Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery, Sex and Betamethasone Exposure

Abstract

The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms.

Fig 1. Comparison of Exon 2 start codons for the NCR31 gene in the guinea pig, Human, Monkey, Rat and Mouse.

Start codons are positioned at methionine residues 1, 27, 86, 90, 98, 316, 331, 336 which result in the translation of GRα-A, B, C1, C2, C3, D1, D2, and D3. GRα-B is not translated in the guinea pig due to the presence of isoleucine at position 27. Alternative start codon (M, AUG) positions of human GR are indicated in the diagram. M27 in guinea pig GR was replaced by an isoleucine (I).

Fig 2. Guinea pig glucocorticoid receptor isoforms in the placentae of pregnancies delivered preterm or at term and in the presence and absence of betamethasone exposure.

Glucocorticoid receptor isoforms were measured in cytoplasmic and nuclear fractions of guinea pig placentae. Human placental tissue was used as a positive control and present in lanes 1 and 2 of Fig 2a and 2b. Fig 2a represents female guinea pig placental tissues delivered preterm and exposed to betamethasone (BTPTF) or term female placentae exposed to betamethasone (BTTF) and compared to control preterm females (CPTF) and control term females (CTF). Fig 2b represents placental tissues of male guinea pigs delivered preterm and exposed to betamethasone (BTPTM) or term male placentae exposed to betamethasone (BTTM) and compared to placentae of control preterm males (CPTM) and control term males (CTM).

Fig 3. A preabsorption control was conducted to identify nonspecific binding of the antibody.

GR antibody was preabsorbed with control peptide prior to Western Blot exposure. The left panel represents binding after GR antibody preabsorption and the right panel is the blot exposed to GR antibody alone. Human placental tissues was included as a positive control.

Fig 4. Summary of GR isoform expression in guinea pig placenta in relation to fetal sex, gestational age and betamethasone exposure.

The circles represent a cell with the smaller circle being the nucleus and the larger being the cytoplasm. Isoforms that were present under each condition are in small font while isoforms that were significantly altered by treatment, or gestational age are in bold. Fig 4a represents female placentae and 4b represents male placentae. This figure was generated from Table 2 and isoforms were included in the figure if the median value was greater than 0 regardless of whether GR expression was significantly altered or not.