Expression of endogenous betaretroviruses in the ovine uterus: effects of neonatal age, estrous cycle, pregnancy, and progesterone

Abstract

The ovine genome contains 15 to 20 copies of endogenous retroviruses (enJSRVs) highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus. enJSRVs are highly expressed in the endometrial lumenal epithelia (LE) and glandular epithelia (GE) of the ovine uterus. The effects of neonatal age, estrous cycle, pregnancy, and progesterone on expression of enJSRVs in the ovine uterus were determined. Expression of enJSRV RNAs was absent from the uterus of ewes at birth, but enJSRV RNAs were expressed specifically in the LE and developing GE from postnatal day (PND) 7 to PND 56. In adult ewes, enJSRV RNAs were detected only in the epithelia of the uterine endometrium, as well as epithelia of the oviduct, cervix, and vagina. In cyclic ewes, endometrial enJSRV RNA abundance was lowest on day 1, increased 12-fold between days 1 and 13, and then decreased to day 15. In pregnant ewes, levels of endometrial enJSRV RNAs were high on day 11, increased to day 13, and then decreased to day 19. In day 17 and 19 conceptuses, enJSRV RNAs were also detected in binucleate cells of the trophectoderm. Immunoreactive JSRV capsid and envelope proteins were detected in the endometrial LE and GE, as well as in the binucleate cells of the conceptus. In transfection assays utilizing ovine endometrial LE cells, progesterone increased transcriptional activity of several enJSRV long terminal repeats. Collectively, these results indicate that transcription of enJSRVs in the endometrial epithelia of the ovine uterus is increased by progesterone and might support a role for enJSRVs in conceptus-endometrium interactions during the peri-implantation period and early placental morphogenesis.

FIG. 1

In situ hybridization analysis of enJSRV mRNA expression in the developing neonatal ovine uterus. Cross-sections of the uterine wall from neonatal ewes (PND 0 = birth) were hybridized with α-³⁵S-labeled antisense or sense ovine enJSRV cRNA probes as described in Materials and Methods. Protected transcripts were visualized by liquid emulsion autoradiography for 1 week and imaged under bright-field or dark-field illumination. S, stroma; M, myometrium. Magnification, ×260.

FIG. 2

In situ hybridization analysis of enJSRV mRNA expression in different tissues of the adult ovine female reproductive tract. Cross-sections of different regions of the female reproductive tract from day 9 pregnant ewes were hybridized with α-³⁵S-labeled antisense or sense ovine enJSRV cRNA probes as described in Materials and Methods. Protected transcripts were visualized by liquid emulsion autoradiography for 1 week and imaged under bright-field or dark-field illumination. Magnification, ×260.

FIG. 3

Effects of day of the estrous cycle or early pregnancy on expression of enJSRV mRNAs in the ovine endometrium. Slot blot hybridization analysis of enJSRV mRNAs in endometrium from cyclic and pregnant ewes was performed as described in Materials and Methods. Steady-state levels of enJSRV mRNAs are presented as LSM TCs with SE.

FIG. 4

In situ hybridization analysis of enJSRV mRNA expression in the uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with [α-³⁵S]-labeled antisense or sense ovine enJSRV cRNA probes as described in Materials and Methods. Protected transcripts were visualized by liquid emulsion autoradiography for 1 week and imaged under bright-field or dark-field illumination. S, stroma; M, myometrium; TE, trophectoderm. Magnification, ×260, except for the 17P panel at the bottom (×520).

FIG. 5

Immunoreactive JSRV capsid (A) and env protein (B) expression in the ovine endometrium. Immunofluorescence staining of frozen uterine sections from cyclic (C) and pregnant (P) ewes was conducted with specific antibodies or irrelevant IgG as a control as described in Materials and Methods. TE, trophectoderm. Magnification, ×230.

FIG. 6

Effects of progesterone on activity of several enJSRV LTRs in transient transfection assays utilizing ovine endometrial LE cells. Results are expressed as fold increases relative to activity of the reporter constructs without the addition of progesterone. All assays were conducted in triplicate with at least two independent experiments. Results are presented as mean fold activation with SE.

FIG. 7

Schematic representation of progesterone levels in the peripheral blood relative to expression of the PR protein in the LE and GE in cyclic and early pregnant ewes. The relative amount of PR protein in the LE and GE is noted as absent (open circles), low abundance (partially [gradient] filled circles), and abundant (solid circles).