Testis is a primary male sex organ performing both exocrine and endocrine functions. Any disturbance in the development and maturation can lead to low reproductive efficiency or even infertility in animals and may reflect as unrecognized defects in the histogenesis of male gonad during critical organizational periods (Kaur et al., 2011). Gaddi sheep is one of the important livestock breed of northern temperate region of India. The morphogenesis of prenatal testis has been studied in goat (Baishya et al., 1987 and Farooqui et al., 2012) and buffalo (Kaur et al., 2011) but scanty literature is available on the prenatal development of testis in Gaddi sheep, so the present work was taken up.

Materials and Methods

The present study was conducted on the testes of 68 Gaddi sheep foetii. The foetii were measured for their crown – rump length (CRL) in cm with the help of a graduated nylon tape (Harvey, 1959) and their approximate age was calculated by using the following formula given by Gall et al. (1994) i.e. Y = 2.74 X + 30.15, where Y is the age of embryos in days and X is the CRL in cm. Thereafter, the foetii were divided into four stages on the basis of their age viz. stage I (31-60 days), stage II (61-90 days), stage III (91-120 days) and stage IV (121- till term).

The right and left testes collected were fixed in 10% neutral buffered formalin. After required fixation period, the tissues were processed with alcohol-benzene schedule for paraffin technique. Sections cut at 5-6 µm thickness were stained with haematoxylin and Eosin method for general histomorphology, Verhoeff’s Elastica stain for elastic and collagen fibres, Weigert’s method for elastic fibres (Luna, 1968); Gomori’s method for reticular fibres (Mallory, 1942) and Masson’s Trichrome method for connective tissue (Lillie, 1948).

Results and Discussion

The outer lining of indifferent gonad was found to be simple squamous type at 37^th day (CRL 2.5 cm) of gestation. No sex cords were observed in the gonads at this age. These findings corroborated well with the observations of Abd – Elmaksoud (2005) and Kaur (2006) in bovine fetuses. Beneath the epithelium, a distinct layer of mesenchymal cells was identified which may develop as future testicular tunica albuginea at 46 days (CRL 5.8 cm) of gestation (Fig. 1).

Germinal Epithelium

The simple squamous epithelium changed to low cuboidal type of epithelium except towards the mesothelium of the mesonephros (Fig. 1) on 46^th day of gestation (stage II). The germinal epithelium became simple cuboidal and had similar characters on 63^rd day of gestation (stage III) as in stage II of gestation. The findings were similar to that earlier reported by Farooqui et al. (2012) in goat.

Testicular Capsule

The tunica albuginea just beneath the germinal epithelium was composed of few layers of mesenchymal cells, fibroblasts and small capillaries (Fig. 1) on 46^th day of gestation. Singh et al. (1979) and Farooqui et al. (2012) observed a distinct tunica albuginea on 47^th day and 44^th day of gestation in goat foetii, respectively.

The tunica albuginea was distinguishable in outer thicker tunica fibrosa and inner thin tunica vasculosa (Fig. 2) on 50^th day of gestation. Kaur (2006) reported that the capsule differentiated into outer fibrous layer (tunica fibrosa) and inner vascular layer (tunica vasculosa) in buffalo foetii at 65 days of gestation.

The connective tissue trabeculae invaginated into the testicular parenchyma dividing it into lobules which contained developing sex cords on 60^th day of gestation (Fig. 3). The two layers of the capsule become more demarcable on 70^th day of gestation (stage II). The outer layer of the capsule became more fibrous on 95^th day of gestation (stage III). The capsule became more folded, compact and compressed in configuration. The number of blood vessels increased. In stage III of gestation the collagen (Fig. 4) and reticular fibers were well differentiated in the capsule. The fibrous layer of the capsule was more folded and compactly arranged on stage IV of gestation (121 days to term) than the previous stage. There was further increase in the vascularity at this stage.

The trabeculae were seen more clearly on 137^th day (CRL 38.9 cm) of gestation. These trabeculae contained large amount of collagen and reticular fibres. Similar observations were made by Abd-Elmaksoud (2005) and Kaur (2006) in bovine and buffalo foetal testis respectively.

