Spermatogonial cells and Reproductive Success in Drosophila melanogaster

Authors: Dougan, Colleen; Walsh, Andrew; Berardo, Caitlin; Haralampoudis, Nicole

Faculty: Guy F. Barbato

Abstract

Fertility, per se, is a poor description of overall reproductive success. Having observed a range of progeny production among isofemale lines of Drosophila melanogaster, we selected 10 populations representing the range of reproductive outcomes. Beginning our investigation among males of the lines, we dissected testes under a dissecting scope and stained with DAPI. Germ cells are easily observed in the testicular tissue and were counted using ImageJ software. Germ cells were counted by three (3) independent students to minimize the observer effect. Correlation analysis between pupal and germ cell counts resulted in a negative correlation of -0.41. Our initial hypothesis was based on the assumption that a greater number of spermatogonia would result in larger numbers of spermatozoa – resulting in higher fertility. This was clearly not the case. Perhaps the males of the low producing lines were slower to mature than the others, or there were other systemic reasons for the inhibition of spermatogenesis.

Introduction

Knowing that sperm are expressed in bundles of 16 sperm in the male Drosophila melanogaster [1], we hypothesized that there might be genetic variation between the maintained populations obtained from a single female fly from across New Jersey. Our first goal was to count the sperm or sperm bundles. It became increasingly apparent that the germ cells specifically varied greatly across the lines analyzed. The sperm cells are known to be found at the apical end of the testis and the mature sperm are in the whirl-like portion of the organ [2]. Since germ cells eventually mature into spermatogonal cells, and then into sperm cells, we assumed there would be a correlation between the numbers of germ and sperm cells. Germ cells were used to compare to reproductive fitness in the 10 lines tested.

Methods

Using pupal counts as a standard for choosing, we selected the lines that represented the high, low, and average. These were taken as our representative lines and 10 male flies were taken from each line and dissected for testes. Testes were stained with DAPI and pictures of the apical end were taken under a fluorescent microscope. Germ cells were counted using ImageJ software and conducted by three (3) students. This was done by using the "Threshold" extension on a greyscale image of the DAPI pictures and the fluorescent frequency was adjusted to only select the germ cells in each of the photos. This software also allows for the particles selected to be counted. This allowed us to specifically count the germ cells. These counts were averaged across all counts to minimize the objective error. A Pearson Correlation was conducted to determine the correlation between the low, average, and high pupal groups and their germ cell count.

Results

The images of the apical end of each population showed varying levels of germ cell counts (Table 1). The low pupal count group (LES5, SOM4, AW4, and SEY1) had an average of 20.2 pupae and 14428 germ cells. The average pupal count group (HA3, LJD3, DAV4, and PR3) had an average of 61.8 pupae and 7023 germ cells. The high pupal count group (LES6 and OX5) had an average of 175.5 pupae and 5913 germ cells. A Pearson correlation of the low, average, and high pupae counts in comparison to germ cells was calculated to be -0.41 (Fig. 1). This is sufficient to explain the correlation between germ cells and reproductive fitness to be negatively correlated.

Table 1:

Germ Cell Counts from each of the three (3) students and an everage across all three counts for each sample.

Figure 1:

The graph above displays the pupal and germ counts for each of the groups assessed.

Complete Testis Picture

A bright field (left) and flourescent (right) images were taken of a testis from the SOM4 line at 10X objective

Low Pupal Count (1)

A bright field (left) and fluorescent (right) images were taken of the apical end of testis from the LES5 line at 40X objective

High Pupal Count (187)

A bright field (left) and fluorescent (right) images were taken of the apical end of the testis from the OX5 line at 40X objective

Discussion

Knowing that our sample size was relatively small for our study in comparison to our total number of populations (88), more sampling, imaging, and calculations need to be conducted to further prove or disprove our findings from this first run through. However, the negative correlation for this first stage of testing might be a predictor for our further research with higher sample sizes. The information we have makes us strongly confident that it is representative of the range of the lines we are maintaining. Being that these results were not our expected outcome, we hypothesize that there may be an issue in the maturing of sperm from germ cells or some lines took longer to mature than others. Further research may need to be done in order to answer those questions.

References

[1] Lefevre G, Jonsson UB. 1962. SPERM TRANSFER, STORAGE, DISPLACEMENT, AND UTILIZATION IN DROSOPHILA MELANOGASTER. Genetics. 47(12):1719–1736. doi:10.1093/genetics/47.12.1719.

[2] Frappaolo A, Piergentili R, Giansanti MG. 2022. Microtubule and Actin Cytoskeletal Dynamics in Male Meiotic Cells of Drosophila melanogaster. Cells. 11(4):695. doi:10.3390/cells11040695.

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