has 2 key components. First, it assumes that the first oocytes to enter meiosis are the first to be ovulated and that those entering meiosis last are ovulated at the end of the reproductive lifespan. Second, it assumes that the first oocytes entering meiosis have more recombination events (crossovers) than do those entering meiosis later in fetal life. Experimental evidence has been consistent with the first tenet of the production-line hypothesis: Radiolabeling studies in rodents have demonstrated that that there is a production line; the first oocytes to enter meiosis are the first to be ovulated. However, the second tenet (the predicted relationship between recombination levels and maternal age), which assumes a difference in recombination levels between oocytes entering meiosis early in fetal life and those entering late in fetal life, remains unproven. The aim of this study was to test the second tenet of the production-line hypothesis by examining meiotic recombination in human fetal oocytes. Molecular cytogenetic analysis of second-trimester human fetal ovaries was used to directly examine the number and distribution of crossover-associated proteins in prophase-stage oocytes. A total of 8518 eggs from 191 ovarian samples were collected from second-trimester elective abortions. Crossover-associated proteins in prophase-stage oocytes were examined with immunofluorescence. A high incidence of abnormal cells was found in aging women, but just as many was found in younger women. There was considerable variation in crossovers within and among women, but no evidence for a relationship of recombination-associated changes with maternal age. This study provides a direct test of the second tenet of the production-line hypothesis and demonstrates that it is not the basis for the maternal-age effect on aneuploidy.

Examining Variation in Recombination Levels in the Human Female: A Test of the Production-Line Hypothesis

The most important risk factor for human aneuploidy is increasing maternal age, but the basis of this association remains unknown. Indeed, one of the earliest models of the maternal-age effect--the "production-line model" proposed by Henderson and Edwards in 1968--remains one of the most-cited explanations. The model has two key components: (1) that the first oocytes to enter meiosis are the first ovulated and (2) that the first to enter meiosis have more recombination events (crossovers) than those that enter meiosis later in fetal life. Studies in rodents have demonstrated that the first oocytes to enter meiosis are indeed the first to be ovulated, but the association between the timing of meiotic entry and recombination levels has not been tested. We recently initiated molecular cytogenetic studies of second-trimester human fetal ovaries, allowing us to directly examine the number and distribution of crossover-associated proteins in prophase-stage oocytes. Our observations on over 8,000 oocytes from 191 ovarian samples demonstrate extraordinary variation in recombination within and among individuals but provide no evidence of a difference in recombination levels between oocytes entering meiosis early in fetal life and those entering late in fetal life. Thus, our data provide a direct test of the second tenet of the production-line model and suggest that it does not provide a plausible explanation for the human maternal-age effect, meaning that-45 years after its introduction-we can finally conclude that the production-line model is not the basis for the maternal-age effect on trisomy.

Oocyte Family Trees: Old Branches or New Stems?

Oocyte Family Trees: Old Branches or New Stems?

Timing of activation of primordial follicles in mature rats is only slightly affected by fetal stage at meiotic arrest.

The objective of this experiment was to test the production-line hypothesis by determining whether timing of activation of primordial follicles at different ages throughout the life of female rats was associated with embryonic age at meiotic arrest of their oogonia. Meiotic arrest of oogonia can occur at any time during mid to late gestation in rats. Pregnant rats received injections of 5-bromo-2'-deoxyuridine (BrdU) on either e17 (treatment 1) or on e19 (treatment 2) (e1 = embryonic day 1 [sperm-positive day 1]). Female offspring (pupping = Day 0) were killed at 30 (6/group), 100 (8/group), 180 (8/group) and 350 (7/group) days of age. Immunohistochemistry was used to determine which primordial follicles contained oocytes and pregranulosa cells that were undergoing DNA synthesis at the time of BrdU injection. The percentage of primordial and primary follicles labeled with BrdU was higher (p < 0.05) at all ages with e17 than with e19 injections. There was a significant (p < 0.001) treatment-by-age interaction in the number of BrdU-containing growing follicles. Growing follicles, however, were found at all ages from both e17 and e19 injections. It is concluded that gestational day of entry into meiotic arrest does influence age of activation of primordial follicles, but it is a minor effect.

