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Throughout life, the ovarian surface epithelium (OSE) undergoes morphogenetic changes that may be hormonally regulated. To investigate this possibility, a population of cells morphologically identical to native OSE cells was isolated from estrous rabbits with collagenase, unit gravity sedimentation, and trypsin-EDTA. Cells were incubated with various concentrations of protein hormones in serum-rich medium or in a chemically defined medium containing fibronectin. Tritiated thymidine was added 24 h before interruption of cultures. Growth-promoting effects of tested hormones were more pronounced and consistent in serum-free cultures. Under these conditions, human chorionic gonadotropin (10,000 mIU/ml) caused a 2.8-fold increase in cell number and a 3.4-fold stimulation of thymidine incorporation. Luteinizing hormone (NIAMDD-oLH-24, 1.0 micrograms/ml) and follicle-stimulating hormone (NIADDK-oFSH-16, 1.0 micrograms/ml) produced, respectively, a 1.7-fold and 1.5-fold increase in cell proliferation, and over 1.4-fold and 1.3-fold stimulations of thymidine uptake. When used together, no growth stimulation by these gonadotropins was seen. Slight but significant increases in cell number (1.4-fold) and in radiolabel incorporation (1.3-fold) were observed with prolactin (NIADDK-oPrl-16, 10 ng/ml). These data indicate that some protein hormones promote the growth of OSE cells. This property may be important in regulating these cells during normal and pathologic states.

The distribution of lysozyme in the endocervix of estrous, pseudopregnant, and ovariectomized rabbits was studied using two different immunocytochemical techniques--the unlabeled antibody enzyme method of Sternberger et al. (1970) and the peroxidase-labeled antibody method of Taylor and Burns (1974). With both procedures, a fine immunostaining precipitate was seen over the entire area of basal mucous granules, while immunodeposits were coarser and mostly located in the outer zone of central and apical granules. A nonspecific staining was noted when tissues were reacted with peroxidase-antiperoxidase complex alone. This troublesome artifact was abolished by preincubating tissues with human IgA. This step did not affect the specific immunostaining for lysozyme yet nonspecific staining was absent from specificity and method controls carried out for both immunocytochemical procedures. The presence of high levels of lysozyme in the endocervical epithelium of estrous rabbits was also confirmed in enzymatically isolated endocervical epithelia using the lysoplate method of Osserman and Lawlor (1966). Mucous granules and immunostainable intracellular lysozyme were abundant during estrus, decreased during early pseudopregnancy, and were absent after long-term ovariectomy. However, they were restored by the administration of estradiol (5 micrograms/12 hours/10 days) to ovariectomized animals. These data indicate a common hormonal regulation and secretory mechanism for endocervical mucous glycoproteins and lysozyme.

Three cytologically distinct cell populations were identified, in addition to ciliated cells, when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit endocervical cells. Two of these cell populations contained histochemically distinguishable (periodic acid- Schiff [PAS]) mucoproteins and were designated vacuolated and granular PAS-positive cells. The third, designated as vacuolated PAS-negative, did not contain secretory granules. Cell integrity was confirmed by trypan blue dye exclusion, [(3)H]leucine incorporation, and ultrastructural analysis. To demonstrate hormonal modulation of endocervical cell morphology, cell distribution profiles were compared from animals in different hormonal states. In the absence of estrogen dominance, PAS- positive cells from 5-d pseudopregnant rabbits were reduced 50 percent, while vacuolated PAS-negative cells increased fourfold as compared with estrous cell populations. The PAS-positive cells sedimented toward the top of the gradient where the bovine serum albumin concentrations were lower, consistent with a reduction in the number of secretory granules. In the sustained absence of ovarian steroid hormones, the number of PAS-positive mucous cells from ovariectomized rabbits was reduced to only 4 percent of the total endocervical cell population. The biosynthetic capacity of isolated endocervical cells was determined by incubating the three nonciliated cell populations from estrous and 5-d pseudopregnant rabbits for 36 h with the mucin precursor, [(14)C]N-acetyl- D-glucosamine. Only PAS-positive cells incorporated significant amounts of labeled precursor. This study indicates that steroid hormones influence cervical secretions by modulating the type of endocervical cells.

The distribution and morphology of rabbit endocervical mucous cells before and after mating were studied histochemically with Alcian blue, pH 2.5, and periodic acid-Schiff (AB-PAS) and by scanning and transmission electron microscopy. Before mating, the lower and upper segments of the endocervical canal contained approximately 75% and 93% of cells engorged with AB-PAS positive granules. Two hours after mating, the percentage of histochemically positive cells decreased to less than 46% and 85%, respectively. Before insemination, mucous cells were prominent among ciliated cells and contained numerous secretory granules some of which appeared to be undergoing release through exocytosis. Granule-engorged apices of mucous cells were rarely seen after insemination. Instead, cells displayed a markedly reduced complement of mucous granules and their apical surface contained numerous exocytotic pits. In many areas, a blanket of mucous material covered mucous and ciliated cells. As for the observed variation in mucous cell distribution, these changes were predominant in the endocervical mucosa of the lower cervix. We have previously shown that both native and isolated mucous cells respond to serum with rapid and massive granule release. Degranulation of endocervical mucous cells in response to semen may play an important role in regulating sperm access to the upper reproductive tract under physiologic and pathologic conditions.

