Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

What are appropriate values of relative krogh diffusion Constant of NO against CO and of theta-NO in alveolar septa?

OBJECTIVES: To propose new physical constants for NO and CO (Krogh diffusion constant ratio (KDNO/CO) and specific blood conductance for NO (θNO)) for calculating DMCO and Vc, according to Roughton-Forster's equation (Roughton and Forster, J. Appl. Physiol. 11: 290-302, 1957) from simultaneous DLNO and DLCO measurements. RESULTS AND CONCLUSIONS: (1) The Graham's law is unacceptable for determining KDNO/CO because CO does not fulfil all the conditions of an "ideal" gas. We have re-estimated KDNO/CO in a new way based on difference in molar volumes of two gases (molar volume theory). The KDNO/CO thus decided is 2.34. (2) θNO measured with rapid-reaction, constant-flow method by Carlsen and Comroe (J. Gen. Physiol. 42: 83-107, 1958) may be underestimated by about 40 % due to unstirred water layer surrounding the erythrocyte. (3) Erythrocyte θO2 can be harvested from O2 release kinetics in presence of high concentration of dithionite, which effectively removes the unstirred water layer-elicited effect. Multiplication of erythrocyte θO2 by erythrocyte KDNO/O2 equals erythrocyte θNO, the value of which is 6.2 mL/min/mmHg/(mL⋅blood). According to the concepts of Kang et al. (RESPNB. 241: 62-71, 2017) and Borland et al. (RESPNB. 241: 58-61, 2017), in vitro θNO decided from rapid-mixing experiments may mirror bulk absorption of NO by erythrocytes. (4) In pulmonary capillaries, NO uptake takes place predominantly in the surface rim of the erythrocyte. This surface absorption of NO increases the θNO 10-fold versus bulk absorption of NO to about 60 mL/min/mmHg/(mL⋅blood).