In pigs, changes in an unidentified endometrial glycoprotein, pGP30, are temporally associated with rapid trophoblast elongation and initial placental attachment on day 12 of gestation. Identification of endometrial pGP30 was undertaken through protein purification, NH2-terminal amino acid sequencing and cDNA sequencing of products generated through reverse transcription-polymerase chain reaction. Sequencing of 35 amino acids from the NH2-terminal end of pGP30 revealed that the 30 kDa glycoprotein is a cleavage product from the C-terminal region of inter-alpha-trypsin inhibitor heavy chain 4 (I alpha IH4), previously known as inter-alpha-trypsin inhibitor heavy chain-like protein. I alpha IH4 is unique compared with the three other inter-alpha-trypsin inhibitor heavy chains as it does not contain a binding site for bikunin that has serine protease inhibitory activity and is sensitive to cleavage by kallikrein. Endometrial gene expression of I alpha IH4 was detected during the oestrous cycle (days 0-18) and early pregnancy (days 10-18). Gene expression of I alpha IH4 appeared to be enhanced during the midluteal phase (days 12 and 15) of the oestrous cycle and the period of trophoblast attachment (days 12-18). Expression of I alpha IH4 was not detected in day 12 conceptus tissue mRNA. Endometrial expression of I alpha IH4 in pigs may function as an acute phase protein for protection of the uterus from the inflammatory response induced by conceptus attachment to the uterine epithelium.

Endometrial polypeptide synthesis, which is regulated through ovarian steroid secretion and steroid production by the developing conceptus, not only provides the necessary secretory components vital to conceptus development but also presents the adhesive changes in the epithelial surface essential for conceptus attachment. In the present study, a 30-kDa, basic endometrial glycoprotein (pGP30) was isolated and characterized during the estrous cycle and early pregnancy of the pig. Uterine flushings and endometrial culture media were obtained from gilts on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and Days 10, 12, 15, and 18 of pregnancy. A polyclonal antibody was generated to pGP30 after isolation of medium from Day 15 pregnant endometrial cultures separated by gel filtration and PAGE. Western blot analysis indicated that the antiserum reacted with isoforms of pGP30 and cross-reacted with a 90-kDa component in serum that was not removed after cleavage of the oligosaccharide chains from the 90-kDa glycoprotein. Antiserum did not detect a 30-kDa band in media from cultures of kidney, fat, heart, muscle, liver, or serum; however, heart and muscle did contain bands of different molecular masses that cross-reacted with the antiserum. Multiple bands of higher molecular mass (35-40 kDa) were detected in the endometrial cultures from gilts on Days 0 through 10 of the estrous cycle. Treatment of ovariectomized gilts with estradiol-17 beta stimulated a similar response. During the mid- to late luteal phase of the estrous cycle (Days 12-18), the 30-kDa band as well as an additional 32-kDa band was present on Western blots. Administration of progesterone for 14 days stimulated the synthesis of both the 30- and 32-kDa products in ovariectomized gilts. However, only the pGP30 was detected on Days 12-18 of pregnancy. Immunocytochemical localization with antiserum to pGP30 indicated that the glycoprotein is present in the endometrial epithelium, with the surface epithelium demonstrating the strongest reaction product. Discrete changes in staining and cellular localization were observed during the early stages of the estrous cycle (Days 0-5) and the midluteal (Day 10) phase. A similar response was achieved with administration of steroids to ovariectomized gilts. Data indicate that discrete changes in epithelial synthesis of the endometrial glycoprotein occur at the time of conceptus trophoblastic elongation and placental attachment in the pig.

Cathepsin L has been proposed to be involved with the endothelial-chorial type of placentation in the cat. Little information concerning the presence and secretion of cathepsin L is available for a species with noninvasive epitheliochorial placentation such as the pig. Cathepsin L activity in uterine flushings and endometrium from gilts during different days of the oestrous cycle and early pregnancy was analysed through specific substrate metabolism and Western blot analyses with antiserum against cat endometrial cathepsin L. This antiserum was utilized to determine the cellular localization of the enzyme within porcine endometrium. Cathepsin L activity within uterine flushings was elevated on Day 15 of the oestrous cycle and early pregnancy, with activity declining on Day 18. Cat cathepsin L antiserum cross-reacted with a group of 46, 40 and 38 kDa uterine proteins and detected a product within the surface and glandular epithelium of the endometrium. The appearance of the 40 kDa protein was first detected on Day 10 of the oestrous cycle with the 38 kDa proteins appearing on Day 15 and 18 of pregnancy. The 40 and 38 kDa uterine proteins appear to be steroid regulated as 12 days of progesterone administration is necessary to detect the proteins and cathepsin L activity.

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