Polani B. Seshagiri

Cellular and molecular regulation of mammalian blastocyst hatching

In mammals including humans, failure in blastocyst hatching and implantation leads to early embryonic loss and infertility. Prior to implantation, the blastocyst must hatch out of its acellular glycoprotein coat, the zona pellucida (ZP). The phenomenon of blastocyst hatching is believed to be regulated by (i) dynamic cellular components such as actin-based trophectodermal projections (TEPs), and (ii) a variety of autocrine and paracrine molecules such as growth factors, cytokines and proteases. The spatio-temporal regulation of zona lysis by blastocyst-derived cellular and molecular signaling factors is being keenly investigated. Our studies show that hamster blastocyst hatching is accelerated by growth factors such as heparin binding-epidermal growth factor and leukemia inhibitory factor and that embryo-derived, cysteine proteases including cathepsins are responsible for blastocyst hatching. Additionally, we believe that cyclooxygenase-generated prostaglandins, estradiol-17beta mediated estrogen receptor-alpha signaling and possibly NFkappaB could be involved in peri-hatching development. Moreover, we show that TEPs are intimately involved with lysing ZP and that the TEPs potentially enrich and harbor hatching-enabling factors. These observations provide new insights into our understanding of the key cellular and molecular regulators involved in the phenomenon of mammalian blastocyst hatching, which is essential for the establishment of early pregnancy.

Molecular insights into the causes of male infertility.

Infertility is a reproductive health problem that affects many couples in the human population. About 13–18% of couple suffers from it and approximately one-half of all cases can be traced to either partner. Regardless of whether it is primary or secondary infertility, affected couples suffer from enormous emotional and psychological trauma and it can constitute a major life crisis in the social context. Many cases of idiopathic infertility have a genetic or molecular basis. The knowledge of the molecular genetics of male infertility is developing rapidly, new “spermatogenic genes” are being discovered and molecular diagnostic approaches (DNA chips) established. This will immensely help diagnostic and therapeutic approaches to alleviate human infertility. The present review provides an overview of the causes of human infertility, particularly the molecular basis of male infertility and its implications for clinical practice.

Regulation of peri-attachment embryo development in the golden hamster: role of growth factors

The molecular regulation of mammalian peri-implantation development is complex and difficult to study in vivo. We successfully cultured hamster blastocysts through hatching and peri-attachment stages, using a chemically defined medium, HECM-2h. Using this system, we showed that a species-specific, embryonic cysteine-like protease is involved in blastocyst hatching and that the process is modulated by growth factors. In particular, heparin binding-epidermal growth factor (HB-EGF) or leukemia inhibitory factor (LIF) enhance blastocyst hatching, and the former also improves attachment and trophoblast outgrowth. We observed interesting changing patterns of expression of mRNA and/or immunoreactive protein for EGF, HB-EGF, LIF and transforming growth factor-beta (TGF-beta) in the embryo and/or endometrial tissue, during peri-implantation development. Together, it appears that hamster blastocyst hatching, attachment and trophoblast outgrowth are regulated by autocrine and/or paracrine growth factors, produced by the embryo-endometrial tissues.

Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, tyrphostin‐A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin‐A47‐treated spermatozoa exhibited circular motility, which was associated with a distinct hypo‐tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000–60,000. In this study, we provide evidence on the localization of capacitation‐associated tyrosine‐phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo‐tyrosine phosphorylated major proteins of tyrphostin‐A47‐treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein‐2 and the 51 kDa protein as tektin‐2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo‐tyrosine‐phosphorylation status of outer dense fiber protein‐2 and tektin‐2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR‐tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa. Mol. Reprod. Dev. 77: 182–193, 2010.

Heparin binding-epidermal growth factor improves blastocyst hatching and trophoblast outgrowth in the golden hamster

The effect of heparin binding-epidermal growth factor (HB-EGF) on the in-vitro development of hamster 8-cell embryos was investigated. Supplementation of HB-EGF to culture medium accelerated zona escape of blastocysts (63 +/- 9% compared with 33 +/- 9% after 36 h; P < 0.05). Complete zona escape of blastocysts persisted even after 48 h (61 +/- 11% versus 30 +/- 4%) and 60 h (75 +/- 6% versus 42 +/- 8%). Addition of anti-HB-EGF antibody drastically reduced the percentage of zona escaped-blastocysts (30.0 +/- 5.0% versus 92.3 +/- 2.8%; P < 0.05). Interestingly, a significant increase in the area of trophoblast outgrowth occurred in the presence of HB-EGF (116 x 10(3) +/- 8 x 10(3) microm(2) versus 74 x 10(3) +/- 8 x 10(3) microm(2) at 48 h; P < 0.05). This, however, was not due to an increased number of trophectodermal cells in HB-EGF-treated blastocysts. Immunoreactive HB-EGF was localized in blastocysts and uterine sections, visible by intense immunostaining in the luminal epithelium, particularly on the apical surface. Moreover, the expression of HB-EGF in the uterus was maximum on day 4 of pregnancy, coinciding with the timing of zona escape and implantation. The receptor of HB-EGF, viz. EGF receptor was also detected in blastocysts and the luminal epithelium of day 4 pregnant uterus. These results show that HB-EGF improves blastocyst hatching and trophoblast outgrowth in hamsters.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.

