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The Distribution of Immunoglobulin-containing Cells in the Goat Mammary Gland: A Morphometric and Immunohistochemical Study.

 ABSTRACT
The distribution of immunoglobulin (Ig)-containing cells in the mammary gland of the goat was studied at different stages of the reproductive cycle using immunohistochemical techniques. Generally, Ig-containing plasma cells (IgA, IgG and IgM) in the mammary gland were abundant during pregnancy, increase in number after parturition and gradually decrease in number, especially in the post weaning period. IgA-containing cells were the predominant cell type among the plasma cells throughout the reproductive cycle. Their number increased at late pregnancy, being maximum at the colostrum period and decreased thereafter. IgG- and IgM-containing plasma cells were fewer at all stages of the reproductive cycle; the former were slightly more than the latter. Both types of cells followed a distribution pattern similar to that shown by IgA-containing cells. The rich existence of plasma cells at late pregnancy and colostrum period suggests that their main role is transfer of passive immunity to the neonates rather protection of the mammary gland.
Keywords: Immunoglobulin-containing cells, Plasma cells, Immunohostochemistry, goat.

INTRODUCTION
The mammary gland not only provides a vital nutrient source to the offspring, but also serves as a necessary source of immunoglobulin. Immunoglobulin in the mammary gland and secretion can be either derived from the blood circulation or produced locally by plasma cells underlying the epithelium (Hurley and Theil, 2011). In ruminants, there is no transplacental shift of immunoglobulin’s to the fetuses but they are passively transferred immediately postpartum (Butler et al., 1972).
Immunoglobulin-containing plasma cells have been demonstrated by immunohistochemical methods in mammary glands of the sow (Brown et al., 1975), cow (Sordillo and Nickerson, 1988; Sordilo et al., 1988), sheep (Lee and Lascelles, 1970), mouse (Weisz-Carrington et al., 1977) and rat (Lee et al., 1978). In addition, many authors have determined the concentrations of different classes of immunoglobulin’s in the colostrum and milk of the cow (Penhale and Christie, 1969; Guidry et al., 1980; Norcross, 1991; Hidiroglou et al., 1992) and human (Chernishov and Slukvin, 1990, Almogren, 2007).
On the other hand, to date, no report is available for immunoglobulin’s or immunoglobulin-containing plasma cells in the goat mammary gland. Hence the present Immunohistochemical study was carried out to reveal the distribution and frequency of occurrence of plasma cells in the goat mammary gland at different stages of the reproductive cycle.

MATERIALS AND METHODS
Animals
Mammary glandular tissue was collected from 4 non-pregnant (resting period), 6 pregnant (mid- and late-pregnancy) and 15 lactating (colostrum period and 15, 30, 60 and 90 days after parturition) Saanen goats. Some animals were euthanized with an overdose of sodium pentobarbital (Nembutal, Abbot laboratories, North Chicago, USA), in other animals, biopsy samples were surgically obtained under local anesthesia (Marx et al., 1963). All animals were clinically normal at the time of sampling.

Tissue preparation
Collected fresh mammary tissues were fixed in Bouin’s fluid or periodate-lysine paraformaldehyde (PLP) (McLean and Nakane, 1974). Paraffin blocks were prepared according to the routine method. Sections, 2-3 μm thick, were cut and stained either for general histology or immunohistochemistry for immunoglobulin-containing cells.

Antibodies and antigens
Anti-goat IgA, IgG or IgM sera were used and their characteristics are shown in Table 1.

Immunohistochemical Staining
Immunohistochemical staining for plasma cells was carried out using peroxidase-anti-peroxidase (PAP) method for the demonstration of 1gG- and IgM-containing plasma cells. Avidin-biotin-peroxidase complex (ABC) method was used for IgA-containing plasma cells. Briefly, the tissue sections were treated with 0.1% hydrogen peroxide and then with absolute methanol for 30 min. in each step to remove endogenous peroxidase activities. Subsequently, non-specific reactions were blocked by incubation with 5% sheep serum in 1% bovine serum albumin (BSA) for 1h. at room temperature (RT), and incubated with primary antibody for 24 h. at 4º C (Table 1). Following rinsing, the sections were incubated for 1h. At RT with either sheep anti-rabbit IgG (non-conjugated) or biotinylated sheep anti-rabbit 1gG for PAP or ABC method, respectively. For IgG and IgM rabbit PAP (E. Y Laboratories, CA. USA) was applied as the third antibody at 1:100 dilution for 1h. At RT. As for IgA avidin-biotin-peroxidase complex (Vector Laboratories, CA. USA) was applied for 30 min. in RT. After rinsing, all tissue sections were developed with 3.3- diaminobenzidine tetrahydrochloride (DAB) solution containing 0.1% H2O2 and slightly counterstained with hematoxylin. As a negative control, either rabbit normal serum or 0.1M phosphate buffered saline (PBS) was used to replace the primary antibody.

Microscopy and Statistical Evaluation
Forty fields per section (each field depicting a rectangle of 0.1 mm2) were examined to obtain an estimate of cells present in tissues at X400 magnification using an eyepiece micrometer of 19x19 squares (Olympus Optical Co., Tokyo) according to the point-count technique (Weibel, 1969). The mean and SD were calculated and comparison between various stages was made using students t-test.