Separation anxiety: the etiology of nondisjunction in flies and people.

Two new studies examine the recombinational history of human chromosomes that nondisjoin at the first meiotic division in females. Our analysis of these studies suggests two possible etiologies of nondisjunction in terms of well-understood properties of chromosome mechanics. For both the X chromosome and for chromosome 21, 60-70% of nondisjoined chromosomes are derived from chiasmate bivalents, many of which display unusual patterns of exchange. The patterns of exchange and nondisjunction observed for human chromosome 21 parallel those exhibited by a mutation in Drosophila that impairs spindle assembly and function. Based on these similarities, we propose that nondisjunction of chromosome 21 in human females results from an age-dependent loss of spindle-forming ability. The recombinational histories of nondisjoining human X chromosomes are quite different from those of chromosome 21, but rather parallel those obtained for spontaneous nondisjunction in Drosophila females. The data for X chromosome disjunction in both species can be explained by a model in which nondisjunction is the consequence of the age-dependent movement of transposable elements. According to this model, nondisjunction is explained as the consequence of the repair of transposon-induced breaks in the DNA. Both models provide reasonable alternatives to biologically implausible explanations such as the 'production line hypothesis'.

A test of the production line hypothesis of mammalian oogenesis

Germ cells in female mammals become committed to meiosis and enter its prophase sequentially in fetal life and, according to the Production Line Hypothesis, the oocytes thus generated are released after puberty as mature ova in the same sequence as that of meiotic entry in fetu. This hypothesis in its original and complete form has a subordinate proposition that concerns chiasma (Xma) frequency; it postulates that Xma number would decrease with fetal age. Consequently, univalents would increase, leading to errors of chromosome disjunction at the first meiotic division (MI), and thus to maternal age-dependent numerical chromosome anomalies. By using an in vitro/in vivo approach, we radioactively labelled the DNA of germ cells at premeiotic synthesis as they sequentially entered meiosis, while the fetal ovaries were in culture. At the end of this in vitro phase, pachytene/diplotene (P/D) stages were studied to determine their labelled fraction. The ovaries were then transplanted to spayed females and, after the in vivo phase, mature ova were harvested and the proportion of labelled first and second meiotic metaphases (MI/MII) determined. By marking the germ cells with label while in vitro during periods equivalent to early and late gestation, and by comparing the observed proportions of labelled MI/MII with those of oocytes labelled at P/D, we concluded that, in the mouse, ova do not mature at random for release, but are formed according to a production line system in which the time of release after puberty is related to the time of entry into meiosis in fetu.

Further examination of the production-line hypothesis in mouse foetal oocytes. II. T(14; 15)6Ca heterozygotes.

Pachytene oocytes from foetal mice heterozygous for the translocation T(14; 15)6Ca were screened for evidence of a "production-line" effect on chromosome pairing. Metaphase I oocytes from adult heterozygotes were also examined to determine whether any such effect on pahytene chromosome pairing is subsequently repeated during adult reproductive life as anticipated by the production-line hypothesis. It was found that as gestation proceeded the proportion of pachytene oocytes with a translocation quadrivalent declined and that with a trivalent and univalent correspondingly increased. That is, there was evidence of variation in pairing behaviour of the translocation at different times of gestation. In contrast, the proportions of metaphase I cells with either a quadrivalent or a trivalent plus univalent did not vary between adult females of different ages. Thus if the variation observed at pachytene was the result of a production-line effect, clearly this was not reflected in the behaviour of the translocation at metaphase I. Our observations therefore do not support the production-line hypothesis for the maternal age effect on nondisjunction.

Chromosome segregation at meiosis I in female T(2;4)1Gö/+ mice: no evidence for a decreased crossover frequency with maternal age.