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.

Endocrine correlates of musth in free-ranging Asian elephants (Elephas maximus) determined by non-invasive faecal steroid hormone metabolite measurements.

The occurrence of musth, a period of elevated levels of androgens and heightened sexual activity, has been well documented for the male Asian elephant (Elephas maximus). However, the relationship between androgen-dependent musth and adrenocortical function in this species is unclear. The current study is the first assessment of testicular and adrenocortical function in free-ranging male Asian elephants by measuring levels of testosterone (androgen) and cortisol (glucocorticoid – a physiological indicator of stress) metabolites in faeces. During musth, males expectedly showed significant elevation in faecal testosterone metabolite levels. Interestingly, glucocorticoid metabolite concentrations remained unchanged between musth and non-musth periods. This observation is contrary to that observed with wild and captive African elephant bulls and captive Asian bull elephants. Our results show that musth may not necessarily represent a stressful condition in free-ranging male Asian elephants.

Assessment of estrus cyclicity in the Asian elephant (Elephas maximus) by measurement of fecal progesterone metabolite 5α-P-3OH, using a non-invasive assay

Reproductive management of the Asian elephant (Elephas maximus) is important for its conservation. To monitor its estrous cyclicity, we earlier used an indirect ELISA to show that levels of fecal progesterone (P(4))-metabolite (allopregnanolone: 5α-P-3OH) in semi-captive females sampled randomly positively correlated with serum P(4) levels [12]. In this longitudinal study (51 weeks), we measured levels of fecal 5α-P-3OH and serum P(4) in seven semi-captive female elephants. Females exhibited three types of hormonal profiles. Four females showed cyclical patterns of fecal 5α-P-3OH and serum P(4) typical of normal estrous cycles, two showed acyclic pattern while one showed high values indicative of a pregnant animal. Values for anestrous or follicular phases were ≤ 0.3 μg g(-1) (5α-P-3OH) and ≤ 0.3 ng mL(-1) (P(4)); for luteal phase 0.32-11.09 μg g(-1) (5α-P-3OH) and 0.32-1.48 ng mL(-1) (P(4)); for pregnancy 1.41-7.38 μg g(-1) (5α-P-3OH) and 0.39-1.6 ng mL(-1) (P(4)). A positive correlation (t = 8.8, p < 0.01, n = 321) between levels of fecal 5α-P-3OH and serum P(4) was observed. A random sample of 30 free-ranging female elephants showed fecal 5α-P-3OH values of 0.06-23.4 μg g(-1), indicating them to be in different stages of estrous cyclicity. This study is the first to assess the reproductive phases of female Asian elephants based on the correlative-patterns of both the fecal 5α-P-3OH and serum P(4) values over multiple estrous cycles. This has a potential application in the reproductive management and conservation of Asian elephants.

Dung as a potential medium for inter-sexual chemical signaling in Asian elephants (Elephas maximus).

Chemical signaling is a prominent mode of male-female communication among elephants, especially during their sexually active periods. Studies on the Asian elephant in zoos have shown the significance of a urinary pheromone (Z7-12:Ac) in conveying the reproductive status of a female toward the opposite sex. We investigated the additional possibility of an inter-sexual chemical signal being conveyed through dung. Sixteen semi-captive adult male elephants were presented with dung samples of three female elephants in different reproductive phases. Each male was tested in 3 separate trials, within an interval of 1-3 days. The trials followed a double-blind pattern as the male and female elephants used in the trials were strangers, and the observer was not aware of the reproductive status of females during the period of bioassays. Males responded preferentially (P<0.005), in terms of higher frequency of sniff, check and place behavior toward the dung of females close to pre-ovulatory period (follicular-phase) as compared to those in post-ovulatory period (luteal-phase). The response toward the follicular phase samples declined over repeated trials though was still significantly higher than the corresponding response toward the non-ovulatory phase in each of the trials performed. This is the first study to show that male Asian elephants were able to distinguish the reproductive phase of the female by possibly detecting a pre-ovulatory pheromone released in dung.