The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from 1 to 80 h after insemination. Penetration through the zona into the perivitelline space (PVS) was seen from 1 to 72 h after insemination. Fusion of human sperm with zona-free hamster oocytes was observed from 1 to 48 h after insemination. Motile sperm were recovered 112 and 120 h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 80 h or more.

1. A densitometric method has been developed for the quantification of azodipyrroles derived from dog bile pigments treated with diazotized ethyl anthranilate. 2. This method was used to estimate the bilirubins in bile and meconium from foetuses of 14-36 weeks gestation. 3. The proportion of the bilirubins in foetal bile changed during gestation. (a) No bile pigments were found until 14 weeks. (b) Between 14 and 15 weeks bilirubin-IX beta was the only bile pigment detected. (c) At 16-17 weeks some unconjugated bilirubin-IX alpha was found in the bile, but up to 20 weeks bilirubin-IX beta was the predominant bilirubin in the bile. (d) At about 20 weeks glucose, xylose, and an unidentified bilirubin-IX alpha monoconjugate were found in the bile. (e) Between 20 and 23 weeks bilirubin-IX alpha glucuronide appeared in the bile. (f) At 30 weeks monoconjugates of bilirubin-IX alpha were the predominant bilirubins in the bile. (g) Only in full-term foetuses was bilirubin-IX alpha monoglucuronide the major bilirubin derivative.

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Fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) were evaluated for use as contrasting fluorescent labels on living human spermatozoa. Unlabeled (control), FITC‐labeled (green fluorescence), TRITC‐labeled (red fluorescence), and mixed FITC‐TRITC‐labeled sperm suspensions were incubated with non‐fertilizable human oocytes for assessing sperm penetration of the zona pellucida and with zona‐free hamster eggs for assessing sperm incorporation into the ooplasm. Fluorochrome‐labeled spermatozoa were as efficient as unlabeled cells from the same donors in penetrating the human zona pellucida and in entering the hamster ooplasm. The middle and principal pieces of spermatozoa undergoing nuclear decondensation within the hamster ooplasm retained their fluorescent label, allowing visual differentiation between FITCand TRITC‐labeled spermatozoa. Videomicrographic analyses of the movement characteristics (percentage of motile cells and mean swimming speeds) of labeled and unlabeled spermatozoa before and after incubation with ova revealed no detrimental effect on the motility of labeled cells. We conclude that the fluorescent dyes FITC and TRITC do not impair the function of human spermatozoa as assessed by motility characteristics and by their ability to penetrate ova in vitro. The contrasting colors of the two fluorochromes make them particularly useful in the competitive assessment of the in vitro fertilizing potential of human spermatozoa from different donors or after different experimental treatments.