The application of a unit gravity sedimentation procedure to monodispersed rabbit tracheal cells resulted in the isolation of enriched (2-fold to 2.5-fold) basal and mucous cell populations. Cellular integrity was confirmed by a trypan blue dye exclusion index of 93%, [3H] leucine incorporation, and ultrastructural analysis. Unit gravity sedimentation is an affective and rapid procedure for obtaining viable, homogeneous preparations of basal and mucous cells that may be used for in vitro studies of cellular proliferation, differentiation, and glycoprotein biosynthesis in respiratory mucous epithelia.

The presence of tubulin throughout what appears to be sperm penetration tunnels of in vivo fertilized rabbit oocytes was demonstrated by both immunofluorescence and transmission electron microscopy, using fluorescein and peroxidase-labeled antibodies, respectively. In approximately half the fertilized oocytes examined, intact microtubules were found at the point of entry of the spermatozoon into the zona pellucida, while amorphous deposits were found throughout the remainder of the tunnel, starting at the point of entry into the matrix of the corona radiata cell layer, and continuing to the perivitelline space. These continuous deposits of tubulin suggest that, in the rabbit, acrosomal microtubule-like structures may perform a role in mammalian fertilization, possibly as an enzyme binding or delivery system, although other functions are possible. No deposition of actin was detectable in these tunnels.

A retrospective study of testicular histology was carried out in 21 prepubertal boys and in 11 intrapubertal or postpubertal boys who died from 1 day to 1 year after treatment with chemotherapy (CT) with or without radiation therapy (RT) for extragonadal solid tumors. Based on autopsy findings, profound histologic changes were found in six prepubertal boys and in all but one of the intrapubertal and postpubertal boys. For three of the prepubertal and five of the postpubertal boys with altered testicular histology, the contribution of pelvic RT could not be excluded. When prepubertal patients were evaluated according to nutritional status, a thickened tubular wall and absent or severely reduced spermatogonia were found in only 1 of 11 adequately nourished boys as compared with 5 of 10 malnourished boys. In intrapubertal and postpubertal boys, the tubular wall was thickened in six and these changes were independent of nutritional status. All malnourished intrapubertal and postpubertal patients demonstrated impaired spermatogenesis (versus 67% of age-matched malnourished controls), whereas similar impairment was noted in 60% with adequate nutrition versus 20% of controls. The diffuse damage of germ cells and tubules in malnourished younger boys suggests that the prepubertal male is not necessarily protected against the reproductive effects of cancer therapy.

Small (60-90 micrometer) and large (100-130 micrometer) preantral follicles were isolated from adult mouse ovaries by a collagenase-dissection technique. These follicles were composed of resting oocytes surrounded either by granulosa cells, only, or by granulosa and undifferentiated theca cells. Further enzymatic dissociation of primary follicles yielded monodisperse cells characterized by abundant rough endoplasmic reticulum, microfilament-rich pseudopodia and only scant lipid droplets. These cells reaggregated, when explanted in stationary culture, forming epithelial cords and structures macroscopically reminiscent of native ovarian follicles. Anticipated association of follicular cells in epithelial-like monolayers was rare (less than or equal to 10% of all cultured cells). Formation and growth of both follicle-like (FLS) and cord-like (CLS) structures occurred within 24 hours of culture, continued for 14 days, and was inhibited by cytochalasin B, but not by neuraminidase. FLS and CLS, as well as cell monolayers, underwent luteinization, as indicated by the presence in the culture medium of radioimmunoassayable progesterone and by frequent cytological features suggestive of active steroidogenesis. The present report indicates that (a) specific cell affinities exist among immature follicular cells which may play a role in folliculogenesis; and (b) follicular cells are endowed, from their early developmental stages with intrinsic steroidogenic capabilities which become phenotypically expressed after escape from the intraovarian environment.

The gonadal histology of 21 prepubertal, intrapubertal, and postpubertal girls who died 1 day to 2 months after cessation of therapy for extragonadal solid tumors was reviewed. In addition to focal or diffuse cortical fibrosis, a reduction in follicle numbers and impaired follicular maturation were observed in cancer patients independent of their pubertal age. These changes appeared to be more severe in malnourished patients and in girls who received multiple agent chemotherapy, with or without irradiation. Both before and after the age of 10 years, most cancer patients had a total number of follicles similar to that of age- and nutrition-matched controls. However, the majority of these girls displayed impaired follicular maturation as demonstrated by reduced numbers of growing and antral follicles compared to controls. Histologic evidence of ovarian damage suggests that the future reproductive performance may be impaired in some female cancer patients treated even before puberty.