Neonatal programming by immunological challenge: effects on ovarian function in the adult rat

Abstract

Neonatal exposure to an immunological challenge (lipopolysaccharide, LPS) increases the activity of hypothalamo-pituitary-adrenal axis and sensitises the GNRH pulse generator to the inhibitory influence of stress in adult rats. We investigated the effects of neonatal exposure to LPS on various reproductive parameters during puberty and into adulthood in female rats. LPS (50 μg/kg, i.p.) or saline was administered on postnatal days 3 and 5. Vaginal opening was recorded, and oestrous cyclicity was monitored immediately post puberty and again at 8-9 weeks of age. At 10 weeks of age, the ovaries were removed and the number of follicles was counted, together with the thickness of the theca interna of the largest antral follicles. Ovarian sympathetic nerve activity was assessed immunohistochemically by measurement of the levels of ovarian low-affinity receptor of nerve growth factor (p75NGFR). In rats exposed to LPS in early life, there was a significant delay in puberty and disruption of oestrous cyclicity immediately post puberty, which persisted into adulthood. The follicle reserve was decreased, the thickness of the theca interna increased and the expression profile of ovarian p75NGFR increased in the neonatal LPS-treated animals. These data suggest that exposure to LPS during early neonatal life can have long-term dysfunctional effects on the female reproductive system, which might involve, at least in part, increased ovarian sympathetic nerve activity.

Figure 1

Effects of neonatal exposure to LPS (LPS, 50 μg/kg i.p.) on postnatal day 3 and 5 on body weight. There was no significant difference in body weight gain between neonatal LPS (neo-LPS)-treated and neonatal saline (neo-saline) controls (n=37–42 per group).

Figure 2

Effects of neonatal exposure to LPS on oestrous cyclicity immediately post puberty and in adulthood. Representative examples of oestrous cyclicity are illustrated in (A) and (B). Normal oestrous cyclicity is defined as having at least two consecutive normal cycles (i.e. cycle length 4–5 days; oestrus phase 1–2 days). The percentage of normal oestrous cyclicity was decreased at puberty and in adulthood of neonatal LPS-treated rats compared with neonatal saline controls (C). P, prooestrus; E, oestrus; M, metoestrus; D, dioestrus. *P<0.05 versus neonatal saline controls (n=37–42 per group).

Figure 3

Effects of neonatal exposure to LPS on the thickness of the theca interna layer of the largest follicles in adult ovary. Representative examples of the theca interna layer of the largest follicle in adult ovaries are illustrated in A and C (neonatal saline controls) and B and D (neonatal LPS-treated rats). The thickness of the theca interna layer of the largest follicles was increased at oestrus, metoestrus and dioestrus in neonatal LPS-treated rats compared with neonatal saline controls (E). CL, corpus luteum; GC, granulosa cell; TC, theca cell; arrows show the theca interna layer. Photomicrographs illustrate dioestrus stage of oestrous cycle. Scale bar=50 μm. *P<0.05, versus neonatal saline controls (n=8–11 for each group).

Figure 4

Effects of neonatal exposure to LPS on the immunoreactivity of p75NGFR in theca interna cells of the adult ovary. Low intensity of immunoreactivity for p75NGFR was detected in theca cells, especially theca interna (arrows) of growing follicles (preantral and antral follicles) from neonatal saline controls (A). The immunostaining for p75NGFR in the theca interna cells of preantral and antral follicles was significantly higher in the neonatal LPS-treated rats (B). (C) shows a higher magnification of the boxed area in (B). (D) is a negative control with no staining in the adjacent section incubated in absence of primary antibody. H-scores of immunostaining intensity for p75NGFR in theca interna cells are summarised in (E) and (F). There was no significant difference in staining intensity between different phases of the oestrous cycle. GC, granulosa cell; TCI, theca interna cell; TCE, theca externa cell. Photomicrographs illustrate dioestrus stage of oestrous cycle. Scale bars: A, B and D=20 μm; C=10 μm. *P<0.05, versus neonatal saline controls (n=8–11 for each group).