Results

A total of 10 fetal testicular tissue samples representing the first, second, and third trimesters of gestation were examined. Progressive developmental changes in the testicular architecture, Leydig cell morphology, and immunoreactivity pattern were observed with increasing gestational age.

During the first trimester, the fetal testis demonstrated prominent interstitial cellularity with abundant Leydig cells arranged as clusters or broad cellular sheets between the developing seminiferous cords. Leydig cells showed intense cytoplasmic immunoreactivity, indicating high expression of the studied marker. The immunopositive Leydig cells were distributed throughout the interstitial compartment.

The developing seminiferous cords showed cytoplasmic immunolabeling involving germ cells and Sertoli cells, producing distinct dark cord-like structures. Compared with Leydig cells, tubular cells exhibited relatively weaker staining intensity. Occasional nuclear positivity was observed in both Leydig cells and tubular components.

The epididymal tissue associated with fetal testis also demonstrated immunoreactivity, particularly involving the apical cytoplasm of epididymal epithelial cells. Numerous immunopositive cells were identified within the fetal testicular tissue during this developmental period. Ki-67 immunostaining demonstrated nuclear positivity predominantly in the epithelial cells of the developing epididymal ducts, with occasional positive stromal cells, consistent with active fetal tissue proliferation.

During the second trimester, Leydig cells demonstrated morphological maturation and appeared predominantly rounded or polygonal in shape. Most Leydig cells exhibited strong cytoplasmic immunoreactivity, although occasional weakly stained or negative cells were observed.

The solid seminiferous (testis) cords showed progressive development with comparatively reduced immunostaining intensity compared with the interstitial Leydig cells. The proportion of immunoreactive cells appeared decreased relative to the first trimester, corresponding with increased seminiferous tubular development and reduction of relative interstitial tissue area.

In the third trimester, Leydig cells appeared more compact and slightly flattened, corresponding to a reduction in the available interstitial space. The intensity of Leydig cell immunoreactivity was variable, with mature Leydig cells showing stronger staining while some immature/regressed Leydig cells demonstrated weak or absent immunolabeling (Figure 1).

Figure 1: 40x magnification showing deep surface of testisKi-67-immunoreactive cells are less numerous, and Leydig cells are slightly flattened due to reduced space within the interstitial tissue

The epididymal epithelium continued to demonstrate strong cytoplasmic immunoreactivity (Figure 2). A distinct reduction in the number of strongly immunoreactive Leydig cells was observed compared with earlier gestational periods.

Figure 2: 40x showing high magnification of deep surfaceThe epididymis tubular cells show strong Ki-67 immunoreactivity

Overall, Ki-67 immunoreactivity progressively decreased with advancing gestational age, reflecting reduced cellular proliferation as the fetal testis underwent maturation.

Ultrastructural findings

During the first trimester of gestation, the testicular interstitium showed fibroblast-like interstitial cells along with fetal Leydig cells. Leydig cells exhibited characteristic steroidogenic features, including abundant sER, mitochondria with tubular cristae, lipid droplets, and electron-dense bodies. Degenerating Leydig cells showed dark cytoplasm, reduced sER, residual bodies, and lipid accumulation. Gonocytes displayed a prominent nucleolus, dispersed chromatin, Golgi apparatus, mitochondria, and polyribosomes. Chromatid bodies were also observed in intermediate cells (Figure 3).

Figure 3: Scanning electron microscopeShowing surface of seminiferous cords (C)

In the second trimester of gestation, the interstitium contained numerous fetal Leydig cells with dark cytoplasm and lipid vacuoles, surrounded by fusiform peritubular cells. Immature Leydig cells and mesenchymal cells were observed in the intermediate region. Blood vessels and basement membranes showed strong immunoreactivity, while primitive spermatogonia and immature Sertoli cells showed positivity. Undifferentiated mesenchymal cells demonstrated triangular nuclei with scant organelles and elongated cytoplasmic processes (Figure 4).

Figure 4: Transmission electron microscopyThe figure shows the nucleus of Sertoli cells with an intact nuclear membrane (E) and dense body in the cytoplasm of fetal germ cells or gonocytes (F)

By the third trimester of gestation, the lamina propria consisted of one to two layers of flattened peritubular cells. Elongated myoid-like cells showed strong cytoplasmic immunoreactivity, with primitive spermatogonia and immature Sertoli cells evident within the developing testicular cords (Figure 5).

Figure 5: Transmission electron microscopyNormal Leydig cells with no cytoplasmic vacuolation (M) are visible

TEM and SEM provided a detailed visualization of the organization and interactions of Sertoli cells and Leydig cells during fetal testicular development.