Molecular analysis of the effects of steroid hormones on mouse meiotic prophase I progression

Background Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis. Methods Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique. Results MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage. Conclusions We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.

Characterization of Seminiferous Epithelium Stages in the Wild Javan Muntjac (Muntiacus muntjak muntjak) Using the Tubular Morphology Method

Stages of the seminiferous epithelium of the testis of the wild Javan muntjac (Muntiacus muntjak muntjak) in hard antler period were characterized based on the tubular morphology method. The number and the relative frequencies of seminiferous epithelium stages and the morphometry of germinal cell nuclei were identified microscopically. We identified eight stages of seminiferous epithelium in testicular tissue of the Javan muntjac and found that the relative frequencies of stages I to VIII were 14.87, 15.12, 17.75, 6.87, 7.37, 12.37, 13, and 12.62%, respectively. The diameter of the nuclei of germinal cells varied in each stage of seminiferous epithelium. Diplotene-stage primary spermatocytes had prominent and large nuclei ~8.97 ± 1.0 μm in stages III and IV. Pachytene primary spermatocytes appeared in most stages, except stage IV, whereas leptotene- and diplotene-stage primary spermatocytes were found in stages I and II, and III and IV, respectively. Round spermatids were observed in stages IV to VIII and in stage I but were absent in stages II and III, while elongated spermatids were observed in all stages except stage I. Our findings show that the stages of seminiferous epithelium in the Javan muntjac are similar to those found in neotropical cervids, small ruminants, and other domestic animals.

Loss of Synaptonemal Complex Protein-1, a Synaptonemal Complex Protein, Contributes to the Initiation of Follicular Assembly in the Developing Rat Ovary

In the rat ovary, germ and somatic cells become organized into primordial follicles 48–72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display of mRNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes synaptonemal complex protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (a nuclear protein whose expression ceases in diplotene) and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited an increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the ability to orchestrate follicular assembly.

Cell–cell interaction and oocyte growth

In most mammals, oocytes initiate meiosis in late fetal life; by the time of birthe they have already entered the diplotene stage of prophase I of meiosis and becaome arrested thereafter at the dictyate state(Baker, 1972). At this stage they became surrounded by a few nonproliferating flat follicle cells forming a unit called the resting or primordial follicle.

Synaptonemal complex transformations in rye microsporocytes at the diplotene stage of meiosis

The process of synaptonemal complex degradation during diplotene was studied in spreads of rye microsporocytes stained with silver nitrate at pH 3.5–4.5 and 6.0–8.0. Two different patterns of the synaptonemal complex degradation process have been observed, depending on the two staining procedures used. Progressive synaptonemal complex fragmentation observable at the higher pH appeared to be absent in staining at pH 3.5–4.5: thin connecting threads have been found in the "gaps" between the synaptonemal complex segments. Complete tracing of the synaptonemal complex degradation process was attempted and revealed the following successive steps: (i) local repulsion of lateral elements; (ii) lateral element looping in the regions of repulsion; (iii) extension of the loops; (iv) transformation of the extended loops into coils of irregular shape with a diameter of about 2 μm and a pitch of about 1.2 μm; and (v) formation of paired beanlike thickenings on a gyral coil. In asynaptic mutant sy-9 unpaired lateral elements are transformed without looping into a similar coil but with single beanlike thickenings. We conclude that synaptonemal complex lateral element loops at diplotene are invisible after the routine silver staining of microspreads (at pH about 6 and higher) and look like gaps between discontinuous synaptonemal complex segments, thus simulating the process of synaptonemal complex fragmentation.Key words: synaptonemal complex, diplotene, rye.