Seminiferous Tubules

The seminiferous tubules were first observed on 46^th day of gestation (stage I) at the gonadal periphery (Fig. 1). Singh et al. (1979) and Farooqui et al. (2012) observed the process of formation of sex cords at 47^th and 44^th day of gestation in goat foetal testis, respectively. The developing sex cords contained small mesenchymal and large cells arranged in cords (Fig. 5). The presence of these cells in the sex cords had also been reported earlier in foetal testis of goat (Singh et al., 1979 and Farooqui et al., 2012) and cattle (Santamarina and Reece, 1957). The sex cords lacked the clear lumina and were filled with an irregularly oriented eosinophilic mass (Fig. 5). Several investigators used the term sex cords for seminiferous tubules until these were luminated (Goyal & Dhingra, 1973 and Chandra Pal, 1976). The testicular parenchyma was subdivided into two zones at 47^th day of gestation. The outer zone contained oval to slightly elongated tubules whereas the inner zone had longitudinal tubules (Fig. 6). In contrast Kaur (2006) reported longitudinal tubules in the outer zone and rounded tubules in the inner zone of developing seminiferous tubules in the initial stages of first trimester of pregnancy in buffalo foetii.

The presence of slight concavity in the sex cords indicated the beginning of convolution in sex cords (Fig. 3) on 61^st day of gestation (stage II). With the advancement of gestation the process of convolution progressed in the sex cords located towards the centre of the parenchyma. The collagen fibers were present surrounding the developing seminiferous tubules. The degree of tortuousness was higher as compared to the previous stage at 99^th day (stage III) of gestation and due to increased degree of convolution the seminiferous tubules appeared “C” shaped at 136^th day of gestation (stage IV). Abundant reticular and collagen fibers were present at the periphery of the tubules in stage IV. The seminiferous tubules surrounded by a distinct basement membrane and a layer of peritubular cells were observed at 47^th day of gestation. These cells were elongated in shape with a rounded nucleus. These cells are responsible for maintaining the structural integrity of seminiferous tubules.

Gonocytes

The seminiferous tubules comprised of large and small cells in initial stages. The sertoli cells were present in between large and small cells.

Large Cells

The large cells were first observed at 46^th day of gestation (stage I) (Fig. 5).These cells were spherical or oblong in shape with distinct cell boundaries. The cytoplasm was vacuolated and weakly eosinophilic (Fig. 7) as reported earlier by Farooqui et al. (2012) in goat. In stage I, large cells were located at the periphery close to the basement membrane and few cells were present towards the centre of the developing seminiferous tubules. That might had been an indication of the movement of the cells towards the future lumen of cords as observed by Farooqui et al. (2012) in goats. In contrast Singh et al. (1979) and Baishya et al. (1987) in goats and Baishya and Vyas (1990) in Surti buffalo reported the presence of large cells in the centre of the tubules. The large cells revealed hypertrophic and degenerative changes with more vacuolations as compared to earlier stages (Fig. 8) on 86^th day of gestation. The large cells decreased in number at stage III of gestation. Cytoplasm of most of the degenerative cells showed large sized vacuoles.

The large cell population was further decreased from 122^nd day of gestation (stage IV). Such cells contained centrally placed nucleus with densely staining chromatin granules (Fig. 9). The degenerative changes, observed in large cells in our study were in agreement with that stated by Singh et al. (1979) and Farooqui et al. (2012) in goat and Baishya and Vyas (1990) in buffalo foetal testis. These authors opined that the primordial germ cells undergo the process of degeneration with advancement of gestation.

Small Cells

These cells were first observed on 46^th day of gestation (stage I) in (Fig. 5). Farooqui et al. (2012) and Singh et al. (1979) observed these cells in goat foetal testis on 44^th and 48^th day of gestation, respectively. These cells were spherical oval or irregular in shape and were located peripherally close to the basement membrane of developing sex cords (Fig. 7). The cytoplasm of such cells was relatively more eosinophilic than that of the large cells. Spherical or oval nuclei of these cells contained evenly distributed, finely granular chromatin (Fig. 8). These cytological characters were in agreement with Farooqui et al. (2012) in goat. The small cells had similar nuclear characters on 69^th day (stage II) of gestation as observed in stage I. The nucleolus showed intense staining with eosin (Fig. 8) on 86^th day of gestation. There was further increase in number of small cells on 95^th day of gestation than the previous stages. They formed a layer along the basement membrane. These cells had granulated eosinophilic cytoplasm and nuclear chromatin exhibited clump formation (Fig. 9).The mitotic figures were present in some of the small cells on 136^th day (stage IV) of gestation. The cytoplasm of these cells became less eosinophilic than that of the previous stages.

