Exploring the Role of Estrogens in Lizard Spermatogenesis through the Study of Clomiphene and FSH Effects

Abstract

Spermatogenesis is a fascinating biological process aiming to generate haploid spermatozoa from diploid spermatogonia through a specific hormonal network between gonadotropins and steroids. Increasing evidence suggests that the primary female sex hormone estrogen plays an active role in this process. This research points out on the role of estrogen during lizard spermatogenesis by using three experimental approaches: (1) exposure to an analogue of nonsteroidal estrogen as Clomiphene citrate that acts both as estrogen agonist and antagonist; (2) exposure to the gonadotropin FSH; and (3) exposures to FSH followed by Clomiphene. Histological and immunohistochemical results demonstrate that in the lizard Podarcis sicula during the mating period, Clomiphene as well as FSH determines the breakdown of spermatogenesis and the epididymal regression, presumably through estrogens input as indirectly demonstrated by the appearance of ERα and vitellogenin in the liver. The ability of Clomiphene to restore the gonadal natural condition after FSH treatment is also demonstrated. Finally, data indicate that lizard testis and epididymis control their morphophysiology regulating the intracellular presence of ERα.

Figure 1

Histology of P. sicula testis. (a) Untreated males, mating period: the seminiferous epithelium is thick (↕) and full of germ cells in all spermatogenic stages, from spermatogonia (○) at the basis of the tubules to spermatozoa (◄) in the restricted lumen (∗∗). (b) Clomiphene-treated samples: the seminiferous epithelium is reduced in thickness (↕); in the lumen, some degenerate cells and oocyte-like structures are evident (↑). (c, d) FSH-treated samples: in the thin seminiferous epithelium, few germ cells and several empty spaces (∗) are present, and oocyte-like structures (↑) are also evident. (e) Detail at high magnification of oocyte-like structure. (f) FSH-Clomiphene-treated samples: all stages of spermatogenesis are evident and the epithelium thickness is increased (↕). The bar is 30 μm.

Figure 2

Histology of P. sicula epididymis. (a) Untreated males, mating period: the epithelium of the corpus is elongated and secreting and a large amount of granules is evident inside the lumen (∗). (b) Clomiphene-treated samples: the cells of the corpus are not secreting; in the lumen, no spermatozoa and secretory granules are evident (∗). (c) FSH-treated samples: in the lumen of the corpus, no granules and spermatozoa are evident (∗). (d) FSH-Clomiphene-treated samples: the cells lining the corpus are secreting and some granules and spermatozoa are evident in the lumen (∗). The bar is 30 μm.

Figure 3

Immunohistochemistry with ERα antibody on the testis (a–e) and epididymis (f–i). (a) Untreated males: ir-ERα is evident only in the final differentiating stage, that is, spermatozoa. (b) Clomiphene-treated and (c) FSH-treated samples: ir-ERα is present in all the cells inside the seminiferous epithelium (SE). The dotted white and black lines indicate the basal (BSE) and apical (ASE) seminiferous epithelium, respectively. Note in (b, c), oocyte-like structure marked for ERα (⇧). (d) FSH-Clomiphene-treated samples: ir-ERα is evident only in spermatozoa. (f) Untreated males: ir-ERα is absent in the corpus (↑↑) but present in the efferent ductules (↑). (g) Clomiphene-treated and (h) FSH-treated samples: ir-ERα is evident in the cells lining the corpus (↑↑) and in the efferent ductules (↑). (i) FSH-Clomiphene-treated samples: ir-ERα is absent in the corpus (↑↑). (e) Negative control prepared omitting the antibody in the reaction. The bar is 30 μm.

Figure 4

Immunohistochemistry with ERα (a–d) or VTG (bottom panel, e–f) antibodies in the liver. (a) Untreated males: no immunoreactivity is detected to anti-ERα antibody; the box (A) shows the negative control of reaction, performed lacking the antibody. (b) Clomiphene-treated and (c) FSH-treated samples: ir-ERα is evident in cells. (d) FSH-Clomiphene: no immunoreactivity is detected as in untreated samples (a). (e) Untreated males: no immunoreactivity is detected to anti-VTG antibody. (f) Clomiphene-treated and (g) FSH-treated samples: ir-VTG is evident in the cytoplasm cells. (h) FSH-Clomiphene: no immunoreactivity is detected, as in untreated samples (e). In (e), (f), (g), and (h), cell nuclei were counterstained with Mayer's haemalum. The bar is 30 μm.