Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities.

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.

Unbalanced Expression of Glutathione Peroxidase 4 and Arachidonate 15-Lipoxygenase Affects Acrosome Reaction and In Vitro Fertilization.

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis.

Examination of the Contributions of Maternal/Placental-Derived Galectin-1 to Pregnancy Outcome.

Galectin-1 (gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, is differentially expressed at the feto-maternal interface and appears to be functionally polyvalent, with a wide range of biological activities. However, the contributions of maternal and/or feto-placental gal-1 to the signaling networks promoting a healthy pregnancy are still being elucidated. This chapter discusses the methods commonly employed to study the maternal or feto-placental contribution of gal-1 during pregnancy in mice. The methods described here can be used to decipher the specific role of each source, e.g., maternal and/or feto-placental derived gal-1 in the orchestration of pregnancy-associated processes.

Reproductive Performance after Unilateral or Bilateral Oviduct Transfer of 2-Cell Embryos in Mice.

Embryo transfer (ET) is a frequent procedure in contemporary animal and transgenic facilities. We compared the reproductive performance of mice after unilateral and bilateral ET of 15 to 18 two-cell embryos per recipient. The genetic backgrounds of the donors were C57BL/6J (B6J), C57BL/6N (B6N), or fewer than 5 generations of backcrossing to B6 (unknown substrain, <5G B6). The pregnancy rate was significantly higher for bilateral than for unilateral ET for B6J lines (85.4% compared with 79%) but similar between modes for B6N (73.7% compared with 77.9%) and <5G B6 (77% compared with 74.5%) lines. The birth rates after unilateral and bilateral ET were 30.8% and 33.0% for B6J lines, 24.5% and 26.9% for B6N lines, and 31.0% compared with 28.2% for <5G B6 lines, with no significant difference between the modes of ET. Birth rate was significantly higher for B6J lines than B6N lines after both unilateral and bilateral ET. For B6J and B6N lines, the number of pups born per litter was significantly higher for bilateral than unilateral ET. Unilateral ET yielded 0.24 to 0.31 pup per embryo transferred compared with 0.27 to 0.33 pups after bilateral ET. Over all genetic backgrounds, 3.03 to 4.09 embryos were required to produce a single pup. The present study provides data to aid in decision-making as to whether unilateral or bilateral ET should be performed. Bilateral ET results in a larger litter but increases pain and discomfort in recipients. However, unilateral ET saves time and contributes to refinement because surgical trauma is reduced.

Productivity of superovulated C57BL/6J oocyte donors at different ages.

Superovulation is often used to increase the number of oocytes that can be collected from donor females for in vitro fertilization. Donor age can affect the quantity and quality of oocytes produced during superovulation, and in some strains of mice juvenile females are optimal donors. The authors reviewed donor and oocyte records from a breeding program to evaluate how donor age affects the number and fertilization efficiency of oocytes collected from C57BL/6J mice. Generally fewer oocytes per donor were collected from females aged > 32 d than from females aged 21-32 d. Fertilization efficiency of oocytes generally declined with donor age when oocytes were fertilized with fresh or with stored sperm. These findings suggest that the use of younger C57BL/6J donors, instead of older donors, can reduce the number of donors needed for IVF procedures.