Sertoli Cells

The sertoli cells were first observed in Gaddi sheep foetal testis on 46^th day of gestation (stage I) located among small cells of the sex cords (Fig. 5). Farooqui et al. (2012) observed the sertoli cells on 56^th day of gestation in goat foetal testis. They were roughly pyramidal in shape with indistinct cell boundaries. The cells showed cytoplasmic extensions from the supra nuclear zone. Farooqui et al. (2012) observed such extensions on 70^th day of gestation in goat. The cells had pyramidal shaped nuclei on 61^st day of gestation (stage II) and some of the cells had elongated nuclei and showed cytoplasmic extensions. At 86^th day most of the cells had pyramidal shaped nuclei and only few cells had elongated nuclei (Fig. 8). The sertoli cells were in close contact with the basement membrane of sex cords on 95^th day of gestation (stage III). The nuclei of these cells were cylindrical or pyramidal in shape with one or 2 nucleoli. These cells had darkly stained eosinophilic cytoplasm in the supra nuclear zone from where the cytoplasmic extensions were going towards the future lumen (Fig. 9) on 121^st day (stage IV) of gestation.

Interstitial Tissue

The interstitial tissue contained blood vessels, loose network of mesenchymal cells, collagen fibers, reticular fibers and interstitial endocrine cells.

Interstitial Endocrine Cells (Leydig cells)

At 46^th day of gestation (stage I) few of the mesenchymal cells located in between the sex cords, started developing into future interstitial endocrine cells, characterized by their roughly polygonal shape and highly eosinophilic cytoplasm. The spherical nuclei of these cells were vesicular in nature and contained a distinct eccentrically placed nucleolus (Fig. 5). Singh et al. (1979) and Farooqui et al. (2012) observed interstitial endocrine cell differentiation at 44^th and 47^th day in goat foetal testis, respectively. The interstitium expanded due to differentiation of mesenchymal cells into leydig cells on 60^th day of gestation and constituted the larger cell population of all the other components of interstitium at 139^th day of gestation. This finding was in agreement with the observations of Orth (1993) and Kaur (2006) who found that the number of mesenchymal cells decreased with the appearance of leydig cells in pig and buffalo foetal testis respectively.

Mediastinum Testis

The mediastinum testis was observed on 46^th day of gestation (stage 1) as an area devoid of sex cords. Singh et al. (1979) and Farooqui et al. (2012) observed the anlagen of mediastinum testis at 48^th and 44^th day of gestation respectively in goat foetal testis. The connective tissue core of mediastinum testis increased further at 61^st day of gestation (stage II) and at this age the area was poorly stained in H & E preparation as compared to surrounding parenchyma of the testis (Fig. 10). The area of mediastinum testis showed improved staining in later stages of gestation.

Rete Tubules

The rete tubules had clusters of cells in the area of mediastinum testis on 70^th day of gestation. Vacuolation was observed in the central area of developing rete tubules marking the beginning of the lumen formation. This observation was in agreement with the findings of Singh et al. (1979) and Farooqui et al. (2012) in goat. The lumen formation was further progressed on 88^th day of gestation and the cells started to rearrange themselves around a small lumen (Fig. 11). The development changes on 108^th day of gestation were similar to the previous stage with slight increase in the size of lumen. Most of the tubules were luminated and were surrounded by coarse reticular fibers which had branching and anastomosing pattern on 122^nd day of gestation. Few of these rete tubules were observed making connections with tubuli recti (Fig. 11).

Conclusion

The differentiation of cortex and medulla in the indifferent gonad was observed on 37th day of gestation. The germinal epithelium was of simple squamous type which became cuboidal type in later stages of gestation. The distinct tunica albuginea was observed on 46th day of gestation. The connective tissue trabeculae from the capsule divided the testicular parenchyma into lobules which contained developing sex cords on 60th day of gestation. The sex cords contained large cells and small cells with sertoli cells present in between these cells. The mediastinum testis was observed on 46th day of gestation as an area devoid of sex cords. The diameter of seminiferous tubules, large, small, sertoli and leydig cells increased with the progression of gestation.

Acknowledgement

The authors are thankful to DGCN college of Animal and Veterinary Sciences, Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur for providing infrastructure and support for the conduct of this research work.

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