In vitro production of naked mole-rats' blastocysts from non-breeding females using in vitro maturation and intracytoplasmic sperm injection.

The African naked mole-rat (Heterocephalus glaber) is an attractive model for cancer and aging research due to its peculiar biological traits, such as unusual long life span and resistance to cancer. The establishment of induced pluripotent stem cells (iPSCs) would be a useful tool for in vitro studies but, in this species, the reprogramming of somatic cells is problematic because of their stable epigenome. Therefore, an alternative approach is the derivation of embryonic stem cells from in vitro-produced embryos. In this study, immature oocytes, opportunistically retrieved from sexually inactive females, underwent first in vitro maturation (IVM) and then in vitro fertilization via piezo-intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultivated with two different approaches: (i) in an in vitro culture and (ii) in an isolated mouse oviduct organ culture system. The second approach led to the development of blastocysts, which were fixed and stained for further analysis.

Multiple neuropeptides produced by sex-specific neurons control activity of the male accessory glands and gonoducts in the silkworm Bombyx mori.

The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.

Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori.

Allatotropin (AT) and related neuropeptides are widespread bioactive molecules that regulate development, food intake and muscle contractions in insects and other invertebrates. In moths, alternative splicing of the at gene generates three mRNA precursors encoding AT with different combinations of three structurally similar AT-like peptides (ATLI-III). We used in situ hybridization and immunohistochemistry to map the differential expression of these transcripts during the postembryonic development of Bombyx mori. Transcript encoding AT alone was expressed in numerous neurons of the central nervous system and frontal ganglion, whereas transcripts encoding AT with ATLs were produced by smaller specific subgroups of neurons in larval stages. Metamorphosis was associated with considerable developmental changes and sex-specific differences in the expression of all transcripts. The most notable was the appearance of AT/ATL transcripts (1) in the brain lateral neurosecretory cells producing prothoracicotropic hormone; (2) in the male-specific cluster of about 20 neurons in the posterior region of the terminal abdominal ganglion; (3) in the female-specific medial neurons in the abdominal ganglia AG2-7. Immunohistochemical staining showed that these neurons produced a mixture of various neuropeptides and innervated diverse peripheral organs. Our data suggest that AT/ATL neuropeptides are involved in multiple stage- and sex-specific functions during the development of B. mori.

Expression of RYamide in the nervous and endocrine system of Bombyx mori.

RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion.

Molecular cloning, expression and identification of the promoter regulatory region for the neuropeptide trissin in the nervous system of the silkmoth Bombyx mori.

Trissin has recently been identified as a conserved insect neuropeptide, but its cellular expression and function is unknown. We detected the presence of this neuropeptide in the silkworm Bombyx mori using in silico search and molecular cloning. In situ hybridisation was used to examine trissin expression in the entire central nervous system (CNS) and gut of larvae, pupae and adults. Surprisingly, its expression is restricted to only two pairs of small protocerebral interneurons and four to five large neurons in the frontal ganglion (FG). These neurons were further characterised by subsequent multiple staining with selected antibodies against insect neuropeptides. The brain interneurons innervate edges of the mushroom bodies and co-express trissin with myoinhibitory peptides (MIP) and CRF-like diuretic hormones (CRF-DH). In the FG, one pair of neurons co-express trissin with calcitonin-like diuretic hormone (CT-DH), short neuropeptide F (sNPF) and MIP. These neurons innervate the brain tritocerebrum and musculature of the anterior midgut. The other pair of trissin neurons in the FG co-express sNPF and project axons to the tritocerebrum and midgut. We also used the baculovirus expression system to identify the promoter regulatory region of the trissin gene for targeted expression of various molecular markers in these neurons. Dominant expression of trissin in the FG indicates its possible role in the regulation of foregut-midgut contractions and food intake.