Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.).

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor ß superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

High, not low-dose of stanozolol (Anabolic - androgenic steroid) impedes embryo implantation by attenuating endometrial receptivity in the mouse, Mus musculus.

The present investigation is aimed at evaluating the efficacy of one of the anabolic -androgenic steroids, stanozolol (ST), on establishment and maintenance of pregnancy in mice. A total of 40 female mice were assigned to three experimental groups. Stanozolol was dosed subcutaneously (low-dose, 0.5 mg/kg bwt; high-dose, 5.0 mg/kg bwt or 1% alcohol-baseline control) for 30 consecutive days. On the 31st day, treatment was withdrawn. The estrous cycle was disrupted in both treatment groups and its resumption was dose dependent. Following estrous resumption, mice were allowed to mate. Results reveal that the low-dose ST-treated mice maintained gestation until term with reduced litter size, while high-dose-treated mice divulged vaginal plug at frequent intervals, indicating conception failure. Because pregnancy failure was noticed in high-dose-treated mice, they were autopsied on GD1.5 and 4.5. Interestingly, neither dose of stanozolol affected early embryonic development or blastocyst hatching. A decrease in the number of corpora lutea in both treated groups suggests it affects either ovulation or recruitment of follicles that occurs in each cycle for maturation. In high-dose-treated mice, decreased serum levels of estradiol, progesterone and increased testosterone along with downregulated endometrial expression of ERα and PR suggest the deficiency of steroid hormones and their respective receptors. Decreased ovarian expression of ERα, hyperexpression of PRLR, AR and abated progesterone secretion led to luteal dysfunction, consequently attenuating endometrial receptivity. Therefore, in high-dose-treated mice, decreased maternal estradiol and progesterone levels and their receptors during implantation hindered signaling to LIF and Hoxa-10, resulting in pragmatic implantation failure.

Acceleration of neutrophil precursors' maturation and immunostimulation of CD3+, CD4+ lymphocytes by stanozolol in mice.

The abuse of anabolic-androgenic steroids (AAS) for improved physical performance is associated with many deleterious effects. The present study aims to evaluate the short-term effect of an AAS compound stanozolol, on lipoprotein profile, granulopoiesis and immune response in adult female mice. The mice were assigned to five experimental groups and different doses of stanozolol (low - 0.05 mg, medium - 0.5 mg, high - 5 mg and highest dose - 7.5 mg/kg bwt or only vehicle respectively) were administered s.c. for 15 days. A decrease in high density lipoprotein cholesterol (HDL-c) as well as total cholesterol (TC) in all the stanozolol treated groups and an increase in low density lipoprotein (LDL-c) in high and the highest dose treated groups indicate that stanozolol alters serum lipoprotein profile. A significant increase in the percentage of myelocytes, metamyelocytes and neutrophils in all the treated mice unveils the stimulation of granulopoiesis through the acceleration of neutrophil precursors' maturation in the bone marrow of mice. The stimulation of erythropoiesis was also noted in all the treated groups. The flow cytometry analysis of lymphocyte subpopulations (CD3(+) and CD4(+)) revealed immunoenhancing response of stanozolol at optimum physiological dose, however, it is immunosuppressive at supraphysiologic level. We conclude that stanozolol accelerates granulopoiesis and stimulates immune response (at physiologic level only), though it alters the lipoprotein profile in mice.

A tropical oviparous lizard, Calotes versicolor, exhibiting a potentially novel FMFM pattern of temperature-dependent sex determination.

Among squamate reptiles, lizards exhibit an impressive array of sex-determining modes viz. genotypic sex determination, temperature-dependent sex determination, co-occurrence of both these and those that reproduce parthenogenetically. The oviparous lizard, Calotes versicolor, lacks heteromorphic sex chromosomes and there are no reports on homomorphic chromosomes. Earlier studies on this species presented little evidence to the sex-determining mechanism. Here we provide evidences for the potential role played by incubation temperature that has a significant effect (P < 0.01) on gonadal sex and sex ratio. The eggs were incubated at 14 different incubation temperatures. Interestingly, 100% males were produced at low (25.5 ± 0.5 ° C) as well as high (34 ± 0.5 ° C) incubation temperatures and 100% females were produced at low (23.5 ± 0.5 ° C) and high (31.5 ± 0.5 ° C) temperatures, clearly indicating the occurrence of TSD in this species. Sex ratios of individual clutches did not vary at any of the critical male-producing or female-producing temperatures within as well as across the seasons. However, clutch sex ratios were female- or male-biased at intermediate temperatures. Thermosensitive period occurred during the embryonic stages 30-33. Three pivotal temperatures operate producing 1:1 sex ratio. Histology of gonad and accessory reproductive structures provide additional evidence for TSD. The sex-determining pattern, observed for the first time in this species, that neither compares to Pattern I [Ia (MF) and Ib (FM)] nor to Pattern II (FMF), is being referred to as FMFM pattern of TSD. This novel FMFM pattern of sex ratio exhibited by C. versicolor may have an adaptive significance in maintaining sex ratio.