The influence of age and hormones on chromosome segregation at meiosis I was studied in female mice heterozygous for the T(2;4)1Gö translocation. Females of two age groups (18-22 and 40-56 weeks old) were stimulated for ovulation with different doses of gonadotropins (1.5 IU PMS/1.0 IU HCG or 10 IU PMS/10 IU HCG). Analysis of metaphase II oocytes revealed the highest level of hyperhaploidy (1.8%) and presegregation (4.4%) in the young females receiving the low dose. Presegregation preferentially affected the small 4(2) marker chromosome. There was no significant interference of the tetravalent with disjunction of the nontranslocated normal bivalents. Moreover, no remarkable difference in the mode of segregation (adjacent I, II or alternate) was observed. Recombination within the interstitial pairing segments of the chromosomes involved in the translocation allowed us to calculate cross-over frequencies in ovulated oocytes. For both the large 2(4) and the small 4(2) marker chromosomes, this frequency was higher in old than in young T(2;4)1Gö/+ females. Our data do not support the production line hypothesis of Henderson and Edwards (1968) which claims that chiasma frequency in oocytes decreases with maternal age.

Further examination of the production-line hypothesis in mouse foetal oocytes. I. Inversion heterozygotes.

Chromosome pairing has been examined in foetal oocytes of mice heterozygous either for an X-linked inversion, In(X)1H, or an autosomal inversion, In(2)2H. The patterns of chromosome pairing have been screened systematically in foetuses of different gestational ages in a search for a "production-line" effect particularly affecting the inversion-bearing bivalents. The proportion of pachytene oocytes with a loop fell with increasing gestational age for both inversions. The decrease was linear for In(X)1H but best described by a quadratic function for In(2)2H. Examination of late zygotene cells and a comparison of loop frequency in early, mid and late pachytene oocytes suggested this age-related decrease to be principally due to synaptic adjustment and not to a production-line effect. However, two particular observations were somewhat at variance with this conclusion. Firstly, in In(X)1H heterozygotes, the presence of an inversion loop and the occurrence of partial pairing of long/long-medium bivalents at pachytene were independent of each other only on day 19. Secondly, although the proportion of oocytes with a loop fell overall, there was a rise at 19 days in In(2)2H heterozygotes. Thus in both inversions there is some evidence of a change in pairing behaviour affecting the inversion-bearing bivalents at the latest gestational age, as would be expected under the production-line hypothesis.

Experiments on chiasmata and nondisjunction in mice.

Meiosis in the female mammal has both an intrinsic interest and a practical one. Intrinsically, it is interesting because of the complexity of its control and because the whole of its prophase, including recombination of linked genes by crossing over, occurs in utero and is followed by a long dormant stage before final maturation of the ova, in readiness for fertilization. Its practical interest lies in the frequency with which errors at meiotic divisions are the source of numerical chromosome aberrations in man and other mammals. Though meiotic errors are documented also in the male, probably more commonly errors occur at first meiotic division in the female by chromosomal nondisjunction and are incremental with her age. There are two alternative explanatory sets of hypotheses for the maternal age relationship; the first, of postnatal aging (for example, the terminalization hypothesis), and the second, which is one of prenatal aging, and is in essence a production line hypothesis. Whichever of these two hypotheses proves in the end to be correct, the important mechanism for the origin of numerical chromosome anomalies through nondisjunction at first meiotic division is failure of formation, or of persistence, of chiasmata: the mechanical properties of chiasmata are essential for normal disjunction. These general points, including older ideas concerning the nature of chiasmata, will be discussed briefly.Because of the largely cryptic nature of the meiotic prophase in female mammals, a technique had to be developed for its experimental study, which would allow a more direct approach to the developing oogonia and oocytes than is possible in vivo during fetal life. More direct control was achieved by tackling oogenesis and meiosis in vitro during embryonic gonadogenesis, well before the final maturation of the ova in vivo. The technique consists in removing the fetal ovaries during early development (often at day 14, taking as day 1 the day of plug detection), and allowing these to mature in vitro, in an organ culture system, for 8 days at 37° C. Subsequently the fetal ovaries, which have by now grown considerably in size, are transplanted under the renal capsule of a previously spayed young adult female. After 19 days, to allow for maturation of the ovaries in vivo, the gonads are removed, the maturing oocytes are harvested and chromosome preparations are made for analysis at 1st or at 2nd meiotic metaphase, at will. The technique we have developed for the experimental study of mammalian female meiosis is versatile in many ways, and one of its applications is exemplified in Polani et al. (1979)KeywordsMeiotic DivisionMeiotic ProphaseRenal CapsuleOrgan Culture SystemFinal MaturationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.