RESULTS
Immunoglobulin-containing plasma cells
Examination of hematoxylin eosin-stained sections of mammary tissue from young goats revealed the presence of many interlobular ducts and aggregation of cells representing the undeveloped alveoli. In spite of the existence of many infiltrating cells, no plasma cells could be observed in the mammary tissue of young goats.
The adult mammary gland at mid-pregnancy was characterized by the presence of many alveoli and ducts. Much development of the mammary gland was noticeable with the advance of the pregnancy and numerous alveoli and ducts were seen at late pregnancy. At this stage both alveoli and ducts were distended with homogeneous secretions in luminal space (Fig. 2 a,b,c,d).
Immunohistochemical staining showed that the number of plasma cells increased from mid-pregnancy, with rapid increase at late pregnancy (Fig. 1A). At pregnancy, IgA-containing plasma cells were the predominant cell type, while IgG- and IgM-containing plasma cells were less numerous, the former being slightly more than the latter (Fig. 1B, 2, 3, 4, 5 and 6). The secretions in the lumen of the alveoli showed an intense positive reaction to antiserum against IgG and a less intense reactivity to the antisera against IgA and IgM. Occasionally, alveolar cells themselves stained positively for all immunoglobulin’s (Fig. 2 a,b,c,d).
With the onset of lactation the mammary gland was more developed, especially between 30 and 60 days of the lactation period. The glandular tissue occupied much area of the mammary gland, while the connective tissue areas were diminished, being represented only by the scanty interalveolar regions. The secretions in the expanded lumen of the alveoli, characterizing the pregnant mammary gland were hardly seen during lactation (Fig. 7 and 11). At the colostrum period the number of IgA-containing plasma cells reached a peak and decreased after Day 15 of lactation, remaining low thereafter (Figs. 8 and 12 ). Statistical analysis revealed that IgA-containing plasma cells were significantly higher than both IgG- and IgM-containing cells during the entire period of lactation (Fig.1 A, B). IgA cells were mainly localized in the vicinity of epithelium or in the interalveolar area (Fig. 8). IgG and IgM cells showed a similar pattern of distribution, but they were more frequently located in the connective tissue (Fig. 9, 10, 13 and 14).
The abrupt cessation of suckling induced involution of the mammary gland. After post-weaning Day 7 the alveoli were distended with secretions and large fat droplets were seen both in the cytoplasm of epithelial cells and luminal contents. Much destruction occurred at post-weaning Day 21 and the alveoli lost their normal structure. The immunoglobulin-containing plasma cells significantly decreased in number with the IgA cells still more than IgG and IgM cells. The luminal contents of the alveoli and some epithelial cells stained positively for the different immunoglobulin.  

DISCUSSION
The data presented here show that IgA-containing plasma cells are the predominant cell type in the goat mammary gland throughout the reproductive cycle. This holds true for the situation in the mammary gland of some mammals including the sow (Brown et al., 1975), mouse (Weisz-Carrington et al., 1977, Van Der Feltz et al., 2001), rat (Lee et al., 1978) and human (Brandtzaeg, 1983). The IgA cells in the goat mammary gland increased in number during pregnancy and their number became maximal at the colostrum period. Their rich existence, especially in the colostrum period, may reflect their contribution to the immunity of the neonates and correspond to the findings that ruminant offsprings are born with low levels of immunoglobulin’s (Larson et al., 1980). The high prevalence of IgA cells in the glandular tissues also supports the idea that IgA in the milk is largely derived from local synthesis (Butler, 1974).
IgA is the major immunoglobulin in the colostrum of humans and rodents (Bistany and Tomasi, 1970; Eddie et al., 1971; Weisz-Carrington et al., 1977; Chernishov and Slukvin, 1990).
IgG- and IgM-containing plasma cells were few in the goat mammary gland at all stages of the reproductive cycle; IgG cells were slightly more than IgM cells. Both cell types followed a distribution pattern similar to that shown by IgA cells, their numbers increased at late pregnancy, being maximal at the colostrum period and decreased thereafter. In contrast, Yurchak et al., (1971) and Lee and Lascelles (1970) reported that IgG-containing plasma cells were predominant in the bovine and ovine mammary glands respectively.
The present investigation showed that the number of plasma cells in the goat mammary gland increased at late pregnancy and early lactation. During the same duration of time T lymphocytes also increased in number (Ismail et al., 1996), suggesting a functional relation between T lymphocytes and plasma cells. Lee et al. (1992) reported that CD4+ cells in the sheep mammary gland play a regulatory role in local B-cell development and antibody production. The paucity of IgG-containing plasma cells in the goat mammary gland in comparison to the high concentration of IgG in the milk (Ismail, 2015, submitted data) lead to the suggestion that IgG in the goat mammary gland is largely serum derived, which is consistent with the finding in the cow and sheep (Nickerson et al., 1985; Larson et al., 1989) and human (Hurley and Theil, 2011).

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