Evidence for a dual source of relaxin in the pregnant rat: immunolocalization in the corpora lutea and endometrium.

Uterine tissue was collected from intact pregnant rats on days 4, 10-15, 18, and 22 of pregnancy and day 2 postpartum and from rats on day 22 of pregnancy after ovariectomy (day 9) and progesterone-estrogen treatment. Placental, cervical, mammary, and pituitary tissues were collected from intact pregnant rats on day 22. Ovarian tissue was collected from intact pregnant rats on days 15, 20, and 22. These tissues were evaluated for relaxin immunostaining at the light microscope level using an antiserum to purified rat relaxin and the avidin biotin immunostaining technique. Since the corpus luteum is the major source of relaxin in the rat, this tissue was used as a positive control. Relaxin immunostaining in the corpus luteum was observed in a discrete region of the cytoplasm. This pattern of staining corresponded with the discrete clustering of relaxin-containing secretory granules found in the rat luteal cells. Relaxin was not observed in the day 22 placenta, cervix, mammary gland, pituitary gland, or day 4-13 pregnant uterus. Relaxin immunostaining was detected in endometrial epithelial cells on days 14-22 of pregnancy and day 2 postpartum. Relaxin immunostaining was more evident at implantation sites than between implantation sites. Antimesometrial epithelial cells at implantation sites contained relaxin immunostaining. The mesometrial surface, which consists of the placenta and decidualized endometrium, did not contain relaxin immunostaining. Relaxin immunostaining was also present in the endometrial epithelial cells of the day 22 pregnant rats that had been ovariectomized on day 9 and given subsequent replacement therapy with ovarian steroids. The presence of relaxin immunostaining in the ovariectomized rat indicates the endometrium is not sequestering relaxin secreted from the corpus luteum. These data provide evidence that the pregnant rat endometrium is a source of relaxin in addition to the corpus luteum. The appearance of relaxin in endometrial epithelial cells after implantation and its prominence at implantation sites may indicate that locally secreted conceptus factors stimulate endometrial relaxin synthesis.

Monoclonal antibodies specific for rat relaxin. VI. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts histological changes associated with cervical softening at parturition in rats.

Relaxin, which is secreted by the corpora lutea throughout the second half of rat pregnancy, promotes the growth and softening of the cervix. The mechanisms at both the cellular and molecular levels by which relaxin brings about these effects remain to be determined. The present study examined the influence of endogenous relaxin on the histological changes associated with cervical softening. A monoclonal antibody for rat relaxin, designated MCA1, was injected iv daily on days 12-22 of gestation. Cervices were removed on day 22, fixed in 10% buffered formalin, embedded in paraffin, and processed for histological staining. Tissue sections (5 microns thick) were stained with Gomori's trichrome stain (collagen), Orcein stain (elastin), or periodic acid-Schiff (polysaccharide). Qualitative and quantitative analyses identified several histological parameters in MCA1-treated rats that differed markedly from those in control rats. Cervices obtained from MCA1-treated rats contained collagen fiber bundles with greater compactness; elastin fibers with greater density, length and interdigitation; arteries with smaller cross-sectional areas; and luminal involutions with smaller areas than controls. The cervices of MCA1-treated rats appeared to contain fewer vacuolated epithelial cells, which secrete polysaccharide-rich material, than did cervices obtained from controls. It seems plausible that most, if not all, of these relaxin-induced modifications of the histological characteristics of the cervix facilitate birth.

Monoclonal antibodies specific for rat relaxin. V. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts development of the mammary apparatus and, hence, lactational performance in rats.

There were two related objectives to this study. The first was to determine the influence of relaxin on development of the mammary apparatus (nipples and glands) during the second half of pregnancy. The second was to determine whether the relaxin-dependent development of the mammary apparatus was required for normal postpartum lactational performance. Both objectives were accomplished by neutralizing endogenous relaxin throughout the second half of pregnancy with a monoclonal antibody specific for rat relaxin (MCA1). MCA1 was administered iv to rats daily from days 12-22 of pregnancy. On day 22 the morphology of the mammary apparatus of MCA1-treated rats differed from that of controls; nipples were dramatically smaller, collagen fibers had significantly greater mean density and consistency, and elastin fibers had greater mean density, length, and interdigitation. In addition, the mean number of alveoli surrounding lactiferous ducts was significantly smaller in MCA1-treated rats than in controls. There were no differences between MCA1-treated rats and controls in the mean thickness of connective tissue surrounding ducts, the height or density of luminal cells lining lactiferous ducts, or the sizes of either adipocytes or arteries. To examine lactational performance, MCA1-treated and control rats were cesarean sectioned between 2100-2400 h on day 22 of pregnancy and given foster pups born of untreated intact donors. Although both MCA1-treated rats and control rats exhibited a high incidence of maternal behavior after cesarean delivery, mean pup weight and incidence of live pups declined markedly during days 1-5 of fosterage in MCA1-treated rats compared to controls. Furthermore, unlike controls, there was no observable postpartum nipple development in MCA1-treated rats by day 5 of fosterage. Mammary glands obtained from MCA1-treated rats on day 5 of fosterage had markedly lower mean weight than controls. This study demonstrates that passive immunization of endogenous relaxin throughout the second half of pregnancy disrupts development of the nipples and mammary glands in the rat. Moreover, it establishes that relaxin's effects on the development of the mammary apparatus during pregnancy are essential for growth and survival of the young